A long recognized tenet of biological systems is that structure gives rise to function. Mechanical force in contrast has emerged only recently as a critical dimension that links form and function, providing the central effector to sculpt the body plan during morphogenesis, as well as a mechanism for cells to sense and respond to local changes in tissue structure and mechanics. Despite the realization that forces, form, and function permeate all living systems, we as a research community sorely lack methods to control the mechanics of the environment, the spatial organization of cells, or the architecture of cell-matrix and cell-cell interfaces, which collectively define the boundary conditions for how forces are transmitted into cells. Here, I will describe our efforts to design and build physical microenvironments that explicitly manipulate and monitor the structure and mechanics of cellular interactions with their surroundings, and how we have used these approaches to gain insights into their role in regulating cell and tissue structure, signaling, and function. I will use our studies to illustrate 1) the multiple means by which cell-material interactions can control cell signaling and function; 2) the importance of novel engineering and materials approaches to understanding cellular decision making; and 3) opportunities and challenges for how to connect these insights to the ultimate translational objectives set by regenerative medicine.

We have designed a lightly crosslinked PEG based copolymer coating with compositional flexibility as well as extended stability for studying human mesenchymal stem cells (hMSC). The copolymer contains a majority of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) as a cytophobic background with poly(ethylene glycol) methacrylate (PEGMA) for peptide coupling, and less than 10% glycidyl methacrylate (GMA) for crosslinking. Copolymer thin films were crosslinked into 30 nm thick mats by either thermal treatment or ultraviolet light and were stable for 35 days in water at 37 °C. The amount of PEGMA in the copolymer was optimized to ~ 11% to minimize non-specific cell-protein interactions while maximizing the amount of total bound peptides. Following the binding of RGDSP to the mat, hMSC were seeded. The hMSC adhesion, spreading and focal adhesion formation was promoted in a concentration dependent manner. Mats coupled with a non-adhesive scramble (RDGSP) maintained their cytophobicity. Competitive detachment experiments further demonstrated that cell adhesion was mediated by receptor binding to the RGDSP peptide. Due to the very thin nature of the mats, XPS was successfully used to quantify the total average peptide. XPS was effectively used to quantify average total peptide concentration as 12.6 ± 6.14 pmol cm-2. A square 2.2 mm N (1s) element map shows an average value of 17.9 pmol cm-2 of RGDSP, which correlates well with the multipoint high resolution data. The stability, compositional flexibility, ease of application and the ability to precisely quantify bound peptides on the mats, make these materials ideal for the study of cellular processes, where stability, functionality and topography of the biointerface are relevant. The large compositional flexibility provided by the copolymer chemistry and the crosslinking make it feasible to present multiple peptide sequences and to investigate proliferation and differentiation in future studies.

Physical factors in the environment of a cell regulate cell function and behavior and are involved in the formation and maintenance of tissue. There is evidence that substrate rigidity plays a key role in determining cell response in culture. Previous studies have demonstrated the importance of rigidity in numerous cellular processes, including migration and adhesion and stem cell differentiation. Atypical response to rigidity is also a characteristic of transformed (cancerous) cells. In vivo, a cell encounters features of different size and stiffness in its environment. Thus, it is important to understand how cells sense rigidity, particularly in the context of tactile cell sensing of discrete localized areas of increased or decreased rigidity.
We have developed a new technique for the creation of biomimetic surfaces comprising regions of heterogeneous rigidity on the micro- and nanoscale. The surfaces are formed by exposing an elastomeric film of polydimethylsiloxane (PDMS) to a focused electron beam to form patterned regions of micro- and nanoscale spots. Finite element analysis of nanoindentation measurements performed on irradiated PDMS films show that in a thin layer near the film surface, where approximately 90% of the electron energy is absorbed, the Young&’s modulus of the elastomer undergoes a significant increase as a function of electron beam dose.
Human skeletal stem cells plated upon electron beam-exposed PDMS in a pattern of spots with diameters ranging from 2 µm to 100 nm display differential co-localization of focal adhesions to the exposed features, with the degree of co-localization depending on both rigidity and feature size. This behavior persists as the area of the exposed regions is reduced below ~1 µm. On spots with diameters of ~ 250 nm and below, co-localization is lost, and cells appear unable to identify the rigid spots. This implies that there exists a length scale for cellular rigidity sensing, with the critical length in the range of a few hundred nanometers.
Understanding the fundamental underpinnings of cellular rigidity sensing can shed light on a variety of cell functions and behaviors. Surfaces that are designed to elicit different cellular responses can be used to guide the design of new types of tissue scaffolds and may be useful in the development of adoptive immunotherapies and treatments for a broad variety of diseases.

A thorough understanding of cell-material interactions is critical for controlled manipulation of these interactions for applications in tissue engineering. Literature shows that changes in mechanical properties within a three-dimensional (3D) scaffold can elicit changes in encapsulated cell function through mechanotransduction. Furthermore, microstructure and mechanical flexibility of a biomaterial scaffold significantly influence cell behavior. For improved scaffold design, it is paramount that researchers are able to fully characterize the mechanical environment surrounding a cell or cluster of cells over time and position within the construct. Bulk rheology and current micro- and nano-scale rheological techniques do not provide sufficient spatiotemporal correlations between microscale mechanical properties and cell function within 3D scaffolds. Thus, there is a critical need for new technologies capable of spatiotemporal quantification of mechanical properties within a 3D cell-seeded scaffold.
To address this need, we employed cavitation microrheology (CMR) with the goal of increasing spatial resolution of mechanical properties (e.g., Young&’s Modulus) within 3D biomaterials. CMR uses mathematical correlations to relate the pressure at the point of elastic instability (by employing bubble formation at the tip of a needle inserted into the hydrogel) to the Young&’s modulus. The length scale probed is determined by the inner radius of the needle applied, which allows CMR to access mechanical properties at very specific locations within a material over a wide range of length scales by simply varying the needle gauge. Our results demonstrate that CMR measures spatiotemporal changes in mechanical properties on smaller size scales than traditional rheology (100-300 µm used here) and with greater precision and accuracy than what is afforded by other techniques.
We use ionically crosslinked alginate hydrogels which supply a flexible scaffold architecture, allowing seeded fibroblasts to reorganize their external environment. Gradients in crosslinking density, and therefore elastic moduli, can be created by modulating calcium diffusion within the construct. Using CMR, we were able to quantify alginate hydrogel degradation in cell culture media, demonstrating radial variation in crosslinking density over 7 days when gels were uniformly crosslinked on day 0. CMR was also used to quantify the mechanical contribution of cell-secreted extracellular matrix within fibroblast seeded alginate hydrogels. Overall, this work demonstrates the power of CMR in quantifying mechanical properties within 3D biomaterials with precise control over measurement position within the construct. Current and future work focuses on the implication of varying microscale mechanical properties on encapsulated cell function, with a long-term goal of improving fundamental and translational knowledge on cellular response to mechanical cues in 3D biomaterials.

Biological phenomena that are difficult to investigate experimentally can often be better understood by developing computational models. Mechanical forces on cells are increasingly becoming understood as playing a crucial role in biological phenomenon in fields ranging from stem cell differentiation to cancer metastases to vasculogenesis. Mechanotransduction has been divided into the three phases of mechanotransmission, mechanosensing, and mechanoresponse yet how the cell performs all three functions using a shared set of components is still poorly understood and a challenge to tackle experimentally. Here, we present a structurally-driven mechanotransduction model that integrates actin network changes under stretch with a mixture model of molecular release to further elucidate the spatiotemporal interplay between network morphology and resultant biochemical signaling.
As stretch is applied to our model of the discrete actin filament network, the distribution of individual bond angles in the network transitions from a more peaked to a flatter distribution. We used our approach to explore various angle thresholding models of how actin filament network deformations might influence rates of release of bound signaling molecules. These thresholding models allow us to project how the cell may interpret an applied mechanical stimpulus into a biochemical response. We train and validate these simulations using published experimental data and use our parameterized model to then test different predictive capabilities of how mechanotransduction may function. Our integrated mechanotransduction model represents a potentially versatile mechanistic platform for examining biophysical systems that link mechanical stimulus at the cellular level to response at the protein level.

This talk will explore platform technologies that are currently being developed
in the KarpLab to tackle some of the most challenging medical problems.
Namely, sealing tissues/closing wounds, achieving long term local drug delivery
for treatment of diseases such as rheumatoid arthritis and glioblastoma,
development of surfaces to separate cells for disposable point of care diagnostics
and for cell therapy, engineered cell therapy, and needles that sense different
levels of tissue for the delivery of cells and drugs. Many of these platforms were
inspired by solutions observed in nature.

Despite transplantation of pancreatic islet cells is a promising treatment for Type 1 diabetes, its clinical application remains limited due to adverse effects of immunosuppression, declining allograft function, and recurrence of autoimmunity. We report on a novel ultrathin cytocompatible coating which displays an inherent anti-inflammatory property to protect living pancreatic islets. The coating consists of a hydrogen-bonded multilayer assembly of a natural polyphenol (tannic acid; TA) and poly(N-vinylpyrrolidone) (PVPON) to allow for conformal coating of individual islets with an ultrathin film of controllable thickness and composition. The coating is conformal over the surfaces of rat, non-human primate, and human islets. The coated islets maintain their viability and β-cell functionality. Coated islets displayed an increased insulin stimulation index after 96 hours in vitro as compared to uncoated islets. Also, the (TA/PVPON) coating material demonstrated immunomodulatory properties showing significant suppression of innate immune pro-inflammatory cytokine and adaptive immune Th1 cytokine responses. The developed material combines high chemical stability under physiologically relevant conditions with capability of scavenging free-radicals, two crucial parameters for prolonged islet integrity, viability, and function in vitro/in vivo. Our study offers new opportunities in the area of advanced transplant materials to be used in Type 1 diabetes treatment as well as in various areas of cell-based therapy.

Most of immunomodulatory materials (e.g., vaccine adjuvants such as alum) modulate adaptive immunity, and yet little effort has focused on developing materials to regulate in-nate immunity (e.g., short-lived effector cells such as neutrophils). Here we show that the incorporation of unnatural amino acids into a well-known chemoattractant—N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF) offers a simple and efficient approach to create a de novo, multifunctional chemoattractant that self-assembles to form supramolecular nano-fibrils and hydrogels. This de novo chemoattractant not only maintains the potent chemotactic activity to human neutrophils as well as mouse neutrophils, but also exhibits dramatically increased biostability against proteolysis. Thus, its hydrogel, in vivo, sustainably releases the chemoattractant and attracts neutrophils to the desired location in a sustainable manner. This work illustrates a novel approach to generate a new class of biomaterials for modulating innate immunity and offers an unprecedented prolonged acute inflammation model for developing various new applications.

With an ever-ageing population in the Western world, the need for regenerative medicine, especially transplantation is increasing, but the number of donors remains static. Therefore improved storage methods for cells/tissues/organs is urgently required to overcome their limited shelf-life and also for improved storage of progenitor cells. Antifreeze glycoproteins (AFGPs) from polar fish slow ice crystal growth and may find application in cryopreservation, but are limited by their extremely high costs, challenging synthesis and potential toxicity.[1] To address this, my team is developing novel synthetic materials which can reproduce the desirable properties of AFGPs (and other glycoproteins[2]), without the side effects, at lower cost. We have recently demonstrated that synthetic polymers designed to inhibit ice recrystallisation (growth) improve cryopreservation, without the need for any toxic organic solvents (traditional method), using 400 x less material.[3,4] A thorough biophysical analysis on the underlying mechanisms of action of these polymers is underway[5,6] alongside biocompatibility and cellular preservation studies. This research is truly interdisciplinary, crossing the boundaries of materials, polymers, chemistry, and the life sciences.
Key References
[1] Gibson, M.I., Polymer Chemistry, 2010, 1, 1141-1152,,[2] Richards, S-J., Jones, MW, Hunaban, M., Haddleton, DM., Gibson, M.I., Angewandte Chemie,.2012, 51, 7812 - 7816; [3] Gibson, M. I., Barker, C. A., Spain, S. G, Albertin, L., Cameron, N. R. Biomacromolecules, 2009, 10, 328-333; [4] Deller, R.C., Vatish, M., Mitchell, D.A., Gibson, M.I. 2012, Submitted; [5] Congdon, TC, Notman, R., Gibson, MI, Biomacromolecules, 2013, 14, 1578 - 1586; [6] Deller, R.C., Congdon, T., Sahid, M., Morgan, M., Vatish, M., Mitchell, D.A., Notman, R., Gibson, M.I., Biomaterials Science, 2013, 1, 478 - 485

The extracellular matrix (ECM) is composed primarily of proteins secreted from support cells such as fibroblasts in connective tissue. The ECM not only acts to physically support cells, but also has been shown to affect cell migration and proliferation. Epithelial cells such as vascular endothelial cells (VECs), however, are also known to secrete and remodel their own extracellular matrix. We mimicked the crowded in vivo physiological microenvironment surrounding VECs by using macromolecular crowding nanoparticles (MMCs). We tracked ECM deposition and organization of confluent VECs over the course of four weeks using immunocytochemistry with and without the presence of MMCs. We stained for collagen type IV, the extracellular collagen found primarily in the basal lamina, and used average angular standard deviation to quantify the orientation and organization of the ECM fibers with and without MMCs. Detectable differences in ECM architecture were identified within one week in vitro . Our results suggest that the ECM remodeling may play a role in quiescence and angiogenesis in vivo. Moreover, our results suggest we can control endothelial cell behavior and the organization of native ECM in vitro through biophysical rather than biochemical tools.

