Symposium SB10—Micro- and Nanoengineering of Biomaterials—From Precision Medicine to Precision Agriculture and Enhanced Food Security

DNA-based self-assembly provides an exciting materials platform with which to build composite nanostructures impossible to fabricate with top-down techniques. While biological DNA is well characterized, much work must be done to develop a mature understanding of DNA as a nanoscale construction material.
In metals defects contribute only a small fraction of the system free energy, but control material microstructure and properties. In DNA origami, where the scaffold molecule is forcibly routed into a predetermined shape, conformational energy contributions are small compared to those of base pairing and base stacking but control assembly properties.
By leveraging the high throughput of qPCR equipment, we examine the thermodynamics of single folds via van’t Hoff analysis of melt curves.
We then examine the effect of staple excess revealing unintuitive results in both cases. In contrast to whole origami, increasing staple excess for a single fold significantly reduces yield. We present these results and show how they can inform an understanding of whole-origami systems.

Silk proteins represent unique protein polymer systems due to their regular peptide repeats with block-copolymer features, along with self-assembly in aqueous, ambient conditions into secondary and higher order structures. In nature, this process leads to unique hierarchically structured and functional fibers with remarkable mechanical properties. Generating insights into the nanoscale features of these systems has generated a new range of material features that can be exploited towards structural and functional biomaterials based on silk. Specific polymer processing paths include exfoliation to generate natural silk nanofibers as building blocks for new materials and generating silk amorphous nanoparticles as feedstocks towards commodity plastics processing using silks. In both cases, the critical features are the silk nanostructures and water content, related to mechanics, processability and functional outcomes. Specific examples will be discussed to highlight these new opportunities with this ancient polymer.

Introduction: With the rising harmful effects of climate change, a novel, emerging field of technology called cellular agriculture has arisen to explore ‘cultured meat’, an artificial meat substitute developed in vitro using animal cells and bioengineered cell scaffolds. Cultured meat is an advantageous alternative to traditional animal agriculture, which is not sustainable in a world with a rising human population and growing meat consumption rates. Plant-based alginate scaffolds can be used to support the development of cultured meat due to their high degree of chemical and mechanical tunability, which can be optimized by adjusting the hydrogel’s molecular weight, chemical composition, and cross-linking process. In this project, our goal was to create a dual-crosslinked alginate-based scaffold with material properties that supported tissue development of primary bovine muscle cells, with the overall goal of achieving a cultured steak for human consumption. Methods: Herein, we synthesized: 1) methacrylated alginate (Alg-MA) using aqueous chemistry to enable covalent crosslinking via visible light exposure, and 2) arginine-glycine-aspartic acid (RGD) conjugates (Alg-MA-RGD) using carbodiimide chemistries to provide cell-binding sites onto the material. Alginate-based hydrogels of different concentrations (1% and 3%, w/v) and cross-linking types were created. Ionically crosslinked hydrogels were made by injecting the hydrogel precursor solutions into custom molds of 8-mm diameter. The molds and solutions were frozen for two hours at -20 °C, and then crosslinking by adding 1M calcium chloride (CaCl₂) dropwise onto the mold. Photo-crosslinked alginate-based hydrogels were made by injecting hydrogel precursor solutions premixed with 1mM eosin Y (photo-sensitizer), 125 mM triethanolamine (photo-initiator), and 20 mM 1-vinyl-2-pyrrolidinone (catalyst) in a custom mold and exposed to green light (525 nm, SuperBrightLEDs) for five minutes. Dual cross-linking was achieved by combining the two types where photo-crosslinking preceded ionic crosslinking. The crosslinked alginate scaffolds were characterized using proton nuclear magnetic resonance (¹H-NMR), Fourier transform infrared (FTIR) spectroscopy, rheometry, and unconfined uniaxial compression tests. Results and Discussion: We demonstrated that modifying the material structure chemically and physically via different crosslinking techniques resulted in differences in the material properties between groups, such as gelation kinetics, viscosity, and scaffold stiffness. For the alginate precursor solution, we were able to obtain low enough viscosity values (0.02 – 0.06 Pa.s) to enable processing via photolithography. The gelation time of the different materials did not vary broadly, although the shear and compression moduli demonstrated significant differences between groups. Ongoing work includes: 1) scaffold structural analysis via scanning electron microscopy (SEM); 2) murine C2C12 and primary bovine muscle cell response and myogenic differentiation on the alginate scaffolds, and investigating the effects of stiffness, pore size, and chemical composition. Conclusion: Our unique method of synthesizing alginate hydrogels and the associated pilot data indicates the efficacy of dual-crosslinked alginate-based hydrogels for the production of cultured meat.

The networked architectures that maintain the structural integrity and function of biological tissues are nanofibrous arrays of proteins. We have developed three manufacturing platforms (rotary jet spinning, immersion rotary jet spinning, and pull spinning) as methods of building nanofiber networks of biological and synthetic polymers to mimic the function of the extracellular matrix. We have used these methods to build 3D tissues for in vitro assays, heart valves for implan, wound dressings for skin, cardiac ventricles, and food. This presentation will review the design principles and lessons learned of these works.

Light and materials interact in natural systems with exquisite precision through a designed interaction between electromagnetic waves and the physical properties of the material, both from the bulk and structural point of view. In particular, structural control over multiple length scales, that span from the nanoscale to the macroscale, defines interesting material responses that enable high-performing functional outcomes in light harvesting, reflection, and thermal management among others. We describe here strategies for light harvesting and optomechanical actuation in hybrid biomaterial-elastomeric composites that leverage rationally designed photonic architectures to obtain wireless, light-responsive material formats. The combination of photonic properties designed at the nanoscale with shaped elastomeric material hosts open opportunities in optomechanical actuators with tunable and complex deformation that respond to simple light illumination. The talk will cover some of the potential opportunities for future development of composite materials that are inspired by Nature's ability to harvest light and explore possible classes of smart optomechanical systems that move with light on demand without the need for external sources of power.

Metal ions play an essential role in many physiological and pathological processes in living systems. Approximately one-third of all proteins currently known require metal ions as cofactors for their structural integrity and biological function. For example, iron is a cofactor of several metalloproteins involved in chlorophyll synthesis and photosynthetic electron transport. Deficiency in iron therefore induces chlorosis and other plant disorder, lowering crop yields drastically.
Peptides are known to exhibit a variety of interactions with metal ions, due to the wide range of functional groups they possess. Metal-binding peptides then attracted a lot of attention as a potential material platform for nanofabrication of organic-inorganic composites built from the bottom up. Besides, coordination with metal ions may induce peptide association into nanostructures that are conformationally and functionally different from the original peptide assemblies. In this presentation, we show that the self-assembly pathway of a silk fibroin-derived peptide can be regulated through addition of differently charged metal ions. Atomic force microscopy (AFM) in combination with a set of correlative light scattering and spectroscopy techniques are employed to investigate the detailed mechanism of peptide assembly facilitated by different metal ions. Factors including the molar ratio of peptides to metal ions and the charge and type of metal ions are found to largely affect the peptide association kinetics and the size and structure of peptide assemblies at equilibrium. Such understanding can hopefully contribute to the design and development of peptide-based nanocarriers for stabilization and delivery of micronutrients in plants.

Globally, rapidly aging human populations are significantly accelerating the occurrence of non-communicable diseases. Understanding the fundamental mechanisms of tissue damage due to aging can provide predictive capabilities that will inform medical treatments. Coupled with this understanding, nature-inspired dynamically responsive biomedical devices and implants can address this major health crisis due to their excellent environmental adaptability and biocompatibility. Developing such novel biomaterials further necessitates multiscale computational modelling and simulations to fundamentally understand the pertinent physical, chemical, and biological material properties. We apply multiscale molecular modelling and machine-learned metamodels to characterize aging-related diseases and engineer various candidate materials on multiple fronts: (I) collagen-related morbidities, (II) dynamically-responsive silk and silk-elastin-like materials, and (III) viscoelastic mechanical properties of soft polymers. Through this approach, we unveil the myriad material properties of mutant collagens, as well as chemically and physically processed silk-based materials. We also rapidly and efficiently determine the material parameters of constitutive models governing the time-dependent behavior of soft polymers.

Controlling the interface of colloidal nanoparticles (NPs) via tethered molecular chains of defined passivation properties is crucial for NP applications in electronics, optoelectronics, catalysis, and nanomedicine. There is an urgent need for a flexible system to vary the chemistry and structure of these interfacial molecules in a systematic and pre-determined way to improve control over NP stability and functionality. Here, we report low molecular weight, bifunctional comb-shaped, and sequence-defined peptoids that effectively passivate gold NPs (AuNPs). Each peptoid (PE) is designed with a multivalent NP-binding domain and a solvation domain consisting of branched oligo-ethylene glycol (EG) and varied in arrangements. We find that a diblock arrangement provides superior colloidal stability in varied salt and ionic aqueous solutions yet form a compact layer on the NP surface only ~1.5 nm thick. We further demonstrate by experiments and molecular dynamics simulations that the PE-coated NPs (PE/NPs) are stable in select organic solvents owing to the strong PE-Au binding and solubility of EG motifs. Finally, the PE/NPs can self-assemble into organized 2D lattices at the air-water interface with small lattice constants (~6 nm) due to the thin PE surface layer. The versatility and designability of peptoid architectures for NP passivation render an attractive approach for nanomaterial-based (bio)technologies, in which precise control over size, surface property and stability are key.

Cephalopods (e.g., squids, octopuses, and cuttlefish) have captivated the imagination of both the general public and scientists alike due to their sophisticated behavior and stunning camouflage abilities. Given such characteristics, it is not surprising that these marine invertebrates have emerged as exciting models for novel materials and systems. Within this context, our laboratory has focused on the development of cephalopod-inspired biomaterials and platforms with unique electrical and optical properties. Our findings hold implications for next-generation bioelectronic devices and biomedical imaging technologies.

Sustainable nanotechnology is an emerging field of interdisciplinary research that focuses on the development of “green” and non toxic nanomaterials and technologies that can be used to tackle major societal challenges in various fields and applications. Maintaining food safety and security for the increasing global population is one of the most important challenges of the 21st century with major societal, environmental and public health implications.
This seminar will present highlights from our current sustainable nanotechnology research in the agriculture and food domain. Projects include among other the development of green, nano-carrier platforms for targeted precision delivery of nature-derived antimicrobials for food safety applications using engineered water nanostructures (EWNS) synthesized using electrospray and ionization processes, EWNS exhibit unique physicochemical properties, are highly mobile due to their nanoscale size and can interact and inactivate food related pathogenic and spoilage microorganisms on surfaces and in the air. In addition, the development of “smart” or stimuli responsive, non toxic and biodegradable antimicrobial packaging materials will be presented. Food packaging plays a key role as it has a great potential to improve food safety and quality and reduce food waste across the “farm to the fork” continuum.

Transport of small liquid volumes (few and sub droplets) is a desired functionality for a wide range of applications, such as microfluidics, lab-on-chip devices, water collection from the atmosphere, etc. In general, liquid transport is usually achieved through the use of pumping devices, or external fields (electromagnetic, acoustic, optical, etc.) working collaboratively with special fluid or surface properties to enable fluid motion (e.g. ferrofluids or photosensitive substrates). On the other hand, passive droplet transport methods are more attractive compared to the active ones, due to the absence of the need for energy input and their inherent facility.
In addition, another common problem at small scales is the natural liquid adhesion of droplets on surfaces, which deters motion, induces high variation in the transported fluid mass, while the adhered fluid residues also cause surface contamination. Most of the developed passive fluid transport approaches, fail to resolve this issue, as they typically transport fluid in the form of a liquid film rather than discretized droplets. Therefore, still, significant advancements are needed before devices for passive liquid propulsion, without the input of external energy and unwanted contamination, become a reality in applications.
In this work, we present an unexplored, bioinspired by the water strider leg surface architecture, scalable, and facile material design approach based on laser micromachining process of copper in the shape of a backgammon-like pattern, followed by chemical growth of copper hydroxide nanoneedles functionalized with a thiol to impart superhydrophobicity. The combined effect of the Laplace-pressure-imbalance and surface superhydrophobicity is able to passively drive droplet motion while maintaining the limited contact of the Cassie-Baxter state on superhydrophobic surfaces. The backgammon-like micro-topography design naturally deforms asymmetrically the menisci formed at the bottom of a spherical droplet and causes a Laplace pressure imbalance that drives droplet motion. We investigate this effect over a range of opening angles (1°, 5°, and 9°) and develop a model to explain and quantify the underlying mechanism of droplet self-propulsion.
We further implement the developed bioinspired topography for applications relevant to microfluidic platform functionalities. In particular, we explore lateral droplet motion and measure the droplet velocity during its self-propulsion (up to 40 mm/s for opening angle 9°), as well as the force acting on the droplet meniscus as it deforms at the solid-liquid interface(up to » 5 μΝ). Additionally, we achieve controlled rebound angles after droplet impact on the substrates up to ω = 8°, droplet merging, and long-distance droplet transport without mass loss. Furthermore, we demonstrate spontaneous droplet merging of droplets that are released simultaneously. The merging occurs very fast (in a few hundreds of ms) and it is aided by the underlying backgammon-like structures. Finally, we achieve horizontal self-transport to distances up to 65 times the droplet diameter and even show significant uphill motion against gravity, which to our knowledge is the longest traveling distance reported for discretized droplets by using fully passive means.

In this study, we designed a unique aptamer-gold(Au) nanoconstruct coated with polymer capable of scavenging reactive oxygen species(ROS) and capturing tumor necrosis factor alpha(TNF-α) as anti-inflammatory agent for the treatment of peritonitis. Polymer-coated Au nanoconstruct equipped with TNF-α aptamer and ATP aptamer is constructed by interaction between ATP and both ATP aptamer and polymeric phenylboronic acid (pPBA). The phenylboronic ester formed in the nanoconstruct can scavenge ROS and TNF-α aptamer can capture proinflammatory cytokine TNF-α. ROS-responsive removing of pPBA from nanoconstruct with high ROS scavenging capacity, and high TNF-α capturing ability of nanoconstruct are investigated. Thus, these combined properties enable the nanoconstruct an additive anti-inflammatory effects. We demonstrated the high anti-inflammatory effects of the nanoconstruct in vitro and in vivo peritonitis model by monitoring ROS and pro-inflammatory cytokine levels.

Cancer immunotherapy is a clinical strategy that acts on antitumor responses by invigorating the immune system to attack cancer cells. In the treatment of cancer, immune checkpoint inhibitors, i.e. mAbs binding to cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD-1), or PD-1 ligand (PD-L1), neutralize the evadability of cancer cells against the immune system. However, this powerful therapy is hampered by expansive cost for the treatment, as well as by limited efficacy only responding to some patients. Here we show the development of hydrogel-based artificial cells (hAC) that potentiate cancer treatment by increasing the efficacy of bispecific immunotherapy targeting to CTLA4 and PD-L1 simultaneously. The conformation of hAC is characterized by a responsive soft hydrogel nanoparticle consisting of N-isopropylacrylamide-co-acrylic acid, antibody binding protein (protein A), and two immune checkpoint inhibitor mAbs (CTLA4 mAb and PD-L1 mAb, respectively). We demonstrate that the use of hAC significantly decreases the survivability of MCF-7 cancer cells in vitro and suppresses the growth of MCF-7 derived xenografts in vivo in comparison to the mAb treatment by itself. Our result also suggests that the bispecific hAC is more potent to treat cancer cells than monospecific hAC which is conjugated with either CTLA4 mAb or PD-L1 mAb alone. The advantage of hAC over current bispecific antibodies arises from their adaptability to link multiple antibodies in easy and fast, enabling the hAC system for rapid validation of bispecific antibodies in cancer treatment. Our finding has an important implication that hAC-based cancer immunotherapy opens a new paradigm not only to treat cancer but also to evaluate bispecific antibodies.

Retinal prostheses are a promising technology in the field of visual restorative medicine for the treatment of patients with severe stages of retinal degenerative diseases. The highly stable light-activated proton pump bacteriorhodopsin (bR) has a great potential to be used in the fabrication of electrodes for artificial retinal implants due to its optical and photoelectric properties. Its usage in the generation of a photoelectrical signal can show remarkable performance in responsivity and quantum efficiency. In the present work, it is shown the development of a bR-sensitized photoelectrode for its future application in a retinal prosthesis. The study is aiming at analyzing the photovoltaic performance of a photoreceptor cell fabricated with a bR photoanode integrated with different low-cost and carbon-based cathodes of 3,4-ethylenedioxythiophene (PEDOT), graphene, carbon nanotubes, and their hybrids. The protein is immobilized on ITO covered by TiO2 by drop-casting and electrophoretic deposition. The cell is tested using a hydroquinone/benzoquinone-based redox electrolyte. Linear sweep voltammetry, open-circuit voltage decay, and electrochemical impedance spectroscopy are measured under dark and illuminated conditions to determine the influence of different parameters in the function of the bR-sensitized photoelectrode. A mathematical model of the bR-cell equivalent circuit is fitted to the experimental data to further elucidate charge transfer resistance and frequency in the different interfaces of the device. The biological photoreceptor could significantly contribute to reduce and replace traditional costly and harmful materials used in conventional photovoltaic technology with renewable carbon-based alternatives; which could decrease the cost, enhance its performance, and improve the device biocompatibility and sustainability.

Knowing the conformational states of amphiphilic molecules at liquid-liquid interfaces is important for understanding the underlying mechanisms governing interfacial phenomena, including adsorption/desorption dynamics, membrane assembly, and domain formation. Previously, using vibrational sum frequency generation spectroscopy (SFG), we found that a series of simple inorganic anions in aqueous solution at a buried hexadecane-water interface profoundly affected the self-assembly of monolayers of charged, amphiphilic oligomers consisting of ionic liquid (IL) headgroups covalently bonded to hydrophobic tails (oligodimethylsiloxane imidazolium cation (ODMS-MIM⁽⁺⁾), J. Amer. Chem Soc. 142, 290-299 (2020)). Elucidating how aqueous counterions alter the interfacial density of the charged IL headgroups will be critical for understanding monolayer formation at the interface. This talk will describe our use of a liquid-liquid Langmuir trough for controlling the interfacial pressure of the ODMS-MIM⁽⁺⁾ monolayer at the oil-water interface in conjunction with SFG and molecular dynamic simulations. This enables us to characterize how changes in molecular area correlate with conformational changes of the tails and packing of the oligomers in the membrane during assembly, with and without electrostatic interactions with the counterions. The ability to control the packing of the charged oligomers with compression independently from specific ion effects and ion pairing interactions offers new insight into the mechanisms for amphiphile adsorption, membrane structural changes, and hydrogen bonding at the interface that can inform strategies for controlling chemical reaction dynamics at liquid-liquid interfaces.

