The aim of the present work is to improve understanding and properties of a hydrogel based on silanized cellulose derivative which serves as a biomaterial for temporary scaffold for articular cartilage reconstruction. The inorganic derivatization of a polysaccharide by siloxane functional group enables us to obtain an injectable gel polycondensating in situ by simple pH reduction. This allows this biohydrogel to adapt perfectly to the bone surface and makes it suitable for micro-invasive surgery. The association with chondrocytes favours the cartilage self-healing.
The addition of only 3 wt% of mesoporous silica nanofibers (NFs) during the hydrogel synthesis increases 5 times the compressive modulus of such nanocomposite Si-HPMC/NFs hydrogels. The NFs can bind to the silanized polysaccharide by the siloxanes bridges and thus enhance the mechanical properties. A good stem cells and chondrocytes viability is observed within the Si-HPMC/NFs hydrogels. Such reinforced nanocomposite hydrogels should increase the cartilage self-healing process.
This talk will be focused on the study of the hierarchical structure of the hydrogel, which shows nanopores as well as micropores, along with the study of the dynamics of confined water within the hydrogel, both aspects being decisive for cell growth and for the transport of nutriments necessary for cell seeding. Diverse tools will be used, such as rheology, cryomicroscopy, calorimetry (DSC), 1H liquid NMR spectroscopy (cryoporometry and PGSE) and quasi-elastic neutron scattering (QENS). We will evidence here, that even though the Si-HPMC hydrogel is considered as a solid (gel), its water dynamics is highly similar to that of bulk water.

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT® (Particle Replication In Non-wetting Templates) technique. Low crosslinking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained, without a significant effect on particle stability, shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1,000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.

Mankind has always been confronted with failing tissues and organs. As a consequence, medicine has tried to offer solutions for conditions which might, in some cases, be life threatening. Throughout the years, nature-inspired biomaterials have been one of the approaches which have been deemed useful.
In our laboratories at Ghent University, we have initiated in 2006 a large research platform in which gelatin and its derivatives are screened for a large variety of biomaterial applications. In collaboration with various national and international research groups, we have been developing and studying porous gelatin or gelatin-coated scaffolds as cell carriers. In the present work, we report on porous gelatin scaffolds to be applied for liver tissue engineering applications.
Due to the solubility of gelatin at physiological conditions, a strategy has been elaborated to chemically functionalize part of the gelatin amino acids with cross-linkable groups enabling permanent cross-linking. While the first generation scaffolds using these gelatin derivatives were developed using a cryogenic treatment of permanently cross-linked gelatin-methacrylamide, technological capabilities have significantly evolved. This has among other resulted in the development of perfectly interconnecting scaffolds using the Bioscaffolder technology. Starting from a scan of a part of the human body, the technology enables the production of patient specific implants.
In addition to the material aspects, the presentation will also cover the biological properties of the developed scaffolds.
Acknowledgement
The authors would like to acknowledge the following funding agencies: Alexander Von Humboldt Foundation, IWT (SBO project HEPSTEM, contract number 990066), FWO and UGent (BOF project 2009-2013 and the UGent Multidisciplinary Research Partnership Nano- and biophotonics 2010-2014).

Gradients in cell types, cell quantities, and extracellular matrix molecules represent a common feature of many biological systems, especially at the interface of two different tissues such as tendon and bone, where the cell phenotype gradually changes from fibroblasts in tendon to osteoblasts in bone. The perturbation of the gradients can trigger a cascade of events that either lead to tissue homeostasis or, more often, scar formation. Therefore, the ability to generate controllable gradients in cell density is critical to the recreation of functionality for many types of tissues. Many methods of generating gradients in cell density had been exploited, most of which relied on the ability of the cells to migrate and adapt the pre-established, template gradients of chemical, mechanical or electrical signals. There are three main drawbacks associated with these methods: 1) long experiment cycle, lasting for months; 2) low reproducibility, compromising the clinical application; 3) foreign stimuli, inducing unnecessary differentiation of the cells. Herein, a simple and versatile method of generating gradients in cell density was developed. By inserting a substrate into a homogeneous suspension of cells at a tilt angle, a gradient in cell density could be generated on the surface of the substrate due to the gradual change in the number of cells available for sedimentation. The pattern of the gradient could be altered by changing the tilt angle. Using this method, gradients in cell density maintaining the original status of the cells can be harvested within 2 hours with identical pattern among every batch. Reversible gradients of two different types of cells can also be generated by multiple seeding processes. In the current study, reverse gradients of tendon fibroblasts and bone osteoblasts were generated to mimic the native cellular distribution at the tendon-to-bone insertion. These advantages make the graded pattern generated using this method a very promising tool to facilitate interface repair.

Due to their potential applications in biomedical and tissue engineering, protein-based hydrogels have attracted considerable interests. Most protein-based hydrogels take unstructured or non-globular proteins as building blocks. Recently, we have begun to use modular proteins, which consist of individually folded protein domains arranged in tandem, as building blocks to construct both physically and chemically crosslinked hydrogels. Using a small protein GB1 and sequences from the insect protein resilin, we constructed an artificial elastomeric protein that mimics the structure and properties of the giant muscle protein titin. Based on this titin-mimetic protein, we successfully constructed GB1-resilin based-hydrogels that exhibit mechanical properties similar with the passive elastic properties of muscles. These work revealed that folded globular domains could endue hydrogels unique macroscopic mechanical properties that can be tailored at molecule level.
Using a de novo designed Ferredoxin-like protein (short for FL), here we report the construction of a novel protein-based hydrogel that show unusual physical and mechanical properties. We constructed a polyprotein (FL)₈ and found that its aqueous solution can be crosslinked into an opaque solid hydrogel using photochemical crosslinking strategy. This hydrogel shows extraordinary de-swelling behavior upon soaking in phosphate buffer saline, losing about 35% of its original weight by expelling water spontaneously. These phenomena suggest that a large fraction of folded FLs are unfolded by the force generated by the swelling process. Subsequently, the unfolded FLs lead to hydrophobic collapse and the formation of large aggregates, which scatter lights and give rise to the opaque appearance. The unfolding of FL in the hydrogel is consistent with the low mechanical stability of FL (~5 pN) as measured by single molecule force spectroscopy techniques. It is also directly confirmed by fluorescent labeling studies that FLs in the solid material are unfolded after swelling. In macroscopic level, the unfolding of FL leads to the intertwining of physical and chemical crosslinking networks and results in unusual mechanical properties. Tensile tests show that (FL)₈-based hydrogel is highly stretchable and tough. It can be extended up to 500% strain with a Young&’s modulus of ~8.5 kPa. The stretching of (FL)₈-based hydrogel lead to large hysteresis and energy dissipation. The fracture energy is ~1700 J/m². This new protein-based hydrogel demonstrates the feasibility of molecular engineering of hydrogels and may open new applications for protein-based hydrogels.

In this talk I will present our recent results on the fabrication of superhydrophobic -superhydrophilic micropatterns on porous polymer surfaces prepared by UV-initiated polymerization and surface grafting techniques. I will show how such polymer substrates can be used to create precise micropatterns of multiple cell lines and to study cell-cell communication. In addition, I will present our recent developments on using superhydrophobic-superhydrophilic microarrays for the high-throughput screening of both adherent and non-adherent cells (DropletMicroarray). The fabrication of high-density arrays of hydrogel micropads for the screening of cells in 3D microenvironments will be also discussed.
References: E. Ueda et al. Lab on a Chip, 2012; A.N. Efremov et al. Biomaterials, 2012; J.S. Li et al. Langmuir 2012, 28, 8286; F. Geyer et al. Angew. Chem. Int. Ed. 2011, 50, 8424; D. Zahner et al. Adv. Mater. 2011, 23, 3030.

Smart surfaces with the capability to dynamically modulate biological functionality have attracted considerable interest due to their diverse applications in the biomedical and biotechnology fields. Here, we report a system with switchable multifunctional ability based on nanopatterned thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm) brushes.
Nanopatterned PNIPAAm brushes were fabricated via interferometric lithography (IL) combined with surface-initiated polymerization. The thermo-responsive conformational changes of the brushes were characterized by atomic force microscopy in water at different temperatures and a significant topographical difference was observed for samples prepared under an optical IL exposure dose. At 25°C (below the lower critical solution temperature (LCST)), the surface was relatively smooth with weak nanopattern features due to the lateral spreading of PNIPAAm brushes. However, when the temperature increased to 37°C (above the LCST), the pattern appeared. This temperature triggered expanded/collapsed conformational change provides the ability to spatially regulate the concealment and exposure of molecules that are immobilized on the intervals between the patterned brushes.
We choose two biofunctional molecules to demonstrate the utility of nanopatterned PNIPAAm brushes to control the interfacial interaction with mammalian cells and bacteria. First, a common extracellular matrix protein, fibronectin was adsorbed on polymer-free regions of the substrate between brushes. It is found that at 37°C, NIH-3T3 fibroblast cells can adhere, spread and proliferate on this surface to form a confluent layer after 3 days. Lowering the temperature to 25°C promotes the detachment of almost all adhered cells from the surface. These results suggested that controllable attachment and detachment of cells can be achieved by the exposure and concealment of fibronectin via changing the temperature. Second, a biocidal agent, quaternary ammonium salt silane (QAS) was integrated into nanopatterned PNIPAAm brushes and E. coli was used as a model bacteria. Similarly, it is found that above the LCST, collapsed and hydrophobic PNIPAAm chains will facilitate the attachment of bacteria and expose the QAS moieties to kill adhered bacteria; while below the LCST, the swollen and hydrophilic PNIPAAm chains will promote the release of dead bacteria. Our results demonstrate that the controllable adhesion, killing and release ability of the surface can be achieved by simply changing the temperature.
In summary, we develop a system which combines the thermo-switchablity of PNIPAAm and biofunctionality of the integrated molecules. Above the LCST, the surface can promote the bioadhesion and/or exhibit biocidal activity; and lowing temperature below the LCST switches the surface to release the adhered cells or bacteria.

Two pathways for activating macrophages exist. One of these pathways is known as the classically activated M1 pathway, which is achieved through exposure to lipopolysaccharide. M1 macrophages are part of the type 1 T helper (Th1) response and are known as pro-inflammatory cells.1 The other pathway is reached through interleukin-4 and is known as the alternatively activated M2 pathway. M2 macrophages produce pro-angiogenic factors.2 Being able to control the polarization of these cells is very attractive for both drug delivery and tissue engineering applications. One such application lies in tumor associated macrophages (TAMs), which promote tumor growth through the release of angiogenic molecules. Our goal is to use polymeric drug delivery systems to reprogram TAMs such that the produce pro-inflammatory molecules, which will kill neoplastic cells. These polymers will be eventually used to delivery anti-cancer therapeutics to the tumor. Our approach has been to examine polymer functional groups and their effect on macrophage polarization.
In assessing the ability of polymers to reverse the polarization of macrophages, we examined secretion of several markers of both the Th1 and Th2 responses. For the M1 macrophages, these included tumor necrosis factor-α, monocyte chemotactic protein-1, and reactive nitrogen intermediates. Arginase and interleukin-10 were monitored as markers of M2 macrophages. Macrophages were activated in both polarizations and were incubated with polymers modified with different functional groups. Several functional groups were identified as being able to reprogram M2 macrophages - the polarization of TAMs - to produce increased amounts of molecules associated with M1 macrophages.
Functional groups determined to reverse M2 macrophages to a Th1 response were grafted onto hydrogels, which were fabricated as nanoparticles. These nanoparticles were delivered to co-incubated macrophages and cancerous cells to determine the efficacy of the functional groups in reducing the viability of malignant cells. The nanoparticles were also laden with doxorubicin and their release was examined. Future plans in this work include expanding the library and using these surface moieties to deliver drugs to tumors in mouse models.
1. Gordon, S. Alternative activation of macrophages. Nature reviews Immunology 3, 23-35 (2003).
2. Schmid, M. C. & Varner, J. A. Myeloid cells in the tumor microenvironment: modulation of tumor angiogenesis and tumor inflammation. Journal of oncology 2010, 201026 (2010).

Polymer brushes, thin films of surface anchored polymer chains and our building block for new structures, are attractive anywhere a tailored, functional polymer surface is needed. Polymer brushes have been used for many years to alter the polarity or chemical functionality of a substrate. Brushes can be bound to surfaces with a range of curvature and they provide sufficiently dense coverage to readily pacify a surface. Brushes are versatile enough they can be functionalized to selectively bind biomacromolecules, or instead they can be constructed to resist non-specific binding of molecules and living cells. We have recently explored the effect of brush structure and thickness on non-specific binding and cellular attachment. Several polymer brush systems are described to control interaction of biomacromolecules and cells by design of specific and non-specific interactions in polymer brush architectures. “Grown from” and block copolymer brushes are described, both of which provide excellent substrates for study of brush surfaces. Examples of polymer brushes used for sensor creation in which the bush is multifunctional and controls attachment and non-specific binding as well as biomolecular action are provided. Separately, examples of brushes which biogenic mimics are introduced for investigation of cellular interaction are described. Finally, brushes used in non-fouling coatings tailored for marine applications and in which amphiphilic structures play an important role are also discussed.

The current paradigm of tissue engineering has not only focused on designing structurally and characteristically similar biomaterials to the extracellular matrix (ECM), but also considered for reproducing its molecular properties including bioactivity, biocompatibility, and ability to bind growth factors. In this aspect, the concept of multicomposite and multifunctional artificial ECM is important approach in tissue engineering fields to mimic the real ECM for tissue repair. However, it is required that development of facile and efficient technique for multicomponent coating on artificial ECM surface. Recently, we reported artificial ECM mimics based on fusion of mussel adhesive protein (MAP) with the biofunctional ECM peptides. Adhesive properties of MAP enabled efficient immobilization of ECM peptides without any protein and/or surface modifications, which significantly enhanced cellular behaviors on each ECM mimics. Here, we focused on the conjugated bioactive peptides related to bone-cell specific active peptides, such as RGD, basic fibroblast growth factor, and human bone morphogenetic protein-2 (hBMP-2). Diverse biological activities such as adhesion, proliferation, and differentiation on artificial ECM mimic mixture-coated surfaces were investigated for several bone cell lines. We found that multicomposite artificial ECM showed superior abilities on cells to single component ECM mimics. The osteogenic differentiation of MC3T3-E1 cells was also characterized by evaluating expression of several osteogenic differentiation markers including RUNX2, osterix, osteocalcin, bone sialoprotein, bone morphogenetic protein-2, and type 1 collagen. After selection of the most effective multicomposite artificial ECM in vitro, we conducted in vivo experiment using multicomposite artificial ECM-coated bone substitutes. Thus, multicomposite artificial ECM based on bone-cell specific active peptide-conjugated MAP can be successfully applied in bone tissue engineering.

An effective surgical procedure for treating periodontal defects involves application of enamel matrix derivative proteins (EMD). Enamel formation is the result of complex extracellular processes that regulate nucleation, growth and organization of its constituent mineral crystals. EMD is thought to favour regeneration of diseased periodontal tissue by mediating the formation of a cellular cementum on the root of the tooth, providing a foundation for growth of the tissues associated with functional attachment.
It is clearly important in this context to understand the response of the EMD to changes in environmental conditions over different time-scales. Various buffers were therefore used to prepare a range of aqueous EMD solutions, whose phase behaviour was studied as a function of heat treatment time at different temperatures. Morphological changes were monitored by optical microscopy (OM), atomic force microscopy (AFM) and transmission electron microscopy (TEM), combined with variable temperature UV-Vis spectroscopy to monitor precipitation. The associated variations the secondary structure of the EMD were investigated by Fourier transform infrared spectroscopy (FTIR) and 3D-cross correlated dynamic light scattering (3D-DLS) was used for systematic determination of the EMD phase diagram.
The EMD solutions were generally found to show a gel-like response at extreme temperatures, but a reduced viscosity under ambient conditions. This behavior could be linked directly to structural changes at the microscopic and mesoscopic scales, e.g. the tendency of the EMD to aggregate, and was also found to be dependent on the method of specimen preparation. These results are expected to provide useful indicators for identifying the effect of specific environmental factors on the in situ physical response of the EMD when employed as a surgical adjunct.

Many products incorporated with nanomaterials are available in the market that may induce adverse health reactions in humans. Recent research have identified toxicity of nanomaterials in human cells and animal models.1, 2 Design and development of functionalized biomaterials that can be used for the removal of toxic nanoparticles (NPs) from environmental samples were reported.3-5 Recently, we have prepared an amine functionalized biomaterial and has been used as a potential adsorbent to remove nanoparticle from aqueous stream. The structure of the surface modified bioadsorbent was established using various physicochemical characterization techniques and the removal efficiency of the adsorbent was analyzed spectrophotometrically. The analytical parameters such as pH, adsorbent dosage, time etc were optimized and the reusability of the adsorbent was studied through regeneration using suitable surface cleaning agent. The prepared biosorbent shows a faster kinetics and the good adsorption capacity with moderate pH, and lesser amount of adsorbent.
Acknowledgement: The authors acknowledge funding support from the National University of Singapore (NUS), SPORE and technical support from Department of Chemistry.
1. Oberdörster et al., Particle and Fibre Toxicology, 2005 (2) 8.
2. Hoet P. H. M. et al., Journal of Nanobiotechnology, 2004(2) 12.
3. Valiyaveettil S. et al., Journal of Material Chemistry, 2012 (22) 1985.
4. Valiyaveettil S. et al., RSC Advances, 2012 (2) 9914.
5. Valiyaveettil S. et al., Nanoscale, 2011 (3) 4625.

Computed tomography (CT) has become an essential tool for everyday diagnosis of numerous diseases and conditions. These contrast agents are highly effective in increasing the contrast of softer tissues around the patient&’s body with few side effects in healthy patients; however, in patients with decreased kidney capabilities, such as the elderly, patients with chronic with kidney disease, and diabetics, are at risk for contrast induced nephrotoxicity (CIN) which further reduces renal function. Additionally, liquid commercial contrast agents have a lack of specificity and exhibit rapid extravasation from blood and lymphatic vessels (typical distribution half-life of approximately 3 to 10 minutes). Nanoparticles have been shown to increase circulation times in comparison to the iodinated liquids in use, and they are removed typically by the liver rather than the kidneys. Ideally, a contrast agent should be highly iodinated, biodegradable, biocompatible, long circulating, and processable. We describe the first example of a material that meets all of these criteria with the added benefit of being cost effective and additionally can be unique radiopaque macroscale sized biomaterials.
We have developed a platform of unique iodinated polyesters materials that show high radiopaqueness, low cytotoxicity, low cost, and high processablity. By using wholly aliphatic iodinated polyesters, we show that we can easily tune polymer properties to give us a multitude of materials with different thermal and mechanical properties with the same base iodinated monomer, which due to its structure is resistant to elimination or substitution. Our iodinated polyesters can then be endcapped with a photo-curable methacrylate group which allows us to easily process the material to crosslinked films or particles. These materials proved to be highly radiopaque (due to weight percent iodine content of over 40% even after processing) and exhibit no cytotoxicity. By using these materials in particle form we are able to avoid renal toxicity issues commonly associated with current contrast agents. Because these polymeric materials are easily processable we can use these materials as radiopaque biomaterials such as implants, components of implants, radiopaque glues, and bone cement, and even shape memory materials.

In this report we develop a complete mathematical model for a porous scaffold from nitinol (NiTi - intermetallic phase) with a shape memory effect (SME), fabricated layerwise via the selective laser sintering (SLS) process. The operation of the SME bio-fluidic MEMS involves such physical process as heat transfer, phase transformation with temperature hysteresis, stress-strain and electrical resistance variations accompanying the phase transformation. The simulations were conducted for electro- and thermo- mechanical phenomena, accompanying SME in porous nitinol structures the simplest shape, which allow to formulate a recommendations for SLS. Previously done the temperature evolution of electrical resistivity was compared with our present calculations as a function of the laser-processing parameters for three dimensional nitinol samples [1]. This model can be used for an estimation of drug delivery system (DDS) route [2] during a porous phase volume changing.
Keywords: selective laser sintering (SLS), drug delivery systems (DDS), shape memory alloy (SMA), nitinol (NiTi intermetallics), micro-electro-mechanical system (MEMS)
[1] Shishkovsky I., Sherbakoff V.,Yadroitsev I., Smurov I. Proceedings of the Institution of Mechanical Engineers, Part C, Journal of Mechanical Engineering Science. 2012. doi: 10.1177/0954406212440766
[2] Shishkovsky I.V. Functional design of porous drug delivery systems based on laser assisted manufactured nitinol. // MRS Proceedings, 2012, Vol. 1415: mrsf11-1415-ii03-10.

Vascular endothelial growth factor (VEGF) is one of the most potent activators of angiogenesis. Although angiogenesis is a requirement during tissue development and wound healing, it is also extremely detrimental in diseases such as diabetic retinopathy, age-related macular degeneration, and cancer metastasis. Currently available anti-angiogenic therapeutics are limited by their rapid clearance rate from the site of administration and require frequent clinical interventions to maintain an effective dose in the target tissues. In order to increase the residence time of molecular entities, we have recently developed a multivalent conjugate technology through the use of hyaluronic acid (HyA), a glycosaminoglycan in the extracellular matrix, conjugated with the protein of interest. As an inhibitor of VEGF signaling, we have synthesized the first 3 extracellular Ig domains of vascular endothelial growth factor receptor-1 (Flt) through bacterial cloning. This soluble fragment of Flt (sFlt) is sufficient to bind VEGF and antagonize its interaction with cell surface receptors. We generated mvsFlt conjugates by reacting sFlt to acrylated HyA through a Michael-type addition reaction at molar ratios of sFlt to HyA varying from 30:1 to 1:1. Then, we evaluated the ability of these multivalent sFlt (mvsFlt) conjugates to antagonize VEGF-induced cellular bioactivity by treating human umbilical vein endothelial cells (HUVECs) with VEGF and mvsFlt at molar ratios varying from 0.5:1 to 200:1. Initial experiments using in vitro tube formation validate the bioactivity of the monovalent unconjugated sFLT with a 40% reduction in human umbilical vein endothelial cells (HUVECs) tube formation at a 1 sFlt : 1 VEGF ratio in comparison to the control. From our preliminary data we can conclude that VEGF is critical for endothelial cell function.. Future work will evaluate the effect of monovalent vs. multivalent hyaluronic acid conjugated sFlt using both in vitro and in vivo approaches.

A wide range of multifunctional molecules are ubiquitously used with biological tissue such as skin to affect the mechanical function. An ideal treatment would utilize these molecules that integrate with the outermost layer of skin, the stratum corneum (SC), and respond to physical environmental stimuli to preserve healthy tissue function. They additionally must be biocompatible, biodegradable, and have multiple functional groups to interface with the protein and lipid components of the tissue. Surprisingly, current understanding of these treatments remains far from complete, and the relationship between the mechanical behavior of human SC, environmental stimuli, and damage alleviating molecules has not been established.
We show how water loss determines SC drying stresses. We investigate the ability of a range of multifunctional molecules to protect the water balance in the SC and characterize how drying stresses develop as a function of time and how they change following the application of classes of multifunctional humectant, occlusive and emollient molecules. In the presence of tissue components, these molecules perform complimentary functions by either forming a barrier to water loss or diffusing into the tissue to interact with SC protein and lipid components. All combinations showed a characteristic peak in stress 1 - 5 hrs after the start of drying followed by a rapid decrease and plateau at a stress of around 0 - 1 MPa. To understand the characteristic shape of the drying stress behavior, water loss kinetics of samples with the treatments applied was examined using Raman spectroscopy. The research demonstrates an ideal example of integration of multi-component molecules with biological tissue where the interaction of the molecules with the tissue critically affects properties.

Biomaterials are used in many implantable applications, from vascular grafts to orthopedic reconstruction. While the first requirement for any implantable biomaterial is that it be biocompatible, in reality most biomaterials are only minimally biocompatible (Kannan 2005), invoking various immune responses. This has led to the use of natural protein-based biomaterials. However, many of these materials are novel and have unknown or incompletely-known mechanical characteristics. This paper reports on mechanical characterization of electrospun tissue scaffolds formed from varying blends of collagen and human tropoelastin, which is an elastic structural protein found in skin, vasculature, and other elastic tissues. The electrospun tropoelastin-based scaffolds have an open, porous structure conducive to cell attachment and have been shown to exhibit strong biocompatibility (Jordan 2007, Woodhouse 2004). These scaffolds can be altered by blending multiple proteins and manipulating the degree to which the protein is crosslinked. Previous studies with electrospun tropoelastin used only crosslinked materials, did not systematically evaluate varying protein blends, and did not consider viscoelastic characteristics of the polymeric materials (McKenna 2011). To address this, mechanical properties were tested for scaffolds consisting of 100% tropoelastin, 100% collagen, and 1:1 tropoelastin/collagen blends. Mechanical properties were tested both at ASTM standard rates as well as 5x the standard strain rate to determine the effects of loading rate on viscoelastic characteristics. The mechanical testing results showed that the materials exhibited vastly different mechanical properties when tested dry vs. hydrated, with a four order of magnitude change in elastic modulus from 2.32 MPa to 0.0003 MPa for 100% tropoelastin. Non-crosslinked scaffolds exhibited 98% less initial stiffness than crosslinked, and exhibited no stiffness at strains >~100%. The elastic modulus of a pure collagen scaffold was 50% higher than that of a pure tropoelastin and the modulus of a 1:1 blend fell between the two. Finally, the scaffolds demonstrated a viscoelastic character, with the materials being ~50% stiffer when strained at five times the ASTM standard rate. This viscoelastic character has not previously been accounted for and may have a significant impact on the materials&’ utility in its end-use application. By systematically varying strain rate, protein composition, and crosslinking, the results demonstrate how protein scaffolds might be manipulated as customized biomaterials, ensuring mechanical robustness and potentially improving biocompatibility through minimization of compliance mismatch with the surrounding tissue environment (Hollister 2002). Future investigation will continue to characterize the mechanical behavior of tropoelastin scaffold, offering the potential to tune tropoelastin tissue scaffolds to specific mechanical characteristics.

Here, we propose to use mussel adhesive protein (MAP) as a natural biomaterial that serves as a blending partner for the preparation of sticky nanofibers which provides a facile, efficient, and multifunctionalized platform for generating novel nanofibrous scaffolds. Nanofibrous scaffolds based on MAPs with various kinds of biodegradable polymer partners were fabricated via a simple electrospinning process. Using PCL as a model blending partner, we identified that incorporation of MAP via the blending strategy strengthened the rigidity of the composite nanofibers and rendered nanofiber surfaces more hydrophilic, which indicated that MAPs were successfully exposed on the surface of the composite nanofibers. In our in vitro cell culture experiments, pre-osteoblast cell adhesion and proliferation were enhanced on the PCL/MAP nanofibers compared to sole PCL nanofibers. Also, we investigated the role of PCL/MAP nanoifibers as multiple coating platforms against many types of biomolecules by simple dipping experiments without any chemical treatment. In conclusion, our novel, multiple biofunctionalized nanofiber platform based on MAPs could be a promising tool for successful tissue engineering applications.