Zinc oxide (ZnO) is a wide-bandgap metal oxide semiconductor that can be excited at room temperature by UV irradiation, and finds applications in a variety of fields and products including paints, pigments, batteries, photocatalysis, foods, to name a few. When in nanometer size range, it may be used in polymer nanocomposites and sunscreens as an efficient UV-filter with high transparency in the visible wavelength range. However, the photocatalytic activity of ZnO causes the degradation of the surrounding polymer rendering it unsuitable for long-term employment. Furthermore, ZnO nanoparticles are highly toxic and may pose risks to the public and the environment. Here, ZnO nanorods are made by scalable flame synthesis and are in-situ encapsulated by an amorphous nanothin SiO₂ layer [1,2,3]. The as-prepared nanoparticles are characterized by electron microscopy (EM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and N₂ adsorption. The hermetic nature of the SiO₂ coating is evaluated by a detailed dissolution study and the photocatalytic activity is monitored by the decomposition of methylene blue dye. The presence of SiO₂ facilitates the dispersion of these nanorods in relevant solutions. The core ZnO nanorods exhibit the characteristic optical properties as determined by UV/vis and diffuse reflectanse spectrometry, while the SiO₂ coating eliminates the photocatalytic activity [4] and minimizes the DNA damage to human cells, as illustrated in cellular studies using multiple cell lines. Therefore, these flame-made ZnO nanorods may be safely used as fillers in polymer UV-filter nanocomposites and in sunscreens exhibiting superior performance while mitigating their impact on environmental health.
References
[1] Sotiriou, G. A., Sannomiya, T., Teleki, A., Krumeich, F., Vörös, J. and Pratsinis, S. E. Non-toxic dry-coated nanosilver for plasmonic biosensors. Adv. Funct. Mater.20, 4250-4257, (2010).
[2] Sotiriou, G. A., Hirt, A. M., Lozach, P. Y., Teleki, A., Krumeich, F. and Pratsinis, S. E. Hybrid, silica-coated, Janus-like plasmonic-magnetic nanoparticles. Chem. Mater.23, 1985-1992, (2011).
[3] Sotiriou, G. A., Franco, D., Poulikakos, D. and Ferrari, A. Optically Stable Biocompatible Flame-Made SiO₂-Coated Y₂O₃:Tb³⁺ Nanophosphors for Cell Imaging. ACS Nano6, 3888-3897, (2012).
[4] Gass, S., Cohen, J. M., Pyrgiotakis, G., Sotiriou, G. A., Pratsinis, S. E. and Demokritou, P. Safer Formulation Concept for Flame-Generated Engineered Nanomaterials. ACS Sustainable Chem. Eng.in press, DOI: 10.1021/sc300152f, (2013).

In the past decade, researchers have sought to develop circular microchannels for cell culture studies that closely mimic in vivo vasculature. 3D cell culture techniques in tubular structures typically involve the physical rotation of the channels to achieve a uniform distribution of cells on the channel circumference. Depending on the rotation frequency, cells may not distribute uniformly and can clog the tube passage. Blockage in the microchannel may potentially prevent fluid flow through the structure, which could impact oxygen and nutrition flow in vascular networks. To address this issue, a low-cost, motorized, hands-free device was built to produce consistent, dynamic rotation of the channeled structures at low speeds. Circular microchannels having an internal diameter of 1mm were fabricated in polydimethylsiloxane (PDMS) using a wire-sacrificed technique, and were dynamically rotated at a speed of 0.7 rotations per second. Optical microscopy showed human umbilical vein endothelial cells (HUVECs) adhering to the surface of the tubular PDMS channel at 2 days in dynamic culture. This device demonstrates potential benefits for use in applications for vascular network fabrication.

For monitoring of biological events within cells, the importance of cell insertion technique is on the rise in various bioengineering fields. To maintain cell viability during such cell insertion by nanoprobe structures, seamless and damage-free insertion of nanoprobe structures through cell membranes is required. Although silicon or metal nanowires were often used as cellular nanoprobes, their fabrication processes are expensive and time-consuming. As a rapid and low cost fabrication method, we introduce a thermal drawing process for vertically-aligned polymer nanoprobes optimized for cell insertion. This thermal drawing allows for rapid fabrication of high aspect-ratio polymer nanoprobes with about 500 nm diameter and 20 mu;m heights. The whole process takes only a few minutes. In this study, we optimized processing parameters for thermal drawing, such as temperatures of substrates and micro pillars, drawing speed, drawing steps, contact depth of micro pillars, and contact time. First, SU-8 2150 was spin-coated on a glass substrate and soft baked for solvent removal. Afterwards, a tungsten pillar was heated above the glass transition temperature (Tg) of SU-8 2150, and polymer substrates were heated just below the Tg. The heated micro pillar was dipped in the heated SU-8 layer for a pre-determined time. Vertical drawing of the pillar created a liquid bridge between the pillar and substrate. Variation of the processing parameters mentioned above indicated that fast drawing after short contact time produced the nanoprobes with smaller diameters. Dipping depth also affected the profile of the nanoprobes. Photosynthetic algal cells, Chlamydomonas reinhardtii, were used to test cell insertion and the inserted algae maintained the integrity of the cell membrane for 2 hours without any apparent damage or leakage.

Alpha-synuclein (aSyn) is a central player in Parkinson&’s disease (PD) and other neurodegenerative disorders collectively known as synucleinopathies. It misfolds and aggregates in the typical pathological hallmarks of these disorders, the Lewy bodies, and is associated with familial and sporadic cases when mutated. However, the mechanisms underlying aSyn aggregation and toxicity are not well understood. One way to gain further insight into the biological effects of normal and mutant aSyn is to use yeast cell models. S. cerevisiae (Sc) has been widely used in biology since it shares basic cellular mechanisms of animal cells and can be genetically modified to produce and correctly fold some human proteins. To study aSyn effects in PD, yeast cells were genetically engineered to produce aSyn fused with GFP. The production of this aSyn-GFP fusion protein is induced by exposing cells to galactose (GAL), and can provide insight into what happens when overexpression occurs.
To study how these cells react when exposed to different concentrations of promoter inducer and known stressors, as well as to follow and compare the behaviour of individual and cell populations over time in different controlled and sustained environments, we have developed a PDMS-based microfluidic device that combines a chemical gradient generator with hydrodynamic single cell traps. The chemical gradient generator is a series of serpentine channels that allow laminar flowing liquids to mix by diffusion. By increasing the number of channels through several mixing stages, we created a 9 step gradient from just 3 initial solutions of different concentrations. This gradient was used to expose 9 chambers to fluid flow with precise chemical compositions. Each chamber has an array of hydrodynamic cell traps designed to capture a single Sc cell. The combination of these two modules allows to expose the genetically modified yeast cells to different chemical environments and, since they are trapped, to follow the accumulation and effects of aSyn on a large set (several hundred) of single Sc cells over time.
The first tests done with this device focused on the calibration and optimization of the gradient generator using FITC. Also, the geometry and spacing of traps was optimized in terms of overall trap occupation and single cell trapping. Experiments performed over 3 h with a gradient of GAL showed that the yeast cells exposed to higher concentrations responded with increased production of aSyn. We will show how individual and a population of yeast cells respond in time to different concentrations of the promoter inducer and then report on the study of the effect of different concentrations of chemical stressors such as hydrogen peroxide and iron chloride on the expression and intracellular distribution of aSyn. Ultimately, this approach will enable us to gain novel insight into the molecular basis of aSyn dysfunction in synucleinopathies.

Novel medical and biological applications drive increasing interest towards development of materials and processes instrumental to probing- and affecting- biological phenomena. We have combined microfabrication technologies with ion beam milling techniques to successfully produce cantilever-type polysilicon and silicon oxide micro-dispensers with 3D etched microchanels. Ion beam polishing successfully produced sharp cantilevers with pre-determined taper angles and smooth edges. The main purpose of this work was to test the piercing efficiency of custom-fabricated, sharp, polished micronozzles on mouse embryos in absence of piezoelectric-assisted drills.
Systematic study of perforation with different angles and in absence of drills was performed over mouse oocytes and embryos. Namely, geometries involved in sharper angles and narrower pipettes were more successful in perforation. Despite nanometer thin tips, no structural damage to the structures was observed upon piercing. Micronozzles were intact after perforation, suggesting this technology could be useful in cell handling. Significantly, pierced embryos are seen to continue their division to blastocyst, hinting at biocompatibility. These results suggest that residual chemical agents or Ga+ ions injected during milling are not arresting cell development.
This innovative technology could prove invaluable in answering fundamental biophysical questions such as intrinsic mechanic properties of bio-layers, since perforation was conducted in absence of vibrational piercing aids. Moreover, focus ion beam made-to-order pipettes could be highly instrumental towards versatile platforms. These platforms are amenable to sensing and actuating capabilities at cellular and subcellular levels, inclusive of targeted drug delivery, owing to their biological-relevant architecture and hierarchical structures: macro (chip-reservoir) to micro (channels) and nano (beam-sharpened tips).

A variety of vaccine delivery systems have been investigated for facile and efficient immunization with patients&’ compliance. Among them, transdermal vaccine delivery might be a strong alternative candidate due to well-designed immune systems in the skin tissues containing Langerhans and dendritic cells. In this work, we developed smart transdermal vaccine delivery systems using hyaluronic acid - ovalbumin (HA-OVA) conjugates as a model system. HA-OVA conjugates were synthesized by site-specific reaction between HA-aldehyde (HA-ALD) and N-terminal amine group of OVA. The successful synthesis of HA-OVA conjugate was confirmed by 1H NMR, GPC and ELISA. Two-photon microscopy clearly visualized the effective transdermal delivery of HA-OVA conjugates labeled with red fluorescence dye (Rhodamine B and Texas Red). Remarkably, the transdermal delivery of HA-OVA conjugate via the ear skin of Balb/c mice resulted in statistically significant immunization according to the ELISA for the concentration of anti-OVA IgG in cardiac blood samples. Taken together, we could confirm the feasibility of HA-based transdermal vaccine delivery systems for further development.

There is wide anecdotal recognition that biological cell viability and behavior can vary significantly as a function of the source of commercial tissue culture polystyrene (TCPS) culture vessels to which those cells adhere. However, this marked material dependency is typically resolved by selecting and then consistently using the same manufacturer&’s product - following protocol - rather than by investigating the material properties that may be responsible for such experimental variation. Here, we quantified several physical properties of TCPS surfaces obtained from a wide range of commercial sources and processing steps, through the use of atomic force microscopy (AFM)-based imaging and analysis, goniometry, and protein adsorption quantification. We identify qualitative differences in nano- and micro-scale surface features, as well as quantitative differences in surface roughness and wettability that cannot be attributed solely to differences in surface chemistry. We also find significant differences in cell morphology and proliferation among cells cultured on different TCPS surfaces, and resolve a correlation between nanoscale surface roughness and cell proliferation rate for both cell types considered. Interestingly, AFM images of living adherent cells on these nanotextured surfaces demonstrate direct interactions between cellular protrusions and topographically distinct features. These results illustrate and quantify the significant differences in material surface properties among these ubiquitous materials, allowing us to better understand why the dish can make a difference in biological experiments.

Natural tissues are composed of multiple cell types, structural extracellular matrix (ECM), and blood vessels. 3D bioprinted constructs are beginning to emerge as viable options in regenerative medicine for situations where the tissue is simple and does not require complex vasculature to function properly, e.g., bladder, cartilage, and the trachea. However, to vastly expand the application of these types of tissues, one must be able to integrate cells, structural, and vascular components concurrently and with precision. Using a custom-designed bioprinter, we are creating 3D multi-material, cell-laden architectures with embedded vascular networks. We will describe the printing platform and enabling ink designs as well as the structure and viability of the printed 3D tissues.

Biohybrid materials integrate synthetic materials with biological systems in order to confer unique properties to existing biological functions. This class of materials can be used in a variety of applications, such as sensing, drug delivery, or biological imaging. One type of biohybrid material is the cell backpack, which incorporates polyelectrolyte multilayer (PEM) patches with cells. Various cargoes, such as small molecules, nanoparticles, or labeling moieties, can be incorporated into the backpacks for the cells to carry, making cellular backpacks a versatile platform for drug delivery. These PEM backpacks are fabricated through a combination of photolithography and layer-by-layer (LbL) assembly. The backpacks are hundreds of nanometers thick, a few microns wide, and attach to part of the cell surface.
The original cell adhesive PEMs produced in our group contained hyaluronic acid paired with chitosan, since CD44 receptors on lymphocyte surfaces bind to hyaluronic acid. However, this receptor-ligand binding interaction is not strong and is specific to certain types of cells, so more robust and versatile techniques for cell attachment need to be developed, such as covalent attachment. This would not only create an irreversible chemical bond between the backpack and the cell, but it would also allow the backpacks to attach to a greater variety of cell types.
Covalent attachment can be achieved by introducing functional groups into the polymers of the cell adhesive multilayers, which would react with proteins on the cell surface. One target functional group includes the maleimide group, which reacts with thiols present on cysteine residues of proteins. Maleimide groups are grafted onto poly(allylamine hydrochloride) using a heterobifunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC). The functionalized polymer is then paired with poly(acrylic acid) for LbL assembly. The cellular backpacks are then reacted with lymphocytes for the covalent attachment reaction. The cells are incubated with both an array of backpacks attached to a glass substrate as well as releasable backpacks that react with cells in solution. In addition to incorporation into cell backpacks, polymers that covalently attach to cell surfaces can also be used to create other types of biohybrid materials for a variety of applications. The development of polymers that covalently attach to cells could thus open a broad area of new ideas to explore.

