Symposium SM01 : Materials for Biological and Medical Applications

Transmission electron microscopy (TEM) is the primary method to image biological cellular ultrastructure, though it is not possible with conventional TEM to label and distinguish different kinds of molecules in a single image. This serious limitation was recently addressed through the development of “multi-color EM,” which uses selective lanthanide ion tagging and electron energy-loss filtered imaging (Ramachandra et al., 2014, Microsc Microanal. 20; Adams et al., 2016, Cell Chem Biol. 23) yielding data analogous to multi-color fluorescence microscopy but at ~100x the magnification. While this technique promises to reveal novel structural information, it is tedious (requiring multiple long exposures with different energy filter settings), terribly inefficient (depending on the small fraction (<1%) of primary TEM electrons that are inelastically scattered), and yields noisy images with significantly lower resolution than is achievable by TEM imaging.

To improve the throughput, efficiency, and resolution of multi-color EM, we have developed a new multi-color EM technique with 4D-STEM, which uses a high-speed pixelated detector to record the vast majority of the primary electrons that interact with the specimen.

In order to achieve the necessary sensitivity for this technique, the pixelated detector must deliver synchronized (global shutter) readout of a large number of pixels (at least 512 × 512 pixels) and it must have single-electron sensitivity for electron counting.

We used the DE-16 direct detection camera in conjunction with the DE-FreeScan scan generator (Direct Electon LP, San Diego, CA USA) to collect a 4D-STEM dataset of a cellular mitomatrix sample, with mitochondria labeled by cerium and containing gold nanoparticles. After correcting for distortions in the diffraction patterns, we were able to develop a metric to distinguish the cerium labels and gold nanoparticles, while simultaneously generating bright-field and dark-field images of the specimen at significantly higher resolution than is possible through fluorescence light microscopy.

Neural electrodes have been widely used to monitor neural signals and/or deliver electrical stimulation in the brain. Currently, biodegradable and biocompatible materials have been actively investigated to create temporary electrodes that could degrade after serving their functions for neural recording and stimulation from days to months. The new class of biodegradable electrodes eliminate the necessity of secondary surgery for electrode removal. In this study, we created biodegradable, biocompatible, and implantable magnesium (Mg)-based microelectrodes for in vivo neural recording for the first time. Specifically, conductive poly-3,4-ethylenedioxythiophene (PEDOT) was first deposited onto Mg microwire substrates by electrochemical deposition, and a biodegradable insulating polymer was subsequently sprayed onto the surface of electrodes. The tip of electrodes was designed to be conductive for neural recording and stimulation, while the rest of electrodes was insulated with a polymer that is biocompatible with neural tissue. The charge storage capacity and impedance of Mg-based microelectrodes and their performance during neural recording in the auditory cortex of a mouse were studied. The results first demonstrated the capability of Mg-based microelectrodes for in vivo recording of multi-unit stimulus-evoked activity and spontaneous activity in the brain.

Small organic molecules exhibiting therapeutic properties are known as active pharmaceutical ingredients (API) and are of great interest in pharmaceutical material science. These API can exist in several crystalline forms as well as in different states of disorder of which the amorphous form is of great interest due to their greater free energy which results in higher bioavailability and exposure in-vivo. Detailed screening as well as subsequent characterization of the different forms with varying degrees of crystallinity is imperative to choosing a suitable lead candidate that will elicit the required therapeutic response in-vivo. Thus the material science aspect of drug development plays a vital role in the pharmaceutical industry since it is responsible for proper physical form selection and its maintenance in the formulation during its shelf life. Polymorphism, amorphization or changes to chemical composition are several ways in which the physical form of the API may be altered which may bring about a change in its pharmaceutical properties and ultimately affect the final drug product and its performance. This talk focuses on the material science part of pharmaceutical drug development and tackles the different challenges faced in characterizing drug substances in both the discovery and development stage as well as in the formulation. Judicious form screening as a salt or polymorph, form changes during processing and unit operations and vitrification of crystalline materials for improving bioavailability will be covered as well as analytical techniques used for such characterization. The focus is on several case studies involving both drug susbtance and drug product with emphasis on experimental (X-ray diffraction, spectroscopy, microscopy, thermal analysis) and computational characterization techniques to showcase advances in pharmaceutical material science encompassing both ordered and disordered materials.

Biosensors and materials for biomedical applications generally require precise chemical functionalization to bestow their surfaces with desired properties, such as specific molecular recognition and antifouling properties. Consequently, tailoring the chemistry at the biosensing interface has a crucial role in obtaining the best selectivity and sensitivity.¹ Especially for DNA biosensor, either biological or artificial probes, as well as antifouling moieties, need a defined type of chemistry to be anchored with respect to their chemical modification and the type of substrate, affecting the biodistribution.² In addition, control of the surface probe density is required for achieving high sensitivity.³ Traditional methods have the applicability limited to specific substrates and aim to control the density at the surface modification step, with the drawback of having to assess the hybridization efficiency each time.
Recently, polyelectrolytes were used in biosensing to tailor the probe type and distribution. Furthermore, the ensemble of substrates for biosensing purpose has been increased thanks to the multiple nature of polymers and their electrostatic interactions. The chemi/physisorption of modified polyelectrolytes provides advantages for the immobilization of biomolecules and for biosensing applications. At physiological pH, poly(L- lysine) (PLL) polymers readily and strongly adsorb onto a variety of metal oxide surfaces through multivalent electrostatic interactions between the positively charged lysine side-chains and a negatively charged surface.⁴ As a result, PLL polymers, which are easy to functionalize thanks to the amino groups in the side chain, allow the accommodation of the grafted functional moieties over the substrate, maintaining their adsorption properties.
Based on this approach, biorecognition surfaces were prepared by deposition of modified PLL polymers grafted with various fractions of oligo(ethylene glycol) (OEG, antifouling) and maleimide (Mal) moieties (PLL-OEG-Mal), so that both the type of functionalization and the control over the density is achieved at the same time, during the synthetic step, verified by ¹H-NMR. PLL-OEG-Mal polymers were self-assembled at the substrate and coupled to thiol-peptide nucleic acid (PNA) probes, forming a real-time DNA biosensor. A linear relationship between the probe density and the PLL grafting density was found monitoring the frequency shift of the hybridization step for the complementary DNA versus the density of Mal group coupled to the PLL backbone. In order to establish the absolute probe density values at the biosensor surfaces, cyclic voltammetry experiments using Methylene Blue-functionalized DNA were performed, providing a density of 1.24 × 10¹² probes per cm² per % of grafted Mal, thus confirming the validity of the density control in the synthetic PLL modification step without the need of further surface characterization.^5,6 Thanks to their advantages, modified PLL polymers can be used to tune the surface properties, accommodating several clickable side groups as linkers and antifouling moieties, and forming different architectures as layer-by-layer and µ-arrays, as well as their application in soft lithography and pillar structures.⁶

References:
1 D. Bizzotto, I. J. Burgess, T. Doneux, T. Sagara, H. Z. Yu, ACS Sensors 2018, 3, 5–12.
2 S. H. North, E. H. Lock, C. R. Taitt, S. G. Walton, Anal. Bioanal. Chem. 2010, 397, 925–933.
3 V. Biagiotti, A. Porchetta, S. Desiderati, K. W. Plaxco, G. Palleschi, F. Ricci, Anal. Bioanal. Chem. 2012, 402, 413–421.
4 S. Pasche, S. M. De Paul, J. Vörös, N. D. Spencer, M. Textor, Langmuir 2003, 19, 9216–9225.
5 J. Movilli, A. Rozzi, R. Ricciardi, R. Corradini, J. Huskens, Bioconjug. Chem. 2018, 29, 4110-4118.
6 J. Huskens, R. Ricciardi, J. Movilli, D. D. Iorio, A. Marti Morant, World Patent WO 2018/222034, 2018.