Biodegradable polymers, which are capable to decompose into small, non-toxic molecules, are of high interest for clinical applications, e.g. as suture materials, medical screws, pins, or drug delivery systems [1-2]. Poly(glycolic acid) (PGA), obtained from the ring-opening polymerization (ROP) of diglycolide [3] is of special interest as it can be easily hydrolyzed and the glycolic acid as degradation product can be metabolized. In addition, biodegradable polymers of high number average molecular weight (Mn) are preferable as the polymer degradation rate strongly depends on the molecular weight [2, 4]. Furthermore, a high Mn is also necessary to provide sufficient mechanical strength. However, synthesis of high molecular weight PGA is a challenge as its thermal decomposition occurs already below the melting point and is accompanied by a brownish discoloration [5]. This decomposition can be avoided by using highly polar solvents providing high boiling temperatures. Nevertheless, most of these solvents are rather toxic, which makes the resulting PGA unfavorable for biomedical applications.
We have explored whether supercritical CO2 could be utilized as reaction medium for the ROP of diglycolide as it possess several favorable characteristics: CO2 is a chemically inert, non-toxic, and environmentally benign gas and therefore could substitute toxic organic solvents. In addition, it can be easily separated from the product by reducing the pressure [6].
To utilize scCO2 as reaction medium ROP were performed in an optical high-pressure cell using pressures of up to 1500 bar and tin (II) ethyl hexanoate as catalyst. Variation of the relevant reaction parameters such as temperature or pressure yielded PGAs of well-defined molecular weight as white powders showing no signs of thermal decomposition. Furthermore, we could demonstrate that this process is even suitable for the synthesis of block-copolymers by using using OH-functionalized poly(ethylene glycol) as macroinitiator. We anticipate that this novel process, which is not limited to PGA, will significantly improve the synthesis of degradable biopolymers.
References
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[2] A. Lendlein, Chemie in unserer Zeit, 1999, 33, 279-295.
[3] S. Kaihara, S. Matsumura, A.G. Mikos, J.P. Fisher, Nat. Prot. 2007, 2, 2767- 2771.
[4] K.A. Athanasiou, G.G. Niederauer, Biomaterials 1996, 17, 93- 102.
[5] K. Takahashi, I. Taniguchi, M. Miyamoto, Y. Kimura, Polymer 2000, 41, 8725- 8728.
[6] T. Meyer, M.F. Kemmere, (2005), Supercritical Carbon Dioxide- in Polymer Reaction Engineering, Wiley-VCH, Weinheim.

Multi-functional chitosan scaffolds as household water filtering medium were prepared by tuning preparation parameters and subsequent freeze-drying conditions. The morphology and pore size of the chitosan scaffolds were studied by scanning electron microscope. It was found that pores in the scaffolds were interconnected with pore diameter around 5mu;m.The surface charge polarities of scaffolds (positive, negative and neutral) were tuned by addition of functional additives. It is advantageous as scaffolds bearing different charges can be used to filter off specific charged species and/or polar chemicals and also micro-organisms (e.g. bacteria) which are charge sensitive. The mechanical strength of scaffolds was improved by cross-linking chitosan with environmentally benign additives. The prepared samples are more versatile than conventional filtering media which based on size-exclusion only. The chitosan scaffolds demonstrated bacteria retention property with 95% retention rate for Escherichia coli. Self-regeneration capability was shown as removal of more than 90% of organic contaminants in water after 10 generation cycles. Heavy metals (Pb2+, Cd2+ and Hg2+) absorption ability were found to be more than 8mg/g respectively by inductively coupled plasma optical emission spectrometer. Filtration efficiencies were correlated to the pore size of the scaffolds and the intrinsic properties of chitosan.

Adhesives play a critical role in most products of electronics, buildings, motor vehicles, and many others. However, adhesives used for the biomedical purpose are under-developed compared to ones used for industries and consumer products, due to the significant loss of adhesive strength in wet environments of a body. For success as medical adhesives, maintaining the strong adhesion strength in the presence of water with the proper mechanical cohesiveness is a major challenge. Here, we report an entirely new class of medical adhesives called TAPE (A combination of the words "Tannic acid (TA)" and "Poly(ethylene glycol)s (PEGs)") that is inspired by a ubiquitous compound in plants, tannin. Tannins are ubiquitous compounds of secondary metabolites present in plants. They have much attention for their biofunctional properties such as antioxidant, antimutagenic, anticarcinogenic, antibacterial, and others. TA, one of the major components in hydrolyzable tannins, shows the ability to form multiple hydrogen bonds as the presence of abundant terminal hydroxyl groups with a central carbohydrate core. TAPE is spontaneously generated by inter-molecular assembly between TAs and PEGs. The inter-molecular interaction forming TAPE was determined as the incorporation of hydrogen bonding, investigated by FT-IR, UV-Vis, and NMR study. TAPE shows 250% increase in adhesive strength compared to the widely used commercial adhesives such as fibrin glue, and the adhesion is also prolonged in presence of water even after 20 repeats of attachment and detachment reversibly. TAPE has effective non-biofouling ability due to the unique property of PEGs, one of main component consists of TAPE, and also it can be used as a potent hemostatic biomaterial, demonstrated by its hemostatic abilities within 30 seconds in a mouse bleeding model. TAPE is immediately formed and settled down after mixing TA with PEGs at both high concentration in water and this simple mixing step can be possible for making ultra-large amount (a few liters) in a day without any synthetic process. We expect its applications for a variety of medical and pharmaceutical purposes such as mucoadhesives and drug depots will open new revenues of TAPE studies.

With high specific surface area and nanoporous structure, electrospun nanofibers membranes are excellent candidates for immobilization of enzymes. In this work, papain enzyme (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in poly (vinyl alcohol) (PVA) nanofibers through electrospinning technique. Morphology of the electrospun nanofibers was characterized through scanning electron microscopy (SEM), obtaining diameters distribution in the range of 80-170 nm. Presence of the enzyme in the PVA nanofibers was confirmed through infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Maximum enzyme catalytic activity was reached at 13% of enzyme loading. Immobilization of papain in the nanofibers membrane was achieved through chemical crosslinking using 50% glutaraldehyde solution. The catalytic activity of the immobilized papain was 88% of the crude enzyme. The optimum crosslinking time was 30 minutes and the immobilized enzyme retained its catalytic activity after six cycles of reuse. Crosslinking samples conserved 40% its initial activity after 40 days storage.

Nitric oxide (NO) release can promote healthy tissue regeneration [1]. In an effort to promote healing in cases of soft tissue injury (i.e. tendinopathy), a controlled NO releasing adhesive hydrogel would be ideal. The purpose of this study was to create a PEG-fibrinogen adhesive hydrogel that would allow for inducible NO release, and mechanical properties that could be tailored to specific applications and tissue types. PEG (4-arm) Succinimidyl Glutarate (pentaerythritol) and fibrinogen were mixed with 10mM Tris, at pH 7.40 in separate vials. These solutions were then mixed in a beaker (total volume = 100mg/mL) at varying ratios of fibrinogen to PEG (3:1, 2:1, and 1.5:1) and sonicated. Mixed solutions were placed into a Teflon mold (dia=18mm) for gelation. After gelation, samples were derivatized with S-nitroso-N-acetyl-D,L-penicillamine (SNAP)-thiolactone and allowed to sit overnight [2]. Light activated NO release was determined using a nitric oxide analyzer (Sievers). Samples were exposed to light at five-minute intervals. Gels were cut into disks (dia=15mm) for rheometric analysis. Swelling behavior was determined by equilibrating samples in diH2O for 24 hours, and imaging to determine a swelling ratio. To assess cell viability, gels were sterilized in ethanol, washed in sterile water, and then seeded with fibroblasts (L929) at a density of 2 x 10^4 cells/cm^2. Cell viability was measured using a Calcein-AM stain and fluorescent microscopy. To establish controlled NO release, gels with varying ratios of fibrinogen to PEG were made, derivatized, and tested. Gels below a ratio of 1.5:1 (fibrinogen: PEG) do not gel, while at a ratio of 1.5:1 gelation occurs and NO release can be induced. Interestingly, the release from 1.5:1 gels was significantly lower compared to 2:1 and 3:1 gel formulations. The average controlled release over a five-minute interval was 46, 258, and 345 PPB respectively. Rheometric data show that 1.5:1 gels are more elastic than viscous, and as the ratio of fibrinogen to PEG increases gels become more viscous in character. Furthermore, derivatized gels exhibited linear elastic moduli, behaving more like other established hydrogels. Swelling data indicates that as the ratio of fibrinogen to PEG increases the swelling ratio decreases. Cells remain viable on both derivatized and non-derivatized gels. PEG-fibrinogen NO releasing adhesive hydrogels were successfully generated. Current work will further characterize material properties, including tissue adhesiveness, as well as assess cell behavior in response to controlled NO-release with the hope of tailoring these hydrogels for specific clinical applications.
[1] Murrell GA. Br J Sports Med. 2007;41:227-31. [2] Frost MC, Meyerhoff ME. J Am Chem Soc. 2004;126:1348-9.

The improvement of our understanding how biomaterial properties affect cells of the immune system represents a major goal in the field of regenerative medicine. Systematic variation of defined chemical properties could help to understand which factors determine such cellular responses. By using poly[acrylonitrile-co-(N-vinylpyrrolidone)]s (P(AN-co-NVP)) as a model system, we could show that surface hydrophilicity can influence the growth of keratinocytes [1, 2]. In this study, a series of copolymers with increasing hydrophilicity, which was related to increasing contents (0, 5, 10, 20, 25, and, 30 mol.%) of NVP, were used to study the response of human primary monocyte derived dendritic cells (DC). DC play a crucial role in initiating immune response and can be activated by microbial products via engagement of Toll-like-receptors. However, they can also be activated by biomaterials. Different P(AN-co-NVP)s were prepared using free radical polymerization under aqueous conditions and processed into chips. It was shown using the LAL-Test as well as a macrophage cell line, which get in direct contact with the material that the materials were free of endotoxin or other microbial contaminations. Such contaminations could bias the readout of the DC studies. The DC were generated (n=6) by cultivation of purified monocytes with IL-4 and GM-CSF for 6 days.
The viability of the DC was not substantially influenced by the different materials. Furthermore, the activation status, determined by the expression of co-stimulatory molecules such as CD80 and CD86 was not systematically altered by the P(AN-co-NVP)s, indicating that increased amounts of NVP in the copolymer do not enhance the activation status of the DC. The analyses of the DC cytokine release confirmed that the cells remained in their immature status after culturing on the different P(AN-co-NVP)s. Similarly, when DC were cultured under inflammatory conditions mimicked by the addition of lipopolysaccharides (LPS), neither the expression of co-stimualtory molecules nor the release of cytokine was influenced by the different biomaterials.
Conclusively, our data show that this class of copolymers does not substantially influence the activation status of dendritic cells, which should be considered for the future development of biomaterials.
[1] G. Boese, C. Trimpert, W. Albrecht, G. Malsch, T. Groth, A. Lendlein, Membranes from acrylonitrile-based polymers for selective cultivation of human keratinocytes, Tissue Eng, 13 (2007) 2995-3002.
[2] N. Scharnagl, S. Lee, B. Hiebl, A. Sisson, A. Lendlein, Design principles for polymers as substratum for adherent cells, J Mater Chem, 20 (2010) 8789-8802.

Micro, nano and crystalline structures were obtained from electrospun polycaprolactone/α-Alumina/Hydroxyapatite-based hybrid fibers (PCL/AA/HA-hf). We proposed for these biodegradable non-woven hybrid nanocomposite fibers a structure-property correlation which is crucial for potential applications in reconstruction or regeneration of bone tissue (bone scaffolding-materials). Analysis by Transmission Electron Microscopy (TEM), X ray Diffraction (XRD), Small Angle Light Scattering (SALS), Polarized Optical Microscopy (POM), Differential Scanning Calorimetry (DSC) and Infrared spectroscopy (ATR-FTIR) enable us to suggest a simple structural model for the series of hybrid nanocomposite fibers. Electrospun PCL/AA/HA-hf were prepared by varying the applied voltage. PCL featured a molecular weight of 70,000 g/mol, and it was dissolved in chloroform. Hydroxiapatite and α-Alumina nanoparticles were dispersed in dimethylacetamide (DMA). Both polymer and nanoparticles solutions were mixed and then electrospun. First, POM exhibited qualitative observations of birefringent regions for all samples. Furthermore, polarized light distinguished between amorphous and micron ordered structures along the hybrid fibers. On the other hand, SALS suggested spherulite-like asymmetric patterns for some hybrid fibers, and their average profiles confirmed the influence of both nanostructured Hydroxyapatite and α-Alumina upon PCL semicrystalline phase behavior. Endothermic peaks detected by DSC agreed with the formation of PCL crystalline structure and showed differences between samples processed with different electrospining parameters. At nanoscale, TEM pictures exhibited monodispersion of both nanoparticles inside the hybrid fibers. Hydroxyapatite nanoparticles featured an average size of 7-20 nm while α-Alumina was 500 nm in average size. The PCL fibers averaged 150 nm in cross section, via TEM. XRD confirmed the crystalline unit cells for Hydroxyapatite immersed in PCL fibers as well as α-Alumina crystalline phase. Molecular identification of the samples was made by ATR-FTIR technique. IR spectra of the hybrid fibers were all similar. Typically functional organic groups for PCL were observed in the region 400-4000 cm^-1. Studies at the micro, nano and crystalline scale will help to control structural parameters needed for applications such as bone tissue regeneration

With the burst of the world population and the rapid development of the global industry, clean water, the most important living element, is getting scarcer and scarcer. Means of translating latent water resource in fog to dominant available water, i. e., fog collection, therefore becomes highly desirable. In nature, multiple biological structures have demonstrated special fog collection abilities, such as the beetle backs with bumps and spider silks with periodic spindle-knots and joints. Many Cactaceae species live in arid environments and are extremely drought-tolerant. Here, we report that one of the survival systems of the cactus Opuntia microdasys lies in its efficient fog collection system. This unique system is composed of well-distributed clusters of conical spines and trichomes on the cactus stem; each spine contains three integrated parts that play different roles in the fog collection process according to their surface structural features: the tip with oriented conical barbs as deposition and collection site, the middle with gradient grooves as transporting site, and the base with belt-structured trichomes as absorbing site. The gradient of the Laplace pressure, the gradient of the surface free energy, and the multi-function integration endow the cactus with an efficient fog collection system. Investigations of the structure-function relationship in this system may help us to design novel materials and devices to collect water from fog with high efficiencies.
Reference
1, J. Ju, H. Bai, Y. Zheng, T. Zhao, R. Fang, L. Jiang, Nat. Commun. accepted
2, A. R. Parker, C. R. Lawrence, Nature 2001, 414, 33-34.
3, Y. Zheng, H. Bai, Z. Huang, X. Tian, F.-Q. Nie, Y. Zhao, J. Zhai, L. Jiang, Nature 2010, 463, 640-643.

Nature has evolved efficient strategies to synthesize complex mineralized structures that exhibit exceptional damage tolerance. One such example is found in the hyper-mineralized hammer-like dactyl clubs of the stomatopods, a group of highly aggressive marine crustaceans. The dactyl clubs from one species, Odontodactylus scyllarus, exhibit an impressive set of characteristics adapted for surviving high velocity impacts on the heavily mineralized prey on which they feed. Our analysis has revealed that the dactyl club consists of a multi-phase composite of oriented crystalline hydroxyapatite and amorphous calcium phosphate and carbonate, in conjunction with a highly expanded helicoidal organization of the fibrillar chitinous organic matrix. These structures display several effective lines of defense against catastrophic failure during repetitive high energy loading events. Here, we have constructed biomimetic composites that incorporate some of the design motifs revealed in the dactyl club that demonstrate effective toughening and resistance to catastrophic failure.

Biomaterial surface modifications via external coating of implant surfaces with hydroxyapatite-magnesium (HA-Mg) composite films have been carried out to improve implant corrosion resistance, wear resistance, surface texture, and biocompatibility. The composite films have been grown on Mg plates using a pulsed laser deposition technique. Mechanical property measurements and analysis have indicated that hardness and young&’s modulus of the nMg-(100-n)HA composite coatings increase with Mg content in the coatings and reach a maximum at a 70Mg-30HA composition. n and 100-n in the nMg-(100-n)HA represent the relative number of laser pulses impinging on Mg and HA targets, respectively. Potentiodynamic polarization study indicates that the corrosion of magnesium decreases with increase in the hydroxyapatite ratio in the composite. the corrosion potential (Ecorr) and corrosion current density (Icorr) for the uncoated magnesium, 30%HA-70% Mg, and 50% HA-50% Mg coated magnesium are -1.59, -1.57, -1.54 v and 2.40×10-5, 2.59×10-6, 5.33×10-7 A/cm2 respectively. The corrosion protective nature of the composites was further confirmed by electrochemical impedance spectroscopy. Cytotoxicity test conducted on the samples showed no adverse effect on human bone marrow stromal cells. The advantage of the composite coatings is the realization of adjustable corrosion and biological properties with a simple maneuvering of composition by means of relative number of laser pulses on a respective target. These coatings are also expected to play favorable roles in cosmetic, therapeutic, preventative, and/or reconstructive applications.

Electro photography (laser printing) has emerged to one of the leading print technologies during the last decades. The xerographic process enables the simultaneous two dimensional alignment of multiple toner materials with high spatial resolution (600 dpi, resolution <100 µm). Due to its solvent-free character, a high solid content can be transferred in a single printing cycle, offering the possibility for a quick layer-by-layer construction of three-dimensional objects. [1] However, the conventional melting process prohibits accurate structure building and in contrast to the well-established ink jet process, three-dimensional electro photography has not yet been examined for the assembly of biofunctional materials. Especially the cytotoxicity of common toner components as well as the requirement of high temperatures during the conventional curing procedure have prevented the approach towards this interesting application. Therefore, a new biocompatible class of organic-inorganic composite particles has been developed to be processed as toners at low temperatures using common laser printing techniques. [2]
In this paper, we provide a detailed kinetic investigation about the controlled surface functionalization of acrylic particles in respect to the hydrolysis time, temperature and the sodium hydroxide concentration. Furthermore, we have studied the subsequent carbodiimidemediated coupling of numerous functional amines onto the generated carboxylic group. Various chemically valuable functionalities, comprising of thiol, alkyne and azide, were bound onto the particles&’s surface and allow for further versatile modifications via huisgen cycloadditions as well as thiol-ene reaction. The functionalization of the acrylic toner surface with thiol-, azide and carboxylic groups has increased the cell viability [3] and might offer an interesting path for new applications using common laser printing techniques.
[1] M. Peltola, F.P.W. Melchels, D. W. Grijpma, M. Kellomäki, Annals of Medicine, 40-4, 268-280, 2008.
[2] C. Speyerer, S. Güttler, K. Borchers, G. Tovar, T. Hirth and A. Weber, Mater. Res. Soc. Symp. Proc. 1340,
(2011), mrss11-1340-t05-09 doi:10.1557/opl.2011.1130.
[3] E. Yoshii, Journal of Biomedical Materials Research, 37-4, 517-524, 1997.

Patients undergoing total joint replacement operations can experience complications during recovery due to failure of the implant to integrate with bone tissue. The use of bonded networks of ferromagnetic fibres as an anchoring technique for bone tissue in-growth has been proposed¹ as an innovative approach to enhance implant fixation. When subjected to an external magnetic field, alignment of the fibres imposes mechanical strains to in-growing bone tissue. Such deformation is known to promote bone cell growth provided the strains lie in the beneficial range. By embedding human mesenchymal stem cells (hMSC) in such a scaffold and initiating osteogenesis, the occurrence of implant failure could be further prevented. HMSC can be cultured initially in the fibre network before implantation in order to synchronise their differentiation towards osteoblasts with the anchoring of the scaffold on the adjacent bone. The biocompatibility of these scaffolds has already been proven for human osteoblasts² and peripheral blood monocytes³.
The aim of the current study is to assess the response of hMSC to 444 ferritic stainless steel fibre networks and fully-dense surfaces. A comparison was made between 444 ferritic (magnetic) and 316L austenitic (non-magnetic) stainless steel (a widely established implant material). Tissue culture plastic (ThermanoxTM) surfaces served as control. hMSC were seeded onto the networks and fully-dense surfaces and cultured in osteogenesis induction medium (Lonza, UK) for 3 weeks. Cellular viability and proliferation as well as metabolic activity were examined using the CyQuant® and AlamarBlue® assays respectively. Scanning electron microscopy (SEM) and fluorescence imaging were used to investigate cellular morphology. Measurement of alkaline phosphatase (ALP) activity was used to determine early osteoblastic differentiation. Gene expression of osteogenic markers was investigated by real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR), while their phenotype was characterized by fluorescence activated cell sorting (FACS). Our results show that hMSC respond positively when in contact with 444 ferritic stainless steel surfaces in terms of compatibility, proliferation and differentiation. Consequently, 444 fibre networks with embedded hMSC have potential to be used for bone implant applications.
REFERENCES
¹AE Markaki and TW Clyne: "Magneto-mechanical stimulation of bone growth in a bonded array of ferromagnetic fibres", Biomaterials 25 (2004), pp 4805-4815
²VN Malheiro, RL Spear, RA Brooks and AE Markaki: "Osteoblast and monocyte responses to 444 ferritic stainless steel intended for a Magneto-Mechanically Actuated Fibrous Scaffold",Biomaterials 32 (2011), pp 6883-6892
³RL Spear, RA Brooks and AE Markaki: "Short-term In vitro Responses of Human Peripheral Blood Mononuclear Cells to Ferritic Stainless Steel Fibre Networks",Journal of Biomedical Materials Research:Part A, 2012, in press

Biopolymers such as proteins and polysaccharides are interesting starting materials for the development of biomaterials, as they often have a low cytotoxicity, can offer cells sites for adhesion, and are degradable, which is important for materials needed temporarily only and are e.g. applied for inducing endogenous regeneration. However, there is a high tendency for self-organization of biopolymers into preferred conformations, which has a large impact on material properties. Therefore, in order to effectively use biopolymer-based materials, their self-organization needs to be controlled.
One particularly effective approach is to form polymer networks by covalent or physical means at temperatures which inhibit the self-organization process. The crosslinking can be performed e.g. by reactions with diisocyanates,[1] photocrosslinking,[2] or functionalization with specifically interacting groups.[3] Although isocyanates do react very well with water, test reactions investigated by ESI mass spectrometry clearly showed the formation of a significant amount of mono- or oligomeric direct crosslinks as well as grafting, both contributing to suppression of collagen-like helices and stabilization of the mechanical properties of the hydrogels. Chain organization in the materials can be studied by wide angle X-ray spectroscopy (WAXS), and gives interesting insights in the molecular structure after synthesis and during degradation. The degradation time in aqueous solution ranged from few days to several weeks depending on gelatin concentration and amount and type of crosslinker. Cytotoxicity of the materials and viability and differentiation of human bone-marrow derived stem cells (MSCs) were investigated,[4] and it could be shown that the support of viable stem cells depends on the mechanical properties of the gels (with gels having Young&’s modulus E = 100 kPa performing superior to gels with E = 600 kPa). Tests on the chorioallantoic membrane of chicks eggs showed no negative effect on blood vessel formation and no induction of hemorrhage or bleeding. These gelatin-based multifunctional materials with tailorable materials properties have therefore a high potential for applications in biomedicine, e.g. for cell culture and tissue engineering.
1. G. Tronci, A.T. Neffe, B.F. Pierce, A. Lendlein, J. Mater. Chem 2010, 20, 8875.
2. B.F. Pierce, G. Tronci, M. Rössle, A.T. Neffe, F. Jung, A. Lendlein, Macromol. Bioscience 2012, 12, 484.
3. A.T. Neffe, A. Zaupa, B.F. Pierce, D. Hofmann, A. Lendlein, Macromol. Rapid Commun. 2010, 31, 1534.
4. B. F. Pierce, E. Pitterman, N. Ma, T. Gebauer, A.T. Neffe, M. Hölscher, F. Jung, A. Lendlein, Macromol. Biosci. 2012, 12, 312.

Sugar-functionalized polymers have been widely used to obtain biologically active materials for such diverse applications as medicinal analysis, drug development and tissue engineering. One important design feature of such polymeric biomaterials is the multiple presentations of the ligands often leading to an increase in binding affinity and specificity compared to the natural systems. This is especially important for glycopolymers as it is well known that due to their weak interactions on a singular level, sugar-protein interactions occur mostly as multivalent binding events. Thus the scaffold presenting the sugar ligands dramatically influences the resulting binding properties. However, the structure-property relations of such glycomaterials, especially in the area of glycopolymers, have not been studied systematically but are mostly derived empirically. In order to elucidate the role of the scaffold mechanistically and formulate more general design rules for new functional glycomaterials, the structure and physicochemical properties of the polymeric scaffold should be highly controlled. Earlier we introduced a new approach towards monodisperse, sequence-controlled polymers based on solid phase synthesis and the stepwise addition of tailor-made building blocks. Through the choice of building blocks applied in each step, we are able to precisely control the physicochemical properties of the polymer and tune from hydrophilic to hydrophobic, flexible to stiff, go from linear to branched chains or introduce chirality at different points in the polymer chain. Recently we expanded this approach towards the solid phase synthesis of multifunctional glycopolymers. Through this combination we are now able to control the number of sugar ligands attached to the polymer scaffold, their position on the scaffold as well as their distancing and to go from homomultivalent to heteromultivalent systems presenting different ligands at different positions.
With this toolbox at hand, we first look at the fundamental principles of multivalent binding and systematically evaluate the influence of different scaffold parameters such as hydration, structure and flexibility using established methods such as surface plasmon resonance (SPR) and NMR but also apply novel techniques such as soft colloidal probe reflection interference microscopy (SCP RICM). The information derived from these fundamental studies is then applied for the synthesis of glycomaterials for biomedical applications such as targeted gene delivery and anti-bacterial coatings.
References:
Mosca, S.; Wojcik, F.; Hartmann, L.; Macromol. Rapid Commun. 2010, 32(2), 197-202.
Wojcik, F.; Mosca, S.; Hartmann, L; J. Org. Chem., 2012, 77, 4226minus;4234.
Ponader, D.; Wojcik, F.; Beceren, F.; Dernedde, J.; Hartmann L.; Biomacromolecules, 2012, 13, 1845minus;1852.
Pussak, D.; Behra, M.; Schmidt, S.; Hartmann, L.; Soft Matter, 2012, 8, 1664-1672.

The practice of medicine is rapidly becoming increasingly complex. Therefore, the biomaterials used in medical interventions must also increase in functionality to meet medical unmet needs. One approach toward the design and synthesis of new polymer-based functional biomaterials is to explore potential monomer building blocks represented within the human metabolome. The metabolome represents thousands of unique structures, all of which are readily recognized and metabolized. The overarching hypothesis for our work is that functional biomaterials derived from biomolecular building blocks can serve unmet medical needs while maintaining biocompatibility profiles commensurate with use in humans.
Dihydroxyacetone (DHA) is the fifth metabolite of glucose as it is metabolized to pyruvic acid. However, while the structure of DHA is very simple (two hydroxyl groups and a ketone), its chemistry is surprisingly complicated. Our group has learned how to harness DHA and use it as a metabolically-derived synthon for functional biomaterials. Of particular interest to our medical use goals was to utilize the ketone functional group of DHA, which readily reacts with primary amines. The initial idea was to use DHA in a polymeric form to act as a bioadhesive to the extracellular matrix.
The first synthetic iteration was a homo-polycarbonate of DHA (pDHA). The polymer was synthesized by ring opening polymerization of a 6-membered carbonate of a protected form of DHA, followed by deprotection to yield to the homopolymer. The resulting polycarbonate was shown to react with primary amines, as designed; however, the polymer was insoluble in all solvents when the Mw exceeded 10,000. To allow manipulation of the material in a medical setting, a second polymer, consisting of a diblock co-polymer of polyethylene glycol (PEG) and pDHA, was synthesized. The resulting material gelled in water owing to the water soluble PEG and water insoluble pDHA. The hydrogel was characterized as a thixotropic non-Newtonian fluid, owing to the physical crosslinks created by pDHA. From a medical perspective, these characteristics are favorable because they allow direct injection into a patient via a needle and syringe. The material was evaluated for its bioadhesive characteristics in a rat model of seroma following radical breast mastectomy. A seroma is an “internal blister” consisting of a space filled with extracellular fluid. It is a postoperative complication that presents in a wide range of surgeries. The data show that upon treatment with the diblock copolymer, postoperative seroma was quantitatively eliminated in the model.