The effort to develop polymeric vectors for gene delivery has been drifted from viral vectors mainly due to the safety and less viable production concerns associated with viral vectors. However, polymeric vectors are impaired significantly with low delivery efficiency compared to viral vectors, and thus to overcome diverse impediments plethora of research activities have been undertaken. Nevertheless, for practical clinical realization it is essential to find out rational design method for polymeric gene delivery system with high efficiency. To develop biologically viable and efficient delivery constructs, it is of paramount importance to decipher the underlying mechanisms of polymer-mediated delivery which comprises several extra- or intracellular events such as circulation in blood stream, cellular uptake, endosomal escape, and dissociation of cationic polymer/gene complex (polyplex). Especially, the dissociation of polyplex is considered as the rate-limiting step for efficient trafficking of gene to nucleus. A study about prompt and opportune dissociation of polyplex bears an immense importance in achieving high gene transfection efficiency, however, only a few work has highlighted this aspect. Therefore, there is a pressing need to develop tools which can monitor the dissociation of polyplex in real-time without compromising the delivery efficiency and the safety issues.
Herein, we employ carbon dot (CD), which has been emerging as a fluorescent nanomaterial with excellent biocompatibility, to monitor the association/dissociation of polymeric carrier/plasmid DNA (pDNA) complex during transfection. To shed light on the underlying post-endosomal events, the adopted strategy exploited the quenching of the fluorescence of CD by Au nanoparticles. The surface of CD and Au was modified with polyethylenimine (PEI) to make cationic surface. Simple subsequent treatment of negative charged pDNA triggered the quenching of the CD fluorescence by reducing distance between CD and Au. The dissociation of pDNA from the complex induced fluorescence recovery due to the increase in distance between CD and Au by charge repulsion. The fluorescence intensity changes during transfection, especially post-endosomal step, were monitored by fluorescence measurement using fluorescence microscope. This nano-assembly system was found to be very effective at monitoring the carrier/pDNA dissociation in a non-labeled manner, providing efficient strategy to study the mechanistic aspect of polymer mediated pDNA delivery.

Silver nanoparticles (nanosilver) have attracted strong interest in biomedical applications [1] for their plasmonic and superior antibacterial properties. The mechanism for the latter depends on bacterial contact with either the nanosilver surface or the released (or leached) Ag⁺ ions from it [2]. Here, nanosilver with closely controlled particle size (7-30 nm) and without any surface functionalization (that could interfere with its ion release or leaching) is prepared, and characterized by X-ray diffraction, nitrogen adsorption and transmission electron microscopy. The release or leaching of Ag⁺ ions in de-ionized (DI) water (pH 6.8) and in low pH (4.65) buffer solution is monitored electrochemically. The presence of Ag₂O on nanosilver surface is detected by optical (UV-vis) spectroscopy and quantified by thermogravimetric analysis and mass spectroscopy. The Ag⁺ ion release in DI water depends on nanosilver size and is traced to the dissolution of one Ag₂O surface layer. [3] On the other hand, higher Ag⁺ ion release occurs in a low pH (4.65) buffer solution stemming from metallic Ag dissolution [4] beyond that of the surface Ag₂O layer. This dissolution of the smallest nanosilver (< 10 nm) occurs almost instantly (within 3 min) while that of the larger ones (> 10 nm) continuously occurs for seven days. Furthermore, the release of Ag⁺ ions from metallic nanosilver is examined by continuous bubbling of O₂ or N₂ though such suspensions. Higher Ag⁺ ion release occurs from metallic Ag at high O₂ concentrations in water, which depends also on nanosilver size, indicating that even metallic nanosilver is oxidized and eventually release Ag⁺ ions. During N₂ bubbling, the smallest nanosilver appear to release some ions but this is attributed to its high surface availability by O₂ that is gradually removed by the bubbling N₂.
1. Chen, X. and H.J. Schluesener, Toxicol. Lett., 2008. 176(1): p. 1-12.
2. Sotiriou, G.A. and S.E. Pratsinis, Environ. Sci. Technol., 2010. 44(14): p. 5649-5654.
3. Sotiriou, G.A., A. Meyer, J.T. Knijnenburg, S. Panke, and S.E. Pratsinis, Langmuir, 2012. 28(45): p. 15929-15936.
4. Liu, J.Y. and R.H. Hurt, Environ. Sci. Technol., 2010. 44(6): p. 2169-2175.

ABSTRACT: Mycobacterium smegmatis is a non-pathogenic microorganism and has been widely used as a model organism to study infections caused by M. tuberculosis and other mycobacterial pathogens. We report that nanoparticles conjugated with selected carbohydrate show a striking increase in the surface adherence and internalization by M. smegmatis. This applies to silica nanoparticles and magnetic nanoparticles ranging from 100 nm to 5 nm. Under the same experimental conditions, minimum adhesion or internalization was observed for unfunctionalized nanoparticles. The synthesis and characterization of the glyconanoparticles will be presented. The finding is applied to imaging M. smegmatis-infected lung epithelial cells, and the results will be discussed.

Introduction
Continuous ultraviolet (UV) radiation to the skin generates reactive oxygen species (ROS) including singlet oxygen (1O2), superoxide anion radical (O2-), etc. The ROS can be a factor of oxidative damage such as rough skin, lipid peroxidation and skin irritation. Therefore, in the field of prevention and treatment such as cosmetics and pharmaceuticals, development of superior ROS scavenging system is desired. In this study, we focus on quercetin and gallic acid, which have been reported to have a high ROS scavenging ability. Experiments were performed under the environmental of lamellar structure model imitating the skin stratum corneum intercellular lipid. The ROS scavenging capacity was measured with high accuracy by monitoring the concentration of ROS with a short lifetime by electrochemical technique.
Experimental
Environment of lamellar structure model was prepared by mixing of ultrapure water, arginine, ethylene glycol, glycerol, oleic acid and linoleic acid. Antioxidants (20mM quercetin, 20mM gallic acid and 10mM quercetin + 10mM gallic acid) were added to the model for the test. A needle-type electrochemical sensor with a platinum working electrode and a stainless steel counter electrode was fabricated for hydrogen peroxide (H2O2) detection, and with an iron porphyrin complex-modified carbon working electrode and a stainless steel counter electrode for O2- detection. H2O2 and O2- were generated in the model systems.
Result & Discussion
We confirmed that the model was prepared successfully by observing a lamellar structure consisting of the lipid and the water layers in the model by a polarizing microscope. Calibration curves for ROS detection were obtained with response current at constant potential electrolysis. From the result of the ROS scavenging capasity test, smaller response current for both H2O2 and O2- was observed in the presence of the antioxidants than in the absence of the antioxidants. From the electrochemical evaluation, the ROS scavenging capacity was higher at the mixed system (quercetin + gallic acid) than at the system with a single antioxidant. This should be due to a synergistic effect of the combination of hydrophobic (quercetin) and hydrophilic (gallic acid) antioxidants.

The physiological properties of polymer brush-afforded silica particles prepared by surface-initiated living radical polymerization were investigated in terms of the circulation lifetime in the blood and distribution in tissues. Hydrophilic polymers consisting mainly of poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA) were grafted onto silica particles by surface-initiated atom transfer radical polymerization that was mediated by a copper complex to produce hairy hybrid particles. The hybrid particles were injected intravenously into mice to systematically investigate their blood clearance and body distribution. It was revealed that the structural features of the hybrid particles significantly affected their in vivo pharmacokinetics. Some particles exhibited excellent stealth properties, for instance, the hybrid particles with SiP cores with a diameter of 15 nm and a shell of PPEGMA brush with an number-average molecular weight of 68000 have an extremely long circulation period with a halflife of about 20 h in the blood, and were found to preferentially accumulate into tumor tissue with the aid of the EPR effect.

Diseased microenvironments, such as tumors release different biological cues as compared to normal physiological tissues. Hence, it is important to develop effective delivery vehicles which can deliver multiple payloads and modulate their payload release in response to these biological cues for enhancing their efficacy. In this regard, mesoporous silica nanoparticles (MSNs) have shown excellent potential owing to their unique porous structure, tunable dimensions, ease of surface functionalization and overall versatility. Futhermore, by aptly modifying their pores with molecular valves, it is also possible to trigger the release of the entrapped drugs in response to various chemical and physical stimuli such as light, pH, redox potential, enzyme and temperature. While there have been numerous reports on the development of stimuli-sensitive MSNs for drug/gene delivery and subsequent intracellular release, monitoring the release of drugs from these MSNs in real-time is still in its nascent stage. Hence, it is essential to develop and integrate a real-time monitoring system within the stimuli-responsive MSNs to investigate real-time monitoring of drug release in complex cellular microenvironments. To address the afore-mentioned challenges, we have developed a versatile fluorescence resonance energy transfer (FRET)-based real time monitoring system consisting of (a) coumarin-cysteine-tethered MSNs as drug carriers, (b) FITC-conjugated β-cyclodextrin (FITC-β-CD) as redox-responsive molecular gate and (c) FRET donor-acceptor pair of coumarin and FITC integrated within the pore-unlocking event to allow real-time monitoring of drug release. Under non-reducing conditions, the intact disulfide bond brings coumarin and FITC in close proximity, thereby turning FRET ON. However, in the presence of reducing environment, such as that encountered by the FMSNs in the cytoplasm of cancer cells, the disulfide bond is cleaved which results in the release of FITC-β-CD, thereby unlocking the pores and release of encapsulated drug. Since the modulation of FRET signal was incorporated into the pore-unlocking event, the triggered release of the drug in target location can be monitored in real-time by simultaneous monitoring the change in FRET signal. Thus, the proposed drug delivery system provides a versatile strategy for real-time monitoring of drug delivery which can be extended to a wide array of biological applications.

The primary objective of this study is to develop a quantitative analytical tool to evaluate the antimicrobial efficacy of Ag/BSA Nanoparticles. Ag/BSA Nanoparticles were prepared by chemical reduction and capping of silver nanoparticles with Bovine Serum Albumin (BSA). The synthesized nanoparticles were characterized by a sleuth of microanalytical techniques to obtain morphological information of nanoparticles. The antibacterial efficacy of silver nanoparticles against E. coli was measured by performing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay (used to measure the viability of cells). Minimum Lethal Concentration (MLC) and the Minimum Inhibitory Concentration (MIC) of the nanoparticles against E. coli were established. Colony forming unit and optical density measurement were used to validate the results from MTS results. Future studies will investigate the optimum Ag/BSA nanoparticle concentrations needed to exhibit MIC and MLC behavior on biodegradable PHBV for potential osteomyelitis application.
Acknowledgment : NSF-DMR, NIH S06 GM 08016 -32

Inflammation, characterized by increased levels of reactive oxygen species, such as hydrogen peroxide (H₂O₂), is involved in various chronic diseases, including atherosclerosis, rheumatoid arthritis, neurodegeneration, and cancer. Targeting drug delivery specifically to these conditions may be possible with a new biocompatible, nontoxic polymeric nanoparticle capable of degrading and releasing its contents upon exposure to biologically relevant levels of H₂O₂. While delivery vehicles designed to respond to inflammatory conditions have been previously developed, none have yet been demonstrated to release cargo in response to sufficiently low concentrations of H₂O₂ to promise likely specificity of delivery in vivo. Our nanoparticles, consisting of a newly designed poly-cresol bearing benzyl ether-linked boronic ester triggering groups in each monomer, release their cargo upon exposure to 50 µM H₂O₂ or activated neutrophils and remain stable in aqueous solution. Release was indicated by quenching of fluorescence of Nile red, LCMS for paclitaxel of supernatants from centrifuged particles (both in vitro), and fluorescence of fluorescein diacetate (in cells; intracellular esterases cleave the dye to a fluorescent form). Nanoparticle degradation was confirmed by transmission electron microscopy; exposure to H₂O₂ induces significant ripping or crumpling in most particles. ¹H NMR and GPC confirm polymer degradation into easily cleared small molecules upon H₂O₂ exposure and stability in its absence. These particles have no significant effects on cell viability, cytotoxicity, or apoptosis at concentrations up to 100 µg/mL. We are currently translating this material into an MRI-based ROS sensor and exploring its ability to enhance the efficacy of antioxidants in models of inflammatory disease.