The oral cavity harbors a wide array of microbiota which if perturbed, would result in the loss of mutualistic/ symbiotic balance leading to diseases. The aggregates of these microorganisms can orderly lay in a protective sheath coined as extracellular polymeric substance (EPS), to form oral biofilm a.k.a. dental plaque. There is mounting evidence that the dental biofilm can be the culprit of several diseases such as dental caries which affects 2.4 billion people worldwide.
In the biofilm, the fermentation of the dietary carbohydrates causes organic acids production (pH~4.5) demineralizing the teeth enamel. The most common carious pathogen involved in the dental biofilm is the gram-positive bacteria, streptococcus mutans (S. mutans), which actively produces EPS and acids.
Unfortunately, the rigorous solution to the dental caries is compounded due to the multifactorial nature of the disease. Nanoparticles (NPs) can offer an unequivocal solution to the biofilm issue with the privilege of multifunctionality, on- demand controlled release of drugs, high loading efficiency, selectivity, and trackability. Importantly, the nanoparticles can be designed to be ‘self-contained’ and exert the therapeutic effects without the utilization of conventional antibiotics . Nevertheless, as a major group of nanobiotics, the experimentation with metallic nanoparticles has raised concerns regarding their accumulation and non-degradation.
Carbon dots (CDots) are the emergent class of carbon family which have attracted a host of research in recent years due to the ease of fabrication, tunable luminescent properties, and the abundance of functional groups. These properties combined with their degradability offer a unique platform for combating bacteria.
Herein, we present for the first time, a ‘particle in particle’ approach for targeting the EPS with its characteristic pH to release the load of inherently therapeutic CDots to kill notorious S. mutans. Specifically, phosphonium containing CDots have been wrapped in a layer of poly(styrene)-b-poly(n,n-dimethylaminoethyl methacrylate) (PS-b- PDMA) which shows pH responsiveness upon encountering acidic pH. Moreover, phosphonium ions have been utilized to confer antibacterial properties to the NPs as with more pronounced inhibitory effect compared to their ammonium counterparts.
The retained load of therapeutic CDots will get liberated only when in touch with the pH of EPS. This approach would not only lead to the biofilm dispersal and enhanced the bacterial susceptibility but would also exert the antibacterial properties in situ without any external drugs. Moreover, the absence of any external stimuli, for example, H2O2 and photostimulation, is a boon which reduces the unintentional toxicity.
We have demonstrated that these NPs could suppress the biofilm EPS and decrease the S. mutans viability in the in vitro human tooth model of the biofilm. The mechanism of action can be classified as ROS generation and fragmentation of genomic DNA on the subcellular level. We have shown, in vivo, in the rat model of dental biofilm that these nanoparticles could successfully decrease the cultivatable S mutans within 13 days using the plate count assay and S. mutans detection kit. It has been recognized through histopathological examination that these NPs did not interfere with the normal activities of the major organs while the dental caries scoring indicated a decrease in the caries development. The gene expression profiling of the oral microbiota from rats revealed that the change in the microbiome diversity was not statistically significant among groups. Therefore, our approach has great implication for the application of nanomaterials as degradable antibiofilm in the dental clinic.

Numerous bacterial stains have become resistant to conventional antibiotics in recent years. Fortunately, an increasing body of research indicates that through the addition of specific metabolites (like sugars), the antibacterial activity of certain drugs can be enhanced. A new type of self-assembled nano-peptide amphiphile (SANPA) was designed in this study to treat antibiotic resistant bacterial infections and to reduce the use of antibiotics. Here, SANPAs were self-assembled into nanorod structures with a nanoscale diameter at concentrations greater than the critical micelle concentration (CMC). Both Gram-positive and Gram-negative bacteria were treated with SANPAs with metabolites supplementation. After various amounts of time metabolites pre-incubation, SANPAs reduced bacteria growth relative to non-matebolite treatments at all concentrations. Cytotoxicity assays indicated that the presence of metabolites seemed to slightly ameliorate the cytotoxic effect of the treatment on model human fetal osteoblasts (or bone forming cells) and human dermal fibroblasts. In conclusion, we demonstrated here that SANPAs-like nanomaterials have a promising potential to treat antibiotic resistant bacteria especially when added to metabolites, potentially limiting their associated infections. By comparision of SANPAs treatments under different communication conditions, the results further provide resource for researcher to design novel agents and treatment methods against antibiotic resistant bacterial infection.

The study of the intracellular compartment requires devices that can not only monitor the bioelectric activity, but also control and observe the biochemical environment at the biomolecular level. Up to now the electrical activity of excitable cells, in particular that of a whole cells network has been studied with multi electrode array (MEA) devices. To overcome the intrinsic limitations of this method, such as low accuracy and low signal to noise ratio, 3D nanoelectrodes were developed on the planar surface in order to improve the interface with cells.¹ In fact, it has been amply demonstrated the strong coupling between these structures and the cellular membrane². A novel fabrication technique for producing 3D vertical nanostructures that can be tuned in material and shape has recently been shown by our group³. This technique enables the fabrication of 3D hollow nanoantennas with high control over geometry and layout by means of a focused ion beam (FIB) procedure. These plasmonic vertical structures have shown to be suitable for enhancing the electrical recording of electrogenic cells, for performing selective intracellular drug delivery^4,5To further increase the capabilities of MEAs, here we combine these 3D hollow nanoantennas on top of MEA electrodes fabricated on thin silicon nitride membranes in order to combine the improved electrical recording with a controlled delivery over the cells. The goal of in vitro devices is to replicate as closely as possible aspects of the true in vivo microenvironment, this platform could be applied to make in vitro assays more realistic and capable of a precise manipulation of the microenvironment to deliver soluble factors to cells. The nanoantenna’s hollow shape, in fact, allows to have a controlled delivery of molecules during cell signal recordings, providing the device with great versatility and allowing the exploitation of different approaches and techniques such as disease diagnosis, single cell analysis, drug screening, proteomics and other biological applications. The device configuration could provide flexibility in controlling the critical biochemical factors that influence cells behavior, allow for the partial differentiation of cells in a single system, provide for the establishment of biochemical gradients in two- or three-dimensional cultures, and at the same time allows for high quality intra and extracellular recordings.
1. Spira, M. E. & Hai,. Nat. Nanotechnol. 8, 83–94 (2013).
2. Dipalo, M. et al. Nano Lett. 9, 6100-6105 (2018).
3. Dipalo, M. et al. Nanoscale 7, 3703–11 (2015).
4. Dipalo, M. et al. Nano Lett. 17, 3932–3939 (2017).
5. Caprettini, V. et al. Sci. Rep. 7, 8524 (2017).