Infection with protozoa of the genus Cryptosporidium is a leading cause of child morbidity and mortality associated with diarrhea in the developing world. As a zoonotic pathogen transmitted from cattle it is an important threat to public health with its potential for waterborne and foodborne dissemination. Research on this parasite has been impeded by many technical limitations, including the lack of cryopreservation methods. Consequently, laboratory strains of Cryptosporidium must be maintained by propagation in susceptible animals every 6–8 weeks, an expensive and time-consuming process. It is parasite sensitivity to formation of ice and limited permeability to cryoprotectants (CPAs) that precludes utilization of classical methods of slow cooling. To address this gap, a vitrification protocol was considered as an ice-free alternative for cryopreservation. Vitrification, unlike slow cooling, utilizes high intracellular concentrations of cryoprotectants combined with rapid cooling rates to achieve formation of an amorphous glassy solid devoid of ice crystals, thus protects cellular systems during cooling. Because of CPA toxicity, the vitrification field has moved toward the development of containers that enable extremely rapid cooling rates and thus vitrify with lower concentrations of intracellular CPA. In consequence of the reduced thermal mass, these containers are restricted to small volumes limiting widespread implementation of vitrification in clinical and research setting. To address this bottleneck, we developed a cryopreservation protocol that enables increased sample volume through the design and manufacture of a novel polycarbonate high aspect ratio specimen container referred to as ‘vitrification cassette’. This strategy increases the volume of a single cryopreserved sample by 50-fold up to 100 μL compared to the previously reported methods using 2 µL microcapillaries. The high surface area to volume ratio of vitrification cassettes allows to maintain cooling rate at ~10,000 °C/min and supports vitrification of 30% DMSO/ 0.8 M trehalose extracellular cocktail solution. Due to the increase of CPA concentration required for glass formation, toxic effects were mitigated by application of step-wise CPA addition protocol. These cassettes were shown to support preservation of viable and infectious Cryptosporidium oocysts. Oocysts cryopreserved using cassettes exhibit viability at ~75%, maintain in vitro infectivity, and are infectious to interferon-gamma (IFN-γ) knockout mice (C. parvum) and gnotobiotic piglets (C. hominis). Importantly, the course of the infection is comparable to that observed in animals infected with unfrozen oocysts. Vitrification of Cryptosporidium oocysts in larger volumes will expedite progress of research by enabling the sharing of isolates among different laboratories and the standardization of clinical trials. Vitrification cassettes allow to obtain multiple inocula from a single cryopreserved sample thus overcoming a major bottleneck in the development and testing of therapeutics and vaccines for the treatment and prevention of cryptosporidiosis. This technology is readily available for translation to other cellular systems.

Routine glioblastoma multiforme (GBM) imaging in clinics are based on using gadolinium-based magnetic resonance imaging contrast agents. However, using these gadolinium-based contrast agents raises major concerns for GBM patients suffering from chronic kidney disease, which can be resolved by using nanoparticle contrast agents that do not show any renal clearance due to their larger size. Here, we will demonstrate how a new imaging technique, called magnetic particle imaging (MPI) can be used for safer GBM imaging. We have investigated our approach for imaging of several types of brain tumors with different levels of aggressiveness, to identify the feasibility of this method in different brain tumor microenvironments. First, we developed methods for tuning iron oxide nanoparticles (NPs) to generate high resolution (i.e., ~600 µm) MPI images with ultra-high contrast agent mass sensitivity of less than ~550pg Fe/µL. Then, we used MPI for three-dimensional targeted imaging of the orthotopic brain tumors in mice after intravenous injection of these NPs. Iron oxide nanoparticles have been approved by FDA for several clinical applications and we hope that this method will ultimately find clinical applications for safer GBM imaging.

The clinic utilization of photodynamic therapy (PDT) to treat cancers and malignant diseases has recently gained increasing attention. However, the shallow penetration of activated light, the concern of biosafety for new light delivery methods, and the risks of stimulating invasion and metastasis because of incomplete phototherapy have limited the further applications of PDT in deep tissues. Here we present a bioluminescence (BL) energy-induced PDT (BL-PDT) platform through conjugating sufficient clinic-approved photosensitizer chlorin e6 (Ce6) into the surface of luciferase, which can capture most of the biphotons and precisely activate Ce6 to produce reactive oxygen species (ROS). After largely entering cells, the conjugate (named Luc8-Ce6) produced very considerable intracellular ROS and induced cancer cell death. At the same time, the therapeutic efficiency is comparable to Laser-PDT using the same PS and moderate power. The further in vivo studies showed that the conjugate-based BL-PDT resulted in complete tumor remission, prevented the following metastasis in lymph nodes and lungs, and obtained potent neo-adjuvant antitumor efficacy for triple-negative breast cancer tumor after intra-tumoral injection. Our results offer a way to efficiently harness the low energic bioluminescence to achieve noninvasive, depth-independent, and widely applicable phototherapy.

Our laboratory at MIT has been interested in exploring the relatively new interface between living plants and non-biological nanostructures to impart the former with new and enhanced functions, which we call Plant Nanobionics. We have developed a theory of subcellular uptake and kinetic trapping of a wide range of nanoparticles, validated in-vivo in living plants. Confocal visible and near infrared fluorescent microscopy and single particle tracking of Gold-Cystein-AF405 (GNP-Cys-AF405), Streptavidin-Quantum Dot (SA-QD), Dextran and Poly(acrylic acid) nanoceria, and various polymer-wrapped SWCNT, including lipid-PEG-SWCNT, chitosan-SWCNT and (AT)₁₅-SWCNT, were used to demonstrate that particle size and the magnitude, but not the sign, of the zeta potential are key in determining whether a particle is spontaneously and kinetically trapped within chloroplasts or the cytosol. We develop a mathematical model of this Lipid Exchange Envelope Penetration (LEEP) mechanism, which agrees well with observations of this size and zeta potential dependence. As an application, we rationally designed a chitosan-complexed single-walled carbon nanotube (SWNT) as nanocarriers to selectively deliver plasmid DNA (pDNA) to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and expression in living mature arugula (Eruca sativa) and watercress (Nasturitium officinale) plants in planta and in isolated Arabidopsis thaliana mesophyll protoplasts. Another application of nanoparticles and nanotechnology to plant sciences is in the form of biochemical sensors that operate in planta and across diverse species. Using non-destructive optical nanosensors, we find that the spatial and temporal H₂O₂ concentration immediately post-wounding follows a simple logistic waveform for six dicot plant species: lettuce (Lactuca sativa), arugula (Eruca sativa), spinach (Spinacia oleracea), strawberry blite (Blitum capitatum), sorrel (Rumex acetosa), and Arabidopsis thaliana, ranked in order of wave speed from 0.44 to 3.10 cm/min. The H₂O₂ wave tracks the concomitant surface potential wave measured electrochemically for the series of plants. We show that the plant NADPH oxidase RbohD, glutamate receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the H₂O₂ waveform upon wounding. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in planta.

New challenges to global food security are posed by rapid population growth, limited access to resources, and changes in climate patterns. Plant engineering is of great significance in current efforts by improving yield and/or resistance to abiotic/biotic stresses. The precise deployment of functional payloads to plant tissues is a new approach to help advance fundamental understanding of plant biology and accelerate plant engineering. However, the morphological and physiological barriers in plants (e.g., epidermis, cuticle) hinder efficient delivery of payloads. Here, a novel silk-based biomaterial is designed to function in planta. A microneedle-like device (named “phytoinjector”), bio-inspired by the stylet of psyllids fed by xylem/phloem sap, is then fabricated from this material, capable of delivering a variety of payloads ranging from small molecules to large proteins into specific loci of various plant tissues. It is demonstrated that phytoinjector can be used to deliver payloads into plant vasculature to study material transport in xylem and phloem and to perform complex biochemical reactions in situ. Furthermore, it is demonstrated Agrobacterium-mediated gene transfer to shoot apical meristem (SAM) and leaves at various stages of growth. In addition, tuning of the material composition enables the fabrication of another device, dubbed “phytosampler”, which is used to precisely sample plant sap. The design of plant-specific biomaterials to fabricate devices for drug delivery in planta opens new avenues to bridge the biotic/abiotic interface with plants, enhances plant resistance to biotic and abiotic stresses, provides new tools for diagnostics, and enables new opportunities in plant engineering.

Biosensors for the rapid detection of low concentrations of pathogens, toxins, contaminants and metabolites in an on-site fashion is of utmost interest for clinical diagnostics, food safety and environmental monitoring. Albeit many differences in these application fields exists, challenges raised for the detection technology are strikingly similar: Low amounts and numbers of analytes have to be specifically identified and quantified within an immensely complex matrix such as blood, saliva, food stuff and environmental samples to name a few. Our research group addresses challenges arising from this complicated task in part through the development of multifunctional nanomaterials that enable significant improvements in signal transduction and enhancement, immobilization, pre-isolation or in-line purification, respectively. In combination with specific biorecognition and signal read-out, on-site biosensor platform technologies are thus developed that provide significantly lower limits of detection while maintaining a focus on low-cost, ease-of-use and high specificity characteristics.
The presentation will focus on nanomaterials designed as functional transducers, i.e. laser-induced graphene (LIG), and nanofibers. We have recently shown that LIG is a superior transducer in comparison to screen-printed electrodes used in most electrochemical on-site biosensors. Since ease and reproducibility-of-fabrication of such transducers are of utmost importance in the point-of-care and on-site sensor fields, we have investigated strategies to generate LIG and carbon nanofibers using laser-induced pyrolysis. Here, polyimide sheets and electrospun polyimide nanofibers are transformed using a CO2 laser in a simple scribing process under ambient conditions. The resulting 3D LIG structures can be transferred to any other surface or in the case of the nanofibers be used as free-standing transducer, and are therefore very easy to make, to miniaturize, to integrate into sensing platforms including microfluidic systems. In addition, this process also enables straight-forward large-scale production. We will present our most recent work on voltammetric, potentiometric and impedance-based biosensors for medical diagnostics and food safety using these LIG and carbon nanofiber materials. Furthermore, we demonstrate that by doping the nanofiber materials with metal precursors, chemical sensing with most significant sensitivity and specificity can be accomplished. Here, morphological and performance characteristics can be directly influenced by metal content and lasing power and hence can be adapted towards desired performance. Most importantly, we can show that this strategy can be adapted to a whole range of metal precursors and hence provide opportunities for such 3D carbon nanofiber nanocatalyst hybrids.
Nanofibers can also be made out of a variety of other polymers for the purpose of analyte capture, pre-concentration or even as optical transducers. We will hence also discuss nanofibers entrapping upconverting nanoparticles and fluorescent dyes, respectively, to demonstrate unique and intricate characteristics applied to food safety testing.

Increased awareness of the long-term impacts of agrochemicals has resulted in the hunt for efficient and sustainable delivery platforms. In order to meet the food demands of an exponentially increasing global population, there is a dire need to establish sustainable technologies for targeted and slow release of agrochemicals with minimal environmental footprints. Furthermore, rising concerns of the global community about the accumulation and environmental effects of microplastics have accelerated the search for alternate delivery vehicles developed from biodegradable polymers. We present a sustainable approach to synthesize aqueous dispersions of biodegradable cellulose derivatives via anti-solvent precipitation. We propose the utilization of these dispersions as controlled and targeted release formulations for a pesticide as a model active ingredient (AI). While the biodegradable nature of our polymer particles and use of water as the dispersant medium justify the sustainable nature of these formulations, we have investigated dispersion stability, leaf adhesion, rainfastness and AI release profile of the formulations to ensure their efficacy for foliar application. Additionally, we have used Isothermal Titration Calorimetry (ITC) to investigate the nature of polymer interactions with the AI to provide a comprehensive picture of the performance and tenability of the formulations.

Ensuring food safety and security requires improvement of the current food quality monitoring system to mitigate risks of foodborne illness and minimize food wastes. Developing a real-time detection platform for bacterial pathogens and spoilage is a compelling goal to achieve this global mission. Incorporating sensors into packaging is a promising approach, but the lack of proper food contacting materials is challenging to enabling all the functionalities required for the smart-sensing sampling. This study presented a platform for detecting pathogenic bacteria in food using a porous microneedle array by utilizing non-toxic and edible silk fibroins extracted from Bombyx mori silkworms. The silkworm fibroins have emerged as promising technical materials for food contact applications due to their non-toxic and edible nature, in addition to their tunable mechanical and chemical properties. The porous microneedle structure allows extraction and delivery of internal food fluid to biosensors on the backside of the needle array by capillary action. We demonstrate food contamination by identifying E. coli in fish fillets within 16 h of injection through the unique patterns of the polydiacetylene-based colorimetric biosensors. This response by E. coli contamination can be distinct from common food spoilage measured via an increase in sample pH. We also show that the microneedle sensor can pierce commercial food packaging, indicating the feasibility of successfully adapting the technology downstream in current food supply chains. This study will contribute to food quality monitoring, ensuring global food safety, and minimizing food loss and waste.

The food packaging market has become an indispensable aspect of our times as it is estimated to reach 450 billion dollars in revenue by 2027. In accordance with the ramping market projections, there has been an increasing demand for improving food quality and guaranteeing food safety. An intelligent and affordable packaging system that can also act as a tracking platform for food quality is the need of the hour. Since the chemical compounds produced inside a package are good indicators of spoilage of non-perishable foods, several active food quality sensors have been developed. However, active sensors are expensive as they need to carry batteries and electronic chips. Battery-less chipless sensors have gained great attention in food packaging and healthcare because of their low cost and wireless sensing capability and are an ideal candidate for pH sensors in food packaging systems. However, the main obstacles along the progress toward commercializing chipless sensors are the shortcomings in their manufacturing process.

Scalable manufacturing techniques such as printing techniques have been widely used for chipless sensors used in item tracking, gas sensors, and healthcare. While scalable manufacturing techniques offer a significant reduction in the number of processes and resources, it introduces tremendous challenges such as the ink formulation for conductive and functional patterns, the need for custom-made screens, and the time required in the drying and sintering processes. We have identified the roll-to-roll laser processing of metalized films as an economically viable low-cost alternative to conventional printing technologies used for chipless sensor development. This approach offers a one-step process that not only patterns the metallic layer to form predefined traces but also leaves remnant nanostructures in the processed region as thin oxide layers which significantly improves the adhesion between the laser ablated region and the functional material.

We propose a novel approach for laser-assisted manufacturing of chipless sensors that can detect the freshness of fish by sensing the levels of hypoxanthine (HX) released by the fish as it undergoes spoilage. A functional material based on xanthine oxidase (XOD), which is sensitive to the HX levels found in fish products, is developed to be coated on the sensor. HX and XOD are optimized to obtain a quick response from the biosensors. The sensor consists of a spiral pattern made of laser ablated Aluminum-based metalized paper that forms an inductor-capacitor tank circuit. The laser ablation of the metalized paper is done to selectively remove aluminum and expose the paper layer underneath. This process also leads to the formation of thin Al₂O₃ layers of the order of nanometers that can provide substantial adhesion to the functional material. The functional material is deposited on the most sensitive region on the inductive coil so that when the functional material dissolves in response to HX, the resonant peak dampens. The sensors are characterized with the help of a portable reader that sends an interrogation signal in the range of 1 MHz to 100 MHz and detects the amplitude of the resonant peak in the spectrum. Since the amplitude of the resonant peak is a function of the dissolution of the functional material, which is concomitant with the HX levels, this wireless sensing approach is employed in the development of a food quality sensor for fish freshness.

This presentation will demonstrate how low-cost printed and flexible graphene circuits can be tailored for agricultural electrochemical biosensing integrated with open microfluidic devices. The use of inkjet and aerosol printing; spin coating; and laser writing (i.e., laser induced graphene) can be used to create such circuits with high resolution (line widths as low as 20 µm). Moreover, the use of laser scribing of the graphene and/or salt porogens added into graphene inks before printing can be used to further improve the electrical conductivity (< 10 ohms/sq.), electroactive surface area (nano/micro structuring), and surface wettability (hydrophilic to superhydrophobic) of the graphene circuits. These printing and laser fabrication techniques are amenable for use on flexible and chemically/thermally sensitive surfaces including polymers and paper.

The graphene-based sensors are subsequently biofunctionalized with enzymes, ion-selective membranes, and antibodies for pesticide and nitrogen sensing in soil/water samples as well as cytokine and histamine monitoring in cattle serum and fish broth. The detection limits of the biosensors so far have achieved 0.6 nM for organophosphate pesticides; 20 mM for ammonium, potassium, and nitrate ions; 25 pg/mL and 46 pg/mL for IL-10 and IFN-g in cattle serum; and 3.41 ppm in tuna broth respectively. Moreover, we demonstrate how the hydrophobicity of the graphene can be tuned to create multifurcating wedge channels with hydrophilic paths surrounded by superhydrophobic walls that transport and split fluid to distinct biosensors via Laplace pressure. We demonstrate how such open microfluidics can be used to develop multiplexed fertilizer ion and pesticides sensors for the field. Such sensing results demonstrate that such scalable graphene sensor technology is well-suited for in-field, low-cost biosensing that is needed for large scale mapping of contaminants in watersheds and farm fields as well as monitoring large volumes of food products.

Pharmaceutical excipients vary widely in function, being used as absorption enhancers, emulsifiers, preservatives, and sustained release matrices. Here the FDA-approved excipient, Poloxamer P188, is explored as a bioactive excipient in the wheat rhizosphere and contrasted with the natural osmolyte, glycine betaine. The P188 triblock copolymer self-assembles into 5.5 ± 0.3 nm micelles whose size does not vary significantly when encapsulating hydrophobic cargo, such as the plant metabolite quercetin. Column transport studies demonstrated loaded P188 micelles delivered cargo intact, supporting assessment in plant studies. Wheat seedlings were grown in a sand matrix for 14 days, subjected to 8 days of simulated drought, and then recovered for 7 days. To better represent wheat grown in drought-prone regions, seeds were inoculated with a model rhizobacterium, Pseudomonas chlororaphis O6 (PcO6) which has been shown to improve drought tolerance in winter wheat. Seedlings were treated separately with 0.2 mmol P188 or 10 mmol glycine betaine per kg of sand matrix, or with the combined nano-formulation. The quantum yield of photosystem II, total growth of plant mass, and relative water content under drought were measured. Both P188 and the P188 / betaine nano-formulation increased total above ground plant mass relative to the control, while the inclusion of betaine alone suppressed tissue growth and photosynthetic activity during periods of simulated drought but improved performance during recovery. P188 addition resulted in significantly higher relative water content in wheat under drought, suggesting activity as a synthetic osmolyte that resists sorption to soil components while carrying additional cargo intact through the rhizosphere.