Cephalopods are known as the chameleons of the sea - they can alter their skin&’s coloration, pattern, texture, and reflectivity to blend into the surrounding environment. Despite much research effort, there are few known strategies (natural or artificial) for emulating the unique dynamic reflectivity and coloration of cephalopods. We have developed an approach to the fabrication of stimuli-responsive cephalopod-inspired thin films with emergent optical properties. We have furthermore enhanced the properties of such films through a synergistic combination of bioconjugate chemistry, bottom up self-assembly, and top down nanofabrication. Our findings hold significant implications for the development of reconfigurable biomimetic camouflage coatings.

The use of bone regeneration procedures is becoming a daily practice in dentistry and clinicians are demanding higher efficient bone substitutes. Beta-tricalcium phosphate (beta-TCP) is a transductive bone substitute that is widely used in maxillofacial and orthopedic field. We hypothesize that iIonic substitution of phosphate ions by silicate ions and calcium by iron could improve and widen the therapeutic efficiency of such ceramic.
Beta- TCP was prepared by the calcinations of brushite and calcium carbonate mixture of a molar ratio of 1.5. Ionic-doped ceramics were prepared by substituting brushite with SiO2 (for silicon substitution) and calcium carbonate with ferric citrate (iron substitution) at different molar ratios.
Si-doped ceramics are mainly composed of beta-TCP and silicocarnotite (Ca5(PO4)2SiO4). While iron doped ceramics are only composed of beta-TCP. The iron ions substitution for calcium is indicated by the systemic shift of diffraction peaks toward higher angles. Porosity and surface area was increased by silicon substitution while lowered by iron doping. Interestingly, iron doping resulting in beta-TCP with magnetic properties. The silicon and iron substitution was proved to improve significantly the osteoblast proliferation and activity which was manifested in improved bone regeneration achieved by filling the created bone defects. This was testified by the histological analysis of bone samples harvested after 8 and 12 weeks of implantation.. In addition, the doped ceramics were loaded with vancomycin and the release of the antibiotic was followed using the absorbance peak of the antibiotic at 280 nm.

In recently years, bone tissue engineering that involves a combination of scaffold, cells and biological signals has attracted widespread attention and has proved a promising approach for the repair and regeneration of damaged bone. Existing artificial scaffold for bone tissue engineering has limitations such as low permeability, poor mechanical strength and osteointegration, therefore the need to produce novel scaffolds is urgent.
In the current study, a method combining the biomineralization approach with controllable freeze casting was developed to prepare a novel biomimetic scaffold for bone tissue regeneration. This method is simple but capable of fabricating bone-like composites with a range of collagen-apatite (Col-Ap) ratios and pore structures. Basically, mineralized collagen fibers were prepared using collagen containing modified simulated body fluid (m-SBF). Then, 3-D macro porous structure was created by controllable freeze casting method. The scaffold was comprised of organized collagen fibers and poorly crystalline apatite nanoparticles. The mineral content in the scaffold was tailored ranging from 0 to ~50 wt% by simply adjusting the collagen content in the m-SBF. The morphology of the scaffold exhibits similar characteristics to those of the extracellular matrix in terms of their nanofibrillar structure and fibrillar density. At the macrostructure level, Col-Ap scaffolds with an isotropic equiaxed structure and a unidirectional lamellar structure were prepared by controllable freeze casting, and the pore size of which could be easily adjusted.
This is the first report of a scaffold that possesses 3-D macro porous structure to facilitate vascularization and nutrient transportation while maintaining the high fibrillar density and biomimetic surface property in the pore walls to enhance mechanical strength and promote cell attachment. Other unique property of the scaffold includes the control of the pore size and pore orientation over a wide range from nano to macro dimensions. The bone forming capability of such prepared scaffolds was evaluated in vivo in a mouse calvarial model. It was proved that the scaffolds well support new bone formation

Fractures of the internal orbit are common facial injuries. They can range in size from small cracks in the orbital floor to extensive multiple-wall defects. Over the years, many different materials have been used to reconstruct the orbit. Autologous bone is often used, but stable synthetic materials like titanium, polytetrafluoroethylene (PTFE), polyethylene (PE) and silicone rubbers (Silastic®, Perthese®) have been employed to prepare orbital floor implants. There is also much interest in the use of degradable and resorbable implant materials such as poly(lactide)s and poly(glycolide)s since they possess a distinct advantage over the life-long risk of complications characteristic for non-resorbable materials.
Ideally, bone is regenerated during healing of the fractured orbit. However, with the currently used materials, this is only the case when autologous bone is used. To develop a synthetic material that will lead to regeneration of bone in bony defects, osteoinductive or osteoconductive properties are required. Composite systems comprising a biodegradable polymer matrix and a bioactive ceramic filler may be interesting substitutes for autologous bone, and find application in reconstruction of the orbital floor.
Our objective was to render polymeric materials based on poly(trimethylene carbonate) (PTMC) osteoinductive, and to evaluate their suitability for use in orbital floor reconstruction. For this purpose, osteoinductive biphasic calcium phosphate (BCP) particles were introduced into a surface eroding polymeric PTMC matrix. After sterilization by gamma irradiation, the sheets were used to reconstruct surgically-created orbital floor defects in sheep. The bone inducing potential of the different implants was assessed upon intramuscular implantation.
The performance of the implants in orbital floor reconstruction was assessed by cone beam computed tomography (CBCT). Histological evaluation showed that in the orbital and intramuscular implantations of BCP containing specimens, bone formation could be seen after 3 and 9 months. Analysis of the CBCT scans showed that the composite PTMC sheets performed well in orbital floor reconstruction.

Bone grafts, commonly performed to augment bone regeneration from autologous or alleogenic sources, carry an enormous cost, estimated at upwards of 21 billion dollars per year. Hydroxyapatite (HAP) bio-ceramic has been widely used in clinic as a bone graft substitute material due to its biocompatibility and the similarity of its structure and composition to bone mineral. However, its applications are limited due to its lack of strength and toughness. Researchers have attempted to overcome these issues by combining HAP bio-ceramics into resorbable polymers to improve their mechanical properties. However, poor bonding between the HAP and the polymer caused separation at the polymer-filler interface. To overcome this, short chains of polymers were grafted directly from the hydroxyl groups on the surface of nanocrystalline HAP. Collagens, being the most abundant proteins in the body, and having suitable properties such as biodegradability, bioabsorbability with low antigenicity, high affinity to water, and the ability to interact with cells through integrin recognition, makes them a very promising candidate for the modification of the polymer surface. In this study, a novel method of synthesizing nano-hydroxyapatite (nHAP)-g-poly(lactide-co-glycolide)-g-collagen polymer was introduced. The synthesis process was carried out in several steps. First, poly (lactide-co-glycolide) (PLGA) polymer was directly grafted onto the hydroxyl group of the surface of n-HAP particles by ring-opening polymerization, and subsequently coupled with succinic anhydride. In order to activate the co-polymer for collagen attachment, the carboxyl end group obtained from succinic anhydride was reacted with N-hydroxysuccinimide (NHS) in the presence of dicyclohexylcarbodiimide (DCC) as the cross-linking agent. Finally, the activated co-polymer was attached to calf skin collagen type I, in hydrochloric acid/phosphate buffer solution and the precipitated co-polymer with attached collagen was isolated. The synthesis was monitored by proton NMR and FTIR spectroscopies and the products after each step were characterized by thermal analysis (TGA and DSC). These composite materials will be tested as potential scaffolds for tissue engineering applications.

Using a novel electrochemical process, we have co-assembled dialyzed collagen molecules and biopolymer nanoparticles (NPs) to form multifunctional, aligned collagen-NP composite fibers. This novel material assembly process takes advantage of isoelectric focusing of collagen and has the benefits of low voltage, room temperature, and environmental friendly set up. We hypothesized that the aligned collagen inside the composite fiber offers a biomechanically competent matrix for stem cell guidance and attachment, while the NPs inside the composite fiber were able to release a cell-mediating growth factor, platelet-derived growth factor (PDGF).
These electrochemically aligned collagen-NP composite fibers were seeded inside tissue culture plate and evaluated for biocompatiblity, cell proliferation, and differentiation of rat adipose-derived stem cells (ADSCs). It was demonstrated that these biocompatible composite fibers enhanced the proliferation of ADSCs, Moreover, compared to random collagen fibers, aligned collagen-NP fibers further promotes the tenogeneic differentiation of ADSCs, as evident by the gene secretion of Scleraxis (BMP-2 Inhibitor), Tenomodulin (TNMN; tenocyte marker), and Tenascin C. On the other hand, the undesirable osteogenic differentiation of stem cells was not observed, as evident by the unchanged level of Osteocalcin (OCN) and Alkaline Phosphatase (ALP). Due to the alignment of collagen, ADSCs attached to the fibers were also aligned, resembling the tendon/ligament fibroblast organization in collagen bundles.
Since tendon/ligament has poor vascularity, limited cell resources and numbers, and is primarily composed of aligned collagen fiber bundles, we envision that our biomimetic aligned collagen-NP composite fiber, once seeded with ADSCs, can be implanted and greatly enhance the healing environment of tendon/ligament. Our study demonstrated that electrochemically aligned collagen-NP fibers can enhance the proliferation and desirable tenogenic differentiation in vitro. This is a first promising step towards the application of these multifunctional, novel composites fibers for in vivo implantation.

Smart polymers are environmentally sensitive polymers that respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties. Shape-memory polymers (SMPs) are a class of smart polymers that have the capability to change from a temporary shape to a memorized permanent shape upon application of an external stimulus. Especially, the use of SMPs as self-repairing or re-writable materials has found growing interest in environmentally-friendly technologies. On the other hand, recent advances in nano technologies pave the way for engineering biomaterial surfaces that control cellular interactions from the nano- to micro scale. The spatial and temporal control of extracellular signaling cues on nano-patterned surfaces is particularly attractive for investigating fundamental mechanisms of adhesion-mediated cell signaling. From these perspectives, we propose a novel technique that explores dynamic cell behavior in response to surface changes in nanotopology using biocompatible poly(e-caprolactone) (PCL) films that actuate on demand under biological conditions.

The molecular structural design learned from natural materials enables synthetic polymers with desirable and unique features. Spider silks have been one of the most extensively studied natural protein materials due to their incredible mechanical properties and intriguing supercontraction feature. On the basis of amounts of works on spider silks, plus our knowledge and understanding of shape-memory polymers (SMPs), we believe that their supercontraction is typical shape memory effect, and propose that hydrogen-bondings within amouphous regions of silks can be interpreted as switches that are responsible for strain fixation, while the β-sheet crystals act as netpoints to stablize the permanent shape. Here, we demonstrate that the β-sheet crystals can be formed and served as the highly strong netpoints in synthetic polymeric networks with suitable switches, thus enable the polymers to exhibit excellect SME and other desirable properties.
Inspired by spider silks, poly(alanine) (PA) of short chains have been introduced into multiblock biopolymers with poly(ε-caprolactone) segments via a coupling reaction. As a result, PA segments in biopolymers form similar β-sheet crystals to that of natural spidroins, which have been confirmed by FT-IR and XRD. These new biopolymers with PA weight content up to 17 wt% have been found to exhibit nearly complete shape recovery (ge; 99 %) and high shape fixity (ge; 92 %) with εm = 100% after the first thermo-mechaincal cycle. They also possess significantly improved thermal stablility, that is, their rubbery plateaus can reach up to 250 oC which is around 100 oC higher than that of the common SMP polyurethane. In addition, the biocompatibility of the resultant polymers have been evaluated by using L929 cells, and can be considered as safe and smart biomaterials. These results provide a clear evidence to support the above-mentioned mechanism of SME in spider silks, that is, the β-sheet structures act as netpoints. This work provides a new insight for the design of novel SMPs with a potential in biomedical applications.

A challenge for materials chemists working in the area of shape memory polymers is the achievement of softness in such materials. We seek the challenging combination of shape “fixability” and softness in the fixed state for applications where softness is demanded, such as conformable seals, soft lithography, and cell-contacting biomaterials. Here, we will report on our latest findings concerning a new soft shape memory polymer nanocomposite derived from a bacterial medium-chain-length polyhydroxyalkanoate, poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU). The polymeric backbone was grafted with polyhedral oligomeric silsesquioxane (POSS), a crystallizable inorganic-organic hybrid nano-filler. The resulting PHOU-POSS nanocomposite, PHOU-POSSw-net (w (= POSS content, wt %) = 0, 20, 25, 30, 38), was either a completely amorphous elastomer (w le; 20) featured nanocrystalline POSS embedded in the amorphous PHOU matrix (w ge; 25). The hybrid nanostructure of PHOU-POSSw-net (w ge; 25) proved to have a reconfigurable nanostructure wherein aggregation and disaggregation of POSS covalently connected to the PHOU network occurred, enabling excellent shape fixing and recovery. Uniquely, the materials exhibited soft and elastomeric mechanical properties even in the fixed state. We report on the demonstrate micron-scale dynamic surface topography of PHOU-POSSw-net, exploiting the combination of shape memory and fixed-state softness of the materials. Comparisons will be made with alternative approaches to soft shape memory polymers, including smectic elastomers and shape memory elastomeric composites.

A shape memory polymer system capable of response to physiological conditions is developed for use as a substrate in less-invasive, chronically-viable neural interfaces. The thiol-ene/acrylate system synthesized through the use of “click” chemistry provides control of the glass transition temperature and subsequently, modulus in physiological conditions. Modulus control is achieved through tailored plasticization of these highly-uniform networks, despite low water uptake (3-10%). Networks with varied monomer rigidity, crosslink density and hydrogen bonding are synthesized. For substrates that are glassy at physiological temperatures, (necessary for penetration of soft tissue) modulus after implantation was controlled from 2 GPa to 6 MPa. In comparison, silicon-based penetrating neural interfaces (140 GPa) have been shown to have limited stability during chronic implantation. This general failure, usually within a year of implantation, has been partially attributed to the extreme mechanical and geometrical mismatch between the silicon substrate and neural tissue. Dynamic mechanical analysis and differential scanning calorimetry are used to identify the extent of plasticization both in vitro and in vivo. A process that allows for the use of substrates in a planar state for processing and subsequent programming of a 3D shape is also described. This allows for high-density electrode fabrication (minimum feature size of 5 µm) on substrates that conform to the complex shapes and low modulus of tissue after implantation. Free strain and constrained recovery characteristics for the system are evaluated. Recording of neural activity from a softening intracortical electrode array has been achieved for 4 months post-implant, and histology indicates the presence of neurons within 10 µm of the implant. Strategies for drug elution and surface functionalization are also explored. The physiological response of these polymers, combined with excellent tolerance to photolithographic processes, makes this a promising system of biomaterials for neural interfaces, and other types of bioelectronics, that decrease the mechanical and geometrical mismatch at the biotic-abiotic interface.

The thermally-induced shape-memory effect (SME) is the capability of a material to change its shape in a predefined way in response to heat. In shape-memory polymers (SMP) this shape change is the entropy-driven recovery of a mechanical deformation, which was obtained before by application of external stress and was temporarily fixed by formation of physical crosslinks. Besides this dual shape effect recently triple and multiple shape effects as well as temperature memory effects have been introduced.
The structural concept of shape-memory polymers requires generally two key components: covalent or physical crosslinks (hard domains) determining the permanent shape and switching domains fixing the temporary shape. In thermoplastic SMP hard and switching domains de-termining segments are combined in one macromolecule, e.g. multiblockcopolymers such as polyesterurethanes. In covalent polymer networks the switching segments are chemically crosslinked. Here the dual and triple shape effects as well as the temperature memory effect of multiblockcopolymers consisting of poly(omega;-pentadecalactone) hard segments and poly(ε-caprolactone) switching segment are compared with the effects obtained for multiphase poly-mer networks, in which star shaped precursors of the same two types of polyesters are cross-linked with each other. Finally general design criteria for multiphase shape memory polymers are derived which will enable a knowledge based design of actively moving polymers.
References:
A. Lendlein, S. Kelch, Angew Chem Int Ed 2002, 41, 2034
K. Kratz, U. Voigt, A. Lendlein, Adv Funct Mater 2012, 22, 3057
M. Behl, M. Razzaq, A. Lendlein, Adv. Mater. 2010, 22, 3388
J. Zotzmann, M. Behl, Y. Feng, A. Lendlein, Adv. Funct. Mater. 2010, 20, 3583

Understanding the complex neurological processes that mediate daily life has the promise to significantly advance not only how we diagnose and treat neurological disorders, but allow us to understand behavior, cognition and those processes underlying mental illnesses and neurodegenerative disease. Modern imaging techniques provide insight into the location of these processes, but lack the spatial and temporal resolution to fully elucidate neural processes. However, implantable neural interfaces have the spatial and temporal resolution required to allow researchers and clinicians to directly communicate with the central and peripheral nervous system: both to input and extract information. Interacting with neural tissue via synthetic implantable devices to establish a reliable communication platform requires control of geometrical, electronic, mechanical and chemical properties. The majority of neural interfaces commercially available to the neuroscience community rely upon engineering materials commonly used in other fields of research such as silicon, microwires, engineering thermoplastics, and elastomers.
Thermomechanical properties of smart polymers can be specifically tuned to address critical problems in neural interfaces. Efforts in the smart polymer community are described toward modulus changing materials, as opposed to the current focus on shape changing materials, as a paradigm that may lead to new discoveries addressing unmet clinical needs. A current limitation in the field of flexible electronics, and more specifically neural interfaces, is the ability to achieve complex 3-D structures using cost-effective, well-studied photolithographic approaches. A class of dynamic, smart materials called shape memory polymers has been used for interfacing with both inorganic and organic conductors, to create devices that change in stiffness as a function of temperature or environmental condition. Using the shape memory effect enables standard lithographic processing on these (temporarily) flat surfaces, which then can recover (permanently) to new complex shapes. This work broadly explores the effectiveness of photolithographically patterned flexible electronics based on shape memory polymers for a variety of bioengineering applications such as neural brain probes, flexible transistors, flexible antennas, cell culture dishes, cochlear implants and flexible prosthetics. Specific neural interfaces include cortical probes, electrocardiography arrays, longitudinal intrafascicular electrodes, regenerative ‘Y&’ electrodes, nerve cuff electrodes and a host of custom made devices to meet the needs of electrophysiologists.

The incorporation of inorganic fillers such as magnetite nanoparticles (mNP) into a polymer matrix can result in enhanced mechanical properties [1]. In addition, the heating of the mNP in alternating magnetic fields can be used to remotely actuate dual-shape polymers (DSP) by exceeding their thermal transition temperature. Such DSP and their composites can change their shape upon application of an external stimulus such as heat or an alternating magnetic field [2,3]. DSP providing crystallizable matrices are of special interest as they provide a shape recovery, which extends over small temperature intervals or intervals of magnetic field strength. In contrast, when amorphous polymer matrices or blends of crystallizable copolymers providing broad thermal transition are used a magnetic memory effect can be realized in analogy to the temperature-memory effect of thermally-induced DSP [4]. However, homogenous distribution of mNP in crystallizable matrices is a challenge. Decoration of mNP with a polymer shell enables mNP, which better integrate in the DSP matrix but still enhance the mechanical properties [5]. Such mNP decorated with a crystallizable matrix can be obtained by the ring-opening polymerization of cyclic lactones onto the mNP surface [4]. Furthermore, the polymer decorated mNP can be used as multifunctional netpoints for the synthesis of polymer networks, which act a covalent netpoints and add the sensitivity towards magnetic fields. Crystalline, thermally-induced DSP actuated under constant stress in a cyclic, thermomechanical experiment are capable of a reversible shape-memory effect based on crystallization induced elongation (CIE) and melting-induced contraction (MIC) [6,7]. In nanocomposites based on semicrystalline DSP matrix with covalently integrated mNP, which are actuated under constant stress conditions such a reversible shape-memory effect can be actuated by alternating magnetic fields. In this way we could show that the combination of mNP with suitable polymer matrices enables manifold possibilities for active movements.
References
[1] S.K. Bhattacharya et al. J. Electron. Packag. 2002, 124, 1.
[2] A. Lendlein et al., Angew. Chem.-Int. Ed., 2002, 41, 2034-2057.
[3] M. Behl et al., Adv. Mater. 2010, 22, 3388.
[4] M.Y. Razzaq et al., Adv. Funct. Mater. 2012, 22, 184-191.
[5] M.Y. Razzaq et al., J. Mater. Chem. 2012, 22, 9237-9243.
[6] T. Chung et al., Macromolecules, 2008, 41, 184-192.
[7] M. Behl et al., Int. J. Artif. Org., 2011, 34, 231-237.

Here we present a new microparticle system for the selective detection and magnetic removal of bacteria from solution. Highly porous PEG microgels were first synthesized by hard templating based on porous CaCO3 microparticles.[1] The synthesis was performed in three steps: loading of PEG macromonomers into the pores of CaCO3 templates, crosslinking via photopolymerization and removal of the CaCO3 template under acidic conditions. Permeation studies and transmission electron microscopy (TEM) showed that the obtained PEG microgels are inverse replicates of their CaCO3 templates and therefore present internal interconnected pores of 20 to several hundreds of nanometers as well as larger pores on the periphery of about 1 µm. This pore size allows for the binding of bacteria not only on the surface of the particles but also for the partial diffusion of bacteria inside the porous particles and should lead to higher loading efficiencies in comparison to non-porous particles.
Two additional features were thereafter introduced to the porous PEG microgels: a) magnetic properties via electrostatic loading of iron oxide nanoparticles into the porous microgels and b) specific targeting of pathogens via functionalization with sugar ligands. PEG microgels were therefore functionalized by radical chemistry and amide coupling to present cationic amine groups and sugar moieties.[2,3] Both features, magnetism and bacteria targeting, were first evaluated separately and finally combined to give magnetic, porous, sugar-functionalized microgels (MaPoS). We will show that these particles are indeed able to specifically detect bacteria, bind them in high efficiency and remove them from solution by using a magnet.[3] Due to their macroporous structure, MaPoS allow for a maximum binding efficiency of around 30 bacteria per particle, 3-4 times more bacteria compared to non-porous systems. We will also show that the use of sugar ligands allows for the differentiation between different bacterial strains. Therefore MaPoS show great potential for biomedical and biotechnological applications e.g. isolation of bacteria from blood samples or purification of water.
References:
[1] Behra, M.; Schmidt, S.; Hartmann, J.; Volodkin, D. V.; Hartmann, L. Macromol. Rapid Commun. 2012, 33, 1049.
[2] Pussak, D.; Behra, M.; Schmidt, S.; Hartmann, L. Soft Matter 2012, 8, 1664.
[3] Behra, M.; Azzouz, N.; Schmidt, S.; Volodkin, D. V.; Mosca, S.; Chanana, M.; Seeberger, P. H.; Hartmann, L. manuscript submitted

The unique photoluminescence of single-walled carbon nanotubes (SWNTs) in the second near-infrared (NIR-II, 1000-1400 nm) region has made them ideal in vivo fluorescence reporters with unconventional deep tissue penetration and negligible background interference, which are unattainable for traditional fluorophores in the visible (400-750 nm) and short NIR (NIR-I, 750-900 nm) regions. In vivo real-time epifluorescence imaging of mouse hindlimb vasculatures in the NIR-II region is performed using SWNTs as fluorophores, achieving high spatial resolution (~30 um) and temporal resolution (<200 ms/frame) for small vessel imaging at >5 mm deep in the tissue. This spatial resolution is unattainable by traditional NIR or microscopic computed tomography (micro-CT), while the temporal resolution far exceeds scanning microscopic imaging techniques. Moreover, using the deep-penetrating NIR-II fluorophores of SWNTs, mouse brain vasculatures can be imaged with 3D perspective through intact scalp and skull, demonstrating the first all-optical fluorescence-based brain angiography without craniotomy. SWNT-based NIR-II brain vascular imaging has also shown the capability of locating ischemia due to stroke in arteries a few mms inside the brain. In both hindlimb and brain imaging cases, arterial and venous vessels can be unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique based on their distinct hemodynamics. Further, the deep tissue penetration, high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both femoral and cerebral arteries, exhibiting clear evidence of reduced blood flow in both hindlimb ischemia and stroke.

In the context of shape memory polymers (SMPs), it is now clear that soft and responsive materials with an actuation property can play an important role in the field of biomaterials. Thermally responsive shape memory polymers have been widely studied, such materials relying solely on heating above and cooling below a transition temperature for deforming and shape fixing, respectively. In contrast, a class of the widely used materials for medical applications includes hydrogels made by crosslinking of poly(ethylene glycol). Despite a number of advantages of such materials, their static nature prohibits use in applications requiring responsivity. Here, we introduce an alternative triple shape memory elastomeric hydrogel consisting of multiple phases with two well-separated transitions that can response to both heat and mass transfer as dual stimuli. Synthesis and characterization will be reported, including tensile testing, linear viscoelastic properties, and quantitative triple shape memory testing. We will show the existence of three distinct thermal transitions: glass transition and two distinct melting transitions. The material reported has excellent shape memory properties with fixing and recovery each exceeding 90%. Additionally, wide- and small-angle X-ray scattering studies were utilized to investigate crystalline structure in both dry and hydrated states before and after shape-fixing, revealing the micro- and nanostructure behavior of both phases present in the material. The unique combination of softness, triple shape memory mechanical action, and responsivity to both heat and water stimulus for shape change can make this material a good candidate for multifunctional biomaterials and open up a wide applicability such as soft and active substrates for cell-material interaction studies.

Harnessing the immense potential shown by stem cells in the field of regenerative medicine necessitates development of robust and efficient methods of genetically manipulating stem cells to control their fate. To this end, inorganic nanoparticle (NP)-based gene delivery is an attractive approach as these NPs can also offer imaging and therapeutic capabilities owing to their unique morphological, optical, chemical and physical properties, in addition to being efficient delivery vehicles. However, the primary requirements to harness the full potential of inorganic NP-based genetic manipulation of stem cells include: i) synthesizing well-defined nanostructures by integrating multiple inorganic components in a single nanocomposite endowed with orthogonal properties ii) surface-modification of these nanoparticles with appropriate ligands for improved solubility and stability in physiological conditions, and iii) efficient delivery methods that do not perturb the sensitive biological functions of stem cells. Among different inorganic NPs, different types of magnetic nanoparticles (MNPs), including metal or metal oxides, metal alloys and more recently, doped MNPs have been utilized for biological applications. While these newer doped MNPs have significantly higher magnetic susceptibility and hence superior magnetic properties, it would be beneficial to integrate an plasmonic modality within the nanocomposite for multimodal stem cell applications and facile surface chemistry to enhance solubility and biocompatibility, while preserving the core properties.
Addressing these challenges, we herein describe the synthesis of well-defined magnetic core-shell nanoparticles [MCNPs], composed of a highly magnetic core surrounded by a thin uniform gold shell and their application for magnetically facilitated delivery of genetic materials (siRNA and plasmid DNA) into neural stem cells (NSCs). The superior magnetic properties of the doped iron oxide core allow us to deliver these constructs using significantly shorter incubation times to stem cells under the influence of an external magnetic field, thereby minimizing the deletarious effects of transfection agents on stem cells. In addition, the gold outer-shell of the MCNPs can enhance their aqueous solubility, biocompatibility and stability while also providing facile surface chemistry which we exploited to generate MCNP constructs to deliver either siRNAs or pDNAs into stem cells As a proof-of-concept experiment for the genetic manipulation of stem cells and the accompanying differentiation studies, neural stem cells (NSCs) were chosen as they are notoriously known to be very sensitive to conventional exogenous lipid-based transfection reagents as well as difficult-to-transfectHence we hypothesize that we can expect significantly higher transfection efficiency of genetic materials without compromising stem cell viability and biological functions using our MCNPs for magnetically facilitated delivery.