More than 2 million End Stage Renal Disease (ESRD) patients receive dialysis to sustain life, with this number likely to represent less than 10% of the actual need. In the United States alone, over 460,000 people are on dialysis. Conventional hemodialysis removes urea and other metabolic waste from the body by running ~120 L of dialysate over hollow fiber dialysis membranes each session, which is typically 3-4 hours and 3 times a week. The intermittent character of hemodialysis results in large fluctuations in blood metabolite concentrations. Observations show that long-term survival in dialysis patients treated by extended hemodialysis are improved compared to conventional hemodialysis. Thus a portable dialysis machine that is working continuously would bring in significant health, quality of life and economic benefits.

To enable portable kidney dialysis for ESRD, the regeneration of the dialysate in a closed loop system is a primary critical technical barrier. Currently the ~120 L (kg) of dialysate per session, far exceeds usable weights for a portable system. We have developed an efficient photooxidation system based on hydrothermally grown TiO₂ nanowires, UV LEDs, and catalytic gas diffusion barriers to decompose urea from the dialysate at rates sufficient to remove daily production of urea at 15 g/day.

A photoelectrical decomposition cell with SiO₂/FTO/TiO₂-nanowire anode, 10 mM urea/0.15 M NaCl electrolyte, and 4 mg/cm² Pt black loaded carbon paper cathode was characterized for urea decomposition efficiency. Under 4 mW/cm² illumination of the 365 nm LED with 40% quantum efficiency, the device yielded a photocurrent density of ~ 1 mA/cm² in a dialysate simulant of corresponding to 40% quantum efficiency in urea decomposition per incident photon. From performance parameters, a feasible portable device with ~0.23 m² active area and a current draw of 11 A is able to decompose a daily 15 g urea production sufficient to regenerate dialysate. Also high selectivity (80%) for urea decomposition over Cl₂ formation is shown. For comparison, prior reports in literature [J. Photochem. Photobiol. A-Chem, 2009, 205, 168] for urea fuel cells of agricultural waste, > 5000 A would be required. Improvements reported here are based on nanowire microstructure to efficiently separate electron holed pairs and efficiently adsorb incident UV irradiation.

Recent advances in molecular biology, single cell manipulation and data analysis techniques have allowed us to look into the molecular states of individual cells with unprecedented detail. These methods have made it possible to investigate how the genotype in combination with internal and external regulatory factors guide the development of complex and diverse phenotype. However, these methods rely on cell lysis and can provide only a snapshot of the cellular state. This makes it challenging to monitor gene expression changes over time. Inferences about dynamic cellular processes are obtained by constructing pseudo-time trajectories from the continuum of molecular states present in a cell population that can reflect the essence of a single cell trajectory. However, the dynamics of cell state may be non-hierarchical or stochastic which the computational algorithms cannot always capture. In order to acquire a holistic understanding of decision-making pathways involved in processes such as cell reprogramming, differentiation and maturation, tracking the same cell in time is necessary. Our long-term goal is to develop a single-cell microfluidics platform that can temporally examine cells by performing precise cellular manipulations (e.g. genetic editing using CRISPR/Cas9) and subsequent analysis of internal cellular biomarkers in a non-destructive manner. Such a system would allow us to monitor the biochemical changes in cells over time and correlate these to the external inputs and perturbations.
Localized electroporation has emerged as an effective technique for introducing the desired changes in cellular systems by delivering foreign molecules such as nucleic acids. Unlike bulk electroporation where the entire cell membrane is exposed to a strong and non-homogeneous electric field, localized electroporation utilized nanostructures (such as nanochannels) to confine the electric field to only a fraction of the plasma membrane. This controlled perturbation allows for efficient transport of molecules into the cells as well as helps maintain high cell viability. As such, localized electroporation is also being used to address the converse problem, i.e. to non-destructively sample the cytosolic contents of living cells.
Although several experimental reports demonstrate the phenomena of localized electroporation, a mechanistic understanding of the different parameters involved in the process is lacking. In this work, we present a multiphysics model that 1) estimates the transmembrane potential developed across the cell membrane in response to a localized electric field, 2) predicts the electro-pore distribution in response to the local transmembrane potential drop and 3) calculates the molecular transport into and out of the cell based on the predicted pore-sizes. Using the model, we identify that cell membrane tension plays a crucial role in enhancing both the amount and the uniformity of molecular transport, particularly for large proteins and plasmids. We also find that a critical voltage range is necessary for efficient electroporation. We qualitatively validate the model predictions by delivering large molecules (fluorescent-tagged bovine serum albumin and mCherry encoding plasmid) and by extracting an exogenous protein (tdTomato) in an engineered cell line on a localized electroporation platform. The findings presented here should inform the future design of microfluidic devices for localized electroporation based sampling and temporal, single cell analysis. Eventually, high throughput temporal analysis of single cells would help us gain insights into fundamental aspects of developmental biology, study the progression of diseases such as Alzheimer’s and Parkinson’s and evaluate the efficacy of drugs over time.

Endogenous electrical fields play a major role in various biological processes from embryonic development to cell division and migration (electrotaxis). The latter has been extensively researched due to its involvement in wound healing processes. A better understanding of electrotaxis induced by external electrical fields (direct current), could hence lead to a breakthrough in wound healing therapy. However, the underlying mechanisms within the cells during electrotaxis remain still unclear.
The main limitation for research, as well as the clinical application of direct current therapy for directed cell migration, lies within the utilized electrodes. Metal electrodes made of platinum or copper, as well as silver chloride, corrode and release toxic by-products under direct currents, which are harmful to cells and in addition, might influence their migratory function. Therefore, all electrotaxis experiments until now require agar salt bridges to connect the electrodes to the cell chamber, which lead to a bulky and inefficient experimental setup. A better solution for the stimulation material could improve cellular research and facilitate the transition towards clinical use.

As an alternative, we propose the use of polyimide-based iridium oxide electrodes coated with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT/PSS). These electrodes are able to maintain stable ionic currents over long periods of time and can drive electromigration of cells without the release of any cytotoxic by-products.

Using a simple electrotactic chip we were able to show that PEDOT/PSS electrodes have sufficient ion filling capacity to drive electromigration of skin cells in an agar bridge free system. The chip was fabricated through soft lithography of polydimethylsiloxane (PDMS) which was chemically bonded to a glass coverslip through surface modification with oxygen plasma. The chamber consists of a channel with a seeding area for the cells and two reservoirs at both ends of the channel for medium and the insertion of the electrodes. The use of polyimide-based electrodes allows the reusability of these for several experiments, facilitates the assembly of the electrotactic chamber and greatly reduces the associated cost.
During the experiments, a constant voltage was applied to the electrodes and the current was continuously monitored. Time-lapse photography documented the directed migration and the cell motility was quantified utilizing circular statistics. It was found that pre-oxidation/reduction of the PEDOT/PSS electrodes increased the current injection and subsequently the possible charge delivery to the cells.
We conclude that PEDOT based electrodes can be efficiently used for direct current stimulation and have the power to influence the migration of cells relevant to the healing of human skin. The combination of polyimide-based reusable electrodes and disposable microfluidics make a cost-efficient analysis of electrotaxis possible at high throughput.