Microcapsules are used in a wide range of products including certain types of fertilizers, plant protection products, leave-on and rinse-off cosmetic products to adjust release profiles, stabilize actives and control environmental fate such as volatilization or wash off. The development of microencapsulation is targeted to minimize active levels and limit negative impact on the environment. However, plastic materials are generally used in manufacturing. There is an increasing concern about microplastics dissemination by the public and legislators due to their potential impacts on environment and human health. Here we demonstrate an approach to fabricate biodegradable microcapsules using silk protein as a sustainable alternative. Silk fibroin is a known natural, proteinaceous fiber that can be tailored into a water-soluble suspension for solution processing. Two fabrication methods are employed to encapsulate different types of payloads, including water-soluble (Vitamin C) and -insoluble actives (agrochemicals). Ultrasonic spraying and freeze drying produce uniform microspheres with controllable porosity. Spray drying produces crumpled, but dense microparticles. Increased crystallinity of silk component is achieved by ethanol-induced beta-sheet transformation. The effects of morphology, loading and crystallinity on release profiles are discussed. The resulting silk-based microcapsules offer a solution to current microplastic pollution while retaining adequate delivery performance.

Food contamination in processing facilities and during fresh produce cultivation can lead to serious public health problems and costly recalls of millions of food units. Currently, rapid sensing technology is lacking for definitive detection of foodborne pathogen in food-processing plants and irrigation water during fresh produce production, which delays initiation of appropriate control measures and increases the likelihood of spreading contamination and foodborne illnesses outbreaks. Herein, we report the fabrication of user-friendly biosensors capable of rapid (17-22 min), enrichment-free detection of foodborne pathogens in food processing facilities and hydroponic systems at levels comparable to current culture based and PCR methods (detection limits ~5 CFU/mL). The sensors are fabricated using graphene or stimuli-responsive polymers, and subsequently functionalized with pathogen-specific antibodies or aptamers. Unique laser processing induces a 3D nanostructured topology in graphene that becomes electroactive and hydrophilic, and polymer actuation protocol, i.e., brushes on the extended conformation for bacteria capture and the collapsed state for electrochemical signal transduction are desirable for enabling high signal-to-noise ratios and test sensitivity. An example for Salmonella Tyhimurium detection is provided. After functionalization with Salmonella-antibodies, the graphene-based biosensors were able to detect live Salmonella in chicken broth across a wide linear range (25 to 10^⁵ CFU/mL) and with a low detection limit (13 ± 7 CFU/mL; n = 3). For Listeria monocytogenes and Escherichia coli spp. detection the use of stimuli-responsive brush actuation is demonstrated. After functionalization with Listeria-aptamer and E. coli-antibodies, the stimuli-responsive brush sensors were able to detect live Listeria monocytogenes and E. coli in vegetable broth and hydroponic water across a wide linear range (10-10^⁶ CFU/mL) and with a low detection limit (as low as 3.3 ± 0.9 CFU/mL; n = 3). These results were acquired using a potentiostat with an average response time of 17-22 minutes without sample pre-concentration or redox labeling techniques. After analyzing nanobrush actuation in stagnant media, we developed a flow through system using a series of pumps that are triggered by electrochemical events at the surface of the biosensor. Sense-Analyze-Respond-Actuate proof of concept may be viewed as a cyber-physical system that actuates nanomaterials using smartphone-based electroanalytical testing of samples. These graphene and stimuli-responsive sensors displayed high selectivity as demonstrated by non-significant response to other bacteria strains. These results demonstrate how graphene and stimuli-responsive electrodes can be used for electrochemical sensing in general and, more specifically, could be used as a viable option for rapid and selective pathogen detection in food processing facilities and hydroponic farms.

The efficacy of COVID-19 vaccination is closely related with the serum level of the SARS-CoV-2 neutralizing antibodies that bind to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Therefore, rapid and quantitative measurement of the SARS-CoV-2 neutralizing antibody (NAb) in the sera of vaccinated people is essential to develop the effective vaccine and further achieve population immunity, i.e., herd immunity. Plaque reduction neutralization test (PRNT), the gold standard of NAb effectiveness in serologic test, is accurate but requiring biosafety facilities due to the use of the virus, which hampers its application for common laboratories and clinical sites. Here, we developed a bioresponsive microgel based surface plasmon resonance (SPR) platform that detects SARS-CoV-2 NAb in clinical samples without complicated pre-treatment. We found that protein multivalent binding (PMB) between microgel conjugated RBD protein and the COVID-19 NA yields significantly enhanced SPR signals than those of nonspecific interference from serum proteins in microgel based SPR assay. The excellence of our microgel based COVID-19 NAb test occurs from its selectivity for the NA only with resistance to all other proteins, allowing rapid detection and quantification of NAbs for each individual. Importantly, our results provide that microgel based SPR assay will be considered as a novel NAb detection platform for COVID-19 vaccination strategy in easy and safely.

Metal-organic frameworks (MOFs) are porous crystalline solids with the ability to selectively concentrate large quantities of gases, offering significant promise as building blocks for energy and biomedical technologies. The surfaces of MOFs are frequently modified by polymers to ensure good compatibility or dispersibility in functional materials or liquids; however, the vast majority of current studies focus on MOF-polymer interactions in solid plastic matrices or organic solvents. Here we show that noncovalent complexation with biological or synthetic polymers enables control over the MOF-polymer interface in water. By understanding and manipulating the interface, properties such as MOF dispersibility and structural stability can be finely tuned—for instance, the degradation rate of a hydrolytically unstable MOF in water can be controlled over 2 orders of magnitude depending on the polymer selection. Present studies provide a path toward controlling the properties of MOFs in water and using MOFs as a platform to understand the behavior of macromolecules at porous interfaces in aqueous media, a ubiquitous situation in Nature that is difficult to study given the sensitivity and complexity of biological systems.

Polydimethylsiloxane (PDMS) could be oxidized by the various stresses of electrical devices, such as heat, ozone, electrical and mechanical stress, leading to loss of elastomeric properties and ultimate failure. To reduce oxidation, various antioxidants could be mixed with PDMS during manufacture processes. Since the effective radius of antioxidant molecules is relatively small, the antioxidants need to be mixed in a molecular level. However, the low surface energy of PDMS makes the mixing in a molecular level difficult. In this study, we mixed tannic acid, naringenin and a commercial antioxidant with PDMS and investigated their characteristic changes after thermo-oxidation. We added microparticles of antioxidants to PDMS precursor, and the particles are retained matrix to reduce oxidation effectively. Although antioxidants are mixed in micron-size, the antioxidants in PDMS effectively reduced oxidation. In thermogravimetric analysis, PDMS showed faster mass losses than the samples containing antioxidants under 400 °C. In the tensile tests of dumbbell shape specimens, the antioxidants provided a 3-fold increase in Young’s modulus, while neat PDMS showed an 88-fold increase in Young’s modulus at the thermo-oxidative condition of 3 days and 250 °C. Even though both the neat PDMS and the PDMS containing antioxidants showed similar values of strain-at-break within the error range before heat treatment, neat PDMS showed a significantly smaller strain-to-break (only 10.2%) than the PDMS with antioxidants (46.8%) after 1 day oxidation at 250 °C. The FT-IR spectroscopy result of PDMS containing antioxidant showed much smaller changes in the peaks of 1060 and 865 than that of neat PDMS after oxidation, which are assigned to Si-R stretching. The Si peak analysis of XPS showed that the bonding of silicon atoms in neat PDMS is more oxidized from SiO2C2 to SiO4 compared to the PDMS containing naringenin. These results offer the effective and nontoxic ways to widen the service conditions of PDMS.

Peripheral nervous system (PNS) injuries often lead to partial or complete loss of sensation, chronic pain, apraxia, and even permanent disability. Current guidelines recommend repairing severed nerves with sutures; however, sutures hinder tissue regeneration and often result in scar tissue formation further preventing functional recovery. To limit the need for sutures, fibrin glue has been proposed as an alternative to reattach severed nerve endings, but current formulations are not mechanically robust to significantly reduce the need for sutures. We have been developing new photocrosslinkable hydrogel systems from bioavailable polymers to be used as a glue for nerve anastomosis. Formulations have been developed to improve mechanical strength, flexibility, biocompatibility, adhesiveness, and electrical conductivity.

Conductive biomaterials can amplify endogenous and exogenous signaling that have been shown to play a crucial role in galvanotropism and galvanotaxis, processes that are crucial for neural repair. However, many of the popular conductive biomaterials are brittle, insoluble, opaque, and non-degradable. Choline acrylate has recently emerged as a biocompatible ionic liquid (IL) that can be incorporated into hydrogels to modify bulk conductivity. This IL is a transparent, degradable alternative to popular conductive materials that are not ideal to be placed as a permanent scaffold in patients. Matching the mechanical properties of the native nerve tissue is a crucial consideration when fabricating a new hydrogel composition. Viscoelastic properties have been demonstrated to play a role in maintaining the extracellular matrix (ECM) and tissue structure during normal movements, as well as cell differentiation. We have investigated the role that the addition of IL has on bulk electrical conductivity, biocompatibility, and dynamic mechanical properties.

Mechanical testing and in vitro cell culture demonstrate tunable physicochemical properties of our hydrogel systems, and that these materials support of the growth of both glial and neuronal cells. Furthermore, results from in vivo implantation models demonstrate that the composite is biodegradability and biocompatible. Finally, we have confirmed that a severed nerve anastomosed by this composite has tensile strengths greater than the commercially available fibrin glue. In this talk will discuss our progress and our future work for hydrogel composites for use in nerve repair.

Introduction: Upon the recent pandemic, the COVID-19 testing process has been plagued by missing components, including swabs. Multiple companies and researchers have been focusing on the development of new swabs to collect clinical samples for COVID-19 testing. In order to validate new swabs, innovative experimental procedures are needed to streamline this process. Therefore, we have developed an in vitro tissue model to mimic nasopharyngeal (NP) and mid-turbinate (MT) nasal passages to perform pick-up and release of biological samples from a physiologically relevant nasal mucosa on the bench. There are currently no tissue models available for this purpose, as companies validate their new swabs by dipping in saline without mimicking any of the physiological aspects of the nasal passage (i.e., architecture, mechanical and physical structures). The tissue model will be used to validate NP and MT swabs and advance the design and optimization of the prototypes before reaching the clinical study stage.
Materials and Methods: A tissue model was generated to mimic the nasopharyngeal and mid-turbinate nasal cavities for the validation of NP and MT injection molded swabs. The physiological architecture was realized by 3D printing the Carleton-Civic Standardized Human Nasal Geometry (from Carleton University’s Aerosol Research Lab), with Acrylonitrile butadiene styrene using a Fused Deposition Modeling 3D printer. The nasal mucosa was recreated by a soft tissue structure saturated with a viscous mucous component, to mimic the native tissue. Specifically, lyophilized silk fibroin sponges from Bombyx Mori formed the inner surface of the model to replicate the nasal passage tissue, and saturated with artificial mucus to mimic both symptomatic and asymptomatic conditions, based on Locust Bean Gum (LBG). Both muci viscosities were measured based on LBG concentration to match previously reported values. The asymptomatic (0.23 w/v%) and symptomatic (0.92 w/v%) muci displayed viscosities at 13 and 1400 cp, respectively. The muci were then spiked with heat inactivated SARS-CoV-2 virus (BEI, NR-52286) to achieve a clinically relevant viral concentration. To quantify swab pick-up, gravimetric analysis was performed by collecting samples from the nasal model following CDC guidelines. Samples were collected using NP and MT injection molded swabs in comparison to FDA-approved flocked swab (Obecare # 20200507). Viral loads were then quantified with a qScript XLT 1-Step RT-qPCR against N1 N2 SARS-CoV-2 probes to quantify swab efficacy.
Results and Discussion: We tested standard flocked NP and MT swabs and novel injection molded NP and MT swab using models saturated with asymptomatic and symptomatic muci containing virus. The gravimetric data indicated that the standard NP, MT swabs with asymptomatic mucus and the standard diseased MT swab picked up significantly more than the injected molded NP and MT swabs for those groups. However, the RT-qPCR data from all swabs demonstrated comparable viral loads.
Conclusions: Despite the reduced pick-up volumes from the injected molded swabs in comparison to standard flocked swabs, the viral detection was comparable. The pick-up and release data from the bench top in vitro model are also in agreement with on-going clinical studies, thus, supporting the validation of the in vitro model for pre-clinical swab validation.
Acknowledgements: NIH Rapid Acceleration of Diagnostics (RADx^SM) program, 3U54HL143541-02

Duchenne Muscular Dystrophy (DMD) is a devastating wasting disease that leads to the continual degradation of muscle tissues and eventually premature death. DMD is caused by a rare genetic mutation in the protein dystrophin, which is believed to act as a molecular ‘shock absorber’, helping stabilize the sarcolemma during muscle contraction. As a rare disease, there are limited curative options available for patients, with current drug trials showing a limited success rate. This is due in part to a lack of viable screening methods, with most pre-clinical testing being done in mdx mouse models, which only partially capture the diseased phenotype. As a result, there is a need for improved model systems using human derived tissues, which can more accurately predict patient outcomes and potentially be used for tailoring individualized treatments. Here we describe how engineered tissue constructs can be used to model the contractile phenotype of diseased and healthy patient cells. Using photolithographically defined muscular thin films, we show that muscle fibers derived from human induced pluripotent stem cell (iPSC) lines exhibit defective contractility when carrying an DMD mutation. This strongly supports the hypothesis that intrinsic contractility defects also contribute to the muscle weakness exhibited in DMD patients, in addition to the classical mechanisms of fibrosis and fatty cell replacement. Additionally, we demonstrate that these contractile defects can be largely rescued using prednisolone treatments, in both patient derived and DMD isogenic cell lines, indicating that prednisolone acts directly on mutant fibers, in addition to its anti-inflammatory or immunosuppressive effects. This work provides an in vitro platform for studying the etiology of DMD in human myofibers. Additionally, this study indicates that engineered microphysiological systems can serve as important tools in modeling rare diseases, as these systems are amenable to individualized patient screening and may offer potential insights into the mechanism of pharmaceutical action.

Proteins are attractive precursors for the fabrication of biomaterials due to their inherent biocompatibility, biodegradability, and low toxicity. Moreover, fabrication of protein-based materials can be conducted sustainably due to their ready availability and high aqueous solubility. A wide variety of functional proteins are naturally available with unique properties which may be harnessed to design complex multifunctional materials for several applications including tissue engineering, diagnostic tools, and sensors. However, the high solubility of proteins often results in lower aqueous stability and poor mechanical properties. Protein films must therefore be treated to enhance their stability. However, it is challenging to develop treatment strategies that enhance aqueous stability, are widely applicable to different proteins and preserve their native properties. We developed an additive-free thermal treatment strategy in fluorous media (hereafter referred to as fluorous-curing) that enhances aqueous stability of protein films, while preserving native protein properties such as surface charge and hydrophilicity. In this work, fluorous-curing was utilized in combination with inkjet-printing of proteins to develop patterned protein films with varying surface properties. We investigated the application of these patterned protein films for controlled cellular adhesion and size-based microparticle sorting.

For our study, we utilized two different proteins - anionic bovine serum albumin (BSA) and cationic lysozyme (Lyso). Aqueous protein solutions (protein inks) were loaded into empty cartridges of an inkjet printer to print protein patterns onto a glass surface followed by fluorous-curing. This resulted in patterns of varying charges along the surface. These surface charge patterns were utilized to dictate adhesion of cells on specific regions of the surface with potential applications such as tissue engineering scaffolds and in vitro co-culture models.

Charge-patterned protein films were also investigated as a platform to size-sort charged microparticles. Rapid size-based sorting of small volumes of analytes is a crucial first step in biomedical diagnostic tools such as microfluidic devices or point-of-care diagnostic systems. We integrated a hypersound-based acoustic streaming system with our inkjet-patterned protein films to provide efficient sorting of microparticles on the basis of particle charge and size. The hypersonic resonator enabled us to generate microvortices in the fluid that exerts a curvature-dependent drag force on the microparticles adhered to the protein surface. This allowed for the translocation of microparticles based on the strength of their electrostatic interactions with the patterned protein surface and the drag force exerted by the microvortices. As the drag force was curvature-dependent, larger particles experienced stronger forces and translocated further on the patterned protein surface. This difference in translocation allowed for efficient (~95%) sorting of microparticles. The combination of inkjet-patterned protein films and hypersound-based acoustic streaming enables precise manipulation of particle movement at the surface and may be used for sorting a variety of chemical or biological analytes or studying interactions of analytes with protein-based surfaces.

Introduction: The immune system evolved mechanisms to respond not only to specific molecular signals, but also to biophysical properties such as binding affinity. Unfortunately, the connections between biophysical parameters and immunological outcomes are poorly understood in the context of disease. Understanding this connection would enable better design and translation of new therapies for autoimmune diseases like multiple sclerosis (MS), where the immune system mistakenly attacks myelin in the central nervous system. We posited that inhibiting inflammatory signaling while myelin self-antigen (MOG) is processed by immune cells could reduce autoimmunity and promote immune tolerance during MS. Here we show electrostatically self-assembling complexes formed from an anionic regulatory ligand (GpG) and a cationic MOG peptide improve paralysis in a mouse model of MS. Because efficacy was dependent on antigen-sequence, we probed how antigen sequence and charge impact electrostatic self-assembly of immune signals and how changes in biophysical features of the complexes link to immune tolerance. We hypothesized increasing cationic charge of MOG peptide sequences would increase the affinity with which MOG peptides adsorb to GpG. We speculated if increased affinity would improve uptake efficiency of signals by immune cells and increase polarization of self-reactive T cells to protective regulatory phenotypes.