Cell membranes regulate the transport of large molecules through endocytosis, which is a pathway for cells to bring substances from the extracellular medium into cytosol. Very recently, however, it was discovered that cationic nanoparticles (NPs) can enter cells in an energy-independent fashion, escaping the traditional endocytosis route. This unconventional translocation happens on the timescale of seconds, significantly faster than endocytosis, and can be somewhat toxic to cells. This has led the field to believe that cationic nanoparticles enter cells by generating holes on membranes, yet direct evidence in cells has not been observed. Meanwhile, cationic cell-penetrating peptides (CPPs) have demonstrated the ability of effectively translocating cargos into cells. Although there have been several different mechanisms proposed for the cell entry pathway of CPPs, there is still much debate and thus no consensus has been reached on the issue. Here, using coarse-grained simulations we show that cationic gold nano-particles, as well as HIV-1 Tat peptide, spontaneously translocate a model cell membrane by generating nanoscale pores in the presence of a transmembrane (TM) potential. The presence of cationic particles alters the electric field across the membrane, forcing the formation of holes, which in turn allow the translocation of particles. We demonstrate that the threshold of TM potential for nanoparticle translocation is concentration dependent, and is of the order of that appearing under physiological conditions. Furthermore, by comparing a linear chain of CPPs and a compact cluster of CPPs, we show that the shape of the cationic object is crucial in determining if it can translocate or not across. After translocation, the TM potential is strongly diminished due to the transport of ions in the local area and the membrane reseals (self-heals) very rapidly (e.g. within a microsecond). The mechanism put forward here provides fundamental information on the uptake kinetics of the cationic nanoparticles/biomolecules as well as their potential cytotoxicity, which is critical for understanding common biological processes such as viral invasion, or the design principles of drug/gene delivery system utilizing cationic agents.

Micro and nanoscale technologies have been increasingly applied in various scientific disciplines from physics and chemistry to biology and medicine. In particular, these technologies have immensely benefited the fields of experimental biology and medicine through designing of complex biomaterials for cell-based studies. In addition, these technologies have allowed for an unprecedented ability to control cell-cell, cell-microenvironment and cell-soluble factors interactions through miniaturized assays for high-throughput cell-based studies. The focus of our research group has been on merging mico- and nanofabrication techniques with innovative biomaterials for tissue engineering and stem cell bioengineering applications. Our research encompasses a wide range of scientific areas from biomaterials design to microfabrication and biology. We have developed novel polymeric and hydrogels based biomaterials and have used ‘bottom-up&’ and ‘top-down&’ fabrication techniques to create three dimensional (3D) vascularized tissue constructs. In addition, we have shown successful incorporation of various nanoparticles and carbon nanotubes within the hydrogel constructs in order to enhance their mechanical and electrical properties for potential applications in bone and cardiovascular tissue engineering. On the stem cell bioengineering aspect, we have created suitable microenvironments, such as high density microwell topographies, to module stem cell behavior and direct their differentiation toward the desired lineages. In this talk, I will present the recent findings in our lab covering biomaterials, tissue engineering and stem cell bioengineering aspects. I will also discuss our major biological findings related to tissue engineering and stem cell differentiation using miniaturized systems.

Spatially defined tethering of individual cells are of great interest in biomedical topics, applied microbiology, environmental research up to forensics and toxicology applications. The methodological possibility to study spatially separated individual cells facilitates simultaneous observation of cell motility behavior and division processes, and allows further to perform in-vivo studies within the cellular environment. In this context, we developed a universal and rapid fabrication of arrays of micro-patches with different shape and size for controlled and localized tethering of living prokaryotic and eukaryotic cells. Human cervical cancer cells (HeLa) and phytopathogen bacteria (Xylella Fastidiosa) have been used for the present study.
Initially, arrays of micro-patches using Ag 5214 photoresist with sizes ranging from 1 µm to 80 µm were fabricated on covalently amino-coated glass substrates which facilitate the cellular adhesion. The substrates were further selectively functionalized with poly(ethylene glycol) (PEG) at the surroundings of the predefined areas which were developed using mask-free laser-lithographic approach. The covalent ligation of the cell-repellant PEG polymer around the tethering areas has been successfully demonstrated via sensitive fluorescence experiments. Within our study we analyze how the cells can be tethered selectively on the predefined areas as a response to different chemical surface compositions and changes in pattern shape and size. While monitoring a single cell, we can able to disentangle the parameters which influence the cell motility behavior, degree of spatial organization and complexity while tethered in a micro environment. Our results unravel the potential use of present methodology for single cell studies as well as developing future biomedical devices in a more versatile and reliable way.

Alpha-synuclein (aSyn) is an abundant protein in the brain that is deeply implicated in Parkinson&’s disease (PD) and other synucleinopathies. PD is characterized by the loss of dopaminergic cells and by the presence of intraneural Lewy bodies, which are mostly composed of lipids and aggregated aSyn. Although the mechanisms behind neural cell death are not yet fully understood it is known that aSyn plays a double role: it is toxic to cells and, in certain aggregated states, activates microglia, the immune cells in the brain. To find long-term therapies for PD, the mechanisms underlying the relation between aSyn, microglia activation, and cell death are currently the subject of intense investigation.
Microfluidic technology can provide new ways to explore these cellular mechanisms and interactions, its key advantage being the fine control that can be achieved over the cellular microenvironment.
In order to further explore the mechanisms of PD, a microfluidic device was built for the co-culture of neural and glial cells and to monitor their interaction through soluble factors. This device has two chambers, one for each cell population, connected by microfluidic channels allowing the exchange of soluble materials while preventing direct cell-cell interaction. The incorporation of micro-pneumatic valves allows to 1) populate the chambers individually; 2) isolate chambers so that both cell populations are exposed to different chemical environments; and 3) control fluid flow to individual chambers or between chambers.
Preliminary tests on the microfluidic device were carried out with HEK cells. In the first series of experiments, cell viability and proliferation tests were performed. By coating the device with fibronectin, an extracellular matrix protein, and supplying cell culture medium with a pH buffer, cell mortality was kept at about 20% and cell proliferation reached 150% after 4 days, which is comparable to typical macroscopic cell culture protocols. In the second series of experiments, HEK cells producing aSyn fused with GFP were seeded on one chamber and HEK cells producing aSyn fused with DsRED were seeded on the other. Confocal microscopy imaging revealed the presence of green fluorescent signal in the chamber containing red fluorescent cells, strongly suggesting the traffic of aSyn between chambers and that cell-cell communication through soluble factors is possible in this device.
We will show that the environment of each cell population can be independently controlled, by testing whether inducing apoptosis in one cell population increases aSyn transmission to the other. We will also show the results of viability and proliferation assays on the co-culture of neural cells and microglia.
Overall, this novel device will contribute to a better understanding of the importance of the traffic of soluble factors between cell populations in normal and pathological conditions, such as PD or other neurodegenerative disorders.

Single cell analysis unmasks information inaccessible in population level data, such as intracellular dynamics and cell-to-cell heterogeneity [1]. In this presentation, the microfluidic manipulation of single motile cells will be discussed, including both trapping and release [2]. The focus will particularly be on motile cells of reduced size, namely bacteria and fungi. The present approach was based on the integration of micron-scale features with sub-micron ones. The realization of such sub-microfluidic structures, including their fabrication, testing and applications in synthetic and micro-biology will be analyzed in detail.
Generally, single cell manipulation methods involve optical tweezers [3], and micromanipulators [4], as well as higher throughput techniques, namely electrophoresis [5], cytometry [6] and surface immobilization [7]. Microfluidics can also enable high-throughput platforms [8], such as the array of PDMS U-shaped protrusions by Di Carlo et al. [9], or the Tan et al. strategy based on a two asymmetric channel junction with asymmetric fluidic resistivity [10]. Both of these techniques have proven very popular in immobilizing mammalian cells, however little has been achieved for the smaller and motile bacteria and certain types of fungi.
To address this, a microfluidic trap of sub-micron dimensions was developed, capable of both trapping and releasing single cells upon demand. To this end, component integration of sizes that vary by more than three orders of magnitude was necessary, such as the nutrient delivery microchannels and the immobilizing sub-micron indentations. The latter integration was possible in a single processing step by developing a hybrid fabrication method based on electron-beam and cast-molding lithography.
The fabrication and its performance will be analyzed, as well as the trap and release of single bacteria and fungi. The trap forms a simple platform for long term imaging of single cells, as well as monitoring cell-material interactions. Applications within the field of advanced biofuel production, namely monitoring the intracellular lipid droplet synthesis and transport, as well as ecology will be discussed.

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[9] D. Di Carlo, L. Y. Wu and L. P. Lee, Lab Chip 6, 1445 (2006).
[10] W. H. Tan and S. Takeuchi, Proc. Natl. Acad. Sci. U. S. A. 104, 1146 (2007).

The settlement of bacteria on submerged surfaces is one of the key steps to biofouling, and poses significant challenges to various areas ranging from biomedical devices to ship hulls and membrane separation processes. Anti-fouling surfaces offer one possible solution to the biofouling problem and a variety of fouling-resistant surface chemistries have been developed, including polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA) and zwitterionic polymers(1). Despite the good performance of these anti-fouling surfaces, the design criteria are largely empirical as the result of lack of knowledge on the cell-material interactions.
Surface bacteria attachment can be evaluated by fluorescent tagging and microscopic imaging(2) or in situ monitoring such as surface plasmon resonance (SPR) or quartz crystal microbalance with dissipation monitoring (QCM-D). These methods are able to track the quantity of surface-adsorbed bacteria over time(3); however, information on the cell-material interactions is missing. In addition, cell adsorption and surface bacteria proliferation cannot be differentiated. Here, we report the application of microfluidic devices to investigate such interactions during the settlement of bacteria.
Microchannels fabricated with polydimethylsiloxane (PDMS) were mounted on standard microscope slides. Glass slides were coated with zwitterionic anti-fouling films with the thickness around 300nm. For the first time, a zwitterionic polymer was designed to resist oxidative damage from chlorine, which enabled the investigation of cell-material interaction in the presence of chlorine. A synergistic enhancement of the anti-fouling performance was observed for the first time, when chlorine and anti-fouling surface coatings were used simultaneously. The microfluidic imaging was enabled by an inverted microscope, which captured the evolving interactions between bacteria and anti-fouling surfaces in the presence of chlorine: reduced attachment, slower surface cell proliferation, and frequent detachment of bacteria clusters. This proves that the biofilm prevention did not result from cell killing by chlorine. Virtually any surface chemistry can be tested with this straightforward method and fast screening of various anti-fouling materials is thus possible. This study sheds light on the dynamics of bacteria surface attachment and facilitates the rational design of the next-generation anti-fouling chemistry.
1. Yang, R.; Asatekin, A.; Gleason, K. K. Soft Matter 2012, 8, 31-43.
2. Yang, R.; Xu, J.; Ozaydin-Ince, G.; Wong, S. Y.; Gleason, K. K. Chem. Mater. 2011, 23, 1263-1272.
3. Yang, R.; Gleason, K. K. Langmuir 2012, 28, 12266-12274.

In this talk I will describe synthetic micro- and nanotechnology tools that we have developed for probing the extracellular microenvironment of tumors as the progress along the metastatic cascade. In combination with genetic engineered mouse models of lung and colorectal cancer, ECM microarrays and nanosensors that emit urinary markers helped to elucidate the role of glycans and matrix metalloproteases in the metastatic niche. Moving forward, these tools may be more broadly utilized to study other biological processes such as wound healing, development and regeneration.

A novel DNA homolog comprising clickable nucleic acids (CNAs) was developed based on thiol-ene ‘click&’ chemistry. Four CNA monomers (A, T, G, C) were designed, synthesized and polymerized via the photo-initiated thiol-ene reaction. The results demonstrate the capacity of CNAs to bind with complementary DNA strands more strongly and specifically than the DNA complements. Melting temperature (Tm) results suggest that the CNA oligomers have higher affinity for the complimentary DNA strand than DNA itself does. Finally, surface modification experiments using CNA-DNA (fluorescence) showed one potential application of CNA-DNA hybrids in biodetection.

Synthetic biology has made tremendous recent strides in constructing artificial cellular systems using minimal cell components ex vivo, creating an experimental platform for characterizing the behavior of isolated cellular modules and a form of biotechnology for the controlled operation of artificial cells. The robustness and efficiency of these systems are nonetheless challenging to control, in part because artificial cells establish an environment that is still very different from that of actual living cells. Here, we present a novel approach towards bridging the gap between artificial and true cell environments by developing artificial cells incorporating controlled macromolecular crowding, mimicking a key feature of natural cells known to dramatically influence biochemical kinetics. We demonstrate the value of our approach by showing that molecular crowding enhances gene expression and confers robustness against perturbations of gene environments. We further elucidate the underlying mechanisms of these phenomena at the single molecule level by demonstrating how large crowding molecules decrease diffusion of T7 RNA polymerase, but increase its binding to a T7 RNAP promoter. Based on single-molecule results, we further show that the impact of molecular crowding on gene circuits is enhanced by weak genetic components and maximized by a negative feedback loop. By bridging a key gap between artificial cell technology and the environment of living cells, we demonstrate the importance of intracellular crowding to efficient and robust function of biological circuits and suggest new engineering principles for controlled modulation of synthetic genetic systems.