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 759655).

Dissolved oxygen [DO] is crucial to environment, industry, life technology, and human health, etc. Hypoxia[1] (≦0.5% O₂, usually at tumor environments) is relevant to cancer, stroke, arteriosclerosis, Parkinsonism, and Alzheimer's disease etc.. Therefore, in vivo dissolved oxygen detection is essential for disease prevention and treatment. For dissolved oxygen detection, phosphorescence based optical oxygen analysis showed superior performance among several ways with noninvasive sensing, no oxygen consumption, high sensitivity, fast responses and single cell scale sensing.
Herein, five kinds of multi-arm block copolymers were synthesized by atom transfer radical polymerization (ATRP) to load the excellent hydrophobic oxygen probe platinum (II)-5,10,15,20-tetrakis- (2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) for optimizing PtTFPP's performance in hypoxia sensing and imaging. Among them, fluoropolymers were introduced with their excellent capacity to dissolve and carry oxygen for investigating carriers' structure-property relationship. Under nitrogen atmosphere, high quantum efficiency of PtTFPP in fluorine-containing micelles could reach to 22% without complicated modification of PtTFPP. Oxygen-nitrogen titration by a gas manipulator showed that fluorine-containing micelles have higher oxygen sensitivity (I₀/I) than fluorine-free ones, and micellar sensors are more sensitive under body temperature (37°C) than room temperature (25°C). Besides, the phosphorescence lifetime of fluorine-containing micelles achieved 76μs under nitrogen, which is among the longest ones in the known literature. This result showed great potential for lifetime sensing and imaging because longer phosphorescence lifetime determines accurate resolution and legible hypoxia imaging.
For biological application, one fluorine-containing PtTFPP micellar sensor was used to detect E.coli JM109's respiration under oil seal by a high throughput plate reader. Based on the Stern-Volmer equation and the dynamic phosphorescence intensity curve, we converted the phosphorescence intensity at each time point to the real-time DO concentration. We also extended the sensor's respiration application to mammalian cells by using J774A.1, MTT assay and confocal imaging were carried out for demonstrating the micellar sensor is nontoxic and extracellular. Similar to the phenomenon of E.coli, higher cell numbers induced faster oxygen consumption. In vivo hypoxia imaging of tumor-bearing mice was also achieved through an In Vivo Imaging System. The phosphorescence imaging was obtained after anesthesia a sensor injected mouse. It can be seen that the phosphorescence image at tumor region is much brighter, and the phosphorescence intensity at tumor region is 1.6 times higher than that of normal region. Moreover, the phosphorescence imaging lasted for at least 10 minutes without obvious decay, indicating that the PtTFPP possessed excellent in vivo stability and strong phosphorescence signal after being encapsulated by our materials. Thus, the sensors reported here might be capable for in vivo tumor imaging. It is expected that the further use of the sensors can be extended to measure the intravascular oxygen contents, which will be more instructive to cancer prevention and cancer clinical analysis.
Reference:
1. Vaupel, P.; Hoeckel, M.; Mayer, A. Detection and characterization of tumor hypoxia using pO2 histography. Antioxid. Redox Signal. 2007, 9, 1221-1235.

Human skin exhibits high stiffness of up to 100 MPa and high toughness of up to 3,600 J m^-2 despite its high water content of 40~70 wt%. Engineering hydrogels have rarely possessed both high stiffness and toughness, because compliant hydrogels usually become brittle when excess crosslinker is added to make the gel stiff. Furthermore, conventional hydrogels usually swell under physiological conditions, weakening their mechanical properties. Here, we designed a non-swellable hydrogel with high stiffness and toughness by interpenetrating covalently and ionically crosslinked networks. The stiffness is enhanced by utilizing ionic crosslinking sites fully, and the toughness is enhanced by adopting synergistic effects between energy-dissipation by ionic networks and crack-bridging by covalent networks. Non-swelling behaviors of the gel are achieved by densifying covalent and ionic crosslinks. The hybrid gel shows high elastic moduli (up to 108 MPa) and high fracture energies (up to 8,850 J m^-2). In vitro and in vivo swelling tests prove non-swelling behaviors of the gel. Live/dead assays show 99% cell viability over a period of 60 days.

Obesity is a global health issue that is suffered by over 700 million people worldwide.[1] Common approaches for treating obesity include non-surgical (excise) and surgical (invasive) treatments, which normally have a high potential of weight rebound or can introduce serious complications.[2] Recent studies demonstrated that vagus nerve blocking and stimulation have multiple physiologic functions related to food intake, energy metabolism, and glycemic control, which can result in a meaningful weight loss. Nevertheless, like many other electrical nerve
stimulation therapies, the electrical signals and corresponding biological activities are very hard to be correlated, particularly for such irregular food taking and stomach peristalsis activities. Therefore, the long-term efficacy may be jeopardized and the constant electrical stimulation may trigger compensation mechanisms.[3] Power supply is also an issue for this type of implanted electronic devices.
Here we present an implanted vagus nerve stimulation (VNS) system that is battery-free and spontaneously responsive to stomach movement. The VNS system comprises a flexible and biocompatible nanogenerator that is attached on the surface of stomach. It generates biphasic electric pulses in response to the peristalsis of stomach. The simulated satiety signals deceive the brain via vagal afferent fibres,[4] and reduces food intake when the stomach is in motion. This strategy was successfully demonstrated on rat models. Within 100 days, the average body weight was controlled at 350 g, 38% less than the control groups. This new VNS system correlated nerve stimulation with targeted organ functionality through a smart, self-responsive device, and demonstrated an outstanding weight control capability better than other electric stimulation strategies. This work also provides a new concept in therapeutic technology using artificial nerve signal generated from coordinated body activities.
References:
[1] Collaborators, G. O. et al. Health Effects of Overweight and Obesity in 195 Countries over 25 Years. N. ENGL. J. MED. 377, 13 (2017).
[2] Bray, G. A. & Tartaglia, L. A. Medicinal strategies in the treatment of obesity. Nature 404, 672-677 (2000).
[3] Schachter, S. C. & Saper, C. B. Progress in Epilepsy Research : Vagus Nerve Stimulation. Epilepsia 39 (1998).
[4] Schwartz, M. W., Woods, S. C., Porte, D., Jr, Seeley, R. J. & Baskin, D. G. Central nervous system control of food intake. Nature 404, 661 (2000

This talk summarizes concepts in materials science that serve as the basis for transient electronic implants, as a new paradigm for treating injuries and tracking recovery. Here, engineered device platforms provide monitoring and/or therapeutic functionality during the time course of a natural biological process such as healing, and then disappear into the body, without a trace, through mechanisms of bioresorption. This mode of operation eliminates unnecessary device load on the body, and associated risk to the patient, in a way that bypasses the need for secondary surgical extraction. Examples of bioresorbable materials and device designs will be presented in the context of (1) electrical stimulators for accelerated neuroregeneration in damaged peripheral nerves, (2) cardiac pacemakers for recovery from heart surgery and (3) programmable drug release vehicles for treatment of disease.