Methods: To vary the charge of MOG peptides, we anchored MOG with 2, 3, or 9 cationic residues of lysine (K) or arginine (R). We used surface plasmon resonance to measure how binding affinity between MOG peptides and GpG changed as a function of total peptide charge. We measured signal loading by gel electrophoresis to assess how self-assembly of MOG and GpG changed as a function of charge balance in solution. To probe how GpG impacts signaling in dendritic cells (DCs), we stimulated DCs with the inflammatory signal CpG, treated DCs with MOG/GpG complexes, and then analyzed expression of inflammatory markers CD80/86 by flow cytometry and MyD88 by RT-qPCR. To determine if treated DCs could polarize T cells towards regulatory phenotypes associated with immune tolerance, we cultured DCs with MOG-specific T cells and measured expression of regulatory markers FoxP3 and CD25 by flow cytometry.

Results: When anchoring MOG with K or R, the dissociation rate constant (K_D) decreased as the number of anchored residues was increased from 2 to 3. This observation indicates that the binding affinity between MOG and GpG increased when MOG exhibited a more positive charge. Signals were fully loaded into complexes when the charge balance in solution was neutral. If the charge ratio of cationic MOG to anionic GpG skewed towards GpG, GpG remained in solution. If the charge ratio skewed towards cationic MOG, then GpG was fully loaded but MOG remained in solution. When stimulated with CpG, DCs treated with MOG/GpG complexes or soluble GpG significantly reduced CD80/86 and Myd88 expression. This result indicates that GpG remained active when delivered in complexes. Intriguingly, DCs treated with MOGK₉ and MOGR₉ complexes expressed significantly more Myd88 relative to DCs treated with other complex formulations. GpG may not have been as readily available to inhibit inflammatory signaling when complexed with MOGK₉ or MOGR₉, possibly due to tighter binding with MOG because of the larger cationic charge per peptide. Furthermore, only MOGK₂ and MOGR₂ complexes polarized MOG-specific T cells towards regulatory phenotypes.

Conclusions: DCs were most effective in polarizing regulatory T cells when treated with complexes containing peptides of lowest cationic charge. Our results suggest self-antigens designed to self-assemble with tolerizing cues without limiting bioavailability of the signals are most effective in polarizing regulatory T cells. This granular understanding of nanomaterial-immune interactions contributes to more rational immunotherapy design.

Obesity and its implication on type-2 diabetes (T2D) have reached epidemic proportions worldwide. The urgent need to find a safe and effective therapies for treatment of these chronic disorders has gained tremendous attention in scientific community. Recent studies have shown that peptide hormones such as Oxyntomodulin (Oxm) and its analogue 2-aminoisobutyric acid oxyntomodulin (Aib2-Oxm) are extremely safe and effective for body weight loss and regulating the blood glucose level to treat T2D patients. Despite their promising therapeutic effects they exhibit a short in vivo bioactivity in serum due to the enzymatic degradation. To address this challenge, it was reported that when these peptides are packed in fibrillar structures they are much less exposed to any degradation and hence their bioactivity is significantly improved. Therefore, it is essential for developing a systematic approach for explore strategies that control the formation of such fibrils and their subsequent controlled dissociation in vivo once injected in patients. An understanding of the fibrillation and dissociation processes of such peptides requires a quantitative knowledge of the kinetics and thermodynamics of fibril formation and dis-assembly; such measurements that to date have remained elusive for these peptides. In this work we report for the first time a through thermodynamic and kinetic study of fibril formation and fibril dissociation using quartz crystal microbalance with dissipation (QCM-D) and bulk experiments for Oxm and Aib2-Oxm. We investigated the speed of peptide attachment and detachment as well as related thermodynamics processes via the Gibbs free energy change for fibril formation as well as fibril dissociation. Our study showed that Oxm and Aib2-Oxm peptides are spontaneously self-assembling into fibrils whilst Oxm fibrils are more stable than Aib2-Oxm fibrils. Therefore, Aib2-Oxm fibrils are more feasible and faster to dissociate in vivo relative to Oxm fibrils which indicates higher bioactivity of Aib2-Oxm in serum relative to Oxm. On the other hand, the higher fibrillation speed of Oxm relative to Aib2-Oxm highlights higher propensity of Oxm for fibril elongation than Aib2-Oxm.

Changes in climate patterns and soil depletion are major challenges that agriculture needs to address to build a sustainable food infrastructure that can feed the growing human population. In semi-arid regions, water is the determining factor for crop production and water stress during seed germination and early seedling growth is the highest cause of crop loss, with dramatic impact on food security for 1 billion people that are threatened by desertification. In nature, some seeds (e.g. chia, basil) produce a mucilage-based hydrogel that creates a germination-promoting microenvironment by retaining water, regulating nutrients entry, and facilitating interactions with beneficial microorganisms. Inspired by this strategy, a two-layered biopolymer-based seed coating has been developed to increase germination and water stress tolerance in semi-arid, sandy soils. Seeds are coated with a silk/trehalose inner layer containing rhizobacteria and with a pectin/Carboxymethylcellulose (CMC) outer layer that upon sowing reswells and acts as a water jacket. Using Phaseolus vulgaris (common bean) cultured under water stress conditions in an experimental farm in Ben Guerir, Morocco, it is demonstrated that the proposed seed coating effectively delivers rhizobacteria to form roots nodules, results in plants with better health, and mitigates water stress in drought-prone marginal lands.

The use of wearable and implantable devices allows continuous monitoring of physiological metrics, personalized drug treatment and rapid sharing of data between patients and caregivers, offering unprecedented diagnosis and treatment opportunities. In this contest, ingestible electronics devices represent a key tool supporting medicine since the ‘60s [1]. To date, these devices are used mainly in diagnostic and administrated in controlled hospital conditions because of costs, need of recollection, and risk of retention.
Nowadays, the research has a chance to move from ingestible to edible electronics to develop safe and self-administrable edible devices[2]. This transformation requires the introduction of new materials coming from food derivatives or approved by FDA/EFSA. In this context, new communication strategies to deliver diagnostic and treatment progress information are required. The most promising communication strategy is the Intra Body Communication (IBC) [3]. IBC is a wireless bioinspired communication platform that exploits body's ionic conductivity to propagate a signal (e.g. nervous system), generating a body area network (BAN). Such technology has the advantage of low power consumption (<10pJ/bit) and the possibility to employ high impedance conductors[4], compatibles with present edible batteries and conductors[2].
To date, IBC has been mainly exploited for wearable applications. The only example of IBC for an ingestible device was proposed by Proteus Digital Health Inc. to monitor the pharmacological adherence[5]. These models of IBC show static systems where the signal is not modulated in time by physiological processes. In this work, we introduce IBC through a fully edible pill employed for tunable and monitored drug release.
In particular a fully organic edible pill is proposed, composed by:
1. A green and large scale processable edible conductor (0.01 S/cm) exploited for the IBC signal transmission;
2. An insulating edible composite, including a transient bioinert matrix and the drug (Metformin and Amoxicillin), enabling erosion triggered drug release mechanism along different parts of gastrointestinal (GI) tract.
Upon ingestion, the IBC signal is triggered by the pill contacts with GI solution. Then, the signal is modulated along the GI tract by the insulating edible composite dissolution and consequent system impedance variation occurring during the drug release. The signal is switched-off by the complete pill dissolution. In combination with a wearable patch the system will provide information on pharmacological adherence and real-time drug release monitoring.
The drug release mechanism has been characterized in vitro employing simulated gastric and intestinal fluids. Multiple and controlled drug release along the GI tract has been obtained tuning the dissolution properties of the insulating edible matrix through its composition, resulting in release time tunable between 20 minutes to 3 hours. Meanwhile, the insulating material dissolution leads to an impedance reduction of 3 order of magnitude, which modulate the IBC signal and describe the drug release profile.
This technology could pave the way to higher personalized medicine being tailored by patients needs, response, and risk.

1. Farrar, J.T., V.K. Zworykin, and J. Baum, Pressure-sensitive telemetering capsule for study of gastrointestinal motility. Science, 1957. 126(3280): p. 975-976.
2. Sharova, A.S., et al., Edible electronics: The vision and the challenge. Advanced Materials Technologies, 2021. 6(2): p. 2000757.
3. Zimmerman, T.G., Personal area networks: Near-field intrabody communication. IBM systems Journal, 1996. 35(3.4): p. 609-617.
4. Das, D., et al., Enabling covert body area network using electro-quasistatic human body communication. Scientific reports, 2019. 9(1): p. 1-14.
5. Hafezi, H., et al., An ingestible sensor for measuring medication adherence. IEEE Transactions on Biomedical Engineering, 2014. 62(1): p. 99-109.

We have developed microbubble ultrasound imaging contrast agents with specific molecularly targeting ligands with the goal of detecting and treating abdominal surgical adhesions. Abdominal surgical adhesions are a major burden to the US healthcare system with estimated annual costs of over $5 billion. During abdominal surgery, the inflammatory response combined with surgically induced ischemia leads to the formation of a fibrin-rich exudate into the peritoneal cavity which can crosslink apposing tissues. Example complications are small bowel obstruction and female infertility. Current FDA-approved strategies to prevent abdominal surgical adhesions are limited because they are site-specific and require the surgeon to predict a priori where the adhesion will form. Moreover, surgical adhesion removal often involves a second surgery, which ironically can lead to additional adhesions. A desired feature of the ultrasound contrast agent is particle and size stability in physiologically relevant fluids. Specifically, an optimal balance must be struck between microbubble stability (maintaining imaging contrast for diagnosis) and reasonable inertial cavitation thresholds (allowing for fibrin breakup). Moreover, care must be taken to ensure no unwanted damage is done to the underlying mesothelium throughout the process. By tuning the crosslink density of the microbubble shell, we show particle and size stability can be controlled. This finding is important for eventual clinical translation because current microbubble in vivo lifetimes are much too short for targeted imaging applications. We will describe our combined in vitro and in vivo approach toward developing a theranostic agent for detecting and treating nascent abdominal surgical adhesions.

The fast-disintegrating oral delivery systems are gaining more interest in the pharmaceutical and food industry [1]. Recently, electrospinning of nanofibrous webs incorporating active agents was shown to be a very favorable approach for the development of fast-disintegrating delivery systems. The very large surface area and highly porous structure along with the amorphous state of the active ingredients make these nanofibrous webs suitable for fast-disintegrating oral delivery systems since they can readily dissolve or disintegrate upon contact with liquid medium [2-4]. Cyclodextrins (CyDs) are classified as cyclic oligosaccharides and they are even being used in drug formulations since hydrophobic drugs become water-soluble by inclusion complexation with the doughnut-shaped cavity of CyD molecules. Moreover, such inclusion complex systems can enhance the bioavailability and stability of drugs [5]. The electrospinning of polymer-free inclusion complex formulations can be yielded into nanofibrous webs with fast-disintegrating character and such electrospun nanofibrous webs would be quite proper for designing fast-disintegrating oral delivery systems for pharmaceutical products and/or dietary supplements [3-4]. Additionally, the electrospinning of nanofibers from aqueous solutions of CyD inclusion complexes has the advantage where it is possible to use only water for the electrospinning without the need of polymeric carrier and without the need of using organic solvents. Moreover, CyDs are highly water-soluble compared to polymeric matrices, hence, CyDs being as a fiber matrix and inclusion complex host, nanofibers solely electrospun from inclusion complexes would dissolve instantly in the oral cavity without the need for water and provide enhanced solubility to active compounds by inclusion complexation. Also, by polymer-free electrospinning of these systems, much higher active compound loadings are possible if needed for the finished dosage forms. In our studies, we have chosen ferulic acid as a model bioactive compound for the electrospinning of polymer-free nanofibrous webs of inclusion complex for the development of orally fast-disintegrating delivery systems [6]. Recent studies have shown that ferulic acid displays a wide range of physiological properties such as antioxidant, anticarcinogen, anti-inflammatory, antimicrobial and neuroprotective. However, the low water solubility and stability of ferulic acid result in a lack of bioavailability and activity which limits further applications. On the other hand, its water solubility and bioavailability can be enhanced by inclusion complexation with CyD. Here, the free-standing inclusion complex nanofibrous webs of ferulic acid were produced using electrospinning technique from two different modified cyclodextrin derivatives of hydroxypropyl-beta-cyclodextrin (HP-β-CyD) and hydroxypropyl-gamma-cyclodextrin (HP-γ-CyD). It has been detected from the in vitro release and disintegration tests that, the amorphous state of ferulic acid based on inclusion complex formation, and the highly porous feature and high surface area of nanofibrous mats have ensured the fast dissolution/release of ferulic acid and disintegration of nanofibrous mats into the liquid medium and artificial saliva. The results elucidated that electrospun nanofibrous webs of ferulic acid/CyD could be promising material as a fast disintegrating oral delivery system.

References
[1] V. A. Saharan, Current Advances in Drug Delivery Through Fast Dissolving/Disintegrating Dosage Forms., Bentham Science Publishers, Sharjah, 2017.
[2] D. G. Yu, J. J. Li, G. R. Williams, M. Zhao, J. Control. Release. 2018, 292, 91-110.
[3] A. Celebioglu and T. Uyar, RSC Med. Chem., 2020, 11, 245-258.
[4] A. Celebioglu and T. Uyar, T. Mol. Pharm., 2019, 16, 4387-4398.
[5] T. Loftsson and M. E. Brewster, J. Pharm. Pharma., 2010, 62, 1607–1621.
[6] A. Celebioglu and T. Uyar, Inter. J. Pharma, 2020, 584, 119395.

Fast-dissolving drug delivery systems are receiving increasing attention lately due to their enhanced bioavailability and physiochemical properties. One technique of such studies is to create an electrospun nanofiber web of an inclusion complex with the drug of study. The porous nature of these electrospun nanofiber webs renders this system readily soluble in liquids, thereby improving the bioavailability of the drug of study. A fast-dissolving system is developed for prednisolone, a drug that is often used to treat allergic reactions [1]. The system aims to create an inclusion complex with hydroxypropyl-beta-cyclodextrin (HPβCD) and generate electrospun nanofibrous webs from this system. Cyclodextrin is a starch derivative often known for its presence in enhancing solubility, shelf life, stability of such products as drugs, essential oils, dietary supplements, flavor/fragrance, etc. [2]. There are mainly three native types of cyclodextrin molecules (α-, β-, and γ-CD) in addition to some engineered varieties, which includes HPβCD [2, 3]. Because HPβCD is a ring-shaped molecule with a cavity in the center [2], it can envelope individual drug molecules, commonly at a CD-to-drug ratio of 1/1 or 2/1 [4]. The usage of HPβCD is known for increasing the physical and chemical properties of various substances [4]. Here, HPβCD is used to improve the aqueous solubility of prednisolone, a drug that is barely soluble in water on its own. The HPβCD/prednisolone inclusion complex allows the drug to dissolve quickly in the presence of saliva so that the consumer does not need to struggle with swallowing a large pill. The inclusion complex with the drug molecules is initially in the form of a spinning dope, which will yield a sheet of flexible, free-standing nanofibers after going through the electrospinning process. By connecting the spinning dope to one end of the circuit and a metal collector on the other end, an application of voltage will create the electromagnetic field that enables the collection of nanofibers [5]. The simple sheet of nanofibers produced here, composed of the inclusion complex, is to be placed on the tongue. The sheet will instantly dissolve when in contact with saliva, thereby eliminating the difficulty of swallowing. On the other hand, pullulan is a common polymer for creating electrospun nanofibers and pullulan/prednisolone nanofibers were produced as a control sample. The use of HPβCD thereby eliminates the need for polymer dopes to provide structural integrity in producing free-standing nanofibers. This fast-dissolving drug delivery system with a freestanding nanofiber web appears favorable and offers new potential for the pharmaceutical industry.

References
[1] Bashar, T., Apu, M. N. H., Mostaid, M. S., Islam, M. S., & Hasnat, A. (2018). Dose-Response, 16, 1559325818783932.
[2] Del Valle, E. M. (2004). Process Biochemistry, 39, 1033-1046.
[3] Celebioglu, A., & Uyar, T. (2021). Food Hydrocolloids, 111, 106264.
[4] Celebioglu, A., & Uyar, T. (2020). RSC Medicinal Chemistry, 11, 245-258.
[5] Greiner, A., & Wendorff, J. H. (2007). Angewandte Chemie International Edition, 46, 5670-5703.

The wound healing process is a dynamic and highly regulated series of events, comprising four main steps: hemostasis, inflammation, proliferation and remodeling. Fibroblasts and keratinocytes represent the main cell types involved in the process, as they synthetize cytokines and collagen to regulate the inflammation process and to build the extracellular matrix, respectively. For this reason, tissue repair mechanisms can be improved by stimulating cell proliferation through the employment of microfibrous scaffolds, that can effectively mimic the 3D conformation of the extracellular matrix, and, when loaded with bioactive molecules, to boost the cellular restoration at the wound area. Nowadays, materials derived from natural resources are becoming an optimal choice to create alternative wound dressing.
In this work we fabricate natural 3D microfiber scaffolds loaded with vitamin C for the treatment of skin lesions, by combining four plant-based molecules: zein, an alcohol-soluble protein derived from corn; low-methoxy pectin, the most abundant hydrophilic polysaccharide in the plant cell wall; soy lecithin, used as emulsifier agent; and vitamin C, able to exert antioxidant activity and stimulate collagen synthesis. The polymeric microfibers were produced via vertical electrospinning of emulsions. Five samples (labeled ZPC0, ZPC1, ZPC2, ZPC3, ZPC4) containing different concentrations of vitamin C (0 – 10 mg/mL) were fabricated and their cross-linking via CaCl₂ was optimized, in order to enhance their water resistance. SEM imaging was conducted to analyze the morphology of the samples and FTIR analysis was carried out for the chemical characterization. An in vitro drug release assay was set up by incubating the samples in PBS (pH 7.4) at 37°C for up to 24 hours, while a DPPH assay was performed to test the radical scavenging activity of the VitC released in the PBS from the microfibers. The antioxidant activity was evaluated in vitro on Cultured Human Keratinocyte (HaCaT) cells, testing their response toward H₂O₂ by DCFH-DA assay. Swelling and degradation rate were monitored by immersing at 37°C for a week the fibrous matrices in PBS alone or with a protease. The biocompatibility was evaluated on Human Dermal Fibroblast adult (HDFa) cells and HaCaT cells by MTS assay.
SEM micrographs showed bead-less and ribbon-like microfibers. The increase of vitamin C content in the samples resulted in an expansion in the fiber average diameter, from 0.84 to 1.19 μm and the CaCl₂ cross-linking strategy helped maintaining the microfibers structure. In vitro drug release indicated a burst release of the vitamin C from the microfibers within 3 hours of immersion. The cross-linking treatment was successful in slowing down the release of the bioactive molecule in the first hour of immersion in PBS. The DPPH assay results indicated that the radical scavenging activity was present both in non-cross-linked and cross-linked microfibers, even if this propriety was higher for the non-cross-linked ones. The DCFH-DA in vitro assay on HaCaT cells showed a response to the H₂O₂ proportional to the amount of VitC loaded inside the samples. Degradation assay showed a higher weight loss of the samples when immersed in PBS with protease, compared to the immersion in PBS alone. In vitro MTS assay on HDFa and Keratinocyte cells resulted in good biocompatibility for each sample under study. Taken together, these results confirmed that our designed plant-based electrospun microfibrous patches represent a suitable, naturally-derived biocomposite for wound healing applications.