This talk will provide an overview of our recent developments in the design of nanomaterials for ultrasensitive biosensing. Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tissue engineering (1). DNA-, protein- or peptide-functionalised nanoparticle aggregates are particularly useful systems since triggered changes in their aggregation states may be readily monitored. Our recent simple conceptually novel approaches to real-time monitoring of protease, lipase and kinase enzyme action using modular peptide functionalized gold nanoparticles and quantum dots will be presented (2). Furthermore we have recently developed a new approach to ultrasensitive biosensing through plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth (3) as well as a “Plasmonic ELISA” for the ultrasensitive detection of disease biomarkers with the naked eye (4). We are applying these biosensing approaches both in high throughput drug screening and to diagnose diseases ranging from cancer to global health applications.
References
[1] Stevens MM, George JH, Exploring and engineering the cell surface interface., Science, 2005, Vol:310, Pages:1135-1138. [2] Aili D, Mager M, Roche D, Stevens MM, Hybrid Nanoparticle-Liposome Detection of Phospholipase Activity., Nano Lett, Vol:11, Pages:1401-1405. [3] Rodriguez-Lorenzo L, de la Rica R, Alvarez-Puebla RA, Liz-Marzan LM, Stevens MM, Plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth, Nature Materials, 2012, Vol:11, Pages:604-607. [4] De la Rica R, Stevens MM, Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye., Nature Nanotechnology, 2012 online. doi:10.1038/nnano.2012.186.

Shape memory polymers (SMPs) are molecular networks that are stimuli-responsive, undergoing a shape change when externally triggered. Applications include biomedical devices, space applications and textiles. A new application of SMPs developed in recent years is utilized for surface engineering “wrinkling.” Here SMPs can be fixed into a temporarily strained state and a thin rigid coating applied. Upon triggering recovery of the SMP, a compressive stress can be induced into the coating, causing it to buckle and form wrinkles. In our study, a substrate with a modulus gradient was prepared for wrinkle formation. We hypothesized that the modulus gradient of the substrate would result in a gradient of wrinkles during the temperature triggering. Thus, a thermosetting glassy polymer was crosslinked on a temperature gradient to yield glass transition (T_g) gradient ranging from 29 to 48 ^oC over 2 cm. An associated elastic modulus gradient spanning ca. 130 to 1730 MPa at physiological temperature was observed. This functionally graded SMP was then used to create gradient wrinkles by uniaxial stretching in a direction perpendicular to the T_g gradient, coating with a thin layer of gold, and thermally recovering the strain. We will report our detailed findings on spatial grading of wrinkle wavelength and amplitude, as well as the role of SMP viscoelasticity in the growth of the gradient wrinkles. Our talk will conclude with a discussion of potential applications for gradient wrinkles, including stamps for micro-contact printing, gradient cell substrates, and temperature sensors.

Flexible electronics that can retain electrical properties through various deformations and can be interfaced with curvilinear surfaces inside the body could enable biosensors that can read body signals on the chronic time scale by providing enhanced device-tissue interfaces. Flexible organic transistors based on dinaphtho-[2,3-b:2prime;,3prime;-f]thieno[3,2-b]thiophene (DNTT), a high-performance air-stable semiconductor, were fabricated on a shape memory polymer (SMP) substrate, which can conform or be adjusted into various shapes by heating the substrate above its glass transition (Tg) temperature.
SMPs are a class of stiffness-changing smart materials that can recover an imparted strain, facilitated by a drop in modulus when heated above the material&’s Tg. By synthesizing a thiolene-acrylate SMP substrate with a Tg near body temperature, organic transistor devices were fabricated that soften and conform to 3D geometries when exposed to body conditions, enabling future biomedical devices that are compliant to surrounding tissue and stationary after implantation. This thiolene-acrylate SMP substrate softens from 600MPa to 6MPa after 24 hours in body conditions, to more closely match the 10kPa modulus of body tissue. The air stable transistors are able to operate at low voltages under 2V and exhibit excellent mobility of up to 1.7 cm^2/V-s and an on/off current ratio of 10^5.
SMPs with 3D recovery states which are activated by body conditions may enable deployable biosensors that can actively grab or wrap around complex 3D geometries such as nerves, blood vessels, or neural tissue.

For ionic polymer metal composite (IPMC) actuators to work, an inner solvent or carrier is indispensable, as it binds to ion, moves to electrode, and causes bending of the actuator. While water is the most commonly used solvent, it limits the stability of IPMC due to its evaporation and electrolysis during actuation at as low as at 1.3 V. Various alternative solvent with higher boiling point or higher electrolysis voltage have been used to enhance the thermal and electrilytic stability. The attempts have not been successful, however, as significant aggravation in performance in terms of displacement or response rate of the actuators accompanied the enhanced stability. The trade-off seemed invariable, as the stability was acquired by the increase in molecular size of the solvent. In the present study a series of mixed solvents were introduced on purpose to enhance the electrolytic stability of IPMC without significant drop in performance. The mixtures of various organic solvent like formamide with water or deuterated water was substituted for water in typical Nafion IPMC, and their electrolytic stability and actuation performance were investigated. Some of the IPMC prepared showed electrolysis voltage over 2.5 V with similar or better actuation performance compared to that containing water. The role of organic solvent in enhancing stability and performance is discussed.

The development of multifunctional nanomaterials with advanced features for combination of diagnostic and therapeutic applications has been one of the key issues in nanomedicinal
research. Theranostic nanomaterials encompass a wide range of organic and inorganic nanoparticles such as liposomes, dendrimers, polymers, quantum dots, inorganic oxides, etc. The main strategy for the design of multifunctional theranostic nanoparticles is the simultaneous incorporation of the dual modality of the diagnostic element and drug delivery motif into a particle. In this work, we report on the dual function of silica-iron oxide hybrid NPs with stimulus responsive gatekeepers as an efficient carrier platform for drug delivery and MR imaging by introducing iron oxide in the core, drug molecules in the pore, and the CD gatekeeper responsive to glutathione (GSH) which is present in the cytoplasm of cancer cells in much higher concentration than in the extracellular environment. In this hybrid system, anticancer drug molecules should be kept in the pore with CD gatekeepers at the mesopore orifices on the surface of Si NPs. Otherwise, drug molecules would be released from the pore immediately after injection into the blood vessel before the hybrid NPs reach tumor sites.We confirmed, from in vitro study, that DOX was released from the internalized carriers due to GSH-mediated cleavage of the CD gatekeeper. Consequently, apoptotic and clonogenic death occurred in the cells treated with Fe@Si-DOX-CD-PEG. The accumulation of Fe@Si-DOX-CD-PEG in the tumors was detectable by in vivo MR imaging. The growth of the tumor in vivo was effectively suppressed by the intravenously injected Fe@Si-DOX-CD-PEG. In addition, the results of the in vivo MR imaging reflected the in vivo inhibition of the cancer growth by Fe@Si-DOX-CD-PEG. These unique properties of the hybrid nanocarriers could provide an opportunity for aid of the decision making process for patient-specific drug administration strategies in the future.

In the last years, nanotechnology has been looking for a good delivery material which be capable of transport and protect nanoparticles that could be used inside an organism with a specific activity. Several materials have been proposed; among them are natural polymers and one of the most used due to its versatility and its recent approval by the FDA is chitosan.
In this work, a series of hybrid materials composed of chitosan gel with silver nanoparticles (8 nm, spherical morphology) and chitosan gel with gold nanoparticles (13 nm, spherical morphology) was prepared and characterized using ESEM, thermal, rheology, and bactericide analyses against S. mutans (which is the principal organism associated with the beginning of dental caries); silver and gold nanoparticles concentration was changed from 0.005 %wt to 0.1 %wt and chitosan concentration was changed from 0.85 %wt to 3.5 %wt.
ESEM observations show that silver and gold nanoparticles have a good distribution into the chitosan matrix, two samples containing silver nanoparticles showed the conformation of dendrites and cubes which could be an indication of a weak interaction chitosan-silver nanoparticles; thermal analysis shows a better thermal stability in chitosan-gold nanoparticles; rheology analyses show differences when silver and gold nanoparticles concentrations change and bactericide results show that these materials show a comparable antibacterial activity.
As conclusion, we can say that chitosan gel with silver or gold nanoparticles is a good hybrid material with antibacterial activity that could be applied to the control and prevention of dental caries.
Funding: CONACYT Ciencia Basica (CB-169020).

The objective of this study is to formulate nanoparticles adsorbed biocompatible polyhydroxy butyrate valerate (PHBV) film for potential bone tissue engineering applications. BSA conjugated silver nanoparticles (Ag/BSA NPs) were synthesized by chemical reduction of AgNO3 and bovine serum albumin (BSA) mixture. Based on AFM, DLS and TEM, the particle size of nanoparticles was found to be 10-15 nm. XPS measurements of freshly prepared and argon sputtered nanoparticles provided evidence that the outer and inner region of nanoparticles are mainly composed of BSA and silver, respectively. The role of BSA outer layer of bio-conjugated nanoparticles in promoting the adsorption of nanoparticles to synthetic and biological substrates were investigated so as to formulate nanoparticle adsorbed PHBV matrix. Real time adsorption of synthesized Ag/BSA NPs on synthetic and model biological immobilized substrates was followed by surface plasmon resonance (SPR). The extent of adsorption of the nanoparticles on the synthetic surfaces were found to be maximum for -NH2 functionalized akylthiol SAMs and least for -OH terminated alkylthiol SAMs. For protein immobilized substrates the retention of NPs to substrate was found to depend on the pKa of the immobilized protein. Desorption of Ag/BSA NPs from the collagen immobilized substrate was most significant under the acidic and basic conditions and least under physiological pH. Collagen was grafted on biodegradable PHBV film to formulate biocompatible matrix and load Ag/BSA nanoparticles. Density of the grafted collagen on PHBV films was determined using Bradford assay. The extent of adsorption of Ag/BSA NPs on collagen grafted PHBV was determined using AAS. Studies are underway to evaluate the release kinetics of nanoparticles from collagen grafted PHBV film and test the efficacy of the Ag/BSA NPs adsorbed collagen grafted PHBV films to inhibit growth of microbes commonly found in the region of bone tissue infection and promote osteoblast cell growth.

Poly(ethylene glycol) (PEG) is a most interesting material due to its solubility in aqueous as well as most organic media, its hydrolytic stability, its low toxicity and low immunogenicity.[1] It has been used for various applications, e.g. in the fields of drug delivery, tissue engineering or surface coatings.[2]
For many of these applications, the number of functional groups per PEG macromolecule is crucial. For the introduction of a high number of functional groups, copolymerization of functional epoxide monomers with ethylene oxide (EO) has been employed, leading to side-chain functionalized PEGs with e.g. alcohol or amino groups.[3] As the number of known monomers for this purpose is limited, we are interested in developing new epoxide monomers for the production of new multifunctional PEGs.
In this study, we aimed to develop a synthetic path to new benzophenone-functionalized PEGs. The benzophenone group acts as a photo-oxidant upon irradiation with UV light. This property has been utilized e.g. for photo-crosslinking of organic materials or graft polymerization from biomaterial surfaces.[4] The combination of benzophenone photochemistry and PEG properties should allow for great use of the new benzophenone-functionalized PEGs as a multifunctional biomaterial.
Results
We successfully synthesized the benzophenone-functionalized epoxide monomer 4-(2,3-epoxypropoxy)benzophenone (EPBP) in a one-step procedure. The identity of the monomer was verified by various methods (NMR, FT-IR, HPLC, elemental analysis).
We studied the ring-opening anionic homopolymerization of EPBP and the copolymerization of EPBP with EO. By performing the homopolymerization under various reaction conditions (i.e. initiator, solvent, monomer concentration), we found by careful MALDI-TOF analysis of the resulting oligomers that an unexpected side reaction severely limits the molecular weights which can be achieved. This side reaction was observed also during the copolymerization of EPBP with EO, its impact on the resulting molecular weights increasing with higher EPBP content. Copolymers with EPBP contents up to 20 % are soluble in water and exhibit a lower critical solution temperature which lies between 15 °C (20 % EPBP) and 60 °C (2.5 % EPBP).
In order to prove that the photoreactivity of the benzophenone groups remained intact after polymerization, a copolymer with 5 % EPBP content was spin-coated onto a silicon wafer and irradiated with UV light for 5 minutes, leading to a crosslinked and insoluble polymer coating.
In conclusion, the presented synthetic path to side-chain benzophenone-functionalized PEGs leads to the formation of photo-crosslinkable and thermoresponsive polymers.
[1] Int. J. Toxicol. 1993, 12, 429-457.
[2] a) Toxicology 2005, 214, 1-38; b) Angew. Chem. Int. Ed. 2010, 49, 6288-6308.
[3] Polym. Chem. 2012, 3, 1714-1721.
[4] a) J. Appl. Polym. Sci. 2011, 122, 168-174; b) J. Photopolym. Sci. Technol. 2010, 23, 161-166.

Gelatin, derived from native collagen, is a very promising matrix material for tissue engineering applications, e.g. due to its native Arg-Gly-Asp (RGD) content. Gelatin has the ability to form triple helices like collagen, yet the resulting gels are thermo-sensitive and not stable at physiological temperature [1]. Various methods have been applied to achieve thermally stable gelatin hydrogels. However, until now its reported mechanical strength is still limited.
Besides the adjustment of mechanical and biological properties of gelatin-derived hydrogels, a control of the viscous behavior of their precursor solutions has become increasingly interesting. Future medical technology based on regenerative medicine needs flexible and effective technologies, e.g. liquid handling techniques. Due to its high viscosity and gelling effects gelatin solutions are prone to clogging pipets and dispensers [2].
The objective of the present study is the preparation of gelatin based biomaterials that constitute both, processable precursor solutions and hydrogels with tunable physico-chemical properties based on photopolymerizable biopolymers. Photocrosslinkable gelatin and chondroitin sulfate were prepared by derivatization with methacrylic anhydride [1]. Hydrogels were gained by photo-induced radical crosslinking in the presence of a water-soluble photoinitiator. The viscoelastic properties and long-term stability of the gels have been analyzed by swelling experiments and rheological measurements. Furthermore, gels were investigated as encapsulation matrices for porcine articular chondrocytes to evaluate their applicability in respect to the generation of artificial cartilage. In regard to their processability by liquid handling techniques, i.e. piezoelectric printing, the precursor solutions&’ properties such as viscosity were analyzed and optimized by further chemical derivatization of gelatin.
The paper discusses our approach to biopolymer-based hydrogels with tunable physico-chemical properties and their use in a biomimetic extracellular matix for three-dimensional cartilage substitutes. The developed precursor solutions hold low viscosities and no gelling effects. Hence, the hydrogel system is suitable for processing by future medical technologies employing printing techniques.
Van den Bulcke et al., Biomacromolecules 1, 31-8 (2000).
Maher et al., Rapid Prototyping J 15, 204-10 (2009).

When a plasmonic nanoparticle is embedding in the evanescent field of a nanofiber waveguide, its scattering cross-section is extremely sensitive to the distance normal to the propagation direction of light. This enhanced distance sensitivity is duo to the strong dielectric-plasmonic coupling effects and the sharp decaying near-field of the waveguide. Recently we have developed a novel force sensing system that leverages near-field light-matter interactions to monitor molecular distance changes and forces. As part of this system, thin(<15nm thickness) compressible polymer coatings are used to provide mechanical feedback for individual plasmonic nanoparticles. Here we present the mechanical characterization results of two studied system. Polyethylene glycol (PEG) and polyelectrolyte multilayers (PEMs), which create uniform and conformal coatings on the nanofibers can be tuned to give the force transducers large dynamic ranges. Atomic force microscopy (AFM) is used to characterize the mechanical properties of the thin polymer coatings and the stiffness of the films is tuned by changing polymer&’s molecular weight, cross-linking density, etc. These hybrid waveguide-nanoparticle structures offer a wide variety of applications including in situ force sensing probes, microscopy and high-throughput nanomechanical analysis.

In biological cells, the membrane continuously changes in response to micro environment through the interplay between lipids and proteins, which play important roles in various cellular tasks. Unlike in biological cells, it has never been realized outside cells to control the membrane curvature and the interface using specific peptides or proteins because it is difficult to control the balance of tension forces at all surfaces and junctions.
Tyrosine is one of the most significant amino acids. They can store the mechanical energy by protein folding and also facilitate proton-mediated electron transport in photosystem II. In our study, we systematically introduced repeating tyrosine units into peptides of various lengths to study the impact of the peptide sequence on self-assembly. The ordering of peptides investigated here has a strong driving force that overcomes the large surface tension of water. We have identified specific sequences of tyrosine containing peptides that can afford densely packed 2D film structure at air/water interface and the resulting structure can withstand the surface tension of water and modify the intrinsic curvature of water droplet. The atomic force microscopy (AFM) analysis along the film edges shows the evidence of film stacking of multiple nano sheets, whoes minimum thickness is 1.4 nm. Additionally, the peptide interface presented here provides a tunable platform to template 2D hybrid materials. Consequently, highly ordered 2D assemblies of polypyrrole that cannot be formed using conventional methods were created for the first time. This self assembled peptide surface could be utilized in many applications such as floating scaffolds for cell culturing, responsive vesicles for drug delivery, ion-selective membranes, catalysts and conductive materials.

Simple and purpose-fit method of surface modification has been explored. Examples of conventional modification method such organosilane chemistry is suitable for particular substances but includes oxidative instability and difficulty of conducting in hydrous condition, which can limit the utilization of living cells in biomedical applications. Therefore, having flexibility in substrates and giving room for post modification with biomolecules, polymer coating with catecholamine is especially being spotlighted. Inspired by mussel, versatile aqueous active surface modification system has been developed with catecholamine. By simple deep-coating method, SAM-like layer of catecholamine is formed, already proven to be useful for both inorganic and organic substrates. This versatile approach not only allows flexible modification in terms of choosing substrate materials but also it is an inexpensive and eco-friendly process.

Norepinephrine and dopamine are both affiliated to a catecholamine family. In alkali condition, they undergo self-oxidative polymerization that allows versatile surface modification. However, unlike poly-dopamine (PDA), the additional alkyl group on poly-norepinephrine (pNOR) gives additional surface chemistry not possible by PDA coating. Herein, we investigated the facilitation of cell adhesion on multifunctional active surface modified with pNOR. The performance of modification with pNOR on a variety of substrates such as metal oxides, ceramic, semiconductors, noble metals and synthetic polymer was successfully demonstrated and characterized by Kang et al. In this study, the instant adhesion of cells was observed on the modified substrates by simply deep-coating method, triggering self-oxidative polymerization in basic condition. Due to the even coating of pNOR, uniformly spreading of attached cell was observed. Furthermore, we investigated cell morphology by staining actin and nuclei, and cell viability with WST-1 cell proliferation assay. Overall, by employing aqueous active and material-independent modification system, we confirmed that the cell adhesion is boosted on pNOR modified substrates as well as the cell proliferation.

The aromatic compounds desaminotyrosine (DAT) and desaminotyrosyltyrosine (DATT) were successfully used to functionalize gelatin in order to form physically crosslinked networks via interaction and hydrogen bonds of the introduced phenols.[1] In this study, the application of this concept to a synthetic polymer was studied which would not exhibit additional interactions such as triple helix formation asin gelatin. As synthetic polymer for the backbone structure we chose oligoethylene glycols (OEG).
Linear OEG (MP = 3 kDa) with amino functionalities as endgroups as well as 4-arm amino OEG (Mn = 5, 10, and 20 kDa) have been functionalized with DAT and DATT (yield up to 90%) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as activating reagent. The compounds have been characterized by NMR and IR spectroscopy. Compared to the unfunctionalized OEG, aqueous solutions of the materials showed a decrease of storage modulus G&’ and loss modulus G&’&’ in rheology. Furthermore, aq. solutions of the OEG-DAT(T) showed lower surface tension than solutions of the starting materials. The critical micelle concentration was determined by a fluorescence spectroscopy using pyrene as hydrophobic fluorescent dye. Due to the structure of the macromolecule with a large hydrophilic core and two hydrophobic end groups, the polymers showed properties of a surfactant, and did not form gels as previously observed for DAT(T)-gelatins.[2] In order to evaluate cellular effects, OEG-DAT(T) solutions were added (0.23 ng/ml - 1000 µg/ml) to murine RAW macrophages. The viability of RAW cells was not significantly impaired, even at highest OEG-DAT(T) concentrations (1000 µg/ml). Since the RAW cells did not show signs of activation, the data indicate that the materials were not contaminated with microbial products and would subsequently not lead to inflammatory responses. This could be confirmed by incubating the polymers with whole human blood and the determination of the production of inflammatory cytokines such as the tumor necrosis factor (TNF)-α and interleukin(IL)-6. Only at high concentrations, the OEG-DAT(T) solution induced low levels of TNF-α and IL-6, indicating that a mild inflammatory reaction could be induced by such high OEG-DAT(T) concentrations. The materials are degraded by H2O2 as was determined by MALDI-ToF MS analyses. This is important for in vivo applications, as macrophages release H₂O₂.
Conclusively, it was demonstrated that DAT(T) functionalized OEGs are compatible with macrophages and induce mild inflammatory response only at very high concentrations, indicating that the materials are suitable as biocompatible, degradable surfactants e.g. to solubilize hydrophobic compounds such as drugs in water.
1. A.T. Neffe, A. Zaupa, B.F. Pierce, D. Hofmann, A. Lendlein, Macromol. Rapid Commun. 2010, 31, 1534-1539.
2. K.K. Julich-Gruner, A.T. Neffe, A. Lendlein, J. Appl. Biomater. Funct. Mater. 2012, in print.

Crosslinking of gelatin with L-lysine diisocyanate ethyl ester (LDI) allows the formation of biocompatible, degradable hydrogel networks with tailorable mechanical properties and swelling behavior [1]. Here, we investigated which influence the different solubility in water and inherent reactivity of the isocyanate groups of butane diisocyanate (BDI), isophorone diisocyanate (IPDI), 2,4-toluene diisocyanate (TDI) and L-lysine diisocyanate ethyl ester (LDI) had on the formation of networks and their properties and functions.
Covalent crosslinking successfully suppressed self-organization of gelatin chains to triple and single helices (< 5% helical content) and resulted in elastic hydrogel systems with Young&’s moduli of 6-740 kPa, water uptake of 200-1400 wt.% and degradation time in aqueous solution ranging from few days to several weeks depending on gelatin concentration and amount of crosslinker, as well as chosen type of diisocyanate.
Investigation of netpoint density clearly showed that the kinetics and efficiencies of crosslinking reactions were strongly dependent on initial reactivity and solubility of the applied diisocyanate compound. Although isocyanates do react readily with water, test reactions investigated by ESI mass spectrometry clearly showed the formation of a significant amount of mono- or oligomeric direct crosslinks as well as grafting, both contributing to suppression of collagen-like helices and stabilization of the mechanical properties of the hydrogels. Formed side products like oligo-urea compounds are typically water soluble and washed out after finishing the synthesis. Overall, diisocyanate crosslinked gelatin-based hydrogels are easily obtained promising candidates for multifunctional materials with high potential for biomedical applications.
[1]G. Tronci, A.T. Neffe, B.F. Pierce, A. Lendlein, J. Mater. Chem 2010, 20, 8875.

Precisely defined multi-dimensional hierarchical structures in nano- or micrometer scale are a requisite for the fabrication of various functional devices in all fields of science and engineering. Conventional lithography techniques (i.e., photolithography, e-beam lithography, dip-pen nanolithography, nanoimprint lithography, and etc.) have been utilized to fabricate various devices for electronics, mechanics, and biomedical engineering. Despite their remarkable attributes and capabilities, those fabrication processes often require complicate procedures as well as considerable labors and expenses. However, in nature many hierarchically organized nanostructures (i.e., diatoms, abalone shell, butterfly wing, and moth eyes) possess exquisite structures and functions, which surpassing the capability achievable by current top-down and bottom-up fabrication methods. Moreover, many of these structures are made of a simple basic building block.
Inspired by nature&’s self-templated assembly processes, herein, we developed a novel biomimetic micropatterning technique to create well-defined two- and three-dimensional hierarchical structures by using elastocapillary forces of helical nanofiber particles at the air/liquid/solid interfaces. We utilized M13 bacteriophage (phage) as a model helical nanofiber building block, due to its&’ monodispersity, liquid crystalline property, and genetic flexibility to display functional peptides. By controlling meniscus forces, we could induce formation of the smectic nanofilament phases of the phage and tune the adhesion properties between the nanofilaments-to-nanofilament and nanofilament-to-solid substrates. The resulting structures possess hierarchically organized two- and three-dimensional periodic structures with exquisite optical properties. These self-assembled multi-dimensional hierarchical structures were tunable by varying parameters that affect the kinetics and thermodynamics of assembly such as pulling speed, pulling time, and ionic concentration. The resulting microstructures could enhance the power of phage-based piezoelectric energy generations. Our facile bio-inspired self-assembly strategy may provide the way to fabricate large-scale advanced micro electronic or optical devices and biomedical applications in the future.

Silk fibroin, extracted from silkworm cocoons, has aroused significant attention due to its biocompatibility, biodegradability, high mechanical strength, and morphologic flexibility. In our study we investigate the capability of silk fibroin to undergo hydrogen-bonded self-assembly with synthetic macromolecules such as poly(methacrylic) acid and poly(N-vinylcaprolactam) as well as with the natural polyphenol, tannic acid in an aqueous environment. We found that silk assembled with poly(methacrylic) and tannic acids on flat templates resulted in pH-sensitive films while silk/poly(N-vinylcaprolactam) films were found to be stable under pH variations. Our results suggest that the intermolecular interactions are primarily driven by hydrogen bonding with a considerable contribution of hydrophobic forces. We also showed that silk preserved its predominant random coil conformation upon assembly as monitored with in situ ATR-FTIR. Finally, we demonstrated that capsules of cubical, spherical and platelet shape were fabricated by using particulate sacrificial templates. Our work introduces new fundamental aspects of silk-based assembly and provides a new platform for designing silk-biomimetic materials with by-design properties.

Brand-new ideas with silk materials have enabled to widen applications of silkworm silk (especially, fibroin) beyond clothes to food, optical device, scaffold, gel, film, and so on. However, relatively poor biodegradation and cell adhesive ability have hindered its wide range of application for cell and tissue engineering. For this reason, many attempts to develop characteristics of fibroin as a tissue engineering material have tried. Here, we attempted to improve characteristics of silk fibroin by introduction of recombinant mussel adhesive proteins (MAPs). Mussel can strongly affix to the wet surfaces by using secretion of MAPs and maintain its adhesive force and flexibility even under harsh conditions. Previously, we successfully mass-produced recombinant MAP in bacterial expression system. We also designed and constructed RGD peptide-fused recombinant MAP as a new cell adhesive which leads better cell attachement, spreading, proliferation, and even differentiation. By co-electrospinning of MAP-RGD with silk fibroin, we fabricated nanofiber sheet as tissue engineering scaffold and investigated cell behaviors on the constructed nanofibrous scaffold. Above all, the constructed nanofibrous scaffold with silk fibroin and MAP has a lot of potentials for functionalization of biomolecules which be used for advanced bioactivity or cell and tissue engineering without complicate modification of scaffold surface.

Organic Thin Film Transistors (OTFT) gated through an aqueous electrolyte have been reported in the past. These devices can operate either in the electrochemical (OECT) or in the field-effect (OFET) mode of operation. For these devices, the voltage signal applied at the gate of the transistor will result in a current modulation at the drain contact. The associated high voltage-to-current gain achieved in these devices suggests using them as sensors in for instance aqueous media. It is possible to introduce analytes directly into the aqueous gate configuration; thus makes them promising as the transducer in single-use devices in chemical, biological and biochemical sensor applications. Here, we report modeling and analysis work performed on water-gated OTFT devices including three different conjugated polymers in the channel. The goal has been to distinguish between the OECT and OFET mode of operation. Three different polythiophene derivatives have been studied including hexyl- and carboxyl side groups with the ratio of 1:0, 6:1 and 0:1, respectively. Carboxyl end groups are of particular interest since these sites can serve as the anchoring sites for biorecognition elements.