TiO₂ nanotube arrays constitute highly versatile scaffolds that are suited for long-term organotypic culture of neuronal tissue, including retina explants and beyond. While tube diameter and surface roughness [1] are central parameters for successful culture and have to be optimized for every specific tissue type, super hydrophilicity ensures nutrition supply by a wetting layer of culture medium even in absence of complicated perfusion systems [2]. Clearly adhesion between tissue explants and nanotube scaffolds plays a key role for maintaining tissue integrity. Lift-off approaches are, on the other hand, desirable for tissue transfer and nanotube regeneration, respectively. Employing extensive environmental scanning electron microscopy (ESEM) as well as laser scanning microscopy (LSM) studies we address both aspects and demonstrate that UV-light exposure and enzymatic treatment are ideally suitable for nanotube regeneration. Based on these findings, we present an assay for concurrent biomechanical straining and in situ imaging employing ESEM and LSM. Thus, we will be able to find correlations between structural components of retina and their variation in tissue mechanics. This will pave the way for in vitro studies on tissue regeneration and surgery techniques in combination with drug testing.
[1] K. Fischer, S. G. Mayr. Adv. Mater. 23, 3838–3841 (2011)
[2] V. Dallacasagrande, M. Zink, S. Huth, A. Jakob, M. Müller, A. Reichenbach, J.A. Käs, S.G. Mayr, Adv. Mater. 24, 2399–2403 (2012)

The study of the restoration of vision is one of the most popular research topics in neuroscience and biomedical sciences. A functional/adaptive retinal prosthesis is highly sought after to combat degenerative eye diseases and build foundational devices for future engineering models. A sub-retinal device requires a specific structure compared to other prosthesis. However, the specifics of this work also require high transparency as a performance metric. Low impedance and high Charge Injection Capacity (CIC) are typical metrics for stimulus electrodes but our chip architecture will place electrode materials anterior to photodiodes, thus a material with high transparency and biocompatibility must be determined.
These electrode characteristics have already been studied extensively in photovoltaics. Therefore in order to provide the best performance for our proposed device it is necessary to extensively test a selection of materials from previous literature. Each of the three categories: Trans-conductive Oxides(TCO), Metal Nanowires(NW), & 2-D materials can yield suitable candidates, such as: Aluminum doped Zinc Oxide (AZO), Boron doped Zinc Oxide (BZO), Indium Tin Oxide (ITO), AgNW(Silver Nanowires) + PEDOT:PSS, Carbon Nanotubes(CNT) + PEDOT:PSS, & Graphene with PEDOT:PSS. The primary materials examined in this study are Aluminum doped Zinc Oxide (AZO), Boron doped Zinc Oxide (BZO), PEDOT:PSS, and AgNW.
The fabrication of test electrodes has two separate wafer designs. The testing substrate used for electro-characteristics has a Ti/Au/Ti sandwich structure on Si wafers; on a SiO2 layer 30nm Ti was sputtered, followed by 500nm Au, and then 30nm or Ti. A photoresist pattern was used with wet etching to create a desired wiring pattern. Next, a 1um SiO2 passivation layer was applied and reserved contact/electrode locations were etched with RIE. Last, an experimental electrode material was sputtered onto the reserved electrode location. For the biocompatibility test and the transparency test, electrode material was sputtered onto 1mm thick quartz glass substrates. The resulting substrate was cut to specifications via optimized saw dicing. Optical testing utilizes the same substrate without dicing measures. Since our device will be mounted in ocular tissue for the long-term biocompatibility concerns become a central issue. An MTS biocompatibility assay test, using Hippocampal neurocytes, was performed on 10mm x 10mm square test substrates on quartz glass.
AZO, BZO, & PEDOT:PSS electrodes were successfully formed and Pt and Ti electrode wafers were fabricated for baseline. In the biocompatibility test. Each conductive material is deposited on quartz pillars. The pillars have dimensions of 50µm x 50µm x 200µm. Each pillar is spaced 200µm X & Y from other pillars in a grid like fashion. This research currently is verifying the bio-compatibility of flagged materials and we will continue to report on electrode performance compared to referenced research. AZO was a promising material but with initial testing was shown to be incompatible. Statistically speaking AZO is less biocompatible than control condition so is incompatible with our application at this point in time.
BZO and AgNW are also being targeted for Biocompatibility testing due to the potential of harmful nanoparticles from the electrode material. PEDOT:PSS is commended for its biocompatibility, electrical, & optical characteristics but process fabrications methods used can lead to undesirable material conditions like optical hazing in the polymer.
The Hippocampal neurocytes in direct contact with our electrode material showed biocompatibility of AZO to be lower than Pt electrodes. Further testing with BZO and AgNW are required because current literature suggests that each of the metallic materials has the potential to release hazardous nanoparticles to the tissue. Optical and electric characteristics have thus far been within expected performance ranges for our desired device.

Hyperthermia (also known as thermal therapy or thermal ablation) has been adopted as one of the cancer treatment approaches in medical surgery. By applying high temperature on the cells, the proteins structure would be damaged and the cell would be destroyed. In general, Radio Frequency (RF) probes are applied onto the target tissues for the ablation process. However, one of the major drawback is difficult to apply when the cancer tissues are on the surface of the organs. Here we propose a novel approach to develop surface hyperthermia technique by using oscillating magnetic field induction on metal thin film. In our device, the heat is transferred from the organ surface to the cancer cells through bioheat transfer process. Heat generation process will be modulated by varying the conductance of the metal thin film, the strength and the frequency of the oscillating magnetic field. Optimal parameters such as film geometry, film thickness, magnetic induction distance and ablation time are identified through the combination of ex vivo experiment and computational modelling. Near-field communication (NFC) temperature sensor is also integrated in the system enabling a wireless interfaces for smartphone to monitor and analyze the treatment performance. Based on the steady state heat transfer model and comparing the measured temperatures by the NFC temperature sensor, we can calculate the temperature profile inside the pig liver. By using a input power of 2000W/m², the highest heater temperature is 443 K in our device which is sufficient for the ablation process. This wireless heater is believed to have high potentials in various kind of in-vivo testing or non-invasive thermal treatments.

Tight interaction between nanophysics, materials science and biotechnology led to the emergence of a new class of bioinspired systems that enables to bring the area of biosensorics e.g. for cell or molecular diagnostics and analytics to the new level. Very promising candidates for the future diagnostics are the electronic nanobiosensors that have attracted great attention in the last years since they provide rich quantitative information for medical and biological assays without pre-treatment and specific optical labelling of the detected analyte. One dimensional nanostructures, e.g. semiconductor and metallic nanowires serving as backbone of the sensor device, have attracted attention as highly efficient elements due to their high surface-to-volume ratio, which simplifies the detection of biochemical species. Use of nanowires enable to ultimately decrease the dimensions of the sensing area of the device and thus increase the resulting sensitivity of the assay. Finally, nanoscale sensors integrated into lab-on-a-chip system offer attractive opportunity of the multifunctional and multiplexed bio- and chemical analysis that can be performed in real time, directly in-flow.
Here we focus on two subsystems for the in-flow analysis at the micro- and nanoscale, represented by (a) silicon nanowires based field effect transistor and (b) metal nanowires assembled as nanocapacitor. We demonstrate the applicability of the systems for the detection single molecules, e.g. influenza or Ebola viruses [1-3], biochemical reactions [4], as well as classify the blood cells in a cytometry format.