Fast-dissolving oral drug delivery systems are gaining more interest in the pharmaceutical industry as a means of administering active pharmaceutical ingredients through tablets, films, and, recently, electrospun nanofibrous webs [1,2]. The large surface area and highly porous structure of nanofibrous webs make nanofibers a promising means of drug delivery, as they readily dissolve in the oral cavity [3-5]. Cyclodextrins (CDs) are classified as cyclic oligosaccharides and are used in drug formulations to increase the water-solubility of hydrophobic drug molecules via inclusion complexation with the doughnut-shaped cavity of CD molecules [2,6]. In this study, an orally fast-dissolving drug delivery system was developed by incorporating inclusion complexes of Griseofulvin/Cyclodextrin with pullulan—a non-toxic, water-soluble biopolymer—to form nanofibrous webs via electrospinning. Griseofulvin is used widely as an antifungal, but it is poorly water-soluble and suffers from low bioavailability. In this study, hydroxypropyl beta cyclodextrins (HPβCD), which have been already used in the pharmaceutical industry due to its high-water solubility, were used to form inclusion complexes [6]. Through inclusion complexation with HPβCD, the water solubility and thus bioavailability of the drug were enhanced. The electrospinning of Pullulan-Griseofulvin/HPβCD solutions yielded defect-free nanofibers collected in the form of self-standing and flexible nanofibrous webs. The formation of inclusion complexes between cyclodextrin and the drug was confirmed using FTIR and XRD analysis, the thermal analysis of the fibers were examined using DSC and TGA, and increased solubility of the drug after complexation was confirmed with UV-Vis spectroscopy. The generated electrospun nanofibrous webs readily disintegrated when wetted with artificial saliva indicating that electrospun Pullulan-Griseofulvin/HPβCD web could be a promising material as a fast-dissolving oral drug delivery system.

References
[1] Muruganantham, S., Kandasamy, R., & Alagarsamy, S. (2021). Critical Reviews in Therapeutic Drug Carrier Systems, 38, 1-35.
[2] Dodero, A., Schlatter, G., Hébraud, A., Vicini, S., & Castellano, M. (2021). Carbohydrate Polymers, 264, 118042.
[3] Ponrasu, T., Chen, B. -., Chou, T. -., Wu, J. -., & Cheng, Y. -. (2021). Polymers, 13, 1-17.
[4] Gao, S., Liu, Y., Jiang, J., Li, X., Ye, F., Fu, Y., & Zhao, L. (2021). Colloids and Surfaces B: Biointerfaces, 201, 11162.
[5] Celebioglu, A. & Uyar, T. (2020). RSC Medicinal Chemistry, 11, 245–258.
[6] Celebioglu, A. & Uyar, T. (2021). Materials Science & Engineering C, 118, 1115142.

Podocytes are highly branched and specialized epithelial cells in the kidney that wrap around glomerular capillaries to form a filtration barrier for the purification and regulation of blood. Many kidney diseases are related to podocyte dysfunction, and limited treatment options make kidneys the most waitlisted transplant organ around the world. Physiologically relevant tissue-engineered models of podocyte function are thus needed to improve our understanding of kidney disease and discover new treatments.

However, in vitro, podocytes have severely limited maturation, reach a plateau of gene expression and morphological development that remains far from native levels, and thus lacks relevance to modeling of the native condition, which is a major bottleneck in research progress. Not only are the cells unable to develop multi-level branching morphology, but there is a lack of reliable read-outs that can assess this complex interdigitating morphology in vitro in a way that cross-references with in vivo practices.

We have taken an interest in the role of fractal structures and how they contribute to the development of cells and tissues in which these patterns are so ubiquitous.

Fractals are structures with features that repeat at multiple scales, like the leaves of a fern or a cascading Russian doll. The fractal self-similarity adds complexity to these structures, in the way that makes a mountain different from a simple cone. Our hypothesis was that fractal complexity in shape cues, akin to those of looping glomerular capillaries on which podocytes grow, will trigger fractal complexity in podocyte maturation, leading to more fractal branching, by patterning relevant machinery on multiple scales.

First, biomimetic fractal substrates were designed by tracing native histology cross-sections
of glomerular capillaries, patterning the designs onto positive photoresist using direct laser writing lithography, and then reflowing to generate curvature. These patterns were then inverse-molded with PDMS to generate 2.5-D scaffolds which were inserted into well plates to use in standard cell culture techniques. Cells cultivated on fractal scaffolds developed higher expression and localization of integrin, cytoskeletal, glycocalyx, and slit diaphragm proteins compared to flat and non-fractal topographical scaffolds, and revealed greater maturation extent by RNA sequencing results. Cells also grew preferentially on topographic features when present.

Remarkably, we observed that the 2.5-D fractal substrates exhibited curvature-dependent patterning of collagen I molecules overtop of the scaffold topography, in perfect correlation with kelvin probe force microscopy measurements of increased charge density and zeta potential on curved versus flat regions of the material surface.

Thus, we present the notion that fractality induces clustering of proteins by concentrating charge in the material interface, which facilitates hierarchical assembly of cell structures resulting in overall advancement of whole cell branching morphology. We observed that 2.5-D fractal shape patterning was dose-dependent, that cells were more sensitive to stimuli when grown on fractal scaffolds, and the morphology could be directly compared with in vivo experiments, thus supporting physiological relevance and reliability. To document platform utility, we demonstrate that this new kidney-on-a-chip model can be used for screening of nephrotoxic drugs, for studies of the effects of coronaviruses on the kidney and screening of factors that lead of kidney rejection in patient serum.

Over the past few years, naturally-derived polymers are being increasingly considered as promising coating materials to obtain novel drug-eluting devices, such as drug-eluting stents (DES). Natural biodegradable polymers include proteins (e.g. zein), and polysaccharides (e.g. alginate), which are not only biocompatible but also release non-toxic degradation products, easily cleared from the body [1]. Moreover, coatings fabricated with naturally-derived polymers may limit adverse effects such as exaggerated inflammatory response and delayed re-endothelialization, which often occur when residual synthetic polymers remain in place [2].
In our study, we developed novel biodegradable zein-based bilayer coatings on stainless steel with improved biocompatibility with respect to synthetic coatings and bare metal cardiovascular devices. Rutin, a plant-based flavonoid, with several biological properties, including anti-inflammatory and antioxidant potential, was loaded into a zein polymer layer. Experiments were carried out to assess the release profile of rutin from single and bilayer coated substrates (up to 21 days) and the resulting antioxidant activities. The fabricated coated surfaces were subjected to extensive physico-chemical characterizations and in vitro biocompatibility testing using human umbilical vein endothelial cells and primary human fibroblasts.
To better mimic the physiological conditions and recreate the dynamic release mechanisms that exist in vivo in a coronary artery, we studied the behavior of our zein-based coating in contact with a flowing liquid. In particular, for these dynamic analyses, we designed and developed a versatile microfluidic platform, which is modeled to mimic the microenvironment of a coronary DES. Experiments were performed to monitor the degradation behavior and drug release from the coated microfluidic channels in contact with several blood-simulated fluids (differing in terms of viscosity and chemical composition), flowing at various flow rates.
Our findings confirm that the proposed plant-based coatings fulfill the key requirements for successful drug-eluting cardiovascular devices. The exclusive use of green solvents and natural polymers can indeed ensure biocompatibility, promotion of cell proliferation, and sustained release. Moreover, our on-chip platform allowed recapitulating the physiological bloodstream microenvironment. Therefore, we consider this study as an important contribution towards a better understanding of polymer degradation and drug release under static and dynamic conditions, which are critical for developing more efficient and optimized local drug delivery systems featuring naturally-derived polymers.

References
[1] G. Suarato et al. Front Bioeng Biotechnol. (2018) 6:137
[2] T. Palmerini et al. J Am Coll Cardiol. (2014) 63(4):299-307

Nanoparticles that can be optically tracked are of interest for cell and organismal biodistribution studies. NPs with surface functionalizations linking or adsorbing dyes or fluorophores may detach or leach, resulting in artifacts. Materials with encapsulated fluorescent molecules or quantum dots (QDs) are of interest for bio tracking while avoiding artifacts due to leaching. NP surfaces altered to carry dyes or fluorophores are also anticipated to affect toxicity profiles, protein interactions, and cell-uptake. We have prepared two types of polymeric nanoparticles with encapsulated quantum dots or fluorescent dyes in order to characterize cell interactions, uptake, and toxicity of polymeric NPs with inert surfaces. These materials have applications in nanoplastics toxicity as well as other applications in which pristine surfaces are necessary. Transparent polymer encapsulated fluorescent molecules or QDs can also be surface functionalized for cell targeting, drug or protein conjugation, while avoiding steric hindrance, electrostatic compatibility issues, and enhanced hydrodynamic sizes imparted by surface bound fluorescent molecules or QDs.

The skin wound healing process comprises multiple mechanisms, as a result of intricated biochemical events and external factors. Any impairment within the correct interplay of these events could lead to the onset of chronic lesions, with potential effects on the patience quality of life. To correctly support the tissue regeneration, the appropriate design of a wound dressing relies on the fine tuning of its mechanical properties and its degradation process, which need to follow the dynamics of the lesion repair, preserve the physiological healing evolution, act as a shield against infections, and release bioactive principles. By providing structural framework and biochemical cues, the extracellular matrix (ECM) is fundamental for cellular organization and tissue formation.
Nature itself can constitute the source of inspiration for the development of fully biodegradable materials, with enhanced bioactive potentialities. In today’s World, the use of natural polymers and materials of biomass origin, either of protein derivation or polysaccharide-based, is becoming a pressing need, to meet the demand of environmentally sustainable technologies to support our future challenges. including applications in the fields of healthcare and wound management. Within this frame, keratin from wool (textile waste, animal biomass) and mycelial materials constitute a valuable source of renewable raw materials.
Keratins are characterized by a high content of cysteine residues, which promotes disulfide inter- and intra-molecular bonding and confers chemical stability and mechanical resistance. Moreover, keratin and its derivatives have gained consideration within the biomedical field, thanks to their good cyto-compatibility and cell adhesion properties. In our recent studies, keratin has been extracted from wool and characterized analytically. From the extracted protein, keratin-based fibrous biocomposites have been obtained using all water-based approaches, suitable for cell culture applications. Thermal crosslinking greatly affected the electrospun matrices biodegradability and water stability, without altering the 3D fibrous network, crucial for primary human fibroblasts attachment. Moreover, encapsulation of active principles within the keratin fibrous structure enhanced the mechanical compliance of the dressing and conferred bioactive properties to the resulting patch.
Another very promising biomaterial alternative is constituted by mycelia, self-growing materials, composed of elongated cells (hyphae), that penetrate a substrate uptaking nutrients and developing a fibrous, 3D interwoven structure. Their chemical constituents can be finely tuned by changing either strains and growth conditions or the feeding substrates. The distinctive porous architecture and the chemical biofunctional groups elect mycelia as potential candidates for the design of novel bio-scaffolds. We therefore conducted an explorative study on the direct use of two edible fungal mycelia, Ganoderma lucidum and Pleurotus ostreatus, as sustainable alternative for the controlled fabrication of an all-natural and low-cost biomedical scaffolds. Several physicochemical properties of the inactivated systems were thoroughly investigated, and biocompatibility in vitrowas tested with primary human dermal fibroblasts, revealing how cells successfully adhered to the 3D matrix. Moreover, in a preliminary in vivostudy of a chronic wound model, rats treated with mycelial scaffolds showed well-formed granulation tissue, collagen deposition and completely regenerated epithelium
The above-mentioned biocomposites can be processed using water-based strategies or by simply tuning their self-growth, in a quest of a more environmentally “green” approach in the biomaterial design.More importantly, their physico-chemical properties and morphologies can be customized to design a new generation of scaffolds for skin wound healing.

Multifunctional nanostructured inorganic materials provide unique advantages in interfaces for applications in medicine and biology. When coupled with organic soft polymer substrates that more closely match the mechanical properties of biological tissues, stable devices can be created for monitoring and maintaining health. This work reports on the growth and integration of self-organized nanotube arrays on biocompatible and bioinspired polymer substrates as a novel drug-eluting platform for controlled drug release from flexible devices. To this end, we developed techniques for the growth as well as the integration of titania nanotube arrays (TNAs) on two types of polymer substrates. Successful growth of TNAs is shown via optimization of electrochemical anodization parameters in anodic oxidation of sputtered coated titanium films to facilitate formation of nanotube arrays on the insulating polyimide substrate. In addition, the microfabrication process for the selective (site-specific) growth of TNAs on the polyimide substrate is discussed. Finally, integration process of TNA membranes with a bioinspired mechanically-compliant polymer nanocomposites (composed of poly vinyl acetate reinforced with cellulose nanocrystals) is demonstrated.
The morphology of the nanotube arrays was investigated using scanning electron microscopy (SEM). By varying the anodization conditions (e.g., applied electrochemical potential and time), we obtained highly-ordered vertically-oriented nanotube arrays with an average pore diameter ranging from ∼30 – 100 nm and a length of ∼0.5 – 20 μm. The nanotube array geometries were defined using either laser micromachining or by selective anodization. Laser-micromachining employed a picosecond laser operated at low power (<2 mW) as a subtractive fabrication method. Alternatively, we optimized a selective-anodization process in which titanium was anodized through a AZ nLOF 2070 negative-tone photoresist mask. By hard baking the photoresist, we were able to maintain a high degree of selectivity even during anodization times exceeding 5 hours. The photoresist was then removed by oxygen plasma exposure, leaving behind defined regions with titania nanotube arrays surrounded by non-anodized titanium.
To demonstrate use in drug-delivery applications, preliminary release tests for the fabricated devices were performed by filling the nanotubes with the food-coloring dye (for the initial tests) and the anti-oxidant resveratrol. Drug release from the resveratrol/dye-loaded TNAs was measured using UV-VIS spectroscopy at various timepoints. After an initial burst phase, the release of both types of molecules follows a highly controlled linear profile (with 47 ng/day as the release rate of resveratrol from 1 mm² sample area) that extends beyond one month. In conclusion, we have demonstrated a facile approach to achieve nanotubes/non-conducting polymer flexible devices with precisely controlled drug release properties. The nano/microfabrication methods developed in this work will enable the development of novel flexible devices with controlled drug delivery systems for use in precision medicine and biological interfacing applications.

There is a significant need to develop novel scaffolding materials for the repair and regeneration of bone. Ideally, these scaffolds should mimic the mineralized extracellular matrix of bone, which is an organic-inorganic nanocomposite consisting primarily of carbonated hydroxyapatite (CHA) and fibrous type I collagen. However, reconstituted collagen gel scaffolds lack structure and mechanical integrity. More importantly, they mineralize poorly in vitro or in vivo.

Our research has shown that bioactive sol-gel derived borate glasses (SGBGs) rapidly convert to CHA, in vitro, which can be attributed to their high reactivity and enhanced textural properties. In particular, a boron substituted Bioglass 45S5 formulation; [(46.1)B₂O₃-(26.9)CaO -(24.4)Na₂O-(2.6)P₂O₅; in mol %] with at least two-orders of magnitude greater specific surface area and pore volume values compared to melt-derived equivalents, has demonstrated rapid ion release rates and conversion to CHA. This CHA formation is also associated with an increase in pH upon SGBG dissolution, a feature that can be used to simultaneously fibrilize and functionalize injectable and 3D-printable dense collagen hydrogels.

In this study, we demonstrate a one-step process wherein acid-solubilized collagen molecules self-assemble in the presence of dissolving SGBGs without the use of NaOH, thereby generating highly hydrated collagen gels. These gels were then subjected to the gel aspiration-ejection technique, which imparts both fibrillar compaction and meso-scale anisotropy, thus forming a SGBG-functionalized dense collagen gel. The resulting bone extracellular matrix-mimicking scaffolds exhibited rapid mineralization along the collagen fibrils within 2 hours of immersion in simulated body fluid, and by day 7, CHA formation in dense collagen gels reached native bone levels, in vitro.

In order to investigate the potential in vivo bone regenerative capability of these rapidly mineralizing scaffolds, they were injected into a critical size defect using a rat tibia model. The short-term mineralization capacity of acellular SGBG-functionalized dense collagen gels was compared to dense collagen gels alone. Micro-CT analysis showed that the bone volume fraction significantly increased in both scaffold implants from weeks 1 to 2. While there was no significant difference between the two scaffolds at week 1, SGBG-functionalized dense collagen gels showed significantly higher bone volume fraction at week 2. In terms of bone microarchitecture, at week 2 of implantation, the SGBG-functionalized dense collagen gels showed less trabecular separation than dense collagen gels alone. These analyses were complemented by histological staining of implants, where Von Kossa, Toluidine blue, alkaline phosphatase, tartrate resistant acid phosphatase, were performed on recovered tissue sectioned samples at weeks 1 and 2 to investigate potential new bone formation in implanted scaffolds. Compared to dense collagen gels alone, the histology of SGBG-functionalized dense collagen gels revealed high levels of positive phosphate staining, suggesting mineralization, and high cellular infiltration with indications of osteoblast and osteoclast activity.

Therefore, the ability to simultaneously fibrilize and functionalize collagen gels via the incorporation of SGBG particles enables the production of injectable scaffolds with promise in bone repair. Moreover, this latest breakthrough allows for the development of bespoke gel bioink formulations, which can be used with a novel biofabrication/3D printing technology based on automated-gel aspiration-ejection. This can facilitate the building of rapidly mineralizable tissue-mimicking structures with varying micro-architectures to mimic native protein fibril density and alignment, as well as cell loading and density according to the intended use.