Self-healing is a vital function of the human skin. However, the human skin&’s repeatable self-healing ability has never been successfully realized on electronic sensor skins until now. Here, we demonstrated the first example of a repeatable self-healing electronic sensor skin by using a composite of a self-healing supramolecular polymer and nano-structured metal particles. By rationally varying the composition of the electrically conductive inorganic fillers, we can obtain both electrodes and piezo-resistive sensor materials. The composite exhibits excellent mechanical flexibility and its electrical conductivity can reach as high as 40 S cm-1. Remarkably, both conductors and sensors display repeatable high electrical and mechanical healing capability at room temperature. We further integrated our self-healing electronic composite into an electronic sensor skin capable of detecting touch and flexion. We anticipate that our strategy of using supramolecular organic-inorganic composites will push forward the exciting application frontier of self-healing, multi-functional electronic composites.
References:
1 C. Wang*, B. Tee*, Z. Bao et al. Nat. Nanotechnol. accepted.

Materials with tunable morphology and mechanical properties show great promise for a wide range of applications in energy, biotechnology, and medicine. Assemblies that rely on highly specific biomolecular interactions are an attractive approach for the bottom-up design of materials with sophisticated properties. Here, we use the intrinsic self-assembling properties of the designed, rod shaped, superhelical consensus sequence tetratricopeptide repeat protein, CTPR, to generate such protein-polymers hybrid materials.
CTPR arrays comprised of 20 tandem repeats self-assemble into uniformly birefringent, transparent films upon solvent casting. X-ray scattering experiments confirm macroscopic alignment of the CTPR molecules within the film and CD and FTIR measurements show that the CTPR protein retains α-helical secondary structure. Individual CTPR arrays in the film are further covalently cross-linked using linear bi-functional photoactive polyethylene glycol. Here we will discuss rheological and thermal properties of resulting networks as a function of polyethylene glycol concentration and length.

Hydrogels are broadly used in bioengineering, but the scope of their applications is often severely limited by the mechanical behavior of hydrogels. Here we report exceptionally stretchable and tough hydrogels made of polymers forming networks via ionic and covalent crosslinks. Although such a gel contains ~ 90% water, it can be stretched beyond 20 times its initial length, and has fracture energy of ~9000 J/m2. Even for samples containing notches, a stretch of 17 is demonstrated. The high fracture energy is attributed to the synergy of two toughening mechanisms: crack bringing by the network of covalent crosslinks, and hysteresis by unzipping the network of ionic crosslinks over a large region of the gel. Furthermore, the network of covalent crosslinks preserves the memory of the initial state, so that much of the large deformation is removed when the load is removed. The unzipped ionic crosslinks cause internal damage, which heals as ionic crosslinks re-zip. We envision that these hydrogels will serve as model systems to explore mechanisms of deformation and energy dissipation, and that hydrogels with enhanced mechanical properties will considerably expand the scope of their applications. This talk draws upon Jeong-Yun Sun, Xuanhe Zhao, Widusha R.K. Illeperuma, Kyu Hwan Oh, David J. Mooney, Joost J. Vlassak, Zhigang Suo. Highly stretchable and tough hydrogels. Nature 489, 133-136 (2012).

Reconfigurable covalent networks confer many unique capabilities in smart materials including shape-memory properties and the capacity for self-healing. In this work, we describe the application of general strategies for reconfigurable polymers for use in medical devices and materials. These efforts utilize concepts of reconfigurable polymer networks across multiple length scales and utilize several exogenous stimuli such as light and heat. The results of these efforts are a new class of stimuli-responsive biodegradable elastomers and gels that will be illustrated in two examples. The first case describes the design and synthesis of elastomeric polyesters with reconfigurable covalent crosslinking. These biodegradable materials can be processed into structures with complex geometries and linear degradation kinetics. The properties of these materials render them ideal for applications in endovascular devices. The second example describes the use of crosslinked networks that are can reconfigured using exogenous light. Photolabile block copolymers are assembled into physically crosslinked networks that can be tuned to form either solid films or hydrogels. The morphological and physical properties of these networks were characterized using several techniques including atomic force microscopy, tensile testing, and rheology. The rapid disintegration of these networks can be triggered through reconfiguration of the physical crosslinks. Taken together, these results suggest that reconfigurable biodegradable polymeric networks have the potential for substantial utility as smart medical materials.

Hydrogels have been long used as cell-entrapping carriers for applications ranging from injectable biomaterials to in vitro cell culture studies. In such scenarios, the cells are passively entrapped in a polymer gel matrix. We demonstrate an alternate scenario where cells serve as active structural elements (crosslinks) in a polymer gel network. The polymers used in this context are hydrophobically modified (hm) derivatives of common polysaccharides such as alginate and chitosan. These polymers are able to rapidly transform a liquid suspension of cells into an elastic gel. In contrast, the native biopolymer (without hydrophobes) does not cause such gelation. Gelation occurs because the hydrophobes extending from the polymer chains get inserted into cell membranes due to hydrophobic interactions. The polymer chains thus bridge the cells, which act as crosslink points (junctions) in a 3-D network. We will show that a variety of cell types, including blood cells and endothelial cells, can be gelled by this approach, and we will discuss the fate of the cells in the network after gelation. Finally, as the crosslinking mechanism is based on hydrophobic interactions, we will show that addition of supramolecules with strong hydrophobic binding pockets can reverse the crosslinking and release the cells.

Biodegradable polymers are clinically used in numerous biomedical applications, but may display a loss in mechanical properties within weeks of implantation. The goal was to use a two-component semi-degradable polymer network to increase the initial mechanical properties during degradation by shifting the network&’s glass transition temperature. The network was formed from a biodegradable poly(β-amino ester) with a low glass transition temperature and a non-degradable (meth)acrylic monomer with a high glass transition temperature (45 wt%/55wt% mix ratio). It was hypothesized that by degrading out the low glass transition temperature component, the network&’s mechanical properties would shift in favor of the high glass transition temperature component. Samples were degraded in vitro in saline and in vivo via subcutaneous implantation in mice. IACUC protocols were followed. After degradation for 8 weeks, samples were strained to failure in tension to characterize bulk mechanical properties. DMA and DSC were used to characterize changes in thermo-mechanical properties, and ATR-FTIR was used to characterize changes in chemical structure. Biocompatibility and inflammatory responses were assessed with histological and immunohistochemical evaluation. By altering the hydrophilicity of the biodegradable component, the mass loss of the network ranged from 3 to 30% over 8 weeks, where 45% would have been the most mass loss expected since semi-degradable. The glass transition temperature increased by 30°Celsius by DSC and 40°C by DMA, and the corresponding elastic modulus increased from 3 MPa to over 200 MPa. The loss of the biodegradable component was verified chemically by a reduction of ester groups via ATR-FTIR and physically by a decrease in crosslinking density via DMA from near 250 to 100 mol/m3. Samples degraded in vivo showed similar increases in glass transition temperature and modulus. Biocompatibility testing showed a favorable response, regardless of the composition, suggesting a shift in thermo-mechanical properties did not negatively affect biocompatibility. The results suggest that these biocompatible semi-degradable networks have the potential for increased mechanical performance during initial stages of implantation. Also, the shape-memory and controlled release behaviors were characterized. The networks displayed near 90% shape recovery under free-strain conditions and up to 35% cumulative molecule release over 4 weeks. These additional properties demonstrate a multifunctional biomaterial with the potential for minimally invasive procedures and drug release with increasing mechanical properties during initial implantation.

Neutral, hydrophilic, polymer-based architectures are widely investigated for a wide range of biomedical applications from drug-conjugates to delivery systems and tissue engineering devices.(1) In most cases, it is crucial that biomaterials provide a blank, inert background in order to hinder unspecific cell-material interactions so that the foreign body reaction is prevented. As an example, poly(ethylene glycol) (PEG) based networks represent one of the most promising classes of non-fouling materials. To this end, we aimed to create novel copolymer networks which might serve as potential antifouling biomaterials. Hydrophilic poly(glycidyl ether) (PGE) networks are a recently developed class of amorphous macroscopic materials, which offer great versatility in design and control of resultant properties.(2)
PGE networks were synthesised via cationic ring-opening polymerization of various glycidyl ethers in the absence of solvent with diphenyliodonium hexafluorophosphate as photoinitiator and acid source. The reaction proceeds in high yield as observed by FTIR spectroscopy and results in thermoset materials of high gel content. The networks are amorphous and exhibit a single well-defined thermal transition corresponding to the glass transition. Detailed dynamic mechanical analyses indicate that copolymers from a range of different glycidyl ether monomers are homogeneous over a wide range of composition ratios with no observable phase separation.
Copolymers investigated were prepared from tritopic glycerol glycidyl ether, ditopic PEG-diglycidyl ethers (of variable average molecular weight), and/or monoglycidyl ethers with short aliphatic sidechains. In order to rationalise the final network architecture, the multitopic monomers can be considered as cross-linking agents whilst monoglycidyl ethers are chain extenders. The polymerized chains are analogous in structure to hyperbranched or linear polyglycerols, which are becoming increasingly established as a class of biocompatible polymers. Thermal and mechanical properties were investigated in detail and it was found in all cases that there is a clear correlation of cross-link density (derived by swelling experiments) with the bulk properties. For example, Young&’s moduli can rationally be tuned from several kPa to several MPa range, and glass transition temperatures can be varied from 26 to -70 degree celsius. Considering the high cross-link densities of the networks, the resultant materials are relatively soft and elastic. The novelty of these systems arises from the exclusively polyether architecture, with a relatively flexible and hydrophilic main chain. The modular design allows for adaptation towards a range of biomaterial applications.
References
1. Chen S.; Li L.; Zhao C.; Zhang J., Polymer, 2010, 51, 5283
2. Ekinci D.; Sisson A. L.; Lendlein A., J. Mater. Chem., 2012, 22, 21100

Biological systems display unique features, such as adaptiveness, homeostasis, motility, reconfiguration, replication and ultimately, evolution. Despite their apparent complexity, these features are ultimately a result of just three molecular processes: molecular recognition, self-assembly and catalysis. We have set ourselves the challenge to harness these features through robust, synthetic analogues of the biological systems based on short peptide derivatives. This is achieved using minimal self-assembling gel-phase systems that are driven by catalysis, and by exploiting both equilibrium (Nature Nano 2009) and non-equilibrium (Nature Chem 2010) self-assembly approaches. We will demonstrate how these systems may adapt to changes in environmental conditions and may be used to identify novel functional charge transfer materials. Molecular biomimetics of this type provides tremendous technological opportunities in biomedicine and nanotechnology.

The swelling-induced shape memory behavior in polymers has inspired interest for their implications for biomedical applications. Absorption of a small concentration of solvent can lead to a dramatic reduction in stiffness and strength as well as loss of shape fixity [1]. For amorphous polymers, these changes in material properties and shape fixity are caused by a large decrease in the glass transition temperature caused by the absorption of a small amount of solvent. In this work, we present a theoretical model of the effect of solvent absorption on the glass transition behavior of the materials. The model is based on a previous thermoviscoelastic model that has successfully described the effect of thermochanical conditions and properties on the recovery response of these materials [2]. The central idea of the model is that the presence of solvent increases the configurational entropy; thus altering the temperature-dependence of the molecular mobility. The model was implemented numerically for finite element simulation. The computational model also considers the effect of diffusion process to describe more accurately the time-dependent effects of solvent-induced shape recovery behavior. To validate the model, we performed isothermal uniaxial tension tests on both the dry and fully saturated materials to measure the effect of solvent absorption on stiffness and strength. Shape recovery performance was measured by observing the shape change of an initially deformed sample in an isothermal water bath using digital image tracking.
[1] K. E. Smith, P. Trusty, B. Wan, K. Gall (2011) Acta Biomaterialia, 7:558-567.
[2] T. D. Nguyen, C. M. Yakacki, P. D. Brahmbhatt, M. L. Chambers (2010) Advanced Materials, 22:34411-3423.

Many materials undergo large deformations in response to a wide range of stimuli, such materials are known as active materials. In recent years thermally active materials are beginning to be used for critical biomedical applications, microsystems, drug delivery, sensors, and actuators, among others. In what follows, I will discuss current research on thermally active materials, specifically thermally-actuated elastomeric gels. An elastomeric gel is a cross-linked polymer network swollen with a solvent, and certain gels can undergo large reversible volume changes as they are cycled about a critical temperature. I discuss the development of a thermodynamically-consistent continuum-level theory to describe the coupled mechanical deformation, fluid permeation, and heat transfer of such thermally-responsive gels. In discussing special constitutive equations, attention is limited to isotropic materials, and is based on a modified Flory-Huggins model for the free energy change due to mixing of the fluid with the polymer network, coupled with a non-Gaussian statistical-mechanical model for the change in configurational entropy --- a model which accounts for the limited extensibility of polymer chains. The theory has been numerically implemented in a finite element program by writing special finite elements that couple mechanical deformation, fluid permeation, and heat transfer. I show that the theory is capable of simulating swelling, squeezing of fluid by applied mechanical forces, and thermally-responsive swelling/de-swelling of such materials.

Microparticle as a cell carrier has received a great attention in field of biological assay because they allow a three-dimensional cell-culture environment and wide versatility for multiplex assays. Especially, for heterogeneous cell-culture environment which is based on cell-carrier technology, it is required that the heterogeneous microparticles as a cell-carriers are assembled to form a multiplex cell-carrier with control.
Here, we developed a new fluidic self-assembly method to generate a multiplex cell-carrier for heterogeneous transfection. With this newly developed method we can assemble particle into another particles in fluid as we desired. This absorptive polymeric microparticle can hold numerous liquid based substances. By controlling size and shapes of particle we have successfully control the locations where heterogeneous smaller particles to be assembled. This method offers very simple fabrication and operation for biological applications.
The micro particle materials for this method are based on combination of photo curable polymer PEGDA 700 with 10% photo initiator. By using OFML (Optofluidic Maskless Lithography) device we can control size and shapes of micro particle for this assembly method. The OFML can be divided into three parts; UV source, Digital Micro Mirror, polymer loading plate. The photo curable polymer becomes polymerized from patterned UV irradiation where this light is created by DMD mirror reflection. Then various kinds of particles can be made with this device. There are two different kinds of particles, base particle with small hole in it and smaller particle which can be fitted into the base particles. To locate heterogeneous smaller particles into desired places, we need two assembly steps. First, we assemble base particles onto the pre made PDMS well plate. Then many different shaped particles are well assembled into each well. Next, smaller particles are assembled into base particles. After these steps various kinds of smaller particles are assembled into base particles.
The main advantage of this method is that we can easily generate an assembled heterogeneous cell-culture environment without precise cell-patterning process, which may increase the process complexity by increasing heterogeneity. This three dimensional multiplex microparticle can be used for cell-cell communication and tissue engineering by providing more complexity and heterogeneity to cell culture platform.

Although naturally occurring membrane lytic antimicrobial peptides (AMPs) and their analogs hold enormous promise for antibiotics-resistance infectious disease therapies, significant challenges such as systemic toxicities, long peptide sequences, poor understanding of structure-activity relationships and the potential for compromising innate host defense immunity have greatly limited their clinical applicability. In our efforts to improve the clinical potential of AMPs, we adopt a facile approach to design a series of short synthetic β-sheet folding peptide amphiphiles comprised of short recurring (X1Y1X2Y2)n-NH2 sequences, where X1 and X2: hydrophobic residues (Val, Ile, Phe or Trp), Y1 and Y2: cationic residues (Arg or Lys), and n: number of repeat units; with systematic variations to the cationic and hydrophobic residues to obtain optimized AMP sequences bearing minimal resemblance to naturally occurring sequences. The designed β-sheet forming peptides exhibit strong and broad spectrum antimicrobial activities against various clinically relevant microorganisms, including Gram-positive S. aureus, Gram-negative E. coli and P. aeruginosa, and yeast C. albicans, with excellent selectivities for microbial membranes. Optimal synthetic peptides with n =2 and n =3 repeat units, namely (IRIK)2-NH2 and (IRVK)3-NH2, effectively inhibit sessile biofilm bacteria growth and lead to biomass reduction. Additionally, (VRVK)3-NH2, (IRVK)3-NH2 and (IRIK)3-NH2 neutralize endotoxins while causing minimal cytotoxicities. Taken together, our findings clearly demonstrate that the rationally designed synthetic β-sheet folding peptides are highly selective, non-cytotoxic and have tremendous potential for use as broad spectrum antimicrobial agents to overcome the problem of multidrug resistance in a wide range of localized, systemic, or external therapeutic applications.

For biosensor devices, functionalizing the surface with covalent linkers is usually employed. However, it is difficult to avoid the loss of activity following the bioreceptor-analyte binding event, which limits the lifetime of the device. The goal of this research is to use phage display to biopan for inorganic binding peptides that are reversible upon applying of an electric field. This can provide dynamic functionalization of surfaces, with applications such as self-cleaning devices. For example, when the bioreceptor becomes clogged, the peptides may be released by triggering an electric field to generate a non-binding state. A fresh surface of bioreceptors can then be applied via a flow through setup. Our group has been panning for peptides that bind strongly to indium zinc oxide (IZO), a transparent conducting oxide, which makes it an attractive electrode for biosensors. An electro-releasing device is being used to collect the strong binding peptides that are released. In an alternative method, because a strong-binding peptide might not be a reversible peptide, our group has also developed a novel phage display biopanning protocol with an electro-elution process instead of the regular chemical elution. Current studies include the comparison of these two approaches for the goal for developing self-cleaning biosensor devices.

Atherosclerosis leads to cardiovascular disease, the number one cause of death in the US. The detection of atherosclerosis poses a difficult problem as plaque formation progresses through multiple stages, each with different upregulated markers. Inflammation plays a large role in the development of atherosclerosis and numerous inflammatory markers are present and expressed by the endothelial cells that line the plaque. These signaling markers of inflammation lead to the recruitment of monocytes and eventually to the growth of the underlying plaque. To address this problem, our work aims to deliver anti-inflammatory drugs to the site of atherosclerotic plaque formation using peptides that target and bind to markers of plaque development. Herein, we present a peptide amphiphile micelle drug delivery system that is multi-modal and, due to its self-assembled nature, allows for easy incorporation of both multiple targeting peptides as well as the ability to carry a drug of interest to the site of plaque formation. Peptide amphiphiles are formed by conjugating a hydrophilic peptide headgroup to a hydrophobic lipid tail. In this work, the hydrophobic tail, DSPE-PEG2000, was conjugated to a peptide that binds specifically to vascular cell adhesion molecule-1 (VCAM-1), a marker of inflammation that is upregulated in early stage atherosclerotic plaques. Dynamic light scattering confirmed the presence of spherical micelles on the order of 20 nm in diameter. The critical micelle concentration of these VCAM-1 micelles, or the concentration at which micelles begin to form, was determined to be approximately 10 mu;M. These VCAM-1 micelles have been shown to be biocompatible and do not significantly result in cell death over a 6 hour period. The micelle half-life of DSPE-PEG2000 micelles is approximately 5 minutes in vitro, using a bovine serum albumin assay to determine the dissociation of these micelles. The dissociation time can be increased fourfold by simply removing the poly(ethylene glycol) component and therefore causing the overall hydrophobicity of the tail to increase. Since VCAM-1 is expressed on endothelial cells, our VCAM-1 micelles are expected to adhere to aortic endothelial cells and are likely to be internalized. Future studies will show that model drugs can be incorporated into the hydrophobic interior of these micelles, demonstrating their potential to both target and deliver drugs for therapeutic applications.

Nanomedicine has offered new solutions for cancer diagnosis and therapy. However, the survival improvement offered by FDA approved nanomedicines remains modest. In order to further advance the development of nanomedicines, it is crucial to investigate and understand the correlation between the properties of nanomedicines, size in particular, and their interactions with biological systems, which lays the foundation for designing and engineering nanomedicines with optimum efficacy against cancers. We systematically evaluated the size effect of the highly size-controlled, monodisperse drug-silica nanoconjugates on the biological responses as well as overall antitumour efficacy in both primary and metastatic tumour models. The 50-nm nanoconjugates had the highest efficacy with 6.0 and 2.1 fold higher apoptosis index to proliferation index ratio in tumors compared to 200- and 20-nm nanoconjugates, respectively. We also showed the great potential of this drug-NC system for clinical translations by demonstrating the excellent long-term biocompatibility and the capability of clearance in vivo.

Regulation of enzyme activity with light is an appealing method for gaining external control over biological processes since light is non-invasive and offers precise spatial and temporal resolution. Most light controlled enzyme systems are constructed by attaching a photoresponsive unit directly to the enzyme of interest, but are often challenging to design because of the large size and complexity of enzymes. We have recently demonstrated a new approach by modifying cofactors, which tend to be structurally simpler and easier to tune than their enzyme counterparts. Using this concept we describe the development of a photoswitchable catalyst system based on Pyridoxal Phosphate (PLP), a cofactor necessary for amino acid metabolism. The integration of a photoresponsive dithienylethene into the backbone of PLP, allows its structure to be reversibly interconverted between two different isomers, each with unique reactivity towards substrate by exposure to UV or visible light. We show how the enzyme cofactor mimic can act as an on/off switch to gate different PLP dependent processes.

Graphene-derivatives (GDs) have emerged as promising nanomaterials for bioengineering applications (e.g., sensors, therapeutics, biomaterials) due to their mechanical strength, thermal/electrical conductivity, optical properties, and low cost. However, because of their poor colloidal stability and lack of inherent affinity towards specific targets, GDs require covalent or non-covalent modifications before they can function in biological systems. Here we describe the one-step non-covalent functionalization of GDs, i.e. graphene oxide (GO), and reduced graphene oxide (rGO), with a genetically engineered, protein-based polymer. Specifically, we used elastin-like polypeptides (ELPs) due to their tunable thermoresponsiveness, biocompatibility, and mechanical properties. ELPs that were genetically engineered to display a phage-display derived graphene-binding peptide motif bound to GDs and stabilized GD dipersions in aqueous and organic solvents. The ELP-GD hybrids exhibited stimuli-responsive behavior both in solution and when incorporated into hydrogels resulting in reversible aggregation/dispersion and swelling/deswelling, respectively. These responses could be induced remotely by infrared light illumination as a result of the GDs' ability to convert IR to heat. Site-specific deswelling of hydrogels resulted in rapid, localized actuation. This effect was used to create light-driven, walking hydrogels. Finally, we demonstrated that further engineering of ELPs with integrin-binding motifs allowed for efficient cell adhesion onto GD surfaces. GD functionalization with genetically engineered ELPs provides a facile and tunable method for imparting multiple functions and stimuli-responsiveness. This strategy can be utilized in the future for theranostics, sensing, and tissue engineering, as well as be adapted for use with other nanoparticles.

In the last decade, microelectrode arrays (MEAs) have become an indispensable tool in the study of different properties of neural networks such as network formation, network dynamics and signal processing. The traditional technology is based on micro-sized metallic electrodes produced through photolithographic etching of physically deposited noble metal coatings. As the size of the electrodes decrease, however, both signal quality and stimulation ability drop due to the high impedance of the metal-tissue interface. Moreover, the fabrication of such devices relies on traditional silicon patterning technology, making commercially available MEAs expensive and restricted to rigid substrates. Conducting polymers such as poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS), show lower impedance and higher capacitance compared to metals, as well as an augmented biocompatibility. Thanks to their soft mechanical properties and processability, conducting polymers can be solution-processed by simple printing techniques on flexible substrates, leading to a new generation of prosthetic devices with unique conformability and form-factors. Here, we present a novel approach to photolithographically define polymer MEAs, patterning at the same time the polymer active sites and the insulating layer. We use the MEAs for in vitro recording of electrophysiological signals from mouse hippocampus slices, demonstrating that our platform is capable of monitoring spontaneous unit activities as well as evoked responses induced by drug perfusion into the nervous tissue. The fabrication process is extremely versatile and can be used to pattern onto a large variety of substrates (both hard and flexible), with virtually any functional material. We further demonstrate the potential of this approach by patterning multilayer structures composed of PEDOT:PSS and proteins or polypeptides, including collagen and poly-L-lysine. In this way, the proliferation of neuronal cells can be controlled with micron-scale resolution and directed towards the electrically active area of the device, allowing for a better transduction of electrophysiological signals.

Glutathione-coated luminescent gold nanoparticles (GS-AuNPs) with diameters of 2.5 nm behave like small dye molecules (IRDye 800CW) in physiological stability and renal clearance but exhibit a much longer tumor retention time and faster normal tissue clearance, indicating that the well-known enhanced permeability and retention effect, a unique strength of conventional NPs in tumor targeting, still exists in such small NPs. These merits enable the AuNPs to detect tumor more rapidly than the dye molecules without severe accumulation in reticuloendothelial system organs, making them very promising for cancer diagnosis and therapy.

Reference:
1. Liu et al, J. Am. Chem. Soc., 2013, 135, 4978.
2. Zhou et al, Angew. Chem. Int. Ed., 2011,50, 3168.
3. Zhou et al, Angew. Chem. Int. Ed., 2012, 51, 10118.

Sulfadiazine (SD) was immobilized onto a wide range of natural and synthetic polymeric materials. The effects of reaction conditions on the immobilization reactions were investigated in detail. In the presence of silver cations, the sulfadiazine moieties in the immobilized polymers could be transformed into silver-sulfadiazine coordination complexes. The resulting polymeric silver sulfadiazines provided potent biocidal functions against Gram-positive bacteria, Gram-negative bacteria, drug-resistant bacteria, fungi, and virus. The antibacterial, antifungal, and antiviral activities were durable for months to years. For even longer application, if the biocidal function was lost, it could be readily regenerated with another silver cation treatment. Additionally, the new polymeric silver sulfadiazines showed excellent mammal cell viability, pointing to great potentials of the new materials for a broad range of biomedical related applications.

The investigations of the efficiency of DNA-transfection to eukaryotic cells are important as a basic research in potential applications of gene delivery. Cationic peptides with positively charged amino acid residues can bind to anionic DNA to form complexes with a net positive charge which enter the cell through disruption of the endosomal membrane. Bovine lactoferrampin is one of such peptides which has strong antimicrobial activity and kills a number of bacteria such as Escherichia coli. The peptide has six basic amino acid residues and has a net charge +5 at neutral pH in solution. This cationic peptide selectively associates with the acidic membrane bilayer to lyse and to kill the bacteria which was precisely investigated by our previous work using nuclear magnetic resonance experiments and molecular dynamics simulation. In this study, we investigated the effect of peptide architecture on DNA binding using atomistic molecular dynamics simulations.

Nanoporous materials have found significant use in biomedical applications, such as fabrication of multifunctional medical device coatings that offer controlled drug delivery, increased surface area for biosensing, and morphological cues for dictating cellular response. Nanoporous gold is a relatively new material that is synthesized by a process known as dealloying, where selective dissolution of silver atoms from a silver-gold alloy lead to an open-pore structure with characteristic features that range from tens to hundreds of nanometers. Large effective surface area, high electrical conductivity, well-developed surface modification by thiol chemistry, tunable porosity, and biocompatibility make nanoporous gold a promising material as a multifunctional device coating that combine electrochemical biosensing and drug delivery. While its function in biosensing applications has been recently initiated, its potential as a drug delivery platform, which is an essential component of multifunctional materials, still remains underexplored. Here, we report on the molecular release phenomenon from nanoporous gold thin films (300 nm to 600 nm-thick) that are loaded with fluorescent probes with molecular weights in the range of 300 Da. The results indicate that nanoporous films sustain molecular release with a Fickian profile up to a week and thicker films non-linearly increase the drug-loading capacity. We discuss the kinetics of loading and release from nanoporous gold thin films, with a particular focus on the effect of loading concentration and duration, molecular size and charge, varying pore size, and film thickness. We expect that this study will provide insight into the drug release from nanoporous materials and their integration into multifunctional stimuli-responsive miniaturized platforms for in vitro and in vivo use.