[1] D. Karnaushenko et al. Adv. Health. Mater. 4 (10), 1517-1525 (2015).
[2] M. Medina-Sanchez et al. Nano Letters 16 (7), 4288-4296 (2016).
[3] B. Ibarlucea et al. Nano Research 11 (2), 1057-1068 (2018).
[4] J. Schütt et al. Nano Letters 16 (8), 4991-5000 (2016).

Highly sensitive wearable sensors that can be conformably attached to human skin or integrated with textiles to monitor the physiological parameters of human body or the surrounding environment have garnered tremendous interest. Owing to the large surface area and outstanding material properties, nanomaterials are promising building blocks for wearable sensors. In this talk I will start with a brief overview of the recent advances in the nanomaterial-enabled wearable sensors including temperature, electrophysiological, strain, tactile, electrochemical, and environmental sensors. Integration of multiple sensors for multimodal sensing and integration with other components into wearable systems are summarized. Then I will present the array of wearable sensors developed in my group using silver nanowire (AgNW) composites. AgNW composites offer outstanding combination of high conductivity and high stretchability. Finally I will discuss a major challenge in the field of nano-enabled wearable sensors – scalable nanomanufacturing – and some recent work addressing the challenge.

In 2018, an estimated 266,120 new cases of breast cancer (BC) will be diagnosed in the United States, and 40,920 cases will die from the disease mainly due to metastasis. For example, according to the American Cancer Society, while the survival rates of BC stages 0-1 are approximately 100% and 93% respectively, metastasized BC has only a 22% survival rate. Understanding circulating tumor cells (CTCs) present a valuable opportunity for possible strategies to reduce dissemination. Currently, the only FDA-approved system (CELLSEARCH^®) enumerates merely a total number of chemically-fixed CTCs and cannot capture CTC clusters.
Microfluidics is an active field of research for isolation of CTCs. Microfluidic technologies such as CTC-Chip, Herringbone chip, CTC-iChip, Vortex, Rarecyte, Fluxion, NanoVelcro, DEP-Array, and JETTA are leading fluidic devices. There are about 400 different microfluidic techniques reported in the literature for CTC capture since 2007. Although promising, there are inherent constraints in large scale production of complex microfluidic based devices and surface functionalization for targeted capture. Further, the rare CTC clusters could potentially be damaged in fluidic devices due to turbulence, vortices and shear forces. Very few devices have been able to successfully capture CTC clusters with 30-40% sensitivity in metastatic patients. Further, some of the leading microfluidic technology has only 5-44% purity which makes genomic sequencing difficult.
By engineering nanomaterials namely carbon nanotubes, and microarray manufacturing techniques, we have been able to successfully develop the world’s first microarray for CTC capture. Advantages of our microarrays include: 1) microarray format enabling large volume of blood to be fractionated into smaller portion that enables better capture sensitivity; 2) variety of antibodies functionalized in the same array, resulting in CTC capture based on multiple markers from a single blood sample; 3) no transfer of cells is necessary to do microscopy, and one can do confocal/optical microscopy on chip; 4) surface architecture lends itself to variety of surface functionalization and instant CTC isolation for further analysis unlike microfluidics where CTCs have to be recovered from sealed chambers and could be lost; and 5) mature manufacturing process resulting in a 99% yield.
Using the mentioned advantages of microarrays, we have demonstrated the capture of spiked breast cancer cells in blood using both antigen-dependent and independent capture with >90% capture rate and elimination of 99% contaminating leukocytes. The microarray devices are electrically conductive, that enables electrical signals and mathematical classification techniques to capture breast cancer SKBR3 cells in blood with 90% sensitivity and 90% specificity. Further, using triple negative breast cancer patient derived xenografts (PDX), we have demonstrated the capture of highly invasive CTCs, CTCs with microtentacles, and the very rare CTC clusters which are metastasis initiating cells. In volumes up to 0.8 ml blood from PDX-mice, we have shown the capture of up to 26 CTC clusters and CTCs of different morphologies. The combination of materials engineering, microarray manufacturing, biochemical surface functionalization and cancer biology has led to this new microarray technology that will help in fight against cancer, the results of which will be presented at the spring 2018 MRS conference.

Autoimmune disorder is a kind of common chronic disease, which is difficult to cure throughout life. Small chemical drugs such as glucocorticoids, immunosuppressors and non-steroidal anti-inflammatory drugs have been widely used for the treatment of autoimmune disorders. However, these small chemical drugs suffer from poor solubility, short circulating half-life and adverse side effects, which lead to poor compliance to patients and limit the widely clinical use. One of the most effective strategies to extend the circulating time is loading drugs into nanocarriers to form nanomedicines, which is of particular interest for cancer and viral diseases therapy but seldom applied in autoimmune disorder treatment. Furthermore, current carriers have many drawbacks such as poor biocompatibility, low stability and over-complicated design. In this study, we developed an easy but general drug delivery platform based on the new polyhydroxyalkanoate terpolymer-poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx). We reported the first example of PHBVHHx nanoparticle loaded non-steroidal anti-inflammatory drug, ultimately being applied in systemic lupus erythematosus therapy. These nanoparticle are biodegradable, stable, and show improved pharmacokinetics, optimized biodistribution, low systemic toxicity and excellent in vivo therapeutic efficacy in a MLR/lpr murine model of systemic lupus erythematosus. This delivery system may provide a new and general platform for the development of nanomedicines with enhanced therapeutic efficacy and reduced side effects.

Antimicrobial resistance is a global health crisis that complicates the prevention and treatment of diseases caused by pathogenic microbes. In particular, the formation of bacterial biofilms on medical implants protects the pathogen from the host’s immune response and antibiotic treatment, causing chronic infections that form the basis of persistent microbial infections in the human body. To date, the treatment of medical device related infections due to biofilms remains a huge challenge in the clinical practice setting, mainly due to the extreme difficulty in the disruption and eradication of bacterial biofilm growth, which is resistant to antibiotics as well as mechanical treatment approaches. Recent advancements on the development of smart hydrogel nanocomposite systems with tailored properties provide promising routes to develop novel treatments that can be used to disrupt and completely eradicate pre-formed biofilms. In this talk, we will present our work on the development of an externally actuated thermoresponsive hydrogel nanocomposite system, containing D-amino acids (D-AAs) and engineered nanoparticles, which can be delivered and transforms from a solution to a gel state at physiological temperature for sustained release of D-AAs and light or magnetic field-actuated thermal treatment of targeted infection sites. The D-AAs in the nanocomposite are known to inhibit biofilm formation and also disrupt existing biofilms. In addition, loading the hydrogel nanocomposite with magnetic or gold nanoparticles allows for combination thermal treatment following magnetic field or light stimulation, respectively. Using this novel two-step approach to utilize an externally actuated hydrogel nanocomposite system for thermal treatment, following initial disruption with D-AAs, we were able to successfully demonstrate the effective disruption and total eradication of Staphylococcus aureus biofilms, which were resistant to conventional antibiotics used in the clinic and were not completely cleared using individual D-AA or thermal treatments.