Biomaterials play a central role in modern regenerative medicine and tissue engineering strategies, where they serve as tunable biophysical and biochemical environments that direct cellular behavior and function. However, one of the biggest obstacles in translating the advances in biomaterials/tissue engineering research to clinical applications has been the lack of sufficient vascular tissue regeneration in current synthetic and natural biomaterials. We develop silk fibroin-based biomaterials for cardiovascular applications, including vascular grafts and cardiac patches, with a particular emphasis on enhancing vascular ingrowth and integration of these devices with the host tissue.

Silk fibroin biomaterials serve as a highly versatile biomaterial platform that allows us to study how biomaterial properties drive biological processes and apply these design criteria to cardiovascular device optimization. In this talk I will discuss our work toward (1) development of 3D silk biomaterials with tunable pore morphology/architecture, (2) effect of silk biomaterial architecture and pore morphology on immune responses, tissue integration, and vascularization of implants, including vascular grafts (3) biofunctionalization of silk biomaterials with basement membrane proteoglycan perlecan to create vascular niche-like environments that support growth factor signaling and angiogenesis.

Microneedles (MNs) are prominent tools for efficient subcutaneous drug delivery in a non-invasive manner. In particular, dissolving MNs have been widely exploited for the rapid transfer of the massive amounts of biomacromolecules from MN to the skin followed by the sustainable release of drugs. However, scaffold materials for dissolving tip should be carefully chosen due to biocompatibility and safety issues. The microfabrication process often needs complicated procedures regardless of the formulation of MNs, limiting the general usage of MNs. Moreover, degradation-susceptible biomolecules such as nucleic acid require careful address and design for intact delivery. Thus, developing a straightforward and universal approach to fabricate MNs to ensure stability of drugs, particularly nucleic acids, with high loading efficiency is inevitable.

To address the concerns, we establish a novel and simple method to fabricate custom-made RNA MNs (RMNs) based on RNA membrane for a universally applicable drug delivery system. The free-standing RNA membrane was fabricated from rolling circle transcription with two complementary circular DNAs, followed by evaporation-induced self-assembly. By coating scaffold MNs with RNA membrane bearing high stability and RNA packing density, RMNs with a large amount of RNA were successfully achieved. RMNs can be customized by controlling material features of the RNA membrane and 3D printer-based fabrication of scaffold MN in a purpose-oriented way. Remarkably, RNA membrane-based fancy coating method enables a full coverage of the scaffold MN regardless of the shape and material. In addition, RMNs facilitate a massive delivery of RNA along with various types of other drugs, demonstrating the universal applicability of RMN-based drug delivery system. Altogether, this novel strategy presents a general platform to fabricate tailor-made MNs for an efficient and universal drug delivery system.

[1] Han, D., Park, Y., Kim, H., & Lee, J. B., Nature communications, 2014, 1-7.
[2] Kim, D., Kim, H., Lee, P. C., & Lee, J. B., Materials Horizons, 2020, 1317-1326.

The blood-brain barrier (BBB) is a barrier preventing the influx of compounds to diffuse from the blood to the brain. It prevents macromolecules penetrate the central nervous system (CNS). Hence, the BBB is a major challenge for drug delivery and a factor to limits the development of new pharmaceuticals for CNS. Rabies virus glycoprotein (RVG) is known for the rabies virus infection that targeting the extracellular surface of neuron and microvascular endothelial cells due to the binding with nicotinic acetylcholine receptor (nAchR). Many CNS therapeutic drugs were designed with the conjugation of RVG to transport the CNS drugs across the BBB, non-invasively. Exosomes are the vesicles comprise of lipid bilayers membrane. The exosomes function as nanocarriers that can be easily loaded with different types of biomolecules. However, the loading of the therapeutic agents to the exosomes poses difficulty. Liposomes are spherical artificial vesicles formed by non-toxic phospholipids and cholesterol. Besides different properties that are greatly affected by the size, surface charge, and sources of a phospholipid, the liposomes are showing biocompatible by nature and promising vesicles for drug delivery. In this study, RVG decorated exosomes were mechanically fused with the drug-loaded liposomes to formed exosome-liposome hybrid nanoparticles. The hybrid nanoparticles were designed to cross the BBB due to the RGV decoration and deliver the CNS therapeutic agents that encapsulated in the liposomes. The fusing of exosome and liposome was confirmed by Förster resonance energy transfer (FRET). FRET is a photophysical phenomenon that energy transfers from a donor fluorophore to an acceptor molecule at a distance in a range of 1-10 nm. FRET intensity, transmission electron microscopy (TEM), dynamic light scattering (DLS), and fluorescence microscopy were used for the characterization of the hybrid nanoparticles.

In this presentation, physical and optical characteristics of the exosome-liposome hybrid nanoparticles will be discussed. Biological characterizations and the preliminary results of the delivery of therapeutic agents across the blood-brain barrier in mice will be presented.

Introduction: siRNA has been received great attention as emerging gene therapeutics due to its superior capacity of specific gene silencing on the post-transcription level. However, the clinical application of siRNA is still hampered due to its inherent properties and delivery problems. Herein, we proposed a novel systemic siRNA delivery system. We synthesized elongated siRNA via rolling circle transcription and developed nano complexes with a glycol chitosan derivative to systemically deliver the elongated siRNAs to cancer tissues. These elongated siRNA nano complexes (ENCs) showed the highly enhanced in vivo stability and less cytotoxicity, compared to conventional polyelectrolyte complexes. Because ENCs were designed to release active siRNA in the cytoplasmic region, they showed high gene silencing efficacy with efficient tumor target ability. Overall, the proposed RCT-based elongated siRNA nano complexes can provide a new platform technology for efficient cancer therapy.
Method: 5’-phosphorylated linear single-stranded DNA oligonucleotides were circulated by hybridizing with the T7 promoter sequence. To synthesize 0.5uM of circular DNA, ligated the nick in the circularized DNA with T4 ligase. Then, closed circular DNA templated was incubated with T7 polymerase at 37 C 5h with rNTPs. Elongated siRNA was complexed with thiol glycol chitosan (tGC). GC was modified by sulfhydryl groups. We analyzed the stability of ENCs by heparin competition assay, cellular uptake and intracellular distribution on human prostate cancer cells (PC3). In vivo biodistribution of ENCs was investigated through PC3 cells were subcutaneously inoculated to the left flank of male.
Results: We developed the periodically repeated siRNA through the RCT process band its delivery system using glycol chitosan derivatives. The synthesized ENCs have stable formation and protect the siRNA therapeutics against polyanion and nucleases attack formation in blood circulation.
Conclusion: In this study, we developed elongated nano complexes, which were synthesized by a uniquely designed RCT method, as an RNAi therapeutics platform technology for cancer therapy. The ENCs formulation can be considered as a potential candidate for tumor-targeted siRNA delivery system.
Acknowledgements: This research was supported by the Intramural Research Program of KIST (2E27940) and the 2016 Research Fund of the University of Seoul (J.B. Lee).
References: Lee, J.B., Hong, J., Bonner, D.K., Poon, Z. and Hammond, P.T.) Nat. Mater., 2012, 316-322. Lee, J.B., Peng, S., Yang, D., Roh, Y.H., Funabashi, H., Park, N., Rice, E.J., Chen, L., Long, R. and Wu, M.. Nat. Nanotech., 2012, 816-820. Lee, S.J., Huh, M.S., Lee, S.Y., Min, S., Lee, S., Koo, H., Chu, J.-U., Lee, K.E., Jeon, H., Choi, Y.et al. Angew. Chem., 2012, 7203-7207.

Phthalates or Phthalic acid esters (PAEs) are one of the most widely used plasticizers in the flexible plastic product manufacturing processes. These chemicals leach, migrate, or evaporate into the surrounding with time, as they do not form chemical interactions with the polymer matrix. As a result, PAEs have become a ubiquitous environmental pollutant that humans and animals could get exposed to and cause health complications even endocrine disruptions. Therefore, qualitative and quantitative detection of PAEs in different environments becomes crucial, especially in water, food, and beverages. Even though there are various PAE detection techniques, the use of complex, time-consuming laboratory processes of advanced technology has emphasized the need for easy, in-situ, and real-time detection technologies. In this study, a Multi-Walled Carbon Nanotube (MWCNT)/Cellulose composite paper was developed as an electrochemical PAE sensing material, where its electric conductivity change after PAE absorption was used as an indicator in qualitatively detecting PAEs in solutions. This technique was developed based on the hypothesis that the absorbed dielectric PAEs would interfere with the electron transfer across the MWCNT network by distancing the MWCNTs due to the presence of their bulky hydrocarbon side chains. The developed sensing material is an electric conductive, flexible, biocompatible, and biodegradable composite paper with potential for recycling and reusing. It was determined that the percolation threshold of MWCNTs in the cellulose composite had to be 250 ppm to form electric conductive papers. These papers were prepared by vacuum filtering an ultrasonicated solution containing MWCNT dispersion in cellulose pulp. The effect of these absorbed PAEs on the electric conductivity of the composite paper was observed using the three-electrode Electrochemical Impedance Spectroscopy (EIS) technique, where the composite paper was used as the working electrode. The electric conductivity of the PAE-absorbed composite paper was analyzed with respect to the pristine composite paper. The reduction in the electric conductivity of the composite paper after PAE absorption was clearly observed through the Nyquist plot, which indicated an increase in the charge transfer resistance between the electrolyte and the electrode, and the Bode magnitude plot, which indicated an increase in the impedance of the composite paper. This novel technique was specifically tested for di(2-propylheptyl) phthalate (DPHP) in methanol solution using a PAE sensing composite paper of 0.001:1 MWCNT:Cellulose composition. The conductivity change of the paper was tested using EIS over a 0.1 Hz – 1 MHz frequency range. The charge transfer resistance of the pristine composite paper was 6130 Ω. It was increased by 1.15 fold for the PAE-absorbed composite paper, when the DPHP methanolic solution concentration was 1000 ppm, whereas an increase of 1.38 fold was observed when the solution concentration was increased by one order of magnitude. However, the charge transfer resistance of the paper was slightly increased (1.46 fold increase with respect to the pristine paper) when the concentration of DPHP methanolic solution was continued to increase by another order of magnitude. Furthermore, it was discovered that the PAE sensitivity of the composite paper varied with its MWCNT composition, showing potential at developing PAE sensing materials with different sensitivity ranges. More studies are conducting to identify the different PAE detecting ranges for different MWCNT compositions. Based on the experimental results, this qualitative technique shows potential to be developed as a quantitative PAE detection technique. Being able to detect the presence of PAE through the conductivity change of the MWCNT/Cellulose composite paper using EIS shows that this PAE sensing material could be used to develop comparably easy, quick, real-time, and in-situ phthalate detecting sensor.

Bulk hydrogels with nanoporous structures have widely been used in tissue engineering and regenerative medicine. These gels are typically made up of randomly crosslinked hydrophilic polymer networks, which have coupled stiffness and porosity. In a simplified model, the stiffer the gel the smaller the pores. Such characteristic limits the applications of bulk hydrogels in 3D cellular engineering wherein stiff scaffolds with large pores are required. In this presentation, we will discuss some of our recent advances in microengineering chemically modified protein microgels based on their orthogonal thermo-chemical response to fabricate biomimetic microporous hydrogels with decoupled stiffness and porosity. We show how these hydrogels support 3D cell viability and proliferation while their bulk counterparts result in cell death. We further demonstrate how the properties of individual microgels regulate the behavior of self-assembled, macro-scale hydrogel constructs. This technology may be generalized for other polymers, opening a new horizon for converting bulk hydrogels to beaded hydrogels with decoupled porosity and stiffness for a broad range of applications in healthcare.

For decades, cellulose nanocrystals (CNCs) have been produced by hydrolyzing the amorphous regions of hierarchical cellulose fiber structures. These colloidal particles are made up of highly ordered arrays of cellulose chains, impeding the physicochemical modifications of inner crystalline layers, which in turn result in limited/compromised dispersion stability, functionalizability and charge, response to external fields, and transport. Nanoengineering cellulose fibrils via controlled oxidation partially disintegrates the amorphous cellulose chains attached to the crystalline body, which yields Janus-like nanoparticles with a needle-shaped crystalline body (similar to CNCs) sandwiched between two highly functionalized disordered cellulose regions (hairs). This newly emerged class of nanocelluloses are called hairy cellulose nanocrystals (HCNC). The protruding soft brushes from HCNC poles impart significant modifications to the colloidal properties of HCNCs, enhancing their functionality, charge density, stability, and self-assembly. In this presentation, we will review our recent progress in engineering HCNCs for advanced applications, such as water treatment, scale inhibition, biomimetic mineralization, rheology modification, nanocomposites, hydrogels, and so forth. We will show how HCNCs enable several technologies that would otherwise be impossible to develop using conventional CNCs. The outcome of our research may leverage universal biomass-based sustainable, green solutions for long-lasting industrial challenges in water, healthcare, food, and energy sectors.

Bacterial diseases in shrimp aquaculture are the major concern to the industry and are typically controlled by eradication of the pathogen. Treatment with antibiotics is a traditional way but it was banned recently because of the concern of antibiotic resistance. To seek an alternative way to control bacteria diseases in shrimp aquaculture, we developed synbiotic (probiotic and prebiotic) microencapsulated beads. We encapsulated probiotic cells in beads (~ 100 mm) prepared through co-gelation of matrix and prebiotic. Compared with physical mixture of synbiotic, microencapsulated synbiotic beads can greatly decrease pathogen viability. In vitro coculture method indicated that both probiotics and pathogen can use prebiotic as their food source. While synbiotic encapsulation could trap probiotic and prebiotic inside of beads, thus blocking the access of pathogens to prebiotic, leaving pathogen to starve while flourishing encapsulated probiotic only. In addition, co-encapsulation of probiotic and prebiotic would promise more success in terms of viability and stability of probiotic when used in shrimp aquaculture.

Non-esterified fatty acids (NEFA) are carboxylic molecule which is worth detecting in both food and health applications. In food, NEFAs are related to the handling, storing, and cooking condition, making them excellent quality indicators. In health, the NEFA concentration in blood plasma can be related to some metabolic disorders, cardiovascular diseases, cancer mortality, and even sudden death. Hence, detection of NEFA is of the utmost importance in both fields. However, fatty acids are difficult to decompose, which hinders the development of portable sensors.
This research aims to develop an electrochemical, enzymatic, flexible biosensor for fatty acids detection. Filter paper and hydrogels are porous, flexible, and cost-effective substrates which can absorb a conductive ink and can also accommodate enzymes. Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) conductive polymer is used due to its biocompatibility and safety for use in health and food application. Regarding the enzyme needed, a multi-enzyme system needs to be designed to detect NEFAs. As mentioned, fatty acids are stable, and their metabolic decomposition is complex. Hence, more than one enzyme is required, obtaining a cascade reaction.
Since many oxidase enzymes produce hydrogen peroxide (H₂O₂) as a by-product, the first step was to be able to detect H₂O₂. Therefore, the first enzyme used is horseradish peroxidase (HRP). As part of the peroxidase family, HRP reacts with hydrogen peroxide while oxidizing a substrate. In this case, HRP uses H₂O₂ – possibly the one produced by other enzymes - to react with PEDOT:PSS. Consequently, a hydrogen peroxide chemiresistive sensor based on an HRP-PEDOT:PSS ink was produced. This sensor is simply produced by 3D printing the sensitive ink on a filter paper, making the manufacturing simple and keeping the cost low. The sensor’s limit of detection for H₂O₂ in liquid is 61.3 nM, and it shows a linear range from 61.3 nM to 61.3 µM. The sensor is also sensitive to hydrogen peroxide vapors. Despite the limitations, such as sensitivity to humidity and a relatively long response time (≈ 1 hour), this sensor could be used in controlled environment applications – e.g. food packaging and face masks.
To improve the drawbacks observed in the paper-based sensor, paper was replaced with a hydrophilic polyurethane hydrogel (HPU). Given their crosslinked structures, hydrogels are characterized by porous structures which allow them to swell when absorbing water. At the same time, the pores can be filled with other compatible materials. By combining HPU with the optimized HRP-PEDOT:PSS ink, it was possible to trap both the conductive polymer and the enzyme in a biocompatible, elastic hydrogel scaffold. Contrarily to what was achieved with the paper-based sensor, the use of HPU enables continuous monitoring of H₂O₂ in liquid. The sensor shows a limit of detection of 613 nM and a linear range between 613 nM and 6.13 mM.
Once developed a suitable mechanism for H₂O₂ detection in two different substrates, lipoxygenase (LOX) was added to the ink. LOX is an enzyme that takes part in the digestion mechanism of polyunsaturated fatty acids (PUFAs). LOX works in the first stage of this process by adding a peroxide group in a specific position of the fatty acid chain. This site is meant to be the position where the carbon backbone is split. However, in the absence of the following enzyme in the sequence, the peroxide group is not consumed and instead is freed as hydrogen peroxide. This hydrogen peroxide is in turn used by HRP as the reagent to interact with PEDOT:PSS, consequently allowing PUFAs detection. This bi-enzymatic system allows the detection of PUFAs in a relatively simple, speedy, and cost-effective way compared to the traditional method of fatty acids detection.

A key initiating step in atherosclerosis is the accumulation and retention of apolipoprotein B complexing lipoproteins within artery walls. In this work, we address this exact initiating mechanism of atherosclerosis, which results from the oxidation of low-density lipoproteins (oxLDL) using therapeutic nanogels. We present the development of biocompatible polyethylene glycol (PEG) crosslinked nanogels formed from a single simultaneous cross-linking and co-polymerization step in water without the requirement for organic solvent, high temperature or shear stress. The nanogel synthesis also incorporates in situ non-covalent electrostatically driven template polymerization around an innate anti-inflammatory and anti-oxidizing paraoxonase-1 (PON-1) enzyme payload - the release of which is triggered due to matrix metalloproteinase (MMP) responsive elements instilled in the PEG crosslinker monomer. Results obtained demonstrate the potential of triggered release of PON-1 enzyme and its efficacy against the production of ox-LDL and therefore a reduction in macrophage foam cell and reactive oxygen species formation.