Like proteins, amphiphilic small molecules self-assemble under different conditions to form supramolecular nanostructures with different morphologies. The kinetically-dependent nature of the nanostructures makes the identification of their cellular target a difficult task. In our work, we developed a hydrogel-based pull-down assay. The assay utilizes hydrogel formed by aggregation of nanostructures as the matrices for selective protein binding between proteins and the nanostructure, and applies protein mass spectrometry for protein identification. This pulled-down assay based solely on the binding affinity between the proteins and the nanostructures, which is intrinsically similar as the non-covalent interactions between the proteins and the nanostructures in cellular environments. Based on the hydrogel based pull-down assay, we found that nanostructure with different morphologies interact with cytosol proteins in a drastically different manner, which, for the first time, confirms that morphology of kinetically-trapped molecular aggregates directly dictates the proteins bound to the aggregates. Moreover, we have successfully identified the protein targets of several types of nanostructure, which suggest that the hydrogel based pull-down assay may play a significant role in identification of cellular targets of supramolecular assemblies.

Cancer cells originating from the epithelium evolve into invasive and metastatic cells through the acquisition of genetic mutations that lead to a highly motile mesenchymal phenotype. The ZEB family of transcription factors potentially plays a key role in regulating migration of MCF-7 breast cancer cells through the repression of cell-cell adhesion junction protein, E-cadherin. Although the migration potential of cells can be assessed with a "wound healing" assay, reproducibility is limited by semi-quantitative image analysis of the wound closure and variability in the mechanically disrupted area in the cell monolayer. Electrical Cell-substrate Impedance Sensing (ECIS) provides an improvement over traditional wound healing assay by providing real-time, noninvasive measurement of in vitro cell behavior as well as reproducible electrical wounding of the cell monolayer. A 1 µA, 4kHz AC current applied across single surface electrodes (250-µm diameter) provided monitoring capability as MCF-7 cells, transfected to over-express ZEB1, ZEB2 or vector, attach and spread on the substrate. The same surface electrode is also used to lethally electroporate cells in the current path by permanently damaging plasma membranes directly above the surface electrode, clearing a well-defined circular area 250 µm in diameter. Based on measured resistance values at 1 kHz, the time to recovery for MCF-7+ZEB1, MCF-7+ZEB2, and MCF-7+vector cells was 9, 10, and 12 hrs, respectively. The ECIS results agreed with traditional, image-based evaluations from scratch-based wound healing assays (migration rate: 10.2 µm/hr (MCF-7+ZEB1), 9.0 µm/hr (MCF-7+ZEB2), and 7.9 µm/hr (MCF-7+vector)). Taken together, the data indicates that ZEB family of proteins play a positive role in inducing MCF-7 cell migration and that ECIS provides a robust and reproducible method for assessing the migration potential of cells.

A better understanding of the physical and biomolecular cues in the stem cell niche has led to a growing interest in the development of material systems for improved 3D culture environments, as well as delivery vehicles to promote cell survival and differentiation. As a result, hydrogels based on both protein components (e.g., collagen and Matrigel) and highly-tunable synthetic chemistries (e.g., PEG) have evolved to address many of these needs. However, as advances in real-time tracking of dynamic cellular functions have emerged, complementary approaches to alter the surrounding extracellular environment in a user-defined and highly-controlled fashion are needed. Such materials systems have the potential to significantly improve our understanding of how cells receive information from their microenvironment and the role that these dynamic processes may play in biological questions related to their differentiation. Towards the goal of developing dynamically tunable scaffolds, this talk will highlight several approaches for in situ hydrogel property manipulation with light, allowing intimate control of a cell&’s microenvironment in both time and space. The synthesis and characterization of gels with photolabile linkers (e.g., nitrobenzyl ether) and photoconjugation reactions (e.g., thiol-ene) will be discussed, along with more recent developments in photoreversible reactions (e.g., addition fragmentation chain transfer reactions). These photoactive hydrogels afford unique user-defined manipulation of the biochemical and biomechanical nature of the extracellular microenvironment. This talk will present several examples where user-triggered changes in the material environment can be used to study study and direct human mesenchymcal stem cell function and by modifying the local hydrogel environment.

Spatiotemporal control over the formation of network cross-links afforded by photopolymerization is a strategy which has proven effective in the development of bioresponsive synthetic polymer hydrogels. This work describes a chemically modified recombinant protein polymer based upon the elastomeric structural protein, resilin, capable of photoinitiated thiol-ene cross-linking. This norbornene-functionalized resilin-like polypeptide (RLP) is cross-linked with a multi-arm PEG thiol to form a covalent network hydrogel. Equipped with a library of RLPs, which incorporate biological domains including MMP degradation sequences as well as cell adhesion sequences, these scaffolds may be adapted to a variety of tissue engineering applications. Expression and purification of the RLP is confirmed via SDS-PAGE and amino acid analysis. 1H-NMR spectroscopy confirmed the chemical modification lysine residues on the RLP with norbornene acid. Oscillatory photorheometry and tensile testing confirm that the mechanical properties of the hydrogels can be tuned upon photo-initiated cross-linking. This RLP-PEG hydrogel harnesses advantages of both synthetic and biosynthetic strategies for the development of hydrogel biomaterials.

Biomimetic materials - man-made materials inspired by natural processes and phenomena - have caught the attention of scientists in many research areas. Specifically, mimicking the adhesives used by maritime mussels to attach to virtually any surfaces are envisaged by us as the base for environmental friendly (photo-)click-able (bio)functional coatings for the attachment of any polymer chain or bioreceptor. In the current work, we fuse poly(dopamine) - a biomimetic polymer and highly versatile coating base - with advanced polymer chemistry able to provide the reversible attachment and detachment of polymer strands to surfaces. Our approaches include the preparation of tailor-made functional surfaces via mild thermally driven cycloaddition as well as photo-triggered reactions as a way to switchable universal surface chemistries and spatially functionalized surfaces for controlled cell adhesion. The described concept offers vast possibilities for the modification of surface properties on demand by both thermal and light triggered signals.
We prepared a dopamine-maleimide derivative which was, initially, conjugated (Diels-Alder reaction (DA)) with a cyclopentadiene-carrying PEG-chain and, secondly, attached to gold and other surfaces via an autopolymerization reaction of the compound itself under maritime conditions (Tris-buffer solution at pH = 8.5). Subsequently, several retro-DA (rDA) and DA sequences, where the PEG-chain was detached and re-attached, were executed on the surface. Thus, we generated a versatile DA-switching system, which possesses the ability to adhere to virtually any kind of surface in a biomimetic fashion and which permits engineering the surfaces chemistry and properties by the attachment and detachment of polymer chains on demand.
One of our most recent studies addresses controlled cell-adhesion on bio-inspired surfaces. In a grafting-from approach, polydopamine (PDA) surfaces were reacted with a photo-click-able group and a maleimide-carrying ATRP-Initiator was attached under UV irradiation. During the irradiation, parts of the surface were covered to achieve a regio-selective attachment of the initiator. In an ATRP-polymerization, MeOEGMA was grown from the surface. Subsequently, cell adhesion studies evidenced the attachment of the cells only in regions where no polymer had been grafted. Thus, we prepared a regio-selective photo-click-system to grow polymer chains in specific areas, which allows controlled and defined cell adhesion.

Short peptides N-protected with hydrophobic moieties have been repeatedly demonstrated to self-assemble into nanofibrillar structures to form gels within aqueous environments upon triggering by a change in pH, temperature, or addition of salts. This mild gelation process makes these materials ideal candidates for injectable biomaterials, and, as the substituents are short peptides of low molecular weight, these materials are potentially bioactive and amenable to renal clearance. However, as these materials are peptide-based, their in situ degradability is likely determined by the protease content of the particular tissue in which they might be injected; peptide bonds are resistant to non-enzyme mediated hydrolysis over the weeks-to-months time scale of wound healing and tissue regeneration. For this reason, our group has sought to develop similar self-assembling materials from depsipeptides, which contain ester substitutions in the backbone. Esters are much more susceptible to hydrolysis than are peptide bonds, and therefore ester-containing materials are able to degrade without the need for tissue specific protease enzymes.
Depsipeptides are synthesized by substituting into synthetic peptides alpha-hydroxy acids with side chain groups identical to those of natural amino acids. Our group recently published a general method for synthesizing di-depsipeptide units that could be further reacted on solid phase using standard solid phase peptide synthesis chemistries. We also found that the di-depsipeptide analog of a simple dipeptide conjugate hydrogelator was also able to form a hydrogel with similar morphological properties. Longer, zwitterionic depsipeptide conjugates were also able to self-assemble into hydrogels. With these results suggesting that the ester substitution may not disrupt the self-assembly process, we focused next on synthesizing a depsipeptide analog of the cell-binding motif, Arginine-Glycine-Aspartic acid (RGD), to investigate the effects of the ester substitution on an important and well-characterized protein-ligand system.
In this work, we describe the synthesis of fluorenxylmethoxycarbonyl (Fmoc)-protected R-Glc-D and several variants with additional residues or alternate hydrophobic N-terminal groups, all of which have the glycolic acid (Glc)-containing sequence analogous to RGD. We demonstrate the conditions under which each variant is able to self-assemble into a hydrogel, and we characterize the degradation rate of the molecules relative to that of the native peptide conjugate, Fmoc-RGD, both in solution and in gel form at physiological temperature. Finally, we show the results of integrin binding assays based on surface plasmon resonance (SPR), in order to compare the integrin binding ability of R-Glc-D variants relative to Fmoc-RGD. Future work will include in vitro studies to quantify any reduction or increase in cell binding to R-Glc-D variants, as well as cytotoxicity and cell proliferation studies.

Although the primary focus to enhance transgene expression in vivo has been the design of the delivery vector, the matrix itself can provide alternative approaches to enhance transfection efficiency as well as promote tissue formation. However, it first needs to be recognized that gene transfer to cells seeded in a three dimensional environment occurs through different mechanisms to cells seeded in a two dimensional environment. Utilizing synthetic hydrogel scaffolds as in vitro gene transfer platforms we were able to elucidate significant differences in the mechanism of gene transfer when cells are seeded inside hydrogel scaffolds than when they are seeded on tissue culture plastic, the traditional surface used to assess the efficiency of gene transfection vectors.

Successful islet transplantation has the potential to cure type 1 diabetes. However, unprotected islet grafts are prone to immune rejection, which leads to the eventual failure of the grafts. To this end, various immunoisolation barriers have been proposed for islet encapsulation to prolong the survival and function of the transplanted islets. We have recently developed a visible light mediated interfacial thiol-ene reaction to form multilayer hydrogels. In this highly efficient visible light photocrosslinking reaction, eosin-Y is used as the single initiator. Using a similar coating technique, we have successfully prepared conformal coating on the surface of beta-cell aggregates and isolated islets. Multi-arm poly(ethylene glycol) norbornene (PEGNB) and dithiol-containing molecules (e.g., dithiothreitol) were used for forming an idealized hydrogel network. Owing to the highly controllable thiol-ene reactions, thickness of the gel coating could be readily tuned via controlling light exposure time and macromer compositions. This unique and simple photochemistry also overcomes major shortcomings associated with prior conformal coating technique using visible light-mediated chain-growth polymerization. First, rapid thiol-ene gelation is maintained without the use of cytotoxic co-initiator (TEOA) or co-monomer (NVP). Second, this reaction is more cytocompatible than the conventional PEG diacrylate (PEGDA) conformal coating. Finally, the step-growth thiol-ene network provides an idealized and homogeneous structure that does not hinder insulin transport. Compared to unprotected islets, the thiol-ene coated islets successfully suppressed high glucose levels in diabetic NOD/scid mice. The thiol-ene conformal gel coating serves as a biomimetic culture platform to preserve the viability of artificial or isolated islets and prevent them from losing islet cell characteristics. Additionally, various photo-conjugation techniques for functionalizing PEG gels are compatible with the thiol-ene reactions for creating biomimetic coating.

Synthetic analogs of the extracellular microenvironment pose as an attractive platform to elucidate complex cellular interactions and potentially guide cellular responses. Although such systems reported thus far are often oversimplified, substantial insight on the effect of extrinsic cues originating from the extracellular matrix, soluble factors, or neighboring cells are paving the way towards directing cell fate. In particular, geometric cues, such as patterns in both the nanoscale and microscale regime, have been demonstrated as a powerful tool to elicit different responses ranging from cell adhesion, migration, differentiation, and morphology. Moreover, hierarchical patterns are emerging as a route to study cell behavior. Approaches that have been pursued for hierarchical patterning of biomolecules include block copolymer (BCP) micelle lithography coupled with photo- or electron-beam lithography, scanning probe BCP lithography, and specialized microfabrication techniques. However, techniques based on BCP micelle lithography are limited to inorganic dot arrays and specialized microfabrication techniques entail expensive equipment and expertise not readily available by many groups. Here, we present a high-throughput method for achieving hierarchical patterns of biomolecules through the self-assembly of a functionalized BCPs coupled with photolithography. The system is based on a BCP of poly(ethylene oxide) end-functionalized with biotin and poly(styrene-co-4-bromostyrene) where the addition of 4-bromostyrene serves as a cross-linking unit upon UV irradiation. Through facile processing, hierarchical patterns were observed by optical microscopy, where post-biofunctionalization was demonstrated through the ubiquitous biotin-streptavidin handle. Two morphologies—lines and dots of biomolecules, are readily accessed with the single BCP by simply altering annealing conditions. Moreover, mixed morphologies on a single substrate may be obtained, where a combination of dots, lines, as an as-cast morphology is achieved by modifying irradiation dose and the order of processing steps. These substrates serve as a straightforward material for investigating the effects of hierarchical patterning of biomolecules on mesenchymal stem cell differentiation.