Rapid progress in nanotechnology allows us to develop a large number of innovative wearables such as activity trackers, advanced textiles, and healthcare devices. However, manufacturing processes for desirable nanostructure are usually complex and expensive. Moreover, materials used for these devices are mainly derived from nonrenewable resources. Therefore, it poses growing problems for the living environment, and causes incompatible discomfort for human beings with long–time wearing. Here, a self–powered cellulose fiber-based triboelectric nanogenerator (cf–TENG) system is presented through developing 1D eco–friendly cellulose microfibers/nanofibers (CMFs/CNFs) into 2D CMFs/CNFs/Ag hierarchical nanostructure. Silver nanofibers membrane is successfully introduced into the cf–TENG system by using CMFs/CNFs as the template, which shows excellent antibacterial activity. Enabled by its desirable porous nanostructure and unique electricity generation feature, the cf–TENG system is capable of removing PM_2.5 with high efficiency of 98.83% and monitoring breathing status without using an external power supply. This work provides a novel and sustainable strategy for self–powered wearable electronics in healthcare applications, and furthermore paves a way for next–generation flexible, biocompatible electronics.

Carbon dots, a new class of zero-dimensional carbon-based nanomaterials, have attracted great interest recently in the nanomedicine because of their versatile merits such as biocompatibility, facile functionalization, and tunable photoluminescence properties. In particular, carbon dots have allowed for their biomedical applications to disease diagnostics and therapeutics such as bioimaging and photodynamic therapy. Here, we present the new design of carbon dots with multiple functions that highly inhibit the aggregation of Alzheimer’s amyloid-β (Aβ) peptides.
The abnormal assembly and aggregation of Aβ peptides in the brain tissue is a major hallmark of Alzheimer’s diseases (AD) that currently affects one in nine people aged over 65 worldwide. Despite numerous efforts made over the past decades, effective suppression of Aβ aggregation is still challenging due to multifactorial pathogenesis involving high levels of metal ions in the brain. Note that copper ions (Cu(II)) have drawn significant attention in AD pathogenesis due to their high activities in binding with Aβ peptides and forming neurotoxic Aβ-Cu(II) complexes.
To provide a new anti-amyloidogenic strategy in the prevention of Cu(II)-associated Aβ aggregation, we have synthesized the multifunctional carbon dots and demonstrated their capabilities on (i) effective Cu(II) coordination, (ii) interruption of Aβ self-assembly, and (iii) photodynamic oxygenation of Aβ residues. The nitrogen-containing aromatic moieties on as-prepared carbon dot’s surface do a key role in interact with Cu(II) and Aβ’s aggregation sites by electrostatic and hydrogen/hydrophobic interactions. Interestingly, the Cu(II) coordination of carbon dots promotes the carbon dots’ absorption and fluorescence enhancement, thus further leads the photoinduced generation of reactive oxygen species (ROS) under light irradiation; the resulting ROS from carbon dots then photodynamically oxygenated Aβ residues (e.g., histidine and methionine) to lose the affinity towards Cu(II) as well as an aggregative property. These carbon dots’ simultaneous and multiple inhibitory effects significantly mitigated the neuronal cytotoxicity of Cu(II)-bound Aβ species, showing 96% of cell survival rate.
In summary, we will introduce the synthesis of multifunctional carbon dots and the various photochemical analyses to validate the suppressing capabilities of carbon dots against Aβ aggregation. Our investigations highlight the distinctive surface and luminescence properties of carbon dots as promising biocompatible nanoagents and their therapeutic application in preventing Aβ-induced Alzheimer’s diseases.

The Nobel Prize in Chemistry 1987 was awarded to Cram, Lehn and Pedersen for their development and use of molecules with structure-specific interactions of high selectivity. Twenty years later in 2016, Nobel Prize in Chemistry recognized supramolecular chemistry and molecular recognition again by awarding Sauvage, Stoddart and Feringa for the design and synthesis of molecular machines. This talk takes cyclodextrin and cucurbituril as examples to develop cancer nanotheranostics that make use of the dynamic and responsive nature of non-covalent interactions. By the formation of host–guest complexes with macrocyclic hosts, the anticancer drugs can be easily formulated to prepare nanomedicines showing satisfactory anti-tumor efficacy and reduced normal organ toxicity.

Collagen (Col) is a well-studied biomaterial, particularly as wound dressing material due to its superior biological and chemical properties [1]. However, the application of collagen in wound healing remains as a critical issue due to poor adhesion, proliferation and migration of cells. In addition, majority of the collagen-based biomaterials are primarily fabricated from mammalian sources, such as bovine and porcine [2]. There have been many considerations in terms of religious issues. Moreover, the extraction process from mammalian animals is complex, laborious, time consuming and expensive due to the stiff and fibrous nature of bovine and porcine tissue [3]. Hence, alternative sources of collagen are highly desirable, particularly for wounds treatment.
In this study, collagen derived from the skin of the American Bullfrog (i.e. Rana catesbeiana) is explored as nature-derived biomaterials for wound healing applications. We report the successful implementation of an innovative method that can significantly shorten the collagen extraction process by ~50% compared to traditional acid solubilisation method with relatively high extraction yield (~35%). Differential scanning calorimetry (DSC) analysis suggest that the collagen has excellent thermal stability (~41°C), suggesting that it is compatible for skin-contacting as well as for in vivo implantation applications. In vitro studies showed that the bioactivity of the bullfrog skin-derived collagen was well-preserved by supporting the attachment and proliferation of human keratinocytes (i.e. HaCaT), compared to commercially-available bovine collagen. Interestingly, our findings demonstrated that the extracted bullfrog skin-derived collagen can significantly enhanced the wound closure rate of HaCaT in an in vitro scratch wound assay as compared to bovine collagen. At the mechanistic level, we showed that this phenomenon may be attributed to the ultra-fine nano-fibers of the frog-skin derived collagen, which could augment the process of re-epithelization by modulating the adhesiveness and chemotaxis of the keratinocytes [4]. Overall, bullfrog skin could be a valuable waste-to-resource material for the production of non-mammalian collagen with high yield percentage and great wound healing capacity.
References
[1] Chattopadhyay, S. and Raines, R.T., 2014. Review collagen-based biomaterials for wound healing. Biopolymers, 101(8), pp.821-833.
[2] Gould, L.J., 2016. Topical collagen-based biomaterials for chronic wounds: rationale and clinical application. Advances in wound care, 5(1), pp.19-31.
[3] Gorlov, I.F., Titov, E.I., Semenov, G.V., Slozhenkina, M.I., Sokolov, A.Y., Omarov, R.S., Goncharov, A.I., Zlobina, E.Y., Litvinova, E.V. and Karpenko, E.V., 2018. Collagen from porcine skin: a method of extraction and structural properties. International Journal of Food Properties, 21(1), pp.1031-1042.