Enzymes enable high specificity in applications such as production of value-added agricultural products, environmental remediation, and diagnostics, but are often limited by challenges in stability and recovery in these complex biological systems. Hierarchical structures, incorporating both macrostructures and nanostructures, can harness the benefits of each size regime for the stabilization of enzymes. Here we describe two case studies. In one, lipase B from Candida antarctica was interfacially assembled with iron oxide nanoparticles into hierarchically ordered structures consisting of a cross-linked core of poly(dicyclopentadiene). Microparticles were characterized for performance in extreme environments (low/high pH, high temperature, solvents) which are denaturing for native lipase. Kinetic analysis revealed a significant increase in the turnover rate following immobilization. After two hours exposure to 50% acetonitrile, there was no measurable leaching of enzyme from the microparticles, while Novozym 435 lost 99.8% protein under the same conditions, suggesting robust design for use in solvent applications. Per unit protein, microparticles outperformed Novozym 435 in direct esterification and transesterification. In another study, we demonstrate the synthesis of copper microflowers in which the enzyme b-galactosidase is incorporated with retained activity. We explore the nuance of considering both biological and non-biological system performance in the pragmatic assembly of these hierarchical biocatalytic nanocomposites. Such understanding of hierarchical biocatalytic systems can permit their application in complex biological systems to improve the safety and sustainability of food, bioprocessing, and diagnostic applications.

Delivery or encapsulation systems can be developed to achieve controlled-, sustained-release and targeted delivery for a myriad of applications, ranging from agri-food, biomedical, nutraceutical to pharmaceuticals applications. In addition, delivery systems simultaneously function as a protective carrier for labile compounds that may otherwise degrade under harsh environments; for instance, when exposed to gastro-intestinal fluids. By incorporating specific design parameters to each functional system, it is hypothesized that bioavailability of drugs or nutrients can be enhanced. In this presentation, we shall fist discuss how oral delivery systems can be designed to improve patient compliance in the delivery of pharmaceutics, using Parkinson’s disease as a model. With the current shift in paradigm in disease prevention through better nutrition, there has been immense interest in augmenting health with nutraceuticals. On this note, we will also report the design of food-based, enteric-coated carriers that can target nutraceutical release along specific parts of the gastro-intestinal tract. Using animal models, we will discuss how this targeted delivery system can improve shelf-life, increase bioavailability and enhance efficacy of nutrients and other nature-derived compounds. In addition, we will also explore alternative oral encapsulation technologies for the development of functional foods, e.g. synbiotics, for agri-food applications.

Cultured meat is artificial meat produced by culturing cells extracted from living animals based on cell engineering technology which allows a continuous supply of meat without unnecessary slaughter. Recently, this slaughter-free meat is coming into the limelight due to its advantages of lowering environmental pollution, protecting animal ethics, and enhancing the safety of meat. In addition, the hunger for a sustainable form of meat has grown even more as the meat industry has been hit by the import and export suspensions caused by the COVID-19 pandemic. However, the fatal limitations of cultured meat production such as using excessive animal-derived materials and high production costs put the brakes on the popularization of cultured meat. To overcome this limitation, we propose the introduction of well-organized biomaterial science into the cultured meat process.
In this research, we first report on a strategy to develop scaffold-free advanced cultured meat using fish gelatin microsphere (GMS) and myoblast sheet. The GMSs are edible and exhibit various morphologies and binding activities based on the degree of crosslinking. We also select insulin-like growth factor 1 (IGF-1) and C-phycocyanin (C-PC), blue algae extract, as reasonable and eco-friendly nutrients to improve the proliferative activity of myoblasts in fetal bovine serum (FBS)-reduced growth medium. Each of these nutrients was incorporated into GMSs with different surface properties. The loading and release behaviors of nutrients for each GMS are analyzed, and subsequently the most effective GMSs to improve the proliferation of myoblasts under an FBS-reduced medium are selected.
GMSs applied to cell sheet culture produce more cost- and time-effective meat-like cell sheets than conventional methods by exerting four important functions. Finally, we prepare two types of cost-effective cultured meat with GMS-based cell sheets. We evaluate the quality of the cultured meat by comparing the tissue properties with soy meat and chicken breast. GMS is a versatile functional material that can be applied to both batch and continuous processes as well as cell sheets. This technology can economically produce cultured meat with high cell content by overcoming the low cell content and high production cost of conventional cultured meat. We believe that this material engineering technology will provide a significant contribution to the commercialization of cultured meat.

Surfactants are materials with hydrophobic and hydrophilic sites that adsorb at the interface between an oil and aqueous phase, allowing immiscible liquids to mix easily and produce stable emulsions. These surface-active materials are of immense significance in the agrochemical industry for the development of adjuvant formulations for pesticides and fertilizers. Surfactants are one of the most commonly used adjuvants that increase the efficacy of crop protection products by enhancing their spreading, sticking and wetting properties. However, most commercially available surfactants in agriculture are derived from petroleum sources, and environmental concerns over toxicity and low degradability are key drivers for the growing market demand of renewable, non-toxic, biodegradable surfactants. Thereby, developing efficient and ecofriendly surfactants is of great interest, particularly in the agricultural sector. Biologically extracted materials, such as cellulose nanocrystals (CNCs), are good candidates to fulfill that need. The surface of CNCs is rich with accessible and hidden hydroxyl groups, lending these renewable, non-toxic and biocompatible nanoparticles, an amphiphilic nature and surfactant-like characteristics. While emulsions stabilized by conventional surfactants have been the norm in most industries for many decades, Pickering emulsions stabilized by solid particles; for example, inorganic particles, such as colloidal silica and graphene oxide, and organic particles, such as nanocellulose, are increasingly becoming favorable choices due to their excellent long-term stability.

In this work, cellulosic Pickering emulsions (CPEs) were developed by blending negatively charged CNCs and cellulosic polymers. The tuning of surfactant-like behavior of CNCs was achieved by salts of magnesium nitrate (Mg(NO₃)₂) and zinc nitrate (Zn(NO₃)₂), respectively. The emulsions were prepared at varied CNC and salt concentrations, without the use of any petroleum-derived surfactants or prior functionalization of the original CNCs with any hydrophobic molecules. The preparation of CPEs was achieved by mixing colloidal CNCs in aqueous phase with oleic acid in oil phase using high-energy ultrasonication. Various interface parameters were investigated, such as surface tension and surface charge as well as shear viscosity, to understand the effect of CNC and salt concentration on the physicochemical characteristics of the CPEs. In addition, the emulsions were also analyzed under an optical microscope to study droplet coalescence and emulsion stability. Results showed that CNCs in the presence of salts influenced the formation and stabilization of the CPEs and were more effective in emulsifying oleic acid compared to when CNCs were used alone. This study not only showed that CPEs can be stabilized by CNCs in the presence of divalent cations, but also established a platform to produce CNC-based PEs with customizable properties and functionalities with new insights into their application as eco-friendly surfactants in agriculture. Such a strategy will be useful to develop advanced products in liquid and solid forms as emulsions and beads not only in agriculture but also in several applications ranging from food and pharmaceutical to paint and coating industries.

This project is supported by the Center for Advanced Surface Engineering (CASE), under the National Science Foundation (NSF) Grant No. IIA-1457888 and the Arkansas EPSCoR Program, ASSET III.

Food waste has far-reaching socioeconomic and environmental impact. Nutrient-rich fresh foods are particularly susceptible to waste resulting from cold chain breaks. Further, the decomposition of wasted food and the subsequent energy and water inputs required for redundant production, processing, and distribution are severely detrimental to the environment. Recent developments have demonstrated the ability of silk fibroin to extend the shelf-life of various classes of foods by minimizing oxidation and dehydration. This work will highlight the physical and chemical features of silk fibroin that allow it to accel as a barrier coating as well as discuss the safety and integration requirements for successful scale up and commercialization within the food industry.

Biodegradable polymers, those synthesized from monomers derived from either the biomass or fossil fuels, or those naturally occurring continue to attract intense research interest given the increased global impetus to address the carbon footprint of plastics and issues around climate change and pollution. The largest single use of plastics is in all forms of packaging, including food packaging and de facto aligned with agriculture, although there are numerous other uses of mostly fossil-fuel derived plastics in agriculture, from pipes to mulch film. Here, we describe two approaches to the development of biodegradable polymer blends and composites that critically can be prepared using sustainable, environmentally friendly processes and at scale in high volumes.
Firstly, focusing on poly(glycolic acid) or PGA, a polymer with a similar structure to poly(lactic acid)(PLA) minus a methyl side group, with the resulting polymer having crystalline content as high as ~55%, high thermal stability (up to 230°C), excellent gas barrier properties as well as high strength (115 MPa) and stiffness (~7 GPa)[1]. However, PGA is 100% compostable and biodegrades rapidly with a profile similar to cellulose. Despite these useful properties, PGA has a number of intrinsic properties, most notably brittleness which have hindered its application. Classically, this has in part been overcome by co-polymerising glycolic acid with lactic acid (PLGA) or to a lesser extent physically blending PGA with other polymers, with the latter strategy being more commercially viable. Here, PGA was melt blended with poly(butylene adipate-co-terephthalate)(PBAT) using a terpolymer of ethylene, acrylic ester and glycidyl methacrylate (EMA-GMA) to promote interfacial interactions between the blend components. The resultant blends had improved ductility (strain at break increased from 10.7% to 145%), reduced oxygen permeability, enhanced water vapour barrier performance (improved by 47%) and thermal stability up to 300°C, properties required for hot food packaging. Further improvements in barrier properties were achieved by treating the surface of blown films of these blends with electron beam treatment with, further manipulation possible for controlled agrochemical release.
A second approach focused on the blends and composites of natural occurring polymers based on chitosan, carboxymethyl cellulose (CMC) and alginate and, 1D/2D materials, both organic and inorganic, including montmorillonite (MMT) and sepolite (SPT). Thermo-mechanical kneading is used to prepare blends and composites of chitosan, a cationic polysaccharide, with negatively charged biopolymers such as CMC, allowing for the preparation of films with excellent dimensional stability and structural integrity [2]. This advance in hydrolytic stability is derived from physical crosslinks due to polyelectrolyte complexation between chitosan and CMC. Furthermore, it is possible to manipulate the properties of these films by the addition of naturally occurring negatively charged MMT or SPT for application in food packaging [3]. The properties of these films were further modified by selective plasticisation using natural molecules and ionic liquids and other 2D materials such as graphene oxide [4].

References
[1] P. Kumar Samantaray, A. Little, D. M. Haddleton, T. McNally, B. Tan, Z. Sun, W. Huang, Y. Ji and C. Wan. Green Chem., 2020, 22, 4055-4081.
[2] P. Chen, F. Xie, F. Tan, T. McNally. Compos. Sci. Technol., 2020, 189, 108031.
[3] P. Chen, F. Xie, F. Tan, T. McNally. Eur. Polym. J., 2021, 144, 110225.
[4] P. Chen, F. Xie, F. Tan, T. McNally. Carbohydr. Polym., 2021, 253,117231.

During photosynthesis, plants use light energy to transform carbon dioxide and water into carbohydrates. The photosynthetic process comprises many steps that can in principle be optimized to have higher efficiency. Rubisco (Ribulose 1,5-bisphosphate carboxylase/oxygenase) catalyzes the carboxylation reaction that is the initial reaction for carbon fixation and conversion of CO2 to sugars. The reaction occurs in organelles called chloroplasts present in leaf cells. However, Rubisco has naturally a poor affinity for CO2 and the transfer of the atmospheric CO2 to the Rubisco enzyme is highly limited by the diffusion of CO2 through the stomata and the intercellular space which ultimately considerably reduces the photosynthesis rate. Polyamines have been shown in-vitro to be able to transfer carbon dioxide to Rubisco for the carboxylation reaction. In this work, we present biocompatible fluorescent polyethylenimine nanoparticles for enhancing the CO2 transfer to the Rubisco enzyme. With in-vitro studies we evaluated the nanoparticles uptake of atmospheric CO2 and transfer kinetics to Rubisco for the carboxylation reaction. Then we introduce the nanoparticles in-vivo into the plant apoplast via agroinfiltration. First, we studied the toxicity of the nanoparticles and results showed no evidence of a direct harmful impact. We then investigated their localization within the plant leaf with fluorescent microscopy and preliminary observations indicate that the nanoparticles are present in the intercellular space but also in the direct centers of photosynthetic activity: the chloroplasts. Finally, we investigated the impact of the nanoparticles on the plant photosynthesis.

The layer-by-layer technique for film deposition presents several advantages for preparing materials for drug delivery, including the mild chemical conditions for handling therapeutics, simple and scalable processing. However, this technique lacks information describing the role of the deposition parameters and film moieties on the drug release process. This study investigated the influence of the assembly parameters (pH, ionic strength, capping layers, and the loading method) on the drug delivery properties in multilayer films. Hyaluronan/chitosan (HA/CHI) films were assembled at different pH (4.5 and 7.2) chitosans with different degrees of deacetylation (40%, 65%, and 85%) to test their role in the loading and release of ionic Rose Bengal dye in the multilayer films. Films assembled at pH 7.2 were rougher and thicker than those deposited at more acidic conditions, presenting superior drug loading capacity and a more sustained release profile. This result was attributed to the more coiled conformation of chitosan during film deposition, which led to a higher number of free amino groups interacting with rose Bengal during drug loading. Drug release data were fitted to the Higuchi model, indicating an increase in the diffusional coefficient with chitosan deacetylation degree. Ritger and Peppas model demonstrated an anomalous drug release mechanism and the
more sustained release profile for films assembled at pH 7.2. This study also investigated the drug delivery properties of the traditional poly (acrylic acid)/poly (allylamine hydrochloride) (PAA/PAH) multilayers, which presented the superior calcein (model drug) loading capacity for films prepared at basic pH (> 8.5) with MgCl 2 or NaCl in the polyelectrolyte solutions. This result was attributed to the higher number of free, no complexed amino groups of the PAH chains in the multilayers, available for interacting with carboxylate groups of calcein molecules
via salt bridges. The drug loading method (post- or during-film deposition) also affected the film loading capacity and release mechanism in the PAA/PAH films, leading to the highest drug loading signal in films prepared using the postassembly method. The limited amount of drug in PAA/PAH films using the during-assembly method may be attributed to the washing out of calcein molecules during film deposition, possibly leading to a matrix with tightly bound drug molecules for this condition. Data fitting to Higuchi and Ritger-Peppas models demonstrated an
increase in the drug release constant with the number of bilayers deposited for films prepared using the postassembly method, whereas the opposite trend was observed for the during- assembly drug loading method. The Berens-Hopfenberg model enabled the decoupling of the individual contribution of different drug release mechanisms (diffusion and polymer chain relaxation), demonstrating an increase of the chain relaxation contribution with the number of bilayers. The stronger interaction between the polymer and the calcein molecules for samples
prepared using the during-assembly method may have hindered the fast, drug diffusion process, enhancing the effect of the matrix swelling in the drug release process and leading to a more sustained drug release profile. PAA/PAH films loaded with calcein were also spin-coated with different capping films (fatty acids and phospholipid monolayers, and ALG/CHI multilayers) to reduce the initial burst release effect. The densely packed phospholipid monolayers led to the most substantial decrease in the release rate with minimal reduction in the drug cargo during the capping layer deposition, highlighting the importance of single-step methods to avoid the drug leaking. These findings illustrate alternatives to modify the drug release profile of small molecules in nanostructure thin films based on the building blocks, film assembly, and drug loading methods, aiming to design drug delivery devices with precise architecture and performance.

Proton transport is essential to many physiological processes. For this reason, devices that deliver a controlled flow of protons to living organisms show promise for advancing medicinal/therapeutic treatments. These bioprotonic devices depend on conductive, biocompatible polymer membranes to facilitate protonic movement. Herein, we present ab initio molecular dynamics (AIMD) simulations to quantify proton transport in a candidate polymer for bioprotonic membranes, m-polyaniline, and its chemically functionalized derivatives. By demonstrating how polar, acidic, and aromatic chemical groups influence protonic movement, our results show that chemical functionalization can be used as a tool for tuning the transport properties of m-polyaniline.

Recently, modified transposases have been used to both fragment and attach end-groups to DNA molecules in solution and on beads. This process, termed ‘tagmentation,’ has been commercialized and used to improve library preparation for DNA sequencing. We report the application of transposase tagmentation to DNA linearly stretched and immobilized onto grating surfaces with micron-sized periodicities. The use of gratings is done to prevent the steric hindrance inherent to DNA strongly adsorbed to flat surfaces. The places where the DNA bridges across the grating provide open regions where tagmentation can occur. Gratings were produced using standard integrated circuit lithography techniques on silicon substrates. The gratings were either coated with polymethylmethacrylate (PMMA) thin films or used to produce polydimethylsiloxane (PDMS) replicas. DNA was adsorbed and stretched over the gratings by withdrawing a substrate from DNA-containing solutions, a method called molecular combing. Loaded Tn5 transposase (Diagenode) solutions in tagmentation buffer were applied to the surface-adsorbed DNA via microliter droplets and reacted at 40°C for 10 minutes, followed by soaking in sodium dodecyl sulfate (SDS) for 5 minutes. Fragmentation of the DNA (stained with SyBr Gold dye) was confirmed by fluorescence microscopy. Surface tagmentation has the potential to maintain long-range order of the fragments for sequencing applications.

DNA is a popular material for constructing complex, multidimensional nanostructures due to its ability to organize in a precise and predictable manner. Popular approaches to create nanostructures from DNA include DNA origami, DNA tile and DNA brick assembly. An alternate approach to direct the assembly of single-stranded DNA (ssDNA) is to conjugate a hydrophobic moiety (i.e., polymer or lipid-like tail) to the ssDNA to form an amphiphilic molecule that spontaneously self-assembles when added to aqueous solutions. However, the majority of structures created by the assembly of ssDNA-amphiphiles have been spherical and cylindrical micelles. In this presentation I will discuss how we design ssDNA-amphiphiles that self-assemble into supramolecular nanostructures such as nanotubes, and how we use them to target glioblastoma (GBM) in an orthotopic mouse model of GBM.