Research in both the physical and biological sciences chronically suffers from the low productivity and reproducibility of experimental studies. This is due in part because the traditional text-based format of science journals cannot provide an adequate description of complex research procedures. This creates a critical “bottleneck” problem of knowledge transfer for research and education. Addressing this challenge, a new generation of science journals employs online video to provide a systematic visualized publication of experimental studies. This presentation will provide an overview of the growing field of video publication and discuss its technical challenges, implications for scholarly communication and acceptance in the academic and library community. Results and recently conducted case studies will be shown in support of video publications as a valid communication venue in scientific publishing.

Introduction
Osteoarthritis afflicts over 50 million individuals in the developed world. Osteoarthritis is characterized by mechanical injury of the cartilage surface, leading to an increase in the surface friction (i.e., coefficient of friction) which leads to progressive deterioration of the cartilage surface. Production of the natural cartilage lubricant, lubricin, is compromised during progression of the disease, leading to increased cartilage damage. Replacement of the lubricin layer via injection of recombinant lubricin inhibits the onset of osteoarthritis, but recombinant lubricin is currently not economically viable and has been largely abandon as a therapeutic product by the industry.
The purpose of the proposed research is to mimic the structure and activity of natural lubricin with inexpensive synthetic analogs and to develop these materials as therapeutic alternatives to osteoarthritis treatment with the ability to prevent disease progression.
Methods
Brush-like polymers composed of polyacrylic acid (pAA) and polyethylene glycol (PEG) side chains were synthesized with a thiol endgroup by RAFT polymerization. A matrix of 27 polymers were synthesized in a full factorial manner to determine the structure-function relationships of the materials. Cartilage explants (nge;4) were removed from the patellofemoral groove of 1-3 day old bovine calves. Native lubricin was stripped from the explants using a 1.5 M NaCl solution. Polymers were tagged with fluoroscein to determine binding kinetics to the cartilage surface. In parallel, cartilage explants treated with polymer were evaluated by tribometry to determine their frictional behavior in the boundary lubrication mode.
Results
Polymer brushes bound to cartilage surfaces with first order kinetics with a time constant of 21 min. Friction coefficients also appeared to decrease exponentially with a time constant of 47 min and a steady state of delta mu (difference in friction coefficient) of -0.07, supporting the premise that the synthetic lubricants both bound and lubricated the articular cartilage surface, approaching the efficacy of the natural lubricant, lubrin.
Acknowledgements
This work was partially funded by the Cornell Center for Materials Research and the New York State Foundation for Science, Technology, and Innovation through the Center for Advanced Technology.

As magnetic resonance is one of the safest, most accurate diagnostic imaging modalities, our group has recently pursued development of two MRI contrast agents. The first is a Gd-chelating hydrogel nanoparticle (nanogel) that, if targeted with peptides or aptamers, may eventually be applied for localization of tumors or other disease sites; the second is a complex in which bioresponsive polymer surrounds Gd oxide nanoparticles to allow disease-triggered activation of MRI contrast. Towards the first aim, we hypothesized that concentrating Gd ions and restricting their rotation by incorporating Gd chelates into nanogels as crosslinkers would yield an especially high-contrast agent, since nanogels allow water access throughout. Polyacrylamide-based nanogels incorporating either acyclic or cyclic crosslinkers display higher relaxivity than clinically used contrast agents, up to 6-fold greater than Dotarem®. Importantly, these nanogels also stabilize Gd against transmetallation by other ions, which should reduce toxicity associated with release of free Gd. We are currently formulating MRI nanogels from biological polysaccharides to create biodegradable agents for faster clearance. In order to enable specific imaging of disease sites by limiting signal from other tissues, our second project has yielded a structure with the greatest contrast difference between “on” and “off” states yet achieved, a full order of magnitude. We demonstrate that the relaxation of Gd oxide nanoparticles encapsulated within a polymer by electrospray is effectively shielded; employing polymers that degrade upon exposure to acidic pH or high concentrations of reactive oxygen species (ROS) yields responsiveness to specific biochemical stimuli characteristic of tumors (low pH) or inflammation (high ROS). We are currently examining the ability of these complexes to detect these conditions in vivo.

Non-invasive imaging of physiological processes like heart rate, breathing motion, blood perfusion and dynamic behavior of the gastro-intestinal tract are of great interest for preclinical research related to cardiovascular disease, hypertension or behavioral sciences.
So far imaging techniques available to study these processes with high spatial and temporal resolution require animals to be fixed and anesthetized. This has great limitations in terms of experiment time. More importantly anesthesia disturbs many physiological processes.
SWIR or NIR-II imaging (between 900 nm and 1700 nm) had been shown promising for non-invasive imaging. However, due to the suboptimal emission properties of the previously used materials (e.g. single walled carbon nanotubes (SWCNTs)) fast imaging rates of 25 to 50 frames per second could not be archived.
Here we use InAs/CdZnSe core-shell quantum dots, which emit in the SWIR region with a quantum yield of 30% after functionalization in physiologic solution. This emission intensity is two orders of magnitude higher than that of SWCNTs used previously for non-invasive SWIR imaging. Moreover, these quantum dots emit enough signal to study physiological processes in active mice opening a novel route to circumvent experimental artifacts generated by anesthesia.
For the first time we can facile image physiological parameters like heart rate, breathing rate, perfusion and energy metabolism in conscious and freely behaving mice.

The importance of microRNA (miRNA) in cancer detection or manipulation of genetic expression has been increasingly recognized. The controlled delivery of miRNA into specific cells constitutes a challenging task. This report describes findings from an investigation of gold nanoparticles (AuNPs) as carriers of miRNA for controlled delivery in cell transfection. Gold nanoparticles were first conjugated with miRNA (miRNA-AuNPs). The immobilization of miRNAs on the AuNPs was detected and the surface stability was substantiated by gel electrophoresis assessment of the highly charged characteristics of miRNA-AuNPs and their surface-exchange inactivity with a highly-charged surfactant. The miRNA-AuNPs were also characterized using surface enhanced Raman scattering spectroscopy, UV-visible spectrophotometry, gel electrophoresis, and transmission electron microscopy. The miRNA-AuNPs were tested in cell transfection using multiple myeloma cells, demonstrating efficient knockdown in the functional luciferase assay. In addition to exploration of controlled delivery, implications of the results for exploring miRNA-conjugated nanoparticles as biosensing or targeting probes will also be discussed.

Monolayer-protected nanoparticles combine the physical properties of inorganic nanoparticles with the chemical properties of their molecular shells. These materials can contribute to solve scientific questions of biological and biomedical interest, especially if we can achieve a high control over the surface chemistry to ensure biocompatibility, stability and targeting. The presentation will be in three stages, starting with our efforts to prepare and characterize biomimetic gold nanoparticles, continuing with applications of those nanoparticles in live cell microscopy and finishing with iron oxide nanoparticles for cell tracking in animal models.
In 2004, we demonstrated the formation of peptide self-assembled monolayers on gold nanoparticles leading to the formation of highly colloidally stable nanoparticles which share some biochemical properties with proteins [1]. Since 2004, we have made some small steps towards the distant challenge of engineering nanoparticles which would, through molecular self-assembly of short peptide sequences on nanoparticles, acquire the advanced functions of proteins: a) evidence for the formation of domains in mixed peptide layers [2] (shown by cross-linking experiments), b) engineering of secondary structure [3] (probed by FTIR and ssNMR), c) fine tuning of interactions with protein partners [4+unpublished work] (enzyme kinetics). We have recently started a project aiming at tuning the nanoparticle properties through photochemical reactions and I will share our progress in this direction.
Recent development in optical microscopy enable detection of single gold nanoparticle as small as a few nanometre to be detected in a live cell. Our technique of choice is photothermal microscopy, initially developed by the Lounis group [5]. We have applied this technique to investigate the entry of functionalized nanoparticles in cells and evaluate the mobility of growth factors at the surface of cells.
A major challenge in regenerative medicine is to be able to track stem cells post injection to better understand their positive effects as well as the potential associated risks. Nanoparticles can be useful to address that challenge because they can be loaded into cells pre-injection and can then serve as contrast agents [6]. I will present our results on the design and characterization of these probes, their uptake and biological impact, and the first results in animal models.
References
[1] Lévy, R et al (2004) J. Am. Chem. Soc. 126, 10076
[2] Duchesne, L et al (2008) ChemBioChem 9, 2127
[3] Shaw, P et al (2012) ACS Nano, 6 1416
[4] Free, P et al (2009) Chem. Commun. 5009
[5] Berciaud, L et al (2004) Phys. Rev. Lett. 93, 257402
[6] Taylor, Aet al (2012) Chem. Soc. Rev. 41, 2707

Nanoparticle-based vectors are fast becoming the main choice for nucleic acid delivery. Fluorescent nanoparticles have the added advantage of tracking the delivery process and also of tracking cells transfected with nucleic acids for cell therapy. Fluorescent upconversion nanoparticles (UCNs) are ideal candidates for tracking since they are excited by NIR light and hence have very low phototoxicity, high signal-to-noise ratio and enable imaging in deep tissues. UCNs coated with a layer of silica and adsorbed with siRNA or siRNA loaded into the mesoporous silica coating on the UCNs have been used for siRNA delivery. However the loading of siRNA is very poor since the silica coating is negatively charged and it repels the negatively charged siRNA limiting the amount of siRNA that can be adsorbed on the surface of nanoparticles. Here we report the use of a layer-by-layer approach to coat the UCNs with Poly-L-Lysine (PLL) and use it for delivery. Highly monodispersed UCNs were synthesized with an average size of 50 nm. They were then modified with PLL and STAT-3 siRNA was adsorbed on to the surface of the modified UCNs. The loading of the siRNA was found to be 60 % more efficient by this approach as compared to silica coated UCNs alone. The PLL-coated UCNs also were minimally cytotoxic as shown by MTS assay. The siRNA coated UCNs also efficiently transfected B16F0 cells and knocked down STAT3 significantly and also enabled cell imaging. Thus, This method shows good promise for siRNA delivery and tracking and this could also be extended to in-vivo transfection and tracking.

Proteins undergo conformational/functional changes as a result of spatial perturbations induced by nanostructured materials. Due to the similarities between the sizes of nanomaterials and proteins, the topographical and other geometric properties of nanostructures may influence the structure, function and stability of adsorbed proteins. Understanding the phenomena of nanostructure-biomolecule interactions is critical to enhance the ability of tailoring protein functions, as well as to enable protein-nanomaterial conjugates to be used effectively and safely in a wide range of biomedical applications.
Among their myriad geometric properties, nanostructures that posses negative curvature enable encapsulation of adsorbed proteins, and thus allow a unique opportunity to explore the effect of spatial confinement and molecular crowding on protein behavior. However, such effects have not yet been extensively investigated. To ameliorate this lack of information, a more precise biomolecular characterization of adsorbed proteins in restricted environments is required, and the synthesis, modification and characterization of appropriate nanostructures is critical.
We are developing a new form of nano-bio conjugates, taking advantage of a previously reported method for creating cage-structure gold nanoparticles, in which proteins are encapsulated, to study protein behavior in such an environment. The hollowed gold nanocages are galvanization-synthesized, and chemically modified for the purpose of biocompatibility and protein affinity. The gold nanocage has a relatively inert surface, which allows the observation of geometric effects on adsorbed proteins; its unique electromagnetic properties also allow further observations of adsorbed protein conformations. These two features render gold nanocages to be a suitable platform to study proteins&’ behavior in restricted concave environments.
During our study, precise morphological controls were incorporated into the synthetic procedure to obtain protein uptake surfaces with appropriate topographies. Lysozyme was used to probe the nanocage&’s efficacy in protein uptake. Similar nanoscale non-hollowed colloidal gold nanocubes were synthesized and employed to compare with the hollowed nanocages, allowing more details of the nanocage-protein interaction to be learned. From a variety of experiments on protein adsorption, conformation, and activity, it has been demonstrated that lysozyme can be immobilized at the internal concave surface of a nanocage, while preserving a significant part of its native activity. Interestingly, such activity was higher than that of proteins adsorbed onto flat surfaces, indicating unique nano-bio interactions between the concave surface and the proteins.
This work is supported by the US National Science Foundation under Grant No. DMR-0642573.

The increased incorporation of engineered nanomaterials (ENMs) in consumer products and industrial processes, the risk of exposure, particularly by inhalation, is inevitably increasing. In order to understand better the fate and transport of ENMs in biological media and their biological properties it is critical to investigate the ENM - ENM and ENM - cell interactions in relevant physiological media. In this project, an Atomic Force Microscopy (AFM) based platform suitable for the assessment of ENM-bio interactions of industrially relevant ENMs in physiologic fluids was developed and utilized to directly measure the ENM-ENM and ENM-cell interactions. Industry-relevant CeO2, Fe2O3, and SiO2 ENMs of various sizes were made by the flame spray pyrolysis (FSP) based Harvard Versatile Engineering Nanomaterials Generation System (Harvard VENGES). The ENMs were fully characterized structurally and morphologically by XRD (X-Ray Diffraction) and TEM (Transmission Electron Microscopy). Their properties in water and biological media were also assessed using DLS (Dynamic Light Scattering). The ENMs were attached on AFM tips and deposited on Si substrates to measure particle-particle interactions. The same modified tips were used to assess the ENM-cell interactions. The ENM - ENM force was measured in air, water and biological media such as . The ENM-cell interaction was measured in the appropriate cell media for fast interaction (<1s) and long-term interactions (>10s) using A549 cell line.
Results indicated that the agglomeration potential of CeO2 ENMs in water and RPMI 1640 (Roswell Park Memorial Institute formulation 1640) was inversely proportional to their primary particle (PP) diameter, but for Fe2O3 ENMs, that potential is independent of PP diameter in these media. Moreover, in RPMI+10% Fetal Bovine Serum (FBS) the corona thickness and dispersibility of the CeO2 is independent of PP diameter while for Fe2O3, the corona thickness and dispersibility were inversely proportional to PP diameter. Similarly in the case of the cells the particles were found to have different affinity towards the cells based both on the PP and the timescale of the interaction. The proposed AFM approach provides a valuable tool for the investigation of the ENM agglomeration state and corona transformations in biological media and will help in improving our understanding on nano-bio interactions.