The pharmaceutical polymer Eudragit^® E100 is a copolymer based on dimethylaminoethyl methacrylate (DMAM), butyl methacrylate, and methyl methacrylate. Today, the polymer is mainly used for the coating of tablets to seal sensitive actives and increase patient compliance by taste masking. However, its chemical properties open the opportunity to use it as a nanoparticulate drug delivery system for the transfection of cells. Its cationic character and solubility under acidic conditions enhance the cellular uptake and the endosomal escape via the proton sponge effect, respectively.^{[1, 2]} The synthesis and characterization of Eudragit^® E100-nanoparticles in the size regime of 200 nm with high encapsulation efficiency of both hydrophobic chemotherapeutics and hydrophilic biologicals is presented.
For the encapsulation of hydrophobic drugs, E100-Nanoparticles were synthesized via a solvent-diffusion method. To trap the hydrophilic, fluorescently marked bovine serum albumine (FITC-BSA) as well as eGFP-DNA into the polymer matrix, a water-in-oil-in-water (W/O/W) microemulsion was utilized.^[3] The cellular uptake by HeLa cells as well as the transfection efficiency of eGFP-DNA was followed by fluorescence microscopy. The cytotoxicity of the nanoparticulate delivery system was determined by the MTT-test.
By both methods, the solvent-diffusion and the W/O/W-emulsion technique, spherical E100-nanoparticles (~200 nm) with a positive zeta potential could be synthesized as shown by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The encapsulation efficiency (ca. 80%) of FITC-BSA and eGFP-DNA was determined by UV-vis spectroscopy. Cellular uptake studies showed that the nanoparticles are efficiently taken up by HeLa cells. The MTT-test showed that the nanoparticles are not cytotoxic.
The solvent-diffusion and the W/O/W-emulsion technique proved to be suitable to synthesize spherical, cationic Eudragit^® E100-nanoparticles in the size regime of 200 nm. Furthermore, cellular uptake studies showed that these particles are capable of inducing the proton sponge effect while being not cytotoxic as shown by the MTT-test. Thus, new polymeric nanoparticles could be synthesized, that act as a delivery system for both, hydrophobic and hydrophilic drugs. In addition, it was shown, that the Eudragit^® E100-nanoparticles avoid the cytotoxic effects of commonly used cationic polymers for the transfection of cells (e.g. polyethyleneimine).
Literature
[1] A. E. Nel, L. Mädler, D. Velegol, T. Xia, E. M. V. Hoek, P. Somasundaran, F. Klaessig, V. Castranova, M. Thompson, Nat. Mater.2009, 8, 543-557.
[2] A. F. Adler, K. W. Leong, Nano Today, 2010, 5, 553-569.
[3] J. P. Rao, K. E. Geckeler, Prog. Polym. Sci.2011, 887-913.

Many target sites of synthetic drug molecules are located inside cells. As larger molecules are typically not able to cross the cell membrane on their own, an efficient carrier is needed. We have loaded calcium phosphate nanoparticles with different synthetic drug molecules, i.e. a polyfunctional anionic polymer, a cationic calixarene dimer and a molecular tweezers. A polyfunctional anionic polymer was developed for selective inhibition of lysozyme as a model of enzyme inhibition. A calixarene dimer due to its chemical and topological characteristics has the ability to specifically bind to the major groove of the DNA duplex that result in cell death. Molecular tweezers inhibit the specific protein-protein interactions that lead to the formation of amyloidogenic aggregates inside the cells. These aggregates are the cause of multiple incurable diseases, for example, Alzheimer&’s disease, Parkinson&’s disease and type-2 diabetes.
Calcium phosphate nanoparticles were prepared by rapid precipitation, followed by functionalization with drug molecules. The polyfunctional anionic polymer and the cationic calixarene dimer were highly charged and therefore able to colloidally stabilize the nanoparticles. In the case of the molecular tweezers, first the cationic polymer polyethyleneimine (PEI) was adsorbed onto the nanoparticle surface, and then the molecular tweezers themselves. All particles were separated from dissolved counter-ions and non-adsorbed molecules by ultracentrifugation and subsequent redispersion in pure water. The amount of the fluorescing synthetic molecules on the nanoparticles was estimated by quantitative UV spectroscopy. All nanoparticle dispersions were characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and scanning electron microscopy (SEM).
The cell experiments were carried out on the HeLa cell line. The cells were incubated with the drug-loaded nanoparticles as well as with controls: the same concentration of the drug molecules, but without calcium phosphate nanoparticles. For quantifying the viability of the cells after incubation the MTT test was performed. Light and fluorescence microscopy along with confocal laser scanning microscopy was used to determine the uptake efficiency. The functionalized nanoparticles had spherical morphology with the size of 150-200 nm. All three drug molecules were easily detectable inside the cells, whereas the synthetic molecules alone were not taken up by cells.
We have demonstrated the loading of calcium phosphate nanoparticles with three synthetic drug molecules of very different chemical structure and charge. The results of cell experiments show that an efficient and non-toxic carrier plays an important role in the cellular uptake of synthetic drug molecules.

Hydrogels are an emergent class of polymer materials especially because their properties/functions such as swellability, mechanical properties, and stimuli-sensitivity can be adjusted to the requirements of specific applications [1]. ABA Triblock copolymer based on oligo(ethylene glycol) segments A and an oligo(propylene glycol) segment B (OEG-OPG-OEG) have drawn interest of researchers for pharmaceutical applications [2]. The micellization of OEG-OPG-OEG in solution upon its critical micellization temperature (cmt) causes its reversible self-gelation [3]. Therefore a drug can be incorporated in a solution and injected, forming a physical gel at 37 °C and allowing a gradual drug release. However, the injected gel has no defined shape and cannot be removed in case of allergic reaction or side effects relative to a drug. This could be avoided by using covalently crosslinked hydrogel based on dimethacrylated OEG-OPG-OEG (OEG-OPG-OEG-diIEMA), providing shape and mechanical stability [4]. The hydrogels prepared from polymer chains of different hydrophilicity (70% or 80% oligo(ethylene glycol)) or distinct molecular weight (Mn = 14600, 12700 or 8400 g.mol-1) showed tunable properties in terms of swellability, swelling kinetics, and mechanical properties. Here we explored how the polymerization temperature, at which the triblock copolymer solutions were crosslinked, affected the polymer network morphology and in this way influenced relevant functions (swellability, swelling kinetics and mechanical properties) for drug release. As materials, we selected OEG-OPG-OEG-diIEMA hydrogels that were prepared via UV-crosslinking at Tcmt in their structured micellar-gelled state. X-ray diffraction investigation revealed the nanostructure of the swollen hydrogels prepared at T>cmt, which exhibited higher gel content and lower degree of swelling compared to the gels prepared at Tcmt showed higher storage and loss moduli. Thus we demonstrated that the polymerization temperature of the hydrogels changed the internal structure which controls functions such as swellability, swelling kinetics and mechanical strength, relevant for drug release applications.
References
[1] Langer R, Vacanti JP. Science. 1993; 260: 920-6.
[2] Escobar-Chavez JJ, Lopez-Cervantes M, Naik A, Kalia YN, Quintanar-Guerrero D, Ganem Quintanar A. J Pharm Pharm Sci. 2006; 9: 339-58.
[3] Alexandridis P, Holzwarth JF, Hatton TA. Macromolecules. 1994; 27: 2414-25.
[4] Tartivel L, Behl M, Schroeter M, Lendlein A. JAB-FM, 2012, accepted.

A vaccine is designed to reproduce infected situations via exogenous pathogens (e.g., bacteria or virus), with being a synthetic tool for immune stimulations. Effective vaccination can be achieved with successive boosting for enhanced immune response. Since several decades, a variety of vaccine studies that utilize either bacterial DNA fragments or recombinant proteins as target antigens have made substantial progresses[1]. To improve the antigens&’ stability, they have been usually entrapped into lipid vesicular compartments. However, conventional vaccine systems using the lipid-supported sacs have suffered from numerous drawbacks such as a short-term stability in plasma. Here, we present a novel multi-boosting vaccine system based on synthetic bacteria, which achieved long-term and successive immune stimulation. The artificial bacteria are composed of three main subdivisions: lipid outer membrane, bacterial genome core, polymer interfaces. The lipid vesicles supported by characteristic interfacial polymers tuned the triggering of vaccine antigens at certain period. In particular, polymer compositions were varied depending on its molecular weight or decomposition rates. The different patterns in polymer hydrolysis altered multi-boosting effects. It induced dendritic cells (DCs) maturation. The unmethylated CpG sequences inside synthetic bacteria were recognized by Toll-like receptor (TLR)-9, being followed by DCs activation via cross-presentation. Transcription levels of TLRs from maturated DCs were determined by qRT-PCR and the specific surface markers, B7-1, B7-2 and CD40 were analyzed by flow cytometry. Interestingly, the surface markers were sequentially expressed in order of PLA, PLGA 75:25 and PLGA 50:50. It is speculated that the synthetic bacteria-based multi-boosting vaccinations may be a promising therapeutic candidate for several unsolved medical issues.
1. Vladimir P. Torchilin. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discovery. 4, 145-160 (2005)

Fibrillar collagens are common tissue scaffolds, and are known to assemble into ordered arrays in matrices such as bones, tendon, and cartilage. They have also been observed to self-assemble in vitro, and the resulting scaffolds have a variety of biotechnological applications that require a better understanding of the collagen assembly processes. Here, we propose a coarse grained model in which the collagen molecules are treated as strings of beads connected via springs, with well defined interactions between beads that belong to different strings. Molecular dynamics (MD) simulations employing this coarse-grained description indicate that the morphology of self-assembled collagen on a flat substrate is governed by the interplay of two competing factors, namely the intermolecular collagen-collagen (c-c) interaction and the collagen-substrate (c-s) interaction. The utility of this model lies in its ability to vary each of these interactions independently, since such a decoupling is difficult to achieve in the laboratory. Our simulations clearly demonstrate that the c-c interactions promote surface diffusion favoring the formation of three-dimensional collagen bundles, while strong c-s interactions lead to random monolayer networks. Furthermore, the configurations predicted by this model were in excellent agreement with atomic force microscopy experiments conducted in the present study and with those available elsewhere.

Hydroxyapatite (HA) [Ca10(PO4)6(OH)2] is the main mineral component in human bone, giving bone strength and ability to regenerate. HA is used in modern medical applications such as hip replacements, thus the existing body of work on HA is mainly experimental and macroscopic in nature. However, improving the implants lifetime also requires a more subtle microscopic understanding. We use density functional theory in our work. Having studied bulk and surface properties previously, we now immerse HA in H2O to mimic physiological conditions. We are interested in the bioactivity of the HA surface, and how the H2O and chemical substitutions on the HA surface affect it. We test Na, Mg, Al, Si, S, and Cl substitutions. Si for example is known to increase HA&’s ability to calcify tissue, the reasons for which being not clear to date. Among our findings are that water forms a distinct mass density distribution close to the interface depending on the substitute. We also find Al, Si and Cl to turn the HA surface hydrophobic, while Si, Mg and Cl induce a strong dipole moment in the H2O layer close to the HA substrate which has not been described before. The combination of our findings may lead to an elementary understanding of bioactivity from a physical point of view.

Textile-enabled interfacial microfluidics, utilizing the capillary force generated by fibrous hydrophilic yarns (e.g., cotton) to drive biological reagents, has been extended to various biochemical analyses recently. However, the restricted capillary-driving mechanism persists to be a major challenge for continuous and facilitated biofluidic transport. In this abstract, we have first introduced a new interfacial microfluidic transport principle to automatically drive three-dimensional liquid flows on a micropatterned superhydrophobic textile (MST) platform in a controllable and continuous manner. Specifically, the MST system utilizes the surface tension-induced hydrostatic pressure to facilitate the liquid motion along the yarns, in addition to the capillary force existing in the fibrous structure. The MST was simply fabricated by stitching superhydrophilic cotton yarns onto a superhydrophobic fabric (contact angle 140±3°) with well-controlled geometrical patterns to establish microfluidic networks. As the extreme wetting contrast presents between the patterned superhydrophilic yarns and the superhydrophobic textile substrate, the liquid-air boundary can be uniquely determined along the triple line. As a result, the sizes of the stitching patterns and the geometric configuration of connecting threads will control the surface tension-induced pressure gradient as well as the flow resistance respectively, from which the transport speed can be defined. As experimental proof of the principles, continuous liquid flow was demonstrated from one bottom inlet to one top outlet on the fabric regardless of the gravitational effect. Moreover, various transport flow rates were achieved by manipulating the geometric relationship between the hydrophilic patterns of the inlet and outlet. The flow resistance of different thread lengths and the bundle structure were measured, from which it can be seen the bundle structure composed by two closely packed yarns reduces the flow resistance significantly. What's more, by directing flow from multiple inlet sites towards one common outlet, we can devise an efficient fluidic collection network, which can be potentially applied to large volume and continuous biofluidic (e.g., sweat or wound exudates) collection and removal.

The influence of material properties on bacterial attachment to surfaces needs to be understood when applying polymer-based biomaterials. Positively charged materials can kill adhered bacteria when the charge density is sufficiently high,1 but such materials initially increase the adherence of bacteria such as Escherichia coli.2 On the other hand, negatively charged materials have been shown to inhibit initial bacterial adhesion,3 but this effect has only been demonstrated in relatively few biomaterial classes and needs to be evaluated using additional systems. Gradients in surface charge can have influence on bacterial adhesion and this was tested in our experimental setup.
Moreover, the evaluation of bacterial adhesion on biomaterials is required to assess their potential for biological applications. Here, we studied the bacterial adhesion of E. coli and Bacillus subtilis on the surfaces of acrylonitrile-based copolymer chips with varying amounts of 2-methyl-2-propene-1-sulfonic acid comonomer (0, 0.5, 1.0, 2.0, and 5 mol.%). This class of polymers was chosen for this study because of its cytocompatibility, processability, and possible applications as an in vitro tissue culture plastic. The biomaterials were synthesized using free radical polymerization methods under aqueous conditions, and the resulting powders were characterized using gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and water uptake studies. The powders were processed into chips that fitted in 24-well plate dishes, which could then be easily studied for their bacterial attachment properties.
Bacterial adhesion was tested on acrylonitrile-based copolymer chips submerged in a synthetic cultural medium and incubated at 37 °C until the biofilm was formed. Inoculation was done with actively growing bacterial suspension of E. coli and Bacillus subtilis (OD 0.03). Before microscopic examination material chips were washed to remove the non-adhering bacterial cells. After fixation of biofilms surface colonization was evaluated by fluorescent and incident light microscopy. Surface colonization was assessed on digitalized images (binary modus) and quantified using ImageJ software. Differences in colonized surfaces of different material chips will be discussed.
1. H. Murata, R.R. Koepsel, K. Matyjaszewski, A.J. Russell Biomaterials 2007, 28, 4870.
2. G. Harkes, J. Feijen, J. Dankert Biomaterials 1991, 12, 853.
3. N.N. Lawrence, J.M. Wells-Kinsbury, M.M. Ihrig, T.E. Fangman, F. Namavar, C.L. Cheung Langmuir 2012, 28, 4301.

Natural Rubber latex (NRL) is a colloidal with complex negatively charged particles. This biomaterial in form of natural rubber membranes can accelerate cells proliferation and induce revascularization, and we also observed similar ability in NRL multilayer films. Herein we evaluate indirect cytotoxicity and cells adhesion onto NRL multilayer films produced by different methods (spraying with and without rinsing steps) and counterpart materials (polyethylenimine - PEI; and polyallylamine hydrochloride - PAH). We used the layer-by-layer (LbL) technique to mount (PEI/NRL)5, (PEI/NRL)15, (PAH/NRL)5 and (PAH/NRL)15 spraying films with 2 sprays of each solution onto the substrate, followed (or not) by 7 sprays of rinsing solution. The percentage of surface covered by NRL and roughness of all samples were determined by AFM (5500 Agilent Inc., tapping mode, silicon tip <10nm), and films growth was evaluated by UV-vis. The cytotoxicity on those films was determined by morphological alterations on Vero cells with indirect contact. The culture were put in contact with extracts eluded from the samples after 3 days of incubation in culture medium, remaining for 24h before observation. For comparison, positive and negative (0.2 % phenol) controls were also made. For the adhesion assay Vero cells were cultured directly on the samples, and adhered and not adhered cells were counted after 24h. Control and castings of the used solutions were also analyzed. All tests were performed in triplicates. Result shows that spraying films made without rinsing steps have greater absorbance, roughness and surface coverage than spraying films made with rinsing steps. Therefore, more material amount is found on films made without rinsing, which results on more irregular surfaces. Apparently, greater amount of material causes more morphological changes on Vero cells, which might lead to a limit on the number of layer pairs for the biomedical application of NRL films. Neither way, no severe cytotoxic effects were detected on films without rinsing, and no toxicity at all were detected on rinsing films. In addition, cells adhesion varied with material, amount of layer pairs and method of the films. However, cells adhesion were greater on the multilayer films rather than on the solution castings. To exemplify, PEI and NBL castings showed 0% of cells adhered to its surface, but (PEI/NRL)n films showed up to 33%. The (PAH/NRL)n rinsing films induced even better adhesion (up to 89%) than no rinsing films (up to 54%) or PAH casting (58%), but it stood below control (96%). It allows one to conclude that bioactivity of NRL multilayer will depend on the method and material used on production, and method that produces smoother and regular surfaces might lead to a nontoxic proliferation inductive support for cells culture.

We introduce a new approach for fabricating hollow microneedles using carbon nanotubes (CNT) for rapid transdermal drug delivery. The CNT Microneedle Array offers rapid advective transport of drugs and is envisioned to be a self-administered, painless alternative to standard subcutaneous injection. Here, we discuss the fabrication of the Array emphasizing the overall simplicity and flexibility of the method to allow for potential industrial use. We also discuss the unique opportunities and challenges presented in using aligned CNT in a delivery device. Finally, we present a feasibility study comparing our CNT approach to current microneedle technologies.

To present, prototypes of the CNT Microneedle Array have been fabricated. The prototype Array consists of nanotubes patterned into blunt hollow needles with a diameter of 150um and an inner cavity diameter of 25um. The Array pierces skin painlessly to a depth of up to 200um. Drugs are then passed from a polymeric reservoir through the hollow cavity of the microneedles. Delivery is initiated by lightly tapping the cover of the reservoir (~0.1N). The Array can support flow rates up to 0.1mL/s giving a near instantaneous, painless subcutaneous delivery of drug.

By capitalizing on the nanoporosity of the CNT bundles, polymer can be wicked into the needles creating a high strength composite of aligned nanotubes and polymer. In vitro penetration of the Array in swine skin is demonstrated. Depending on the physical properties of the polymer such as surface tension and viscosity, the polymer can be cured either thermally or through photolithography to ensure that the inner cavity of the microneedle is preserved. In the photolithography approach, CNT act as a masking element to prevent polymerization in the inner cavity. We will discuss the application and challenges of several classes of polymers including polyoxazolidine, epoxy, and polyimide.

By using a self-assembled nanomaterial as the skeleton of our device, we can directly and rapidly alter the geometry of our needles through catalyst patterning and can achieve a wide range of needle gauges ranging from 250um to less than 1um with minimal alterations to the fabrication. While fabricating CNT requires a large initial investment, increased commercial options to buy CNT and CNT equipment will in the long run reduce the cost of working with CNT. The specific transform from CNT to microneedle is easily implemented with thermal cured polymers consisting of simply spin coating and hard baking. Furthermore, by achieving skin penetration with the as grown blunt CNT tip, we forego extraneous fabrication steps such as RIE or FIB to sharpen the tip. While the ultimate biocompatibility of CNT remains unknown, its incorporation in polymer significantly reduces direct exposure with the body. Our method also minimizes the use of secondary materials such as metallic masks and liquid etches that can leave residues on the needles.

3D architectures have advantages as good candidate for sensitive sensing system owing to the high surface-to-volume ratio. The characteristics of high surface system have large diffusion area and short diffusion time for target molecule. Furthermore, controlling flow around the target surface, momentum-transfer boundary layer thickness and mass-transfer boundary layer thickness formed between target surface and flow can be reduced. This phenomenon has merit that time to reach target surface of mass flux is reduced. Thus it is possible to detect target molecules rapidly.
In this study, we have developed 3D artificial cilia for the rapid detection. Firstly, we have fabricated 3D cilia using Capillary Force Lithography. After immobilizing probe DNA on the cilia, we controlled reaction flow of the hybridization solution containing the target DNA. Having confirmed that hybridization time is reduced, this system can be applied for rapid and reliable on-site detection applications.

Multifunctional materials (hydrogels, polymers and sol-gels) have become ubiquitous for biochemical sensing and actuating systems. They provide many useful functions in same materials system and make system design much easier. These materials not only facilitate the in-situ functionalization of integrated micro/nano electrodes but are also used to tune the sensor response for a particular environment and application. In this talk, we will describe the techniques used to pattern these multifunctional materials at micro/nano scale without using harsh chemistries to avoid damage to their functionalities during fabrication. These techniques provide very thin but stable layers to provide the sensitivity and specificity required for many sensing applications in real-time. We will also show that the combination of a top-down and bottom-up hybrid fabrication technique (for structured electrodes) with the techniques for fabricating the multifunctional materials results in completely integrated sensors on chip alongwith microelectronic circuitry to provide full system-on-chip solutions . Furthermore, we will also discuss the incorporation of suitable metal particles (micro or nano based upon application) in the functionalization matrix to form a pseudo sol-gel that enhances the sensor performance and results in stable devices for long periods of time. We will also show that coating the entire electrochemical sensor with these multifunctional materials (rather than just the working electrode as in conventional schemes) incorporating the biochemical transducer molecules (e.g. enzymes) provide much stable integrated sensors and make their fabrication much easier. We will also present a completely new technology for interfacing sensing systems with the blood stream, via the creation of an artificial capillary bed grown in vivo. Again, multifunctional materials act as matrices to hold the biomolecules required to facilitate this growth in vivo. Finally, we will describe a completely wireless implantable system which utilizes all these aspects of multifunctional materials for long term in vivo sensing of blood analyte.

In nanotechnology, there are a lot of efforts for developing smaller and more compact devices to meet our multiple demands. To improve the performance of nanodevices, chemists have been molecularly designing novel multifunctional materials since nano technology is a part of the chemical domain, which produces new materials at the nano-scales. However, there are some limitations to produce multifunctional materials through conventional synthesis. New synthetic approaches such as microfluidic synthesis have gotten a great attention to prepare materials/particles/polymers at the nano-scales. Microfluidic approach allows us to produce multifunctional materials, which can satisfy our diverse demands. It is also dynamic and continuous productions with small reagents and high yields. In this study, we demonstrate a microfluidic synthesis of molecularly imprinted polymeric (MIP), which can be prepared by “molecular imprinting technique. The molecular imprinting method is a general protocol for the creation of “synthetic receptor sites” with specific molecular recognition functions for bio or chemical sensors in cross-linked network polymers. Synthesis of “high affinity receptor sites” in MIPs systems is a key contribute to achieve high performance in their molecular recognition functions. Microfluidic synthesis of MIP produces specific advantages that conventional method can&’t achieve.

In this study, we report the development of all-solution-processed multifunctional organic devices, comprising reduced graphene oxide (rGO) and dexamethasone 21-phosphate disodium salt (DEX) drug loaded poly(3,4-ethylenedioxythiophene) (PEDOT) microelectrode arrays on indium tin oxide glass, that can be used to manipulate human mesenchymal stem cell (hMSC) differentiation. In our devices, the rGO micropatterns were used as the adhesive coating to attract the adhesion of hMSC cells whereas PLL-g-PEG coated PEDOT electrodes served as the anti-adhesive coating where no hMSC cells can attach. In addition, the PEDOT electrodes also work as drug releasing components where control DEX release from PEDOT matrix can be achieved via cyclic potential stimulation (CPS). The hMSC cells are found to adhere on the graphene micropatterns. This result confirms that our approach of using adhesive and anti-adhesive coating can effectively confine the location of hMSC cells on chip. Since osteogenetic inducer, DEX, could lead to osteogenic differentiation for the hMSC, our devices may be used to manipulate the differentiation of hMSC with spatiotemporal control, if we can release DEX by electrical stimulation. The drug releasing mechanism of our devices is based on the swelling and deswelling process of PEDOT polymers upon electrical stimulation. During the CPS, the PEDOT-DEX film undergoes a fast swelling and deswelling process caused by ion and water movement in and out of the film, which may act as a pump to push the anions out and dramatically speed up the drug release (seconds or minutes). The fabrication process of the drug releasing devices indicated that the DEX release profiles from various rGO-PEDOT devices (Device 1; Device 2; Device 3) in PBS solution were measured by UV spectrometry at 242 nm. As expected, without the poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) blocking layer on the PEDOT:DEX surfaces (Device 1), a significant release of DEX from rGO-PEDOT microelectrode arrays was detected without electrical stimulation due to diffusion. However, when a layer of PEDOT:PSS was used, DEX could be sealed very well in Device 2 and Device 3 without electrical stimulation. Upon electrical stimulation, the DEX was released linearly with time in Device 2 and Device 3. The long-term DEX release profile of Device 3 in PBS solution has been studied where almost no DEX was released without electrical stimulation. Upon electrical stimulation, mu;g of DEX can be released in our device after staying in incubator for six days.

The functionality of implanted devices is dependent on the foreign body reaction that occurs in the tissue surrounding the implant. A number of cells contribute to the foreign body reaction in vivo, including lymphocytes, monocyte-derived macrophages and neutrophils. Following implantation and protein adsorption, one of the first cellular responders in an inflammatory response are human blood mononuclear cells [1]. The adhesion and response of these cells depends upon the adsorbed protein layer, which in turn depends upon surface chemistry, topography and roughness. In our study, we have varied the surface area available for protein adsorption and cell attachment by comparing two fibre networks made of 444 ferritic stainless steel. Our previous work [3] has demonstrated that fully-dense 444 is compatible with human osteoblasts [2] and monocytes as assessed in short-term cultures. The aim of the current work is to investigate the corrosion of 444 fibre networks in biological solutions and the short-term in vitro behaviour of human blood mononuclear cells cultured on these materials. The responses are compared with 316L austenitic stainless steel fibre networks, of the same fibre volume fraction, which served as a medical grade control.
Cyclic voltammetry in biological conditions (pH 7.4 solutions, 37 °C temperature and 5% CO₂ atmosphere) was used in electrochemical studies of the fibre networks when immersed in a series of modified phosphate buffer solutions. Peripheral blood human monocytes, which are a subset of the human blood mononuclear cells, were used to evaluate short-term in vitro responses. Scanning electron microscopy was used to observe the morphologies of the cells and fibres, while the Brunauer-Emmett-Teller (BET) gas adsorption isotherm method was used to measure the specific surface area of the networks.
Electrochemical results showed that 444 fibre networks exhibited better resistance to corrosion in biological solutions compared with fibre networks composed of clinical grade 316L. Monocyte studies further indicated that 444 ferritic stainless steel fibre networks do not cause significant inflammatory or cytotoxic responses from human monocytes in short-term cultures. Consequently, fibre networks composed of 444 ferritic stainless steel have potential for biomedical applications requiring ferromagnetic implant materials.
REFERENCES:
[1] Anderson JM, Rodriguez A, Chang DT.Seminars in Immunology 2008; 20: 86-100.
[2] Malheiro VN, Spear RL, Brooks RA, Markaki AE. Biomaterials 2011; 32: 6883-6892.
[3] Spear RL, Brooks RA, Markaki AE. Journal of Biomedical Materials Research: Part A. 2012. in press
ACKNOWLEDGEMENTS:
Financial support for this work comes from the European Research Council (Grant No. 240446).