Nanofiber membranes can be prepared rapidly and inexpensively using the electrospinning process. These fibers have very high surface to volume ratio and a wide range of surface functionalities have been developed for purifying, capturing and mixing fluids within a microfluidic channel. Specific examples of nanofibers with persistent and switchable positive or negative surface charge and biotinylated surfaces for biotin-streptavidin sandwich style assays and their use in microfluidic devices will be presented. Additionally,two strategies for functionalizing electrospun nanofibers for specific capture of biological molecules will be discussed. First, silver-doped carbon nanofibers (SDCNF) are used as the base material for the selective capture of Escherichia coli in microfluidic systems. Polyacrylonytrile nanofibers containing silver nitrate were electrospun and carbonized to form carbon fibers with silver particles at the surface. Antibodies are immobilized on the surface via a three-step process. The negatively charged silver particles present on the surface of the nanofibers provide suitable sites for positively charged biotinylated poly-(L)-lysine-graft-poly-ethylene-glycol (PLL-g-PEG biotin) conjugate attachment. Streptavidin and a biotinylated anti-E. coli antibody were then added to create anti-E. coli surface functionalized (AESF) nanofibers. Functionalized fibers were able to immobilize up to 130 times the amount of E. coli on the fiber surface compared to neat silver doped fibers. To demonstrate selectivity and functionalization with both gram negative and gram-positive antibodies, anti-Staphylococcus aureus surface functionalized (ASSF) nanofibers were also prepared. Experiments with AESF performed with Staphylococcus aureus (S. aureus) and ASSF with E. coli show negligible binding to the fiber surface showing the selectivity of the functionalized membranes. Second, biotin-cellulose nanofiber membranes are successfully fabricated via “click” chemistry. Cellulose acetate (CA) is electrospun, deacetylated and substituted with an alkyne group in either a one or two step process. Azide-biotin conjugate is then “clicked” onto the alkyne-cellulosesurface via Copper-catalyzed Alkyne-Azide Cycloaddition (CuAAC). FTIR,Scanning ElectronMicroscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX), and X-ray Photoelectron Spectroscopy (XPS) are used to confirm addition of biotin without damage to the fiber structure. The biotin-cellulose nanofiber membranes are used in example assays (HABA colorimetric assay and fluorescently taggedstreptavidin assay) where streptavidin specifically binds to the pendant biotin without requiring blocking agent. Both methods yield durable attachment of the specific capture functionality at the fiber surface.

The novel coronavirus disease, COVID-19, has spread rapidly around the world. Its causative virus, SARS-CoV-2, enters human cells through the physical interaction between the receptor-binding domain (RBD) of its spike protein and the human cell receptor ACE2. As an increasing number of variants of SARS-CoV-2 circulates globally, estimates of infectiousness and lethality of newly emerging strains are important. Here, we provide a novel way to develop a deeper understanding of coronavirus spike proteins, connecting their nanomechanical features – specifically the vibrational spectrum and quantitative measures of mobility – with virus lethality and infection rate. The key result of our work is that both, the overall flexibility of upward RBD and the mobility ratio of RBDs in different conformations, represent two significant factors that show a positive scaling with virus lethality and an inverse correlation with the infection rate. A quantitative model is presented to make predictions on the infectivity and lethality of SARS-CoV-2 variants based on molecular motions and vibrational patterns of the virus spike protein. Our analysis shows that epidemiological virus properties can be linked directly to pure nanomechanical, vibrational aspects, offering an alternative way of screening new viruses and mutations against high threat levels, and potentially exploring novel ways to prevent infections from occurring by interfering with the nanoscale motions.

Introduction: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which is still endemic in many parts of the world including Southern Texas. The two polar clinical forms of leprosy, termed tuberculoid and lepromatous, have polarized cellular immune responses, with complex immunological distinctions.
Dendritic cells (DCs) are the primary antigen presenting cells in the immune system. TiO2 nanoparticles have shown to induce maturation of these cells leading to an inflammatory response.
Objective: We aimed to evaluate the effect of potassium incorporated TiO2 nanostructures, namely KTiOx, in the response of human monocyte-derived-DCs to M. leprae.
Methodology: THP-1 human monocytes were differentiated and maturated into DCs using commercially available media, and then treated with KTiOx for 24 hours. Following, cells were infected with M.leprae at an MOI of 10:1 for 24 hours. Secreted human cytokines were measured in the culture supernatants by a multiplex ELISA system.
Results: We observed an increase in the levels of secreted IFN-a and TNF-b upon M. leprae infection in cells treated with KTiOx. IFN-β and its downstream gene IL-10 are preferentially expressed in disseminated and progressive lepromatous lesions, while TNF-β gene is identified as a major risk factor for early-onset leprosy. Interestingly, the levels of cytokine secretion differ comparing 1 M KOH treated Ti and 10 M KOH treated Ti. This can be explained by a wider surface area of the 10M preparation compared to 1 M KOH treated Ti. It is possible that this wider KTiOx area can activate more DCs due to an increase in the contact area.
Conclusion: This study demonstrates the effect of nanostructures of KTiOx and the usefulness of nanoparticle technology in the in vitro activation of human DCs against an infectious disease with a puzzling immune spectrum. Our findings may prompt future therapeutic strategies such as DC immunotherapy for disseminated and progressive lepromatous lesions.

Foodborne pathogens have led to many hospitalizations and deaths. They have been responsible for several outbreaks in recent years. Bacteria are able to survive on a range of surfaces and environments. The risk of bacterial infection from a contaminated surface has increased exponentially due to the formation of biofilm. Bacteria have been able to form a resistance to the modes of action of current antibacterials. This study utilized a hot water treatment (HWT) method for introducing antibacterial property to aluminum surfaces relevant to food processing and packaging applications. The HWT process produces a nanostructured oxide layer on a wide range of metallic materials by simply immersing the metal in water at relatively low temperatures, which can enhance the antibacterial properties. In this work, aluminum foil was hot water treated at 75°C for 30 minutes. Concentrations of E.coli (192 cells) and of S.epidermidis (185 cells) were placed on aluminum foil for different times, ranging from 1 minute to 60 minutes. The survival time was measured and the results indicate, the longer the bacteria was on the HWT aluminum foil surface, the more bacteria was killed. As the temperature of the HWT increased to 85°C and 95°C we observed a decrease in bacterial growth, which is consistent with the more pronounced nanostructures at higher HWT temperatures. Overall, the HWT aluminum foils were 95-100% more effective in killing the bacteria within 1-60 minutes than the untreated foil. HWT aluminum oxide nanostructures are believed to utilize multiple mechanisms to deactivate bacteria, ranging from contact killing to the production of reactive oxygen species (ROS), which requires the bacteria to form multiple defenses in order to maintain vitality.

RNA nanotechnology has advanced extensively for its potential biomedical applications by leveraging a wide range of biological functions of RNA. Especially, RNA microstructures fabricated by rolling circle transcription (RCT)-based enzymatic RNA self-assembly has gained much attention as one of the promising approaches to enhancing the loading capacity and delivery of therapeutic RNA. In this respect, we controlled the enzymatic RNA polymerization by regulating viscosity and studied how viscosity affects the RNA microstructures fabrication by altering the enzyme activities and molecular interactions between reaction components. The solution viscosity was manipulated by increasing a glycerol contents. The increment of viscosity in reaction conditions resulted in reduced RNA polymerization rate, which means that the polymerase activity is regulated by viscosity. Furthermore, the inorganic crystallization of magnesium pyrophosphate, which has an important role as a structural basis of RNA microstructures, decreased as the solution viscosity increases. As a results, the size of the RNA microstructure was decreased as the solution viscosity was increased. Also, the flower-like structure was disappeared in highly viscous conditions. Finally, the size of the RNA microstructure was fine-tuned by regulating viscosity with other previously reported size reduction method that modulates template DNA to monomer rNTP ratio. The required size of the RNA microstructures would be different depending on the biomedical application. Therefore, we suggested new approaches to manipulate the size of RNA microstructures to satisfy diverse biomedical demands precisely.

Chitosan is known to have a high capacity for metal absorption, though it exhibits low mechanical strength. Cellulose, on the other hand, has high mechanical strength but low metal absorption capacity. We propose combining the two materials to form a composite that has both metal-absorbing abilities and high mechanical strength. An abundant source of cellulose is in waste cotton fabric, due to the 10 million tons of fabric the textile industry disposes of each year.^[1] Citric acid was used to deweave cotton fabric into fibers and also to cross-link chitosan and cellulose through esterification, which produces a stronger and more chemically active cotton-chitosan composite while retaining its biodegradability.^[2] One application of cellulose-chitosan composites is the absorption of heavy metals, such as chromium. Chromium pollution is largely caused by industries in energy, mining and metal works, fertilizer, paints, and more. Chromium pollutants, especially chromate, dichromate, and chromium trioxide, have toxic effects on plants, animals, and microorganisms and pose risks to human health ranging from skin irritation to DNA damage and cancer development. Existing methods for removal include chemical reduction, adsorption on porous surfaces with ion exchange, and electrocoagulation, but such methods often produce sludge, require large amounts of chemicals, and pose a risk of secondary pollution.^[3] Cellulose-chitosan composites, being fully biodegradable, provide a greener method for heavy metal cleanup.

A 12.5 g/L solution of chitosan was prepared at a pH of 5 (adjusted by HCl). Pieces of fabric, cut across the weave in a diagonal to maximize the number of fibers exposed, were placed in 40 mL of 0.5 M citric acid and stirred at 800 rpm and 60 °C for 15 minutes. The fibers were then placed in a beaker, mixed with approximately 20 mL of chitosan solution, and cured with two different methods: ultraviolet irradiation for 10 minutes and heat lamp for one hour. The UV-cured composite exhibited gel-like properties, suggesting how it can be used as a hydrogel. The heat lamp-cured composite was a stiff material and far more rigid than both the UV-cured composite and the cotton raw material. Scanning electron microscopy (SEM) of the heat lamp-cured composite showed the cotton fibers supporting the chitosan matrix, suggesting that cross-linking was successful and able to provide mechanical reinforcement.

To test the chitosan composite’s ability to absorb heavy metals, the heat lamp-cured composites and a control of untreated fabric were immersed in 15 mL of 0.01 M potassium dichromate solution for one hour. The samples were then retrieved from the solutions and dried overnight. The chromium content of the two samples, before and after absorption, was analyzed with X-ray fluorescence (XRF). XRF showed that the chitosan composite contained 149 900 ppm of chromium, which was significantly higher than the 38 000 ppm in untreated fabric and 50 300 ppm in deweaved fibers. To use the composite as an in-line water filtration system, we demonstrated that it was also possible to grind the material into a fine powder after cooling with liquid nitrogen, which can then be contained in a packed-powder filter or incorporated into a porous nanocomposite membrane.

[1] Mohamed S., et. al, Polymers, 2021, 13, 626
[2] Akpomie, K., et. al, Ecotoxicol. Environ. Saf., 2020, 201, 110825
[3] Tumolo, M., et. al, Int J Environ Res Public Health, 2020, 17, 5438

Surface-enhanced Raman scattering (SERS) sensor has been recognized as one of the most versatile tools for ultrasensitive detection of trace chemical and biological analytes. It is now well understood that the plasmonic coupling effect at the nanometer-sized gap junction between particles, that is so called ‘nanogap’ or ‘hot spot’ induces enormous electromagnetic enhancement that allows SERS signal to be detected with single-molecule sensitivity. However, inhomogeneous distributions of the location and intensity of the nanogaps lead to varying and non-uniform Raman intensities over substrate, and consequently provides irreproducible measurements. It seems to be practically impossible to obtain both of high sensitivity and good signal reproducibility in a large area.
Currently, SERS sensor is actively employed for the detection of diagnostic biomarkers which provides information about disease from biological fluids. Blood is the most important biological fluid which stands as the medium for transporting nutrients, oxygen and immune cells throughout the body and also maintains body temperature and pH. Therefore, it is an active indicator of various pathological disorders. For the analysis of blood, however, the separation of blood plasma has to first be accomplished to remove red blood cells. The conventional process of blood plasma separation is performed in macroscale centrifugation which requires several milliliters of blood and bulky and expensive apparatus. For patients, it is quite inconvenient due to the high volume of sample blood and transportation time.
To address this problem, we propose a novel strategy for simultaneous separation and SERS sensing of whole blood without additional blood plasma separation. The SERS-active porous metal film is integrated onto the blood plasma membrane. Then, blood plasma is separated from whole blood through the membrane and reached at the surface of SERS-active porous metal film through its pores. For the SERS-active porous metal film, a commercially available pluronic P123 block copolymer was used as a pore former, and physicochemical properties such as porosity and pore size of the metal thin film were controlled by electrodeposition process. As a result, mesoporous palladium thin film with homogeneously perpendicular 6-nm-sized channels was formed onto the blood Pd separation membrane. The homogeneous and perpendicular nanopores was very advantageous for the reproducibility of SERS signals and the facile diffusion of blood plasma. Also, Au layers with the thickness of 1 nm were carefully deposited on the mesoporous Pd thin film by electroless plating process. The obtained mesoporous Pd@Au film integrated with blood plasma separation membrane successfully detected various chemicals and biomarkers from whole blood without interfering with red blood cells.

The ability to detect bacteria in complex matrices with a rapid, low-cost, and simple method is paramount to improving food and water safety worldwide. Bacteriophages (phages), which are natural predators of bacteria and have been used to detect and treat bacterial infections, are a potential solution for bacteria separation and detection. Phages immobilized on magnetic nanoparticles can separate bacteria from their surroundings and then concentrate and detect low concentrations of bacteria in a short time. Our bacteria separation and detection system consists of T7 phages with modified capsid proteins that give a specific and directional binding to silica-coated magnetic nanoparticles. No modification of the phage genome is required; instead, phage capsid modification was accomplished by modifying the bacterial host, which was confirmed with sequencing. Expression of the modified phage capsid protein and assembly on the phage capsid was confirmed by western blotting. The immobilization of modified phages on the silica-coated magnetic nanoparticles was confirmed by infectivity studies, where the infectivity of the modified T7 was higher than wild-type T7. The phage-nanoparticle construct could then separate and detect bacteria in relevant water conditions. Expanding the range of complex conditions in which phage-based detection platforms can perform is an essential step towards improving food and water safety and, in turn, global public health.

Encapsulation of bioactives like nutrients, probiotics, and drugs is of great interest in the aquaculture industry recently. For oral delivery systems, scalable microencapsulation techniques using food-grade materials, protection of the nutrients against harsh environments, and targeted delivery in a certain portion of the Gastro-Intestinal (GI) tract are considered important. In an aquaculture setting, the nutrient delivery system must be able to prevent the leaching of the nutrient in the water environment and ensure release at the specific site of the fish’s GI tract, usually the intestine. Alginate-based microparticles are one of the most studied microencapsulation systems for oral delivery applications. Traditionally, crosslinked alginate microparticles are fabricated using a multi-step technique that involves wet extrusion-gelation using calcium salt solution, followed by additional drying steps. The multistep fabrication makes the process less feasible for large-scale production and application in the food and agriculture industry. In this study, a one-step, and scalable fabrication process was designed using spray drying to fabricate crosslinked alginate microparticles. The presence of the crosslinking between the calcium ion and the alginate polysaccharide network was studied using different material characterization techniques. Additionally, the microparticles were studied for encapsulation of a carotenoid, β-carotene— a vitamin A precursor and an antioxidant. The process enabled a high yield production (~60.0%) of the crosslinked microparticles with high encapsulation efficiency (~50.72%) of β-carotene. The microparticles were found to be stable against disintegration in the aqueous environment due to the crosslinking effect. Additionally, the release studies in the simulated gastric and intestinal fluids showed that the crosslinked microparticles had a minimal release in the gastric fluid (3.42% in 2 hours), while a faster and complete release of the β-carotene in the next 4 hours of dissolution in the simulated intestinal fluid. While typical release behavior comparable to crosslinked alginate microparticles prepared using the traditional extrusion-gelation technique was observed, the optimized process is superior in terms of ease of fabrication, scalability, and cost-effectiveness for the use in bioactives encapsulation in the aquaculture industry.

Bioprocessable and biodegradable polymers can be used as functional material for assessing the metabolic activity of microbes within their environment. Current methods for detecting, assessing, and quantifying microbial activity in soil are limited to time consuming and expensive procedures requiring skilled labor, and can rarely be done directly on site. Simultaneously, as the world faces a growing population, the demand for IoT technology that allows for real-time sensing is crucial in precision agriculture to achieve maximum yields. Previous work has shown the feasibility of using an impedimetric method of detection to monitor the degradation of a cellulose acetate film, cast on interdigitated electrodes (IDEs), to assess the cellulolytic activity of microbes directly in soil. Cellulose degradation, which is a plant product specifically degraded by microbes and fungi in soil, can be directly linked to the metabolic activity of microbes, which has been previously used as a marker for soil health and could provide insight into crop yields and soil performance. Other biopolymers found in soil, such as lignin and chitin, can be used as representative to characterize the diverse metabolic activities of microbes. Similarly, monitoring the degradation of similarly relevant parameters is possible. Materials with pH-dependent solubility, such as cellulose acetate phthalate (CAP) and several methacrylates, can be used to detect sudden changes in pH in several environments.
In this new concept, we have developed a wireless method for assessing microbial activity based on the same system of material degradation. Wireless sensing tags based on RF backscattering (0.5 – 1.5 GHz) have been designed using simulations done on CST microwave studio to achieve miniaturized sensing systems that can be buried and monitored directly in soil. In this method, the wireless reader transmits an interrogation signal to the sensor tag buried in the soil and collects the backscattered signal from it. The sensor tag embeds its resonance as an electromagnetic signature on the frequency spectrum of the reflected signal. Since the resonant frequency of the sensor tag is a function of the dielectric constant of the surrounding media, variations in the thickness and dielectric properties of the soil as well as the coatings on the sensor tag influence the resonant frequency. The sensor tag consists of a sensing unit and reference unit of size 2 cm x 2 cm each. By passivating the sensing unit with the biodegradable material, shifts in the resonance frequency as the material is decomposed can be measured and correlated to the microbial metabolic activity within the environment. The reference unit is coated with an anti-microbial film which makes it insensitive to microbial activity but sensitive to all the other environmental stimuli the sensing unit is exposed to. This differential pair configuration helps in extracting microbial activity by eliminating the effects of other soil parameters, such as volumetric water content (VWC) and temperature. These small, low-cost, battery-less sensing tags have been manufactured using scalable processes like 3D printing and laser cutting. By making sensing tags from extruded polylactic acid (PLA) filament, the meander-structured antennas using Zinc tape, and using a biodegradable polymer as functional material, we’ve achieved a fully biodegradable environmentally-friendly sensing system that can be buried in the soil and left for natural resorption without any concerns. These tags have been successfully used to monitor VWC (5% - 30%) in soil and the decomposition of materials with pH-dependent solubility with applications in food packaging and storage.