While cell migration in 3D environments has now become a standard, lack of robust models continue to hamper our ability to investigate tumor level behavior and Epithelial-to-Mesenchymal Transition (EMT) in 3D environments. This limitation significantly affects our ability to understand the signaling cascades and mechano-chemical regulators of metastasis and cancer progression. Using an integrated strategy using cell derived and naturally occurring matrices, we have developed millimeter sized tumoroids of highly invasive breast and bone cancer and have investigated the response of these tumors to external mechanical and chemical perturbation, as well as therapeutic interventions. Our method allows us to create a biomimetic environment to investigate cellular level signaling upon surgery, incision and chemotherapy. Our results with MCF-7 and U2OS cells demonstrate the fidelity and robustness of our approach and demonstrate how matrix mechanics, in conjunction with porosity and availability of adhesion ligands regulates tumoroid size, shape and EMT. Additionally, we note that matrix environment plays a strong role in mediating cellular response to both bolus and nanoparticle based chemotherapeutics. Overall, our approach represents a new strategy in creating stable, scalable and organized tumoroids and have demonstrated how matrix properties regulates tumor progression and response to therapy in native like 3D environments.

Tumor invasion and metastasis are ultimately responsible for over 90% of cancer-related fatalities, but remain poorly understood. One signature of malignant tumor progression is the detachment and dissemination of individually invading tumor cells from a collectively invading front. These complex and emergent behaviors are thought to occur as a consequence of the epithelial-mesenchymal transition (EMT), which converts adherent epithelial cells to a motile mesenchymal phenotype. Here, I integrate engineered microenvironments with high-content imaging to comprehensively measure heterogeneous invasion dynamics in populations of tens of thousands of cells. Based on their observed behaviors, cells can be clustered into distinct subpopulations that display individual or collective invasion. Remarkably, individually invading cells are associated with faster velocities as well as straighter trajectories, enabling efficient scattering and dispersal. These spatial and temporal dynamics are then physically modeled based on the solidification of binary alloys, suggesting that the governing mechanisms are differences in motility as well as phenotypic interconversion. Finally, these behaviors are perturbed using migration-inhibiting compounds, revealing that cells with different invasion phenotypes also exhibit pronounced differences in drug sensitivity.

Embryonic development consists of a complex series of cell signaling, cell migration and cell differentiation processes that are coordinated during morphogenesis. Collective cell sheet migration is an important process that sculpts the shape of an organism and its internal tissues during early development. The fate of the tissues and their ultimate physiological function relies on integrating genetic programs and processing external cues to provide positional information to guide cell migration and induce cell differentiation. By contrast, embryonic development and tissue-self-assembly requires the integration of cell movements within multiple cell layers composed of different cell types. Considering the important role cell mechanics plays in tissue self-assembly it is surprising that little is known about the mechanical response of the multi-layer tissues to chemical cues. One of the reasons of this knowledge gap is the lack of the technologies to analyze the individual responses of epithelial and mesenchymal cell sheets in a multi cell layer tissue to mechanical cues. To investigate the processes that guide collective movements of multiple cell layers our group has focused on developing a novel microfluidic technique capable of producing complex patterns of laminar multicellular structures. We call this technique "3D tissue-etching” by analogy with the techniques used in the MEMS field. We use tissue etching to shape a complex multi-layered embryonic tissue and explore the dynamic collective responses of epithelial and mesenchymal cells in a single tissue. We use a custom-designed microfluidic control system to deliver a range of tissue etching reagents over Animal Cap tissues of Xenopus laevis. Microfluidics provides the ability to control chemical stimulation of biological systems using laminar flow patterning. To deliver precise chemical stimulation to a multicellular tissue, we have developed a system for controlling the inlet pressures to a microfluidic device by modulating fluidic resistance and capacitance. Using microfluidics, we were able to construct in a controlled multi-step etching process a complex tissue. Using etching we produce free-edges of epithelial cells over mesenchymal cells and free-edges of mesenchymal cells. We followed the process of etching and the resulting tissues with time-lapse microscopy to understand the effects of tissue etching on the mechanics of the tissue microenvironment and the impact of these changes on collective cell migration. This allows us to study multi-layer coordination of epithelial and mesenchymal cell layers and acute mechanical and behavioral response of intact epithelial and mesenchymal cell sheets to removal of neighboring or overlying tissues. The ability to control the form of multicellular tissues potentially will have a high impact in tissue engineering and regeneration applications in bioengineering and medicine

Traditional methods of quantifying cell movement in response to a chemotactic factors provide either a binary count of cell migration in response to a known concentration of the factor of interest in solution, as in Boyden chamber assays, or a method of tracking cells to determine velocities across a solubilized protein gradient where exact concentrations vary over time and are difficult to define, as in the Ibidi chemotaxis gradient assay. Using a silane self-assembling monolayer (SAM)-based procedure pioneered by V Hlady, et al. we have developed an assay capable of covalently binding a wide variety of proteins to an optically transparent surface in a 2D pattern via amine linkages. This new assay provides greater control of protein concentration and gradient intensity than possible in assays using solubilized proteins.
To achieve a smooth and continuous gradient, a monolayer of beta-mercatopropyltrimethoxysilane was deposited unto a quartz substrate in an ultra-high purity Nitrogen-filled glovebox (>1.9 mm Hg) under anoxic anhydrous conditions. The silane polymerization reaction was performed by immersing an acid etched quartz slide in 99.9% anhydrous toluene and catalyzed with the addition of 5 uL of water to 100 mL of toluene. Polar/non-polar emulsion was achieved by addition of .51 mg of sodium dodecyl sulfate /mL water and sonication. Chemical gradients were formed by selectively inducing the oxidation of surface thiol groups present in the silane monolayer via UV irradiation in the presence of atmospheric oxygen, using a mask and translating stage. This process results in conversion from thiol to sulfonate groups. Protein bonding is achieved through the use of a heterobifunctional crosslinker (SMPEG12) that contains maleamide and NHS groups capable of binding selectively to thiol and amine groups respectively.
Results from preliminary investigations into the silane monolayer chemistry using contact angle goniometry and X-ray photoelectron spectroscopy (XPS) demonstrate that a monolayer of silane is formed, and for specific irradiation times, there is a linear relationship between the total irradiance and conversion from thiol to sulfonate groups. Specific protein and crosslinker concentration in units of ng/um2 will be verified by XPS. Initial cell microscopy studies planned include time lapse phase contrast and fixed focal contact immunostaining (vinculin, actin, etc.) of chondrocytes and mesenchymal stem cells.

Perhaps one of the most remarkable and synthetically desirable properties of some biological cellular systems is the process known as chemotaxis. Chemotaxis is the ubiquitous biological process by which a cell is able to detect the concentration gradient of a particular chemical signal in an environment and then moves accordingly to that gradient. Bacterial E.Coli exhibit “run and tumble” chemotactic behavior in order to move towards regions that are rich in nutrients or to avoid regions of high toxicity. Here we have designed functional assemblies that can mimic biological systems that undergo chemotaxis and are sensitive to both temporal and spatial gradients. The microswimers or walkers we have designed are unique in that our walker&’s propulsion mechanism is essentially a hybrid design incorporating both biological propulsion mechanisms, such as cilia and flagella, and molecular motors, which transport cargo inside cells along the cytoskeleton. This duality enables the locomotion of the walkers themselves while still propelling the surrounding fluid, which has unique advantages for micro-scale manipulation through mimicked chemotaxis. This novel approach was realized by utilizing functionalized superparamagnetic particles or beads that spontaneously self-assemble due to magnetic forces and form chains. A rotating magnetic field drives the chains to turn and walk across the surface. The streptavidin coated particles are coupled to a surface with a gradient in the density of biotin binding sites and the velocity of the particle is modulated as it walks across the surface. As the particles encounter more binding sites the chain tends to “hinge” and translate more than it would in an area with a low density of binding sites where it tends to “slip” and translation decreases. Thus by controlling the surface-bead interactions, we can control the velocity of the walkers and detect inhomogeneous patterns on surface, effectively creating chemotactic walkers.

A key issue in the use of nanomaterials is controlling how they interact with themselves and with the outer world. Our research program focuses on the tailoring of nanoparticles of surfaces for a variety of applications, coupling the atomic-level control provided by organic synthesis with the fundamental principles of supramolecular chemistry. Using these engineered nanoparticles, we are developing particles for cancer therapeutics and diagnostics. This talk will focus on the interfacing of nanoparticles with biosystems, and will discuss our use of nanoparticles for delivery applications including our studies of small molecule, nucleic acid, and protein delivery. This presentation will also feature the use of nanoparticles for diagnostic applications, including the use of array-based sensing paradigms for the detection and identification of cancer.

Nanoparticles have emerged as a promising tool in the biomedical field, where they serve as delivery carriers of imaging agents or drugs for nanomedicines. After intravenous injection, nanoparticles show higher accumulation in angiogenic disease sites than normal tissues due to the enhanced permeability and retention (EPR) effect. To further improve their specificity to disease sites, active targeting strategies have been developed using biological targeting moieties such as antibodies, aptamers, or peptides which bind proper receptors on the surface of target cells. Efficient binding of nanoparticles to the cell surface can increase their accumulation at the target site, while unbound nanoparticles are cleared from the tissue and eliminated from the body. However, saturation of these receptors limits the capacity of targeted nanoparticles. In addition, targetable receptors are rarely unique to the disease, so nanoparticles can accumulate in other healthy tissues via these receptors resulting in reduced therapeutic efficacy and unintended side effects. Herein, we introduce a new in vivo active targeting strategy for nanoparticles via metabolic glycoengineering and bioorthogonal copper-free click chemistry in the living body. This technique is expected to enable dose-dependent and temporal generation of chemical groups on the target tumor site just like biological receptors, controlling the tumor-targetability of nanoparticles.

Targeting nanoparticles (NPs) to interact with over-expressed surface receptors on cancer cells, commonly known as active targeting, has shown potential in increasing selective cellular uptake, diminishing systematic toxicity, and enhancing therapeutic efficacy. These factors in combination have the capacity to reform cancer treatment; but thus far success of targeted drug delivery systems has been limited. There are a number of variables (e.g. nanoparticle composition, targeting ligand type, targeting ligand density, etc.) that contribute to in vivo behavior which may lead to a diminished therapeutic effect if not optimized. In this account, we studied the effects of targeting ligand density on high aspect ratio polymeric hydrogel nanoparticles. Utilizing the nanoparticle fabrication technique PRINT (Particle Replication in Non-wetting Templates), 80 nm x 80 nm x 320 nm PEG-based hydrogels were synthesized with surface amine substituent groups for post-fabrication functionalization. A FITC-tagged Z^EGFR binding domain affibody was conjugated to the nanoparticle at various loadings via poly(ethylene glycol) cross-linker. Standard plate-reader analysis was used to determine ligand density, ranging from ~1000 to ~100 ligands per NP. In vitro studies in multiple cell lines indicated that particle internalization was dependent on time, NP dose concentration, and targeting ligand density. Pharmacokinetic analysis of EGFR-targeted particles indicted a strong relationship between ligand density, tumor accumulation, and circulation half-life. Overall, we have described how ligand density affects key parameters in the active targeting of NP drug delivery systems. In future work we will incorporate chemotherapeutics into our targeted nanoparticle system in order to determine therapeutic efficacy in a number of cancer models.

We herein present a miniaturized holographic imaging system for high-throughput detection and molecular profiling of rare cancer cells. In this portable imaging system, the sample is illuminated by a partially coherent light source and in-line holograms are recorded by a digital sensor array. A new computational algorithm is developed to reconstruct original images and count target cells. To detect and profile cancer cells, we first label blood samples with molecular-specific microbeads. Such labeling enables 1) a reliable differentiation between cancer cells and host cells (e.g., leukocytes); and 2) quantitative profiling of target marker expression through bead-counting. The bead-labeled blood sample is then flowed through a fluidic channel placed directly on the digital sensor array, and holographic images of cells are recorded. From these holographic images, the computational algorithm recovers the original cellular images. This reconstruction algorithm is able to retrieve both the intensity and the phase information of the original image. It also recognizes and counts the number of target cells and microbeads. By counting the number of beads attached on cells, we could measure the expression levels of different cancer markers. The profiling results agree well with that of flow cytometry and fluorescence microscopy. For real-time image reconstruction and cell counting, we have implemented parallel computing algorithm that utilizes multicore graphics processing unit (GPU). The resulting imaging system enables high throughput cellular detection - up to 1000 cells/µL could be imaged over a wide detection area (20 mm2), and cellular images could be reconstructed in real time (20 frames/sec). Furthermore, the assay could be performed without any dilution or washing steps, minimizing cell loss and simplifying the assay procedure. Cost-effective, portable, and easy-to-use, this imaging system could be readily applied in clinical settings for cancer diagnosis.