Lysosomal storage diseases (LSDs) are a group of inheritable genetic diseases caused by mutant lysosomal enzymes, leading in the accumulation of undigested macromolecules in the lysosomes and causing increases in lysosome size and number, cellular dysfunction, clinical abnormalities, and premature death. These LSDs can be treated with Enzyme Replacement Therapy (ERT) through intravenous administration of a recombinant enzyme in replacement of the defective enzyme. However, this is an expensive and inefficient method with adverse sides effects associated with the high enzyme amounts required for the treatment, the need of post-translational modification of the enzyme and the host immune system response.
We hypothesize that nanocarriers composed of Polyethylene glycol and Polycaprolactone (PEG-PCL) block copolymers can enhance ERT by eliminating the need of enzyme modification and protecting it from host immune system until lysosomal target is reached. We have designed these nanocarriers because of their capacity to remain stable at physiological pH and destabilize at acidic pH, making the nanocarrier reach the cell intact and degrade once inside the acidic lysosome, thus releasing therapeutic cargo into the affected cellular organelle.
In order to obtain the appropriate nanocarrier for ETR application, we synthesized a group of PEG-PCL nanocarriers, varying their production process and surfactant concentration. The most suitable combination was found by performing dynamic light scattering analysis (DLS), gel permeation chromatography (GPC) and several spectroscopic techniques. We are currently working on the reproducibility of the syntheses and their cytotoxic profile in cultured cells.

Titanium (Ti) is the material of choice for orthopaedic applications because it is biocompatible and encourages osteoblast ingrowth. It was shown that the biocompatibility of Ti metal is due to the presence of a thin native sub-stoichiometric titanium oxide layer which enhances the adsorption of mediating proteins on the surface [1]. The present studies were devised to evaluate the adhesion, survival, and growth of cells on the surface of new engineered nano-crystal films of titanium and titanium oxides and compare them with orthopaedic-grade titanium with microcrystals. The engineered nano-crystal films with hydrophilic properties are produced by employing an ion beam assisted deposition (IBAD) technique. IBAD combines physical vapor deposition with concurrent ion beam bombardment, in a high vacuum environment, to produce films (with 3 to 70 nm grain size) with superior properties. These films are “stitched” to the orthopaedic artificial implant materials with characteristics that affect the wettability and mechanical properties of the coatings.
To characterize the biocompatibility of these nano-engineered surfaces, we have studied the osteoblast function including cell adhesion, growth, and differentiation on different nanostructured samples. Cell responses to surfaces were examined using SAOS-2 osteoblast-like cells. We also studied a correlation between the surface nanostructures and the cell growth by characterizing the SAOS-2 cells with immunofluorescence and measuring the amount alizarin red concentration produced after 7, and 14 days. The number of adherent cells was determined by means of nuclei quantification on the nanocrystalline Ti, TiO2 and microcrystalline Ti and analysis was performed with Image J. Our experimental results indicated that nanocrystalline TiO2 is superior to both nano-and microcrystalline Ti in supporting growth, adhesion, and proliferation. Improving the quality of surface oxide, i.e. fabricating stoichiometric oxides as well as nanoengineering the surface topology is crucial for increasing the biocompatibility of Ti implant materials
[1] F. Namavar, R. Sabirianov; et al. Orthopaedic Research Society San Francisco Feb 2012

The clinical success of many chemotherapeutics and biotherapeutics intimately depends on the development of non-toxic and efficient tumor-targeting nano-carrier systems. The ideal drug and protein carriers should be able to retain and protect the payloads under extracellular settings (e.g. in cell culture and during circulation) while dump them once arriving at the site of action. This site-selective drug and protein release might on one hand maximize therapeutic effects and on the other hand minimize systemic side effects. However, despite significant progress has been made over the past decade, current biodegradable nano-carriers are still challenged with inadequate in vivo stability, low tumor-targetability, premature release, and/or sluggish release inside the tumor cells. Here, I will present our strategies to achieve active intracellular drug and protein delivery. For example, we have developed bio-responsive, in particular reduction- and pH-responsive, degradable micelles and polymersomes for efficient loading and triggered intracellular release of anti-cancer drugs and proteins. We have also developed stimuli-responsive reversibly crosslinked micelles and polymersomes to resolve the stability and intracellular drug release dilemma.
References
1. C. Deng, Y.J. Jiang, R. Cheng, F.H. Meng, and Z.Y. Zhong*, Biodegradable Polymeric Micelles for Targeted and Controlled Anticancer Drug Delivery: Promises, Progress and Prospects, Nano Today 2012, 7, 467-480.
2. F.H. Meng, R. Cheng, C. Deng, and Z.Y. Zhong*, Intracellular Drug Release Nanosystems, Materials Today 2012, 15, 436-442.

Heterotopic ossification (HO) is the formation of bone in non-osseous connective tissues including muscle, ligament and tendon, as a result of traumatic orthopedic and brain injuries. HO is one of the major causes of postoperative pain and disability in amputees and in patients after total joint arthroplasty where it often severely limits range of motion. Unfortunately, in most cases the condition is never diagnosed until irreversible progressive bone growth occurs. Furthermore, there is currently no effective prophylaxis available for the treatment of HO. Traditional treatment includes surgical excisions to remove excessive bone mass, followed by radiation therapy and non-steroidal anti-inflammatory drugs to prevent recurrence of abnormal bone growth and to alleviate pain.
Post-transcriptional gene silencing based on the principle of RNA interference (RNAi) represents a potential treatment modality for heterotopic ossification. In this approach, a synthetic short interfering RNA (siRNA) encoded for an osteogenic messenger RNA target is introduced into cells susceptible to osteogenesis.
In the present work, we have developed an effective polymer/lipid-based nanoparticle delivery system for siRNA that has targeted affinity for bone tissue. The pH-sensitive, endosomolytic polymer poly(propylacrylic acid) (PPAA) was first modified by grafting poly(ethylene glycol) (PEG) chains (1 mol % of PPAA) onto the PPAA backbone to impart amphiphilic character to the polymer. Subsequently, the grafted PEG chains were conjugated with the amino-bisphosphonate drug alendronate (ALN) (0.16 mol % of PPAA) in order to achieve active bone targeting. Analysis of the graft copolymer by NMR and FTIR confirmed the successful modification. A high extent (~87%) of binding was observed for the interaction of PPAA-g-PEG-ALN with hydroxyapatite (HA), one the major constituent of bone mineral.
The ALN conjugated graft co-polymer was then used in association with dioleoyl-3-trimethylammonium-propane (DOTAP) cationic liposomes to form self-assembled ternary nanocomplexes with the anti-Runx2 siRNA. Delivery of the siRNA was evaluated using a cell culture model of HO in which mouse myoprogenitor C2C12 cells were stimulated with bone morphogenic protein-2 (BMP-2) which causes a distinct switch from a myogenic to osteogenic phenotype (Mishra et al., Integr. Biol., accepted for publication). The PPAA-g-PEG-ALN/DOTAP/siRNA nanocomplexes exhibited co-localization (measured by Cy-5 labeled siRNA) at mineral-rich regions in BMP-2 stimulated C2C12 cultures. Confocal microscopy and flow cytometry measurements showed that the nanocomplexes successfully delivered siRNA intracellularly and that the expression of Runx2 was suppressed by ~40% in the C2C12 cells. The ternary PPAA-g-PEG-ALN/DOTAP/siRNA nanocomplexes are therefore a promising bone targeting system for the intracellular delivery of siRNA to osteogenically active cells in trauma-induced in vivo HO studies.

The parallel developments in the design of pharmaceutical drugs and in the controlled manipulation of materials at the nanometer scale have recently begun to merge in order to produce new generations of diagnostic and therapeutic agents. Many agents used for pharmacotherapy, such as antitumor drugs, show side effects and limited effectiveness that restrict their clinical application. To maximize therapeutic efficacy and minimize side effects, numerous attempts have been made in the design of target-specific drug delivery systems that can securely transport the medications to targeted cells and tissues, without degradation or untimely release.
Of the various drug nanocarriers explored, polymer assembly have been widely used in drug delivery, especially for the controlled release, such as pH responsive hydrogels, thermal responsive microspheres and enzyme responsive micelle, because of the low toxicity, high degraded ability and excellent compatibility to biosystem. However, conventional polymer-based drug delivery systems can suffer from inherent problems, such as limited capacity for drug loading, poor stability in blood after injection or difficulty in temporally controlling the release of therapeutics. These drawbacks motivated us to combine polymers with inorganic nanoparticles which has high drug loading capacity, high stability and ‘zero premature release&’ property. Mesoporous silica nanoparticle (MSN) materials have been shown to be excellent candidates to fulfill the above-mentioned requirements .
Herein, we report the design, synthesis, and operation of a novel, biocompatible controlled-release motif we call pH and enzyme double responsive polymer covered silica nanocontainer. The porous mesostructure is templated by cetyltrimethylammonium bromide (CTAB) surfactants, and particle synthesis is accomplished using a base-catalyzed sol-gel procedure. Methods for derivatizing silica are well-known and are used here to functionalize the nanoparticle surfaces with fluorescein (5-Carboxyfluorescein diacetate, which has no fluorescence before esterase break the ester bond) and polymer (hydrazine conjugated PEG-b-PCL). When closed, there is no fluorescence emitted and the polymer contains guest molecules stored within the pores. However release of guests happened following cleavage of the acylhydrazone bond (pH control) and enzyme-mediated hydrolysis of ester bond (enzyme control) when the pH is lower than 4 plus enzyme in present. Meanwhile, fluorescence also appeared exposing us which part is releasing drugs. Because the cancer cells have a more acidic environment compared with the normal cells, so cancer cells are the only place contain both enzyme and low pH value, which provides an efficient way for our system to control the drug release in just predesigned cancer cells.

Stimuli-responsive block polymeric systems, including micelles and hydrogels, have attracted an extensive attention as "smart" materials for biomedical applications. In this abstract, we would like to introduce the design of some pH and/or thermo responsive block copolymers which can form micelles and hydrogels for theranostics.
In our system, the pH-responsive polymeric micelles were composed of block copolymers, including hydrophilic methyl ether poly(ethylene glycol) (MPEG) and pH-responsive poly(β-amino ester) (PAE). This block copolymer, which could self-assemble into nano-sized micelles with core-shell structures under a physiological environment (around pH 7.4), showed a pH-dependent micellization-demicellization behavior. Some antitumor drugs or molecular imaging agents can be loaded into the pH-responsive polymeric micelle for theranostics based on the acidic pH environment of cancer areas. From the in vitro and in vivo results, the polymeric micelle can successfully deliver antitumor drugs and imaging agents to the envisioned tissue locations due to the pH influence. Also, the pH/thermo sensitive injectable block copolymer hydrogels were investigated. The hydrogels can controlled release drug/protein for theranostics by changing pH and/or temperature in the surrounding environment. The cationic nature of PAE is also used as the second function to make the ionic complexes with anionic proteins, such as insulin. Eventually, the cumulative release of the protein from the complex hydrogels was investigated through in vitro and in vivo experiment.

Development of smart nano-drug delivery systems, capable of modulating their release in response to specific stimuli, have become a subject of intense research worldwide. By a careful selection of the constituting materials, these systems can be designed to respond to a wide array of internal and external stimuli. Although external stimuli such as light, magnetic field, ultrasound etc. allow greater temporal and spatial control over drug release, they also present drawbacks such as inability to deliver in occluded locations and potential risk of injury to the surrounding tissue. Hence an ideal system would be one which can release the drug under physiological conditions when exposed to an internal stimulus that is present specifically at the target site.1
In an attempt to develop such systems, we have designed and fabricated nano-drug delivery systems fabricated from biopolymers such as protamine, heparin, hyaluronic acid and chondroitin sulphate that can release the encapsulated drug when exposed to specific enzymes such as proteases, hyaluronidase etc. present at target sites within the body. Upon exposure to these biological triggers, the nano-systems disintegrate releasing the encapsulated drug. The workability of these systems in an in-vitro scenario has been evaluated using various human cell lines.
Reference
[1] Krishna Radhakrishnan, Ashok M. Raichur, Biologically triggered exploding protein based microcapsules for drug delivery, Chem. Commun., 48, 2307-2309. 2012.

Cardiovascular disease is one of the leading causes of mortality in the United States and atherosclerosis remains to be the major source of cardiovascular disease. Atherosclerosis is an inflammatory disease that is complicated by progressively unstable plaques and can result in acute coronary syndromes and sudden cardiac death. Although noninvasive diagnostic imaging technology including intravascular ultrasound, computed tomography, and magnetic resonance imaging have made great strides in the last two decades to provide a means to detect such plaques, none of these existing technologies can meet the sensitivity and accuracy standards that are required for any realistic clinical application for preventive measures.
In order to intervene during the early pathology of plaque vulnerability, we are engineering monocyte-targeting, fluorescently-labeled peptide micelles (MFPMs) through the incorporation of the chemokine receptor CCR2-binding motif (residues 13-35) of the monocyte chemoattractant protein-1 (MCP-1). One of the first markers of plaque formation is the inflammatory activation of endothelial cells which then secrete MCP-1, recruiting monocytes in large quantities through their CCR2 receptor. Therefore, a molecular imaging tool that binds specifically to monocytes may provide a noninvasive, detection system for early-staged plaques.
The MCP-1 peptide or Cy-7 was conjugated to DSPE-PEG-2000 and mixed to form fluorescently-labeled, monocyte-targeting peptides. The critical micelle concentration of MFPMs was determined to be approximately 15 mu;M with an average diameter of approximately 20 nm, confirmed via dynamic light scattering and transmission electron microscopy. Studies using MFPMs and a scrambled, negative control peptide micelle will determine the chemoattractive efficacy with monocytes and biocompatibility using aortic endothelial cells and smooth muscle cells in vitro. Since peptide micelles can be synthesized with multiple functionalities (i.e. targeting element, imaging dye, etc.), our investigations will lay the ground work for peptide micelle-mediated drug delivery at the site of early plaque formation in future studies.

A series of dual stimuli-responsive copolymers containing biocompatible and zwitterionic poly(sulfobetaine methacrylate) (PSBMA) and pH-responsive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) has been synthesized via free radical polymerization. The stimuli-responsive phase transition behaviors of P(SBMA-co-DPA) copolymers and PSBMA homopolymer dissolved in aqueous solutions with different pH values were measured by UV-visible spectrophotometer. Both the solutions of P(SBMA-co-DPA) copolymers and PSBMA homopolymer showed pH-dependent upper critical solution temperature (UCST). For the P(SBMA-co-DPA) copolymer with PSBMA/PDPA molar ratio of 15/1 (P1), the UCST of copolymer solution reached a maximum at pH 6.5 and decreased significantly when the pH value shifted above or below pH 6.5. This might be attributed to the cations or anions that introduced antipolyelectrolyte effect and significant influence on their intra- and intermolecular electrostatic interaction and chain conformation. Below the UCST, P(SBMA-co-DPA) copolymers formed nanoparticles due to electrostatic attraction and their morphology in acidic ( pH < pKa(DPA) = 6.3) and neutral solution were investigated by Dynamic light scattering (DLS), Scanning Electron Microscope (SEM), and Transmission Electron Microscopy (TEM). P1 nanoparticles in acidic solution showed the average sizes of 181 and 13.5 nm below and above its UCST, respectively. Through the delicate adjustment of the molar ratios of PSBMA/PDPA, the stimuli-responsive phase transition could be suitable for physiological environment. These indicate that the new series of biocompatible zwitterionic sulfobetaine copolymers has the potential to be applied as drug delivery system.

This communication focuses on organic polymer structures containing sulphur (II) atoms, hereafter termed polysulfides.
Typically, polysulfides can be obtained in the form of well-defined macromolecules by using a living polymerization mechanism, the anionic ring-opening polymerization of episulfides. By combining episulfide polymerisation with appropriate and quantitative end-capping reactions (e.g. Michael-type additions) or with other controlled polymerization mechanism (e.g. ATRP), it is possible to obtain complex architectures, e.g. with branched, multi-block and amphiphilic structures and/or conjugated to active enzymes.
We here will specifically focus on amphiphilic structures, which allows the preparation of a number of self-assembled aggregates in a water environment.
A number of oxidants can convert the sulphur (II) groups (thioethers) of polysulfides into higher oxidation states groups (e.g. sulfoxides); this transition dramatically changes the polarity of the aggregates, and typically leads to their swelling/solubilization; this can also trigger the release of loaded molecules.
However, the speed of the oxidation process and even the likelihood of its occurrence depend on the nature of the oxidant, which need to combine an appropriately high oxidation potential and sufficient solubility in the generally hydrophobic polysulfides. For example, polysulfides are stable towards molecular oxygen; they are also stable towards some anionic, biologically relevant oxidants (Reactive Oxygen Species, ROS) such as superoxide, although they can be made superoxide-responsive via decoration with superoxide-scavenging enzymes. On the other hand, polysulfides readily respond to ROS such as hydrogen peroxide or hypochlorite, also when they are generated enzymatically,although the chemical details depend on the individual ROS: hypochlorite does cause also depolymerization reactions.
Using therefore appropriate polysulfides derivatives, it is possible to produce systems with a broader or narrower response to ROS. The effect of these systems on activated inflammatory cells (macrophages, dendritic cells, microglia) has been studied, showing significant effects on their signalling.

Electrically responsive hydrogels are desirable materials in robotics, microfluidics, sensors, optics and drug delivery due to their potential to be precisely controlled using simple electronics. However, they have been plagued by poor responsivity and robustness which severely limits their utility. We hypothesized that the inclusion of macropores would greatly enhance the electro-responsivity of polyanionic hydrogels by allowing for more efficient movement of water and ions through the gel. Additionally, apparent reductions in gel volume would be related to macropore collapse rather than rearrangement of polymers within the chemically cross-linked gel matrix. These attributes would not only enhance the responsiveness of gels at a given electric field, but also enable one to subject hydrogels to much stronger electric fields (without destroying the gel), thus further enhancing electro-responsivity.
A cryopolymerization approach was developed to create electrically-responsive macroporous hydrogels from acrylic acid, acrylamide, and bisacrylamide (BA). In cryopolymerization, monomer solutions are first placed in a semi-frozen state, concentrating monomer in the non-frozen solvent, resulting in a cross-linked gel structure containing embedded ice crystals following polymerization. The cryogels are then thawed, leaving voids, or pores, in the space formerly occupied by ice. We first monitored the electrical collapse of 19-mm-diameter poly(acrylic acid-co-acrylamide) hydrogel disks using BA as a cross-linker, following exposure to 0-250 V in deionized water. Hydrogels formed at room temperature (i.e., nanoporous gels) generally lost structural integrity within a minute of electrical exposure. However, cryogels exhibited significant volume change while remaining intact. The rate and degree of cryogel collapse was dependent on the polymer weight percentage in the gels, and the extent of cross-linking. Extremely rapid collapse (e.g,. 65% volume reduction in less than 1 minute, and a 97% volume reduction after 10 minutes) could be readily achieved with specific cryogel formulations .
These highly electro-responsive cryogels have a number of potential applications in optics and drug delivery. For example, one can rapidly modulate the optical properties of a two-dimensional array of pigmented cryogels, in terms of both color and spatial configuration, simply by addressing low voltage to specific gels in the array. To demonstrate the use of these gels in drug delivery, poly(acrylic acid) cryogels were fabricated using poly(ethylene glycol) dimethacrylates as a cross-linker. In biological media, these cryogels were capable of delivering large amounts of a model drug in a voltage-dependent manner, while exhibiting excellent drug retention when unstimulated. One can also integrate these gels in a drug-loaded array to deliver multiple drugs in a precisely timed, sequential manner.

Thermoresponsive multilayer hydrogels provide a new platform for designing novel stimuli-sensitive coatings and microcontainers for controlled delivery. We present novel nanothin multilayer hydrogels of poly(N-vinylcaprolactam) (PVCL) with the reversible thermoresponsive behavior. The PVCL hydrogels were produced by selective cross-linking of PVCL in multilayers of PVCL-NH2 copolymers assembled with poly(methacrylic acid) (PMAA) via hydrogen bonding. Non-cross-linked two-component films did not exhibit any temperature responsive shrinkage due to the presence of PMAA. We demonstrated that capsules of cubical and spherical shapes with PVCL hydrogel walls could be fabricated as hollow hydrogel replicas of inorganic templates. The temperature-triggered capsule size changes were completely reversible. The cubical capsules retained their cubical shape when temperature was elevated from to 50°C exhibiting the size decrease. Our work opens new prospects for developing biocompatible and nanothin hydrogel-based coatings and containers for temperate-regulating drug delivery, cellular uptake, sensing, and transport behavior in microfluidic devices.

Elastin-like polymers (ELPs) are genetically engineered protein polymers with unique, tunable stimuli-responsive and elastic properties, making them a promising material for technologies such as stretchable electronics or biomimetics. ELPs are distinct among proteins for their reversible temperature-dependent self-assembly, which occurs at specific transition temperatures dependent on individual polymer composition and solvent conditions. In order to confer ELPs with optical and electrochemical properties, we designed and created ELP scaffolds for incorporation of optically responsive poly(phenylenevinylene) (PPV) oligomers or redox-active metal-ligand complexes. A library of ELPs was designed to explore the effects of several variables on the behaviors of hybrid materials in aqueous solvent: polymer length, which is inversely correlated with transition temperature; polymer polarity, which is correlated with transition temperature; and the density and spatial distribution of chemical modification. We used in vitro combinatorial DNA sequence assembly techniques to incorporate randomly positioned conjugation sites at different ratios and verified successful gene construction by sequencing. We expressed ELP constructs in E. coli and purified proteins to homogeneity by inverse temperature cycling, a strategy enabled by the reversible temperature-sensitive aggregation behavior of ELPs. Purity was assessed by gel electrophoresis and mass spectrometry. Using dynamic light scattering and optical spectroscopy, we have systematically characterized the integration of the temperature- and pH-sensitive behavior of ELPs with the optical properties of conjugated functional moieties.

Silk from silkworms is composed of two key proteins—fibroin, a mechanically strong core protein, and sericin, a glue protein responsible for cementing fibroin fibers together in the cocoon. Silk proteins have novel use in renewable biomaterials design, due to high tensile strength of fibroin, and the favorable aqueous properties and unique degradation kinetics of sericin. However, typically silks and silk proteins are passive materials that are cast into different structures such as membranes and films. We present the use of chemical conjugation techniques to functionalize silk proteins, and create stimuli responsive biomaterials that can change morphology in response to external stimuli including pH, heat or light. These novel building blocks allow the formation of active structures from the micro to the macro scales. By controlling functional groups and crosslinking densities, the biophysical properties of the materials can be precisely tuned. High-resolution atomic force microscopy and scanning electron microscopy are used to characterize topography as well as measure nanomechanical properties of these silk structures. The ability to form active microstructural architectures that are high strength, biocompatible and biodegradable present a new set of materials that can be used in biomedical applications.

Nanodiamond powder is one of the most promising carbon nanomaterials for drug delivery applications [1]. Diamond nanoparticles of ~5 nm in size offer a large accessible surface and tailorable surface chemistry; have unique optical, mechanical, and thermal properties; and are non-toxic. Although the potential of nanodiamond in drug delivery has been demonstrated, fundamental mechanisms, thermodynamics and kinetics of drug adsorption on nanodiamond are still poorly understood. To fully exploit the potential of nanodiamond in drug delivery, attention must be paid to its purity, surface chemistry, dispersion quality, as well as to temperature, ionic composition, and other parameters of the environment that may influence drug adsorption and desorption. Surface modification of nanodiamond and its effects on adsorption equilibria of several important drugs representative of different classes, including antibiotics and anticancer chemotherapeutics, such as tetracycline, polymyxin B, doxorubicin and others, will be discussed. In drug delivery, rational surface modification allows for enhanced adsorption and chemical binding of drugs with sustained or triggered release. The knowledge of fundamental mechanisms of drug adsorption and desorption on nanodiamond will be required in the development of nanodiamond enabled drug delivery platforms. We anticipate that the understanding of adsorption mechanisms on nanodiamond will provide a basis for rational design of ND adsorbents with controlled surface chemistry and agglomeration behavior. This is not limited to drug delivery systems and includes novel materials for chromatographic separation, enterosorption, agglutination of viruses and bacteria, and other applications where adsorption plays an important role.
1. Mochalin, V. N.; Shenderova, O.; Ho, D.; Gogotsi, Y. Nature Nanotechnology, 2012, 7, 11-23

Silk fibroin has been successfully used as a matrix for enzyme entrapment and stabilization. Previous studies have shown that free-standing, enzyme-loaded films drop-cast from aqueous silk fibroin retain activity even when exposed to adverse environmental conditions such as heat, UV, and organic solvents. We found that increasing the surface area of the films lead to an increase in enzyme activity. To further explore this phenomenon, we utilized the electrospinning process with silk fibroin/polyethylene oxide blends to produce extremely high surface area, enzyme-loaded nano- and micro-scale silk fibroin mats. In order to form a more complete understanding of the behavior of enzymes within electronspun fibers, this study will focus on the incorporation of three enzymes: organophosphorus hydrolase, beta-galactosidase, and alkaline phosphatase. We will determine the contribution of fiber morphology, pore size, enzyme size and enzyme distribution on the catalytic activity of the fiber mats. Finally, the effect of fiber entrapment on enzyme resistance to adverse environmental conditions and activity retention after long-term storage will be analyzed.

Polymers possessing antimicrobial activity have been used for self sterilization surfaces as well as agents for treating viral and fungal infections.
Cationic polymers based on quaternary ammonium or guanidine groups have shown high inherent antimictobial activity where the activity is related to the disruption of the micro-organism cell wall. A range of antimicrobial polymers in the form of nanoparticles possessing active quaternary ammonium groups with one of the alkyl is a C4-C8 alkyl chain have been synthesized. These nanoparticles were incorporated in dental restoration compositions to form self sterile composites [1]. Polymers releasing active halogens have been prepared and tested for microbial deactivation in drinking water. Guanidine based polymers have demonstrated efficient antimicrobial activity when added to contaminated water.
Amphotericin B (AmB) a polyene macrolide antifungal agent is the drug of choice for the treatment of mycotic infections caused by a wide range of fungi. New nanoparticles containing polysaccharide-oleyl amine conjugates of amphotericin B (AmB) were synthesized on the inspiration of novel arabinogalactan (AG)-AmB highly water soluble conjugate characterized by reduced toxicity. AG is a highly branched natural polysaccharide with unusual water solubility (70% in water). Particles were conjugated with Amphotericin B by a Schiff-base reaction with oxidized arabinogalactan followed by the same reaction with oleylamine, using DDW as a solvent. Nanoparticles showed narrow size diameter with mean particle size of 200 nm and high antifungal activity [2].
[1] Nurit Beyth , Ira Yudovin-Farber , Michael Perez-Davidi , Abraham Domb ,Ervin Weiss, Polyethyleneimine nanoparticles incorporated into resin composite cause cell death and trigger biofilm stress in vivo, PNAS, 2010 Dec 21;107(51):22038-43285.
[2] Elgart, A.; Farber, S.; Domb, A.J.; Polacheck, I.; Hoffman, A., Polysaccharide pharmacokinetics: amphotericin B arabinogalactan conjugate-a drug delivery system or a new pharmaceutical entity? Biomacromolecules, 2010. 11, 1972-7

With the increased prevalence of antibiotic-resistant infections, there is an urgent need for development of innovative antimicrobials. Macromolecular antimicrobial agents such as cationic polymers and peptides have recently received increasing attention because they can selectively target and disintegrate bacterial membranes via electrostatic interaction and insertion into the membrane lipid domains, avoiding potential bacterial resistance. As a result, a plethora of bio-inspired synthetic polymers have been proposed and are achieving considerable success in overcoming many drawbacks found in using peptides. Unfortunately, biocompatibility and/or biodegradability have presented significant problems during in vivo administration.

In this talk, a new class of antimicrobial polymers will be discussed. These antimicrobials are based on biodegradable cationic polycarbonates, which are synthesized by our organocatalytic living ring-opening polymerization approach. This synthetic platform yields polymers with well-defined molecular weight and structure, which is crucial in the future clinical applications as individual molecular weight fractions of a polydisperse system are expected to exhibit distinct pharmacological activities in vivo. Polymers with various molecular configurations (e.g. linear, branched, star-like, random and block) have been designed and synthesized. The polymers with optimal hydrophilicity/hydrophobicity balance have strong activities against multidrug-resistant Gram-positive and Gram-negative bacteria as well as fungi without inducing significant toxicity both in vitro and in vivo. Therefore, these antimicrobial polymers hold potential for use in the prevention and treatment of multidrug-resistant infections.

Polymer-based, degradable microparticles (MP) are an attractive approach as carrier for vaccine delivery, since polymer and MP properties can be specifically tailored. The co-encapsulation of antigen and adjuvants in the same MP could combine two functionalities and provide improved vaccination strategies. By selection of suitable adjuvants, strong immune reactions should be achieved after MP phagocytosis by antigen-presenting cells for formation of immunity. Interesting adjuvant candidates are ligands of cytosolic receptors sensing nucleotide and oligomerization domains (NOD), such as N-acetylmuramyl- L-alanyl-D-isoglutamine (MDP). Achieving strong immune reactions against the antigen is favored in regards to the formation of a protective long-term immunological memory and therefore the microparticles need to be phagocytosed by antigen-presenting cells. Dendritic cells (DCs) are the major professional antigen-presenting cells, which phagocytizes pathogens, present antigens for downstream activation of T-cells and subsequently expresses important co-stimulatory molecules such as CD86. Therefore DCs are an attractive target for vaccine MP and their efficient microparticle uptake is mandatory.
Here, the uptake efficiency of fluoresceinisothiocyanat-labelled MDP-loaded poly [rac-lactide)-co-glycolide] (PLGA) microparticels by DCs and the activation status of each individual cell was evaluated. The PLGA particles had endotoxin levels below the limit of the U.S. Food and Drug Administration and did not induce unwanted activation of immune cells, indicating that they are free of immunogenic impurities [1]. Monocyte-derived DCs where incubated for 24 hours with increasing concentrations of MP varying from 1.56 µg/ml to 800 µg/ml.
We found a concentration dependent increase of microparticle uptake and, remarkably, the DC viability was not impaired, even at the highest particle concentration. The expression of CD86 was evaluated as a co-stimulatory molecule, which is upregulated by DCs under inflammatory conditions and an indicator of the DC activation status. We only found upregulation of CD86, in cells which have phagocytosed the microparticles, indicating that sufficient amounts of MDP were released from the PLGA carriers into the cytosol of the DC leading to NOD-receptor activation.
Conclusively, we showed that our PLGA microparticle carrier system is suitable to deliver encapsulated MDP, which specifically activates DCs. Since the MDP only acts on DC which have phagocytosed the MP, minimal bystander effects are expected, providing the basis for specific activation of the adaptive immune response and thereby establishing protective long-term immune memory.
[1] C. Wischke, S. Mathew, T. Roch, M. Frentsch, A. Lendlein, Potential of NOD receptor ligands as immunomodulators in particulate vaccine carriers, J Control Release, July 2012,
DOI 10.1016/j.jconrel.2012.06.034.

Shear piezoelectricity is a property present in collagen which is believed to result from hexagonal packing of collagen molecules. Collagen piezoelectricity is a functional property, which may be associated with bone remodeling, although, the biofunctional implications of piezoelectricity in collagen and other biomaterials has yet to be clearly demonstrated. A preliminary step towards this goal is to determine if collagen is piezoelectric in physiological conditions, which involves understanding the role of pH and moisture content. Piezoelectric properties of collagen can be measured using piezoresponse force microscopy (PFM), which is a voltage-modulated contact mode atomic force microscopy technique that allows bias-induced surface deformations via the converse piezoelectric effect to be detected. Thus, PFM makes it possible to investigate electromechanical coupling in biosystems at the nanoscale. To date, on collagenous samples, PFM has been employed to investigate piezoelectricity primarily in D-periodic collagen type I. Here, piezoelectricity has been investigated in two types of assembled type I collagen membranes, with and without D-periodicity, via PFM. Both collagen membranes are formed by aligned collagen fibrils self-assembled from an acidic collagen solution. The structure and piezoelectric properties of the membranes are measured before and after a neutralization process, which alters the pH from acidic to neutral conditions. Before neutralization, shear piezoelectricity has been observed in both D-periodic and non D-periodic collagen fibrils, which may force a re-evaluation of how piezoelectricity manifests in collagen type I. The neutralization process itself leads to a distinct change in the structure of the D-periodic collagen membrane, namely the formation of crimps perpendicular to the fibril alignment direction, while the non D-periodic collagen membrane remains largely unchanged. Both membranes retain their piezoelectric properties following neutralization. Electromechanical properties of the D-periodic membrane have been further investigated under controlled humidity conditions using band excitation PFM to probe whether collagen is piezoelectric in moisture rich environments.

Glucose metabolism is the vital process for living system. It provides not only energy, but also metabolites for biomasses which are required in proliferating cells. Glucose and oxygen, which are consumed at different rates due to the corresponding stimulus and proliferative states, are the two key factors in glucose cell metabolism; therefore, simultaneous measurement of glucose and oxygen level is important for the continuous monitoring of glucose, diagnosing diabetes and hypoxia (low oxygen) related diseases and cancer, and understating of biological processes of the metabolism. For this purpose, a new dual glucose and oxygen sensor with a tri-color emission has been synthesized. The sensors are in thin-film states (membranes) with a glucose probe (a blue emitter), an oxygen probe (a red emitter), and an internal build-in reference probe (a yellow emitter) which does not respond to glucose or oxygen. Polyacrylamide was used as a matrix, where the optical probes are chemically grafted and immobilized in it. The sensor has a higher selectivity to glucose than to fructose, mannose, and galactose. The sensor has a suitable dynamic range for glucose from 4mM to 30mM and for an oxygen concentration (0-40ppm). An application of the sensor for simultaneous real-time monitoring of the consumption of glucose and oxygen with eukaryotic and prokaryotic cells is in progress.

INTRODUCTION:
Proteases are fundamental to diverse physiological processes from generalized protein digestion to more specific regulated processes. However, despite major advances in our understanding of proteases, their substrates and biological functions are not fully understood. The objectives of this research are to develop a generic biosensor system for “real-time” monitoring of protease activity and to define substrate specificity of these enzymes. We describe a highly versatile approach for enzyme immobilization to a porous solid support through DNA-directed immobilization (DDI). The crucial step in DDI development is to synthesize a distinctive DNA-enzyme conjugate with a well established architecture. The advantage of DDI method lies in the regeneration of the surface by removal procedure of the DNA-enzyme conjugate, preparing it for additional biosensing analysis.
RESULTS AND DISCUSSION:
The porous SiO2 (PSiO2) optical transducers are synthesized from a highly doped p-type Si wafer, using anodic electrochemical etch. A synthetic approach is being used for the immobilization of capture single-stranded DNA (ssDNA) through standard silane chemistry. The immobilized strands serve as a structural template for the anchoring the enzyme-conjugate through DDI. Two classes of enzymes are studied, horseradish peroxidase (HRP) and trypsin from bovine pancreas. To confirm the attachment of the biomolecules onto the PSiO2 we used fluorescence labeling and reflective interferometric Fourier transform spectroscopy. The latter technique is sensitive to small changes in the average refractive index of the thin film induced by the different bioconjugation events. To assess the enzymatic activity of the anchored enzymes, the oxidation and proteolysis products of HRP and trypsin were analyzed spectrophotometrically. Both, HRP and trypsin reveal high relative activity (74% and 59%, respectively, compared to the activity of the conjugates in solution). An important advantage of the DDI approach is the ability to regenerate the surface by mild dehybridization conditions or temperature increase (above melting temperature of the DNA pair). Indeed, applying these conditions allow for complete surface regeneration. In addition, we investigate the potential of this platform to serve as an optical biosensor for monitoring protease activity in real-time and analyzing the retrieved fragments by MS and tandem MS techniques. We demonstrate that our biosensing platform is compatible with these common proteomic methods.
CONCLUSIONS:
A biosensing platform was designed for investigation of the proteolytic activity of proteases. The specific and reversible nature of the enzyme immobilization enables the use of minute sample quantities for monitoring the reaction. The generic design of the biosensor will potentially allow tailoring unlimited experimental setups for systematic analysis of the protease of interest.

Along with the tremendous success of commercial glucose oxidase-based electrochemical sensors, enzyme-based dopamine sensors have been proposed to detect abnormal dopamine release. Dopamine imbalance can lead to severe psychological diseases such as schizophrenia, bipolar disorder, and clinical depression. Unfortunately, enzyme-based dopamine electrodes employing tyrosinase, catechol oxidase, or lactase, have low specificity, sensitivity and especially short lifetime. As dopamine co-exists with the other electrochemically active species such as ascorbic acid (AA) and uric acid (UA) in cerebrospinal fluid (CSF), the blocking of more abundant interfering species to the electrode surface is a major roadblock. Furthermore, the dopamine sensor should have sub-micromolar sensitivity. We developed a simple method of incorporating simple redox ions in a polyelectrolyte to develop a highly sensitive electrode with mediator. This polymer-based sensor is highly versatile since the redox potential can be tuned by choosing the proper ions for mediation. The oxidation current increased by 1030% for a dopamine concentration changes from 1mu;M to 1mM, while only a 70% increase was observed at the same concentration change for ascorbic acid. We will also discuss the fabrication method of making the redox polymer films using a special electro-optical on-line monitor for end-point determination that is critical to make the sensor device. The key result is that the sensor device does not utilize enzymes for specificity and is robust.

Tuberculosis (TB) is one of the most common infectious diseases, affecting as much as one third of the world&’s population. Although TB can be treated and prevented with the aid of antibiotics and vaccines, diagnosis of TB is also of primary importance as 90% of TB infections are latent and asymptomatic. The most common TB diagnostic method is a Tuberculin skin test, where a small dose of TB antigen is intradermally injected through a syringe (within the dermis layers) followed by reading the resultant induration. The test, however, is mostly utilized in clinics or only by patients who have received special trainings on correct injection protocols, as deeper injections lead to test failures. Moreover, the use of hypodermic syringes is known to be associated with other issues such as pain, needle-phobia, and even the spread of bloodborne pathogens. Here, we report on novel microneedles fabricated from chitin [poly (β-(1-4)-N-acetyl-D-glucosamine)] and discuss their applicability as a transdermal diagnostic tool for TB. Chitin is a naturally abundant polysaccharide which is well known for its excellent biocompatibility, non-toxicity and biodegradability. Due to these properties, it has attracted a great deal of attention along with chitosan (the deacetylated analog of chitin) particularly for biomedical and pharmaceutical applications. In this study, we have adapted common microfabrication techniques to fabricate chitin microneedles, where concentrated solutions of chitin/hexafluoro isopropanol were micromolded with PDMS replica molds. As a proof-of-concept, the chitin microneedles were coated with TUBERSOL®, a protein-based antigen for TB diagnosis, and tested on the skin of guinea pigs.

Optical sensors are important tools for cellular imaging and analysis, for probing cellular metabolism, for understanding pathways and biological and physiological processes, and for diagnosing diseases and cancers. We have developed several series of fluorescent optical sensors (pH, oxygen (O2), potassium ion (K+), and glucose sensors) suitable for intracellular and/or extracellular imaging and analysis. We integrated individual sensors as dual sensors for simultaneous multi-parameter measurements. Of particular value for application in the complex biological environment, some of the sensors emit two colors in response to the same excitation wavelength, enabling ratiometric measurements and providing superior measurement accuracy. For example, we have prepared a dual pH/O2 sensor with three emission colors. The tri-color sensor composed of a blue emitter as an internal build-in reference probe, a green emitter as the pH probe, and a red emitter as the oxygen sensor. The build-in reference probe is unresponsive to pH or O2. The potassium ion sensors we prepared have high selectivity to potassium ions and controllable dynamic ranges specifically for intracellular and extracellular analysis, respectively. The potassium ion sensors were demonstrated to be suitable for monitoring potassium influx and efflux.

The biomaterial-associated infection (BAI) occurs at significant rates (0.1-15.5%) in all kinds of spinal disc implants. Standard systemic antibiotic therapy is usually ineffective to eliminate such infection because of the limited local antibiotic concentrations. In the current study, berberine-loaded nanofibrous material (B-NFM) with antimicrobial properties was fabricated to achieve much higher local antibiotic concentrations and result in fewer BAIs. Non-berberine-loaded nanofibrous material (NFM) was also fabricated as a control. The testing hypothesis was that the B-NFM could provide both a physical matrix (nanofibrous structure) for spinal disc cell proliferation, and a biological protection (berberine) against microbial pathogens. The antimicrobial activity, the drug release profile, the wettability and the biocompatibility of the materials were evaluated. Antimicrobial activity assessment (inhibition zone test) showed that the B-NFM has good antimicrobial activity against gram-negative bacteria E. coli (strain 15597) and gram-positive bacteria S. aureus (strain 6538). Contact angle test indicated that berberine can significantly increase the wettability of electrospun nanofibrous material, suggesting an improved surface biocompatibility of the B-NFM as compared with the NFM. The drug release profile of the B-NFM showed an initial burst release of 30 ± 0.71% of berberine on the first day followed by a sustained release over the period of 33 days. Primary annulus fibrosus (AF) cells were isolated from porcine spinal disc and used to evaluate the in vitro biocompatibility of the materials. After seeding with AF cells, the in vitro biocompatibility was determined by measuring cell proliferation on the B-NFM and the NFM. In comparison with the NFM, a higher level of cell proliferation was observed in the B-NFM group throughout the experimental period for up to 14 days. The results from this study suggest B-NFM as a potential antimicrobial material for spinal disc implants.

Poly(ε-caprolactone) [PCL] and its copolymers are extensively explored as implant materials for medical devices or drug carriers [1] since their degradability as key functionality enable their removal from the body. By photocrosslinking of ε-caprolactone based cooligoesters functionalized with methacrylate end groups in the melt, multifunctional polymer networks with tailorable degradation rates can be obtained [2] [3] [4]. Recently, crosslinking of nascent PCL microparticles (MP) in aqueous suspensions was suggested with photoinitiators using PCL with several pendant methacrylate groups [5]. However, the applied long-term irradiation and the presence of photoinitiators may have gradually increased the reaction temperature. Additionally, under such solid-like conditions, polymer chain mobility is reduced with unknown effects on network architecture.
For exploring the feasibility of such an approach to prepare defined networks, crosslinking of oligo(ε-caprolactone) [oCL] based MP in their solid state was performed in a controlled regime with well-defined precursors either with or without photoinitiator. The MP (~40 µm) were prepared by an oil-in-water emulsion process from linear ²oCL or 4-arm star-shaped ⁴oCL (8 kDa) with methacrylate end groups (degree of functionalization 98 mol.%), in some cases supplemented with 3 wt.% photoinitiator (Esacure One). After washing and freeze-drying, the MP were resuspended in water for crosslinking by exposure to laser irradiation (308 nm; intensity 26 mW/cm²) at room temperature between quartz glass plates. Successful crosslinking was confirmed by a lack of MP dissolution in dichloromethane. The melting temperatures slightly decreased upon crosslinking. In a quantitative evaluation of swelling by dynamic light scattering, higher particle swelling ratios (²oCL: 360 vs. 300 vol.%; ⁴oCL: 480 vs. 370 vol.%) were detected in the presence of photoinitiator and compared to photoinitiator-free conditions. Importantly, wrinkled particle surfaces and distorted particle shapes were observed by light microscopy in the swollen state. This suggested inhomogeneities of the MP network, probably due to a limited laser light penetration to the MP core. The limitations of solid-state crosslinking of MP may be overcome by experimental conditions that allow higher chain mobility during laser irradiation.
References
[1] Dash TK, Konkimalla B, J. Controlled Release 2012, 158, 15-33.
[2] Kelch S, Steuer S, Schmidt AM, Lendlein A, Biomacromol. 2007, 8, 1018-1027.
[3] Wischke C, Steuer S, Neffe AT, Lendlein A, Eur. J. Pharm. Sci. 2010, 41, 136-147.
[4] Friess F, Wischke C, Behl M, Lendlein A, J. Appl. Biomat. Funct. Mat. 2012 in press.
[5] Vaida C, Mela P, Kunna K, Sternberg K, Keul H, Möller M, Macromol. Biosci. 2010, 10, 925-933.

“hellip;biology is largely the study of fibershellip;” wrote Joseph Needham in Order and Life in 1936. Cells, tissues and organs rely on polymeric nanofibers as supporting structures, and thus polymeric fibrous scaffolds have a major role to play in the burgeoning fields of tissue engineering and regenerative medicine. Also, cell interiors and surfaces are endowed with nanofibers (the cytoskeleton) which play key roles in defining mechanical properties and various important cellular functions. For more than a decade, we have been involved in the development of electrostatic spinning (electrospinning) to create bio-mimicking fibers in a diameter range (ca. 20-100 nm) difficult to access by conventional fiber processing methods. We employ electrospinning as a method of fabrication of scaffolds for tissue engineering, drug delivery, and more recently the development of nanofiber constructs as models for functional biological systems. The talk will focus on recent studies dealing with collagen nanofiber fabrication and applications in wound healing, delivery of enzymes for degradation of scar tissue associated with central nervous system damage, and Ca2+-responsive poly(acrylic acid)-based nanofibers as crude models of the cortical gel layer of nerve.

Nanocrystalline cellulose (NCC) is a renewable "nano-resource" derived from plants, tunicates and algae. The nanocyrstals have high tensile strength and show a tendency to self organize into a liquid crystal chiral nematic phase under certain conditions. They exhibit properties of 1-dimensional photonic crystal Bragg reflectors when the helicoid axes are uniformly aligned. But even the polydomain material exhibits stunning iridescence. We report on results of grafting of coumarin dye molecules by "click" chemistry to the surface of NCC. This provides a probe of solvent-NCC interactions through dye molecule solvatochromism. Under well defined conditions, combinations of NCC with oligoethylene glycol methacrylate yield birefringent and iridescent films when the chiral nematic phase is locked in by photopolymerization. The resultant films show enhanced toughness, glass transition temperature and changes in mechanical properties. The ultrastructure of the polymer nanocomposite reveals chiral nematic order reminiscent of the bulk NCC phase material. Films fabricated from NCC alone give rise to bright iridescence and selective reflection of left circularly polarized light. This optical response can be used to make an hierarchical encryption material when combined with coumarin dye molecules. Moreover, NCC incorporated into optical waveguides serves to enhance TE-TM mode conversion, suggesting a role for the material in "green" photonics.

Soft active materials such as responsive hydrogels and shape memory polymers have recently drawn great attention because they can sense, react, and adapt to various environmental changes such as pH, light, and temperature. Because of the active nature of material behaviors, they hold great potential in the development of autonomous and multifunctional devices and systems. Recently, many researchers have found various applications of soft active materials in broad fields of science and engineering including soft robotics, metamaterials, drug delivery, and tissue engineering. Unique properties and advantages of these materials, however, have not been fully exploited primarily because manufacturing and material processing for this new class of materials still rely on conventional methods, especially photolithography and soft lithography.
This study presents a multi-material micro 3D printing technique for soft active materials. Projection micro-stereolithography (Pmu;SL) is a 3D micro-stereolithography technology capable of rapidly building highly complex microstructures by converting photocurable resin into solid layer-upon-layer. As opposed to existing stereolithography, an entire layer is rapidly photopolymerized within seconds by a single light illumination using a digital dynamic display. Various material properties such as elastic modulus, permeability and swelling ratio can be spatially encoded in the structure through digitally controlled light intensity distribution. More importantly, we introduced microfluidic material feed module for facile switch of constituent materials during the fabrication process. As a result, multiple active materials can be heterogeneously integrated in 3D micro space. This allows for diverse geometries with higher complexities and advanced functionalities which would not be made possible otherwise. Applications that will be discussed include soft robotic micro-devices, zero thermal expansion materials, low-density/high-stiffness materials, and shape memory polymer vascular stents.

Nerve injury in both central and peripheral nervous system is a major health problem. Spontaneous axonal regeneration is limited to small lesions within the injured peripheral nervous system and is actively suppressed within the central nervous system. Axons are guided along specific pathways by gradients of attractive and repulsive chemical and physical cues. To understand the effect of guidance cues on axon growth rate, development of in vitro platforms that are capable of producing precisely controlled shape guidance cues is essential. Conducting polymers have been widely used in biomedical applications, in particular, for drug delivery systems and neural interfaces. Conducting polymers have the ability to response to electrochemical oxidation/reduction by changing their color, conductivity, wettability, and volume. Previously we developed a novel method for fabrication of randomly oriented conducting polymer nanotubes for controlled release of an anti-inflammatory drug. We hypothesize that the aligned conducting polymer nanotubes will provide both physical and chemical guidance for axonal regeneration.
Here we report a novel method for fabrication of multifunctional aligned conducting polymer nanotubes for axonal regeneration. The fabrication process involves (1) electrospinning of nerve growth factor (NGF) loaded poly(lactic-co-glycolic acid)(PLGA) fibers on a rotating gold substrate (angular velocity ~1200 RPM) to create aligned PLGA-NGF nanofibers; (2) Electrochemical polymerization of conducting polymer poly(3,4-ethylenedioxythiophene)(PEDOT) on the gold substrate and around the aligned PLGA-NGF nanofibers to create NGF-loaded aligned PEDOT nanotubes. Electrochemical deposition of PEDOT was carried out by an applied current density of 0.5 mA/cm2. We characterized surface morphology and electrical properties of the PLGA-NGF nanofibers and PEDOT-NGF nanotubes using scanning electron microscopy and impedance spectroscopy, respectively. The diameter of core PLGA nanofibers and thickness of PEDOT nanotubes were 100 ± 23 nm and 30 ± 5 nm, respectively. In order to release the NGF from PEDOT nanotubes in a controlled fashion, we actuated PEDOT nanotubes by applying a bias voltage 1 V. Preliminary results showed that NGF was precisely release (~65ng/ml) from PEDOT nanotubes after electrical actuation. We are investigating the effect of PEDOT nanotube alignment and precisely release of NGF on axon guidance and growth rate using in vitro cell culture assays (dorsal root ganglion and PC 12 cells). In Future, we will design a multifunctional conduit using aligned PEDOT-NGF nanotubes and we will examine the rate of axonal regeneration in 15 mm nerve gap in rats.

Multifunctional polymer-based biomaterials, which combine degradability and shape-memory capability, are promising candidate materials for biomedical implants. An example are degradable multiblock copolymers (PDC), composed of poly(p-dioxanone) (PPDO) as hard and poly(ε-caprolactone) (PCL) as switching segments. PDC exhibit a unique linear mass loss during hydrolytic degradation as well as an excellent thermally initiated dual-shape effect [1-2]. PDC can be synthesized by co-condensation of the two oligomeric macrodiols (PCL-diol and PPDO-diol) using different aliphatic diisocyanates as the coupling agent, e.g. 2,2(4),4-trimethylhexane (TMDI) [1-2] or 1,6-diisocyanatohexane (HDI) [3].
Here, we investigated, whether different phase-segregated morphologies could be obtained for PDC synthesized from identical oligomeric macrodiols (PCL-diol with Mn = 2000 g/mol and PPDO-diol with Mn = 5500 g/mol) with TMDI in 1,3-dioxolane and HDI in dimethylcarbonate. More specifically, atomic force microscopy (AFM) was utilized for in-situ investigation of the surface morphologies in spin-coated thin PDC films within one heating and cooling cycle. In combination with the results obtained in wide-angle X-ray diffraction experiments (WAXD), it could be demonstrated that the choice of the coupling agent and synthesis solvent can be utilized to control the phase morphology of PDC. This research provides some thoughts for choosing a suitable coupling agent to tailor the required morphologies and properties of SMPs for specific applications.
References:
[1] Lendlein, A.; Langer, R., Biodegradable, elastic shape-memory polymers for potential biomedical applications. Science 2002, 296, (5573), 1673-1676.
[2] N'Goma, P. Y.; Malz, F.; Ziegler, H. J.; Zierke, M.; Behl, M.; Lendlein, A.; Radke, W., Characterization of multiblock copolymers by chromatographic techniques. International Journal of Artificial Organs 2011, 34, (2), 110-117.
[3] Kratz, K.; Habermann, R.; Becker, T.; Richau, K.; Lendlein, A., Shape-memory properties and degradation behaviour of multifunctional electro-spun scaffolds. International Journal of Artificial Organs 2011, 34, (2), 225-230.

The United States dependence on fossil fuels has become mandatory over the past few decades. The fuel shortage during the 1970s and after Hurricane Katrina has catalyzed a need for creating alternative energy sources, improving the efficacy of these alternative energy sources, and enhancing energy sustainability. The U.S. Department of Energy has set goals to replace 30% of the liquid petroleum transportation fuel with biofuels and to replace 25% of industrial organic chemicals with biomass-derived chemicals by 2025. In the southeast United States, subterranean termites are prevalent and present microbes in their gut that degrade wood particles and produce the simple sugars that can be used to produce level one biofuels, such as bioethanol. Upon seasonal change, subterranean termites undergo less enzymatic activity and wood-eating capability limiting the amount of sugars that may be produced. This limited activity sparks an interest to investigate this poorly understood phenomenon of how temperature may affect the enzymatic activity in subterranean termites guts. In this study, we report the development thermo-responsive biomaterial nanofiber mats containing cellulose to model cellulase activity. Using electrospinning techniques, poly(N-vinylcaprolactam) celluose fiber mats have been prepared via alkaline hydrolysis and labeled with fluorescent tags. Subterranean termites (reticulitermes species) were fed fiber mats for 10 consecutive days to assess enzyme matriculation and kinetics. Fluorescent microscopy images confirmed spaital and temporal localization of cellulase enzyme throughout the termite gut upon time and temperature change. Moreover, simple sugar production was also determined using infrared spectroscopy. These novel high affinity enzyme detection membranes show promise towards future biofuel production.

The creation of large, complex tissues in vitro remains a challenge in materials science and tissue engineering. The formation of 3-dimensional positive and negative space in biomaterials is required to engineer large tissues and spatially-organize multiple cell types. Here we report a technique for generating bio-homologous microstructures in thick (>1 mm) biomaterials to create defined cellular architectures. We employ a sacrificial fiber technique to make 3-dimensional arrangements of microchannels with diameters 100 and 500 µm. Polylactic acid (PLA) fibers are tensioned in predefined patterns and set in a tissue-compatible hydrogel. The fibers are dissolved with trifluoroethanol to produce round microchannels that are a high-fidelity inverse replica of the PLA fiber pattern. These luminal microstructures act as scaffolds for seeding and culturing cells. We demonstrate the formation of a 3D hexagonal array of microchannels to make a vascularized tissue-mimic. A central channel is seeded with endothelial cells to form a vessel and the six surrounding channels are loaded with tissue-specific cells. Our method is the first to control the 3D spatial distribution of cells and generate bio-homologous structures that extend beyond the diffusion limit of oxygen.

Controllable architectures that combine biological building blocks with synthetic materials have potential applications in nanofabrication and fundamental studies of hybrid biomaterials. Here it is shown, that cowpea chlorotic mottle virus(CCMV) particles can be assembled into well-defined micrometer-sized objects using poly[3-(3&’-N,N,N-triethylamino-1&’-propyloxy)-4-methyl-2,5-thiophene hydrochloride](PMNT) at pH 5.0 and then converted into well-separated viral particles at pH 7.5. At pH 5.0, the association of PMNT with virus particles is dominated by electrostatic interactions, leading to the formation of a hierarchically ordered self-assembled complex. At pH 7.5 well-separated virus particles are formed. This pH-driven assembly of virus particles with PMNT can be monitored by the “naked-eye” through color changes of the PMNT and the increasing turbidity of the samples in aqueous medium. Viral nanoparticles based on plant viruses such as CCMV can be functionalizated by chemical conjugation and genetic modification, potentially allowing a broad range of biomedical applications. The resulting PMNT/CCMV particles at physiological pH were taken up into a human cervical cancer cell line (Hela) more efficiently compared to native particles. Our study provides a facile control for the formation of well-separated virus particles and initiates the groundwork for cell imaging of viral particles.