The development of both normal tissues and tumors depends on concerted interactions between cells and their local microenvironment. In the case of a tumor, this microenvironment includes not only the tumor-associated chemical and mechanical factors, but also the surrounding host cells. Here we used tissue engineering strategies to mimic these aspects of the tumor microenvironment in order to parse the relative roles of each in tumor development. We used a 3D microfabrication-based approach to normal epithelial ducts containing individual tumor cells and tumor aggregates, and microfluidic strategies to control the mechanical properties of the tissue. We found that tumor cell phenotype depended entirely on these parameters, regardless of underlying genetic abnormalities. Tumor cell invasion depended on the host mechanical stress, as shown both computationally and experimentally, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining micro-computed tomographic analysis with finite element modeling confirmed that regions of high mechanical stress correspond with regions of tumor formation in vivo. This work suggests that the mechanical tone of the non-tumorigenic host epithelium directs the phenotype of tumor cells, and provides additional insight into the instructive role of the mechanical tumor microenvironment. Our study also suggests that tissue engineering strategies can be used to unveil novel aspects of tumor development.

Cancer stem cells (CSCs) are a small fraction of cancer cells in the tumor bulk that drive the tumor initiation, progression and recurrence. CSCs, also called tumor-initiating cells, are characterized by properties including capabilities of self-renew, sphere forming, drug resistance, and tumor forming in vivo. Therefore a better understanding of the regulation of CSCs will facilitate the development of therapeutic CSC-targeting strategy for the successful eradication of cancer. However, isolated CSCs based on cell surface marker spontaneously differentiate into non-stem cancer cells in traditional two dimensional culture condition with tissue culture plastics. In our study, we have demonstrated that synthetic fibrous scaffolds generated by electrospinning can mimic the extracellular matrix in structure and provide three dimensional culture condition. A variety of cancer cell lines cultured in fibrous scaffolds significantly increased the properties of CSCs, which indicates the CSCs in those cell lines expanded in the three dimensional scaffolds. These results suggest electrospun fibrous scaffolds itself without protein components can enrich the CSCs in tumor cell lines. These fibrous scaffolds have a great potential as an in vitro platform to screen anti-CSCs drugs, model the tumor growth, as well as study the CSCs dynamics and the cell-matrix interaction.

Hydrogels are widely employed as cell culture platforms to study cellular phenomena in a 3D tissue-mimetic context. Engineered hydrogel networks have been designed to aid in understanding the influence of biophysical properties on cell behavior. In order to understand more complex cellular phenomena that take place in wound healing, disease, and development, hydrogels need to be engineered to mimic dynamic changes that occur in the microenvironment. For example, in mammary tumor progression, the extracellular matrix progressively stiffens from 100 Pa to over 1000 Pa. There is evidence that abnormally stiff matrices lead to loss of epithelial character in mammary epithelial cells and an increase in invasion. It is hypothesized that the dynamic changes in extracellular matrix properties can contribute to disease progression. Thus, it is our objective to develop a 3D dynamic hydrogel culture platform that allows for modulation of gel stiffness over time.
Our approach utilizes three essential components; i) alginate, an anionic biopolymer that progressively crosslinks in the presence of divalent cations, ii) temperature-sensitive liposomes encapsulating calcium, and iii) gold nanorods that locally heat when irradiated near the absorption peak in the near-infrared region. Thus, irradiation of the gold nanorods induces a release of calcium that crosslinks the alginate hydrogel.
We have demonstrated that the calcium loaded liposomes can be mixed with alginate solution and gelled upon irradiation. When distributed within a weak 3D gel and irradiated, the stiffness can be linearly increased from 91 Pa to 1548 Pa. The extent of stiffening is a function of irradiation time, which allows for temporal modulation to intermediate values to mimic gradual tissue stiffening. Alginate crosslinks are reversible, thus chelators such as citrate can be loaded in the liposomes to temporally soften gels. Using this strategy, we generated a decrease in stiffness from 1104 Pa to 467 Pa. 3T3 cells seeded within a gel consisting of alginate, calcium loaded liposomes, and Matrigel as a cell-adhesive moiety were irradiated for 30, 60 or 120 s or left unirradiated as controls. No loss of viability due to irradiation was observed through either MTS or Live/Dead assays compared to controls. Cells in the unirradiated gels adopted elongated morphologies while cells in the irradiated samples became more rounded with increasing irradiation time (thus, increasing stiffness). Finally, we have demonstrated the ability to induce gelation transdermally in a mouse model due to the high penetration depth of NIR light in tissue.
We have developed a novel light-triggered system for temporal modulation of hydrogel stiffness over a physiologically relevant range. Ultimately, we intend to use this system as a tool to explore the influence of dynamic microenvironments on cell phenotype in a highly controlled and directed manner.

Despite significant progress in the development of new chemotherapeutic agents and drug delivery methods for brain tumors, malignant gliomas (high grade brain tumor) remains deadly with a median survival period of only about a year. The high dosage of chemotherapeutic agents required for penetration through the blood brain barrier during chemotherapy not only kills cancer cells but also damages healthy tissues and causes adverse side effects. Hence, a major unmet challenge in the treatment of malignant gliomas is the development of effective and targeted local delivery of chemotherapeutic agents at the cellular level. Drug-loaded biodegradable microcapsules with high drug encapsulation efficiency and controlled shape and size are attractive candidates for a more precise control of anticancer agent delivery at the tumor sites.
Here, we report the results of a systematic study of the size, shape, and drug release profiles of Poly(lactic-co glycolic) (PLGA) microcapsules produced and loaded with the anticancer agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) using an electrojetting technique. We report production of BCNU-loaded PLGA microcapsules in the form of flattened microspheres, microspheres, and microfibers with significantly (1) higher drug encapsulation efficiency, (2) more tunable drug loading capacity, and (3) narrower size distribution than those generated using other encapsulation methods. To prepare BCNU-PLGA solutions with PLGA concentrations ranging from 1 to 10 wt%, BCNU/PLGA mixtures (5 to 25 wt% with respect to PLGA) dissolved in chloroform were electrojetted on gold substrates. We quantified the shape and size distribution of BCNU-loaded PLGA microcapsules as a function of the polymer concentration and flow rate used during electrojetting, and characterized drug release profiles for microcapsules of three different morphologies: flattened microspheres, microspheres, and microfibers. Flattened microspheres were produced from 1 and 2 wt% PLGA solutions, while microspheres and microfibers were formed as the PLGA concentration increased to 3-5 wt% and 10% wt%, respectively. We investigated the effect of flow rate (i.e. 0.25, 0.5 and 1 mLh-1) on the size and monodispersity of BCNU-loaded PLGA microcapsules. Microcapsules of 1 wt% PLGA, 4 wt% PLGA, and 10 wt% PLGA formed at a flow rate of 0.25 mLh-1 had narrower size distributions (CV = 4.6%, CV = 4.5%, and CV = 4.8%, respectively). A commonly accepted definition of monodispersity is CV < 5%. We also measured drug release profiles and developed mathematical models to estimate the effective diffusion coefficient of BCNU in different PLGA microcapsules. The BCNU release profiles for all three microcapsule morphologies were found to be in good agreement with model predictions for drug release as a result of drug diffusion and degradation of PLGA microcapsules.

Hydroxyapatite (HA)-containing microcalcifications (MCs) are routinely used for detection of breast cancer via mammography and associated with malignant disease. However, the role of MCs in the etiology of osteolytic bone metastasis, the leading cause of deaths in breast cancer patients, remains unclear. We have previously developed a polylactide-co-glycolide (PLG)-based scaffold system containing commercially available HA nanoparticles to evaluate MDA-MB231 breast cancer cell behavior in response to HA. Our results using this system suggest that interactions with HA promote the expression of bone-metastatic genes in breast cancer cells and regulate their secretion of the pro-inflammatory and osteolytic cytokine interleukin-8 (IL-8). However, to what extent these changes are related to specific HA materials properties (e.g., particle size, morphology, and crystallinity) remains unclear given the large heterogeneity of HA in commercial preparations. To overcome this limitation and analyze HA-breast cancer cell interactions as a function of varied materials properties at high-throughput, we have developed a biomimetic approach based on HA-forming simulated body fluid (SBF). More specifically, we have incubated PLG coated 96-well plates with SBF, a solution containing inorganic components of human blood plasma, and have analyzed the resulting coatings by SEM, FT-IR, and XRD to assess morphology, composition and phase of coatings, respectively. Our results suggest that SBF carbonate concentration is inversely correlated with the crystallinity of the HA surfaces formed. Furthermore, MDA-MB231 cultured on variations of these mineralized surfaces up-regulated IL-8 secretion in response to increased HA crystallinity, while exhibiting decreased adhesive and proliferative capacity. These results are corroborated with similar findings from PLG scaffolds incorporated with synthetic HA of high and low crystallinities, which validates the relevance of our approach. Taken together, these data suggest that biomimetic mineral-coatings are an effective tool with which precisely defined cell-mineral interactions may be efficiently assessed.

Two major promising applications of nanotechnology towards the treatment of cancer are the control over molecular binding sites and the detection of binding phenomena, which could lead to better therapies and earlier detection. Recent advances in the ability to control matter at the nanoscale have enabled unprecedented capabilities using semiconductor and polymeric materials. Bringing together expertise in nanoscience, chemistry, and biology, our laboratory is developing these two classes of materials both alone and in combination to control molecular recognition. This work has a wide range of potential therapeutic and diagnostic applications.
One focus of our lab is the development of fluorescent single-walled carbon nanotubes as optical sensors to create monitors for cancer biomarkers. These materials allow the detection of binding events down to the single-molecule level, in real-time, and in a minimally-invasive way. Encapsulation of nanotubes in synthetic polymers and biopolymers creates a handle for the transduction of analyte binding. Small molecules, such as reactive oxygen species and other genotoxins, can be detected by transduction of the binding event to the polymer or the nanotube itself. Multiplexed analyte identification is possible by observing variations in the nanotube&’s spectral response, resulting in distinct optical fingerprints. Nanotube emission can undergo both wavelength and intensity modulation, permitting the identification of analytes which are difficult to differentiate via conventional methods. The analyte responses can be spatially mapped in live cells and tissues, measured with sensitivity down to the single-molecule level, and detected in real-time, facilitating new and unprecedented biological measurements.

We have recently developed a miniaturized microfluidic chip-based technology, the micro-Hall detector (uHD), that can perform rapid, highly sensitive, and quantitative measurement of individual cells in unprocessed biological samples. The uHD detects the Hall voltage induced by magnetic moments of cells in-flow that have been magnetically tagged with magnetic nanoparticles (MNPs) and bio-orthogonal chemistry. The entire assay is performed on a single microfluidic chip without the need for washing and purification steps, thereby allowing cellular diagnostics to be conducted in point-of-care settings. As a proof-of-concept, we previously implemented the first uHD prototype that had 8 micro-Hall elements. The system was used to detect MNP-labeled Gram-positive bacteria. The detection limit was similar to that of culture tests (~10 bacteria), but with a 50-times faster assay time. The application was further extended to detect and profile circulating tumor cells (CTCs) in the whole blood of cancer patients.To improve the assay throughput and sensitivity, we designed the next generation uHD for practical applications. The new system incorporates a larger array of micro-Hall elements within a wide fluidic channel to increase sensing area, thus flowing higher sample volume. Signal processing circuits are integrated in the same chip, which helps to maximize signal-to-noise ratio (SNR). With such capabilities, the new uHD could be a comprehensive and universal diagnostic platform with potential for broad applications in resource-limited, point-of-care settings.

Surface plasmon resonance (SPR) biosensors have been widely used to study the interactions of proteins, small molecules, and nucleotides. Their sensing range (50 ~ 200 nm) parallels the size of exosomes, which are nano-sized membrane-bound vesicles released from cells. Here, we demonstrate a label-free and sensitive approach for molecular profiling of exosomes using a novel SPR nanohole platform. The device comprises arrays of nanoholes, patterned onto a thin gold film on a glass substrate. With a multi-channel flow cell constructed on top of the sensor arrays, each nanohole array was functionalized with a different type of antibody. Target-specific binding of exosomes on the array induced spectral shifts in the resonance wavelength. Such shifts were proportional to the number of bound exosomes and correlate sensitively with overall cancer antigen abundance, therefore enabling quantitative molecular profiling. The nanoholes displayed high detection sensitivity; using CD63 as a pan-exosomal marker, we established a detection threshold of ~ 2,000 exosomes. We also profiled the expression level of molecular markers (e.g., EpCAM, HER2, EGFR) in exosomes isolated from cancer cell culture, whose profiling results showed an excellent match with those of parental cells. This indicates that cancer-derived exosomes reflect the molecular signature of their primary tumor. With its capacity for sensitive, label-free molecular detection, the plasmonic nanoholes would be a promising new platform for comprehensive exosomal molecular analyses.