The complexity and heterogeneity of cancer and subsequently developed multidrug resistance (MDR) seriously undermines the therapeutic potential of chemotherapy. Herein, we developed a biodegradable redox-heat assisted cancer killing (RHACK) nanoparticle for enhanced treatment of cancer in vitro and in vivo. Unlike conventional anticancer strategies, our hybrid nanoparticle based drug delivery system (DDS) not only target-specifically delivers anti-cancer reagents, but also targeting heterogeneous MDR pathways to achieve sensitization of cancer cells. More specifically, our RHACK nanoparticle-based drug delivery system (DDS) reduces intracellular glutathione (GSH) level, induces ROS generation and simultaneous provide photothermal (PTT), thereby synergistically sensitizing breast cancer towards chemothreapy. Remarkably, our RHACK nanoparticle is constructed from an iRGD conjugated hollow iron oxide carbon core and a manganese dioxide shell (Fe₃O₄-C@MnO₂-iRGD NPs) and shows unique benefits for synergistically overcoming MDR: i) a strong NIR absorption for effective laser ablation of tumor and sensitize the tumor to further chemotherapy; ii) a tunable glutathione-reactive MnO₂ shell; iii) a redox-based endogenous biodegradability; iv) a hollow structure for efficient anti-cancer drug loading; v) Fe³⁺ as degradation product for induction of intracellular Fenton reaction and more toxic ROS species (hydroxyl radical); vi) Mn²⁺ as the second degradation product for T1 MRI imaging; and vii) capabilities of breast tumor targeting and penetration. Based on this unique platform, we successfully demonstrate a robust co-sensitization of a malignant drug-resistant breast cancer towards a clinical applied anti-cancer drug in vitro and in vivo by combining PTT, glutathione reduction and Fenton reaction on a single platform. A detailed mechanistic study on the molecular biology pathways further reveals the synergy originate from the regulation over several key genes and proteins including HSF-1and MDR-1/P-gp, TP53, BCL-2, BAX, Caspase 3/9. Given the heterogenous tumor microenvironments and the highly complicated drug-resistance pathways in breast cancer, our multifunctional hybrid nanoparticle-based DDS could provide a promising therapeutic alternative for more efficient treatment of breast cancer in clinical applications.

Materials engineering is typically divided into “bottom-up” and “top-down” methodologies. While, top-down engineering has been common practice for centuries, these approaches are rarely used with respect to biological systems. We have applied a top-down approach for generating a mineral gradient scaffold in trabecular bone. Mineral gradients are found in a variety of biological systems that link soft tissue and bone. Some of these systems are primarily mechanical, such as the attachments of ligaments and tendons with bone or the interface between cartilage and bone. Other soft tissue-to-bone interfaces are necessary for development, such as the growth plate or the interface present during endochondral ossification. These interfaces are further relevant to cancer metastasis, as tumors have been found to localize to the growth plate. These systems are complex, requiring a method for producing the mineral gradient present at the interface. With these goals in mind, we fabricated a mineral gradient scaffold through the spatially controlled removal of mineral from bone.
To fabricate this scaffold, cylindrical trabecular bone biopsies were extracted from neonatal bovine femurs. These biopsies were decellularized, followed by partial submersion in a demineralizing solution. Scaffolds were removed from the demineralizing solution at time points up to 6 hours, finding that 4.5 hours represents the median point in the mineral removal process. The main factor driving the amount of mineral removed from the biopsy was observed to be the porosity in the trabecular bone. This observation was confirmed by plotting porosity vs. time vs. the fractional demineralized content and fitting a surface to these data. Scaffolds were segmented into quarters, and X-ray diffraction was performed on these segments, confirming the presence of a mineral gradient within the scaffold.
To examine the cellular compatibility of the resulting mineral gradient, mesenchymal stem cells were seeded onto these scaffolds. The seeded mineral gradient scaffolds were cultured in osteogenic media, and a live/dead stain was performed. The cells attached to the exterior of the trabecular structure with a viability of ~91%. The scaffolds exhibited high viability and with no differences in viability were observed in the mineralized or demineralized portions of the scaffolds.
Immunohistology was performed to examine the effects of mineral content on cellular behavior. Seeded scaffolds were cultured, fixed, and sectioned for staining. Stains were chosen to determine if stem cells were showing any chondrogenic or osteogenic behavior. Staining profiles revealed differences in cellular behavior between the mineralized and demineralized ends as well as on the exterior and interior of the scaffold. Type I collagen staining was observed throughout the scaffold. Type II collagen staining was consistent on the interior of the scaffold but appeared higher in the exterior regions of the mineralized end of the scaffold versus the demineralized end. Alkaline phosphatase staining was consistent on the exterior of the scaffold but appeared higher on the mineralized interior vs. the demineralized interior. These findings reveal that the presence of a mineral gradient drives stem cells to produce proteins associated with chondrogenic and osteogenic cellular behavior.
Overall, we fabricated mineral gradient scaffolds using a top-down engineering approach. These scaffolds, generated using a novel approach to biomaterials synthesis, drive cellular behavior and will be useful in a variety of fields, including orthopedics, development, and cancer research.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, characterized by a rapid loss of upper and lower motor-neurons resulting in patient death from respiratory failure within 3-5 years of initial symptoms onset. Although at least 30 genes of major effect have been reported, the pathobiology of ALS is not well understood. There is a critical need to develop a system which can accurately diagnose this rapidly deteriorating disease. Herein, we report on graphene’s phonon vibration-energies as a sensitive measure of the composite dipole moment of the components of the interfaced cerebrospinal fluid (CSF) to specifically identify patients with ALS disease. The second-order overtone of in-plane phonon vibration energy (2D) of graphene shifts by 3.2±0.5 cm^-1 for all ALS patients studied in this work. Further, the amount of n-doping induced shift in phonon energy of graphene, interfaced with CSF, is specific to the investigated neurodegenerative disease (ALS, Multiple Sclerosis and Motor Neuron Disease). By removing a severe roadblock in disease detection, this technology can be applied to study diagnostic biomarkers for researchers developing therapeutics and clinicians initiating treatments for neurodegenerative diseases.

Cells are continuously performing chemical computations based on a range of inputs from their immediate environment. Two fundamental phenotypes that result from these computations are cell adhesion and migration, which play central roles in development, wound healing and diseases such as cancer. The degree to which cells adhere or migrate is dependent upon a combination of chemical and physical properties of the substrate. However, the complex coupling amongst these physical and chemical cues has made it challenging experimentally to deconvolve their individual contributions. Furthermore, cells dynamically alter their environment by the secretion of proteins. To address these complexities, we have employed a combination of electron beam and optical lithographic techniques to fabricate substrates which incorporate independently tunable topographical and chemical signaling cues, as well as nanosensors for detecting cell secretions. The approach works by patterning two materials on a single chip, one for topography, and the other for chemical functionalization. The first material, quartz, is light microscopy compatible and can be dry etched for 3D topography formation. The second material, gold, is patterned independently of the quartz etching process into nanostructures for use in either interfacing with the cell membrane or detecting protein secretions. I will present results on a range of cell types cultured on multiplexed substrates that integrate nanosensors for quantifying single cell secretions, topographical patterns for directing cell migration and biofunctionalized nanostructures for targeted adhesion studies. Cells at the boundary of two patterns experience a surface-induced competition that can be tracked in real time as they decide to adhere to one surface or migrate to the other. These chips enable quantitative insight into the biology of how local environments influence cellular decisions as well as potential guidance in the design of next generation wound healing materials and implants.

The dynamic response of cells when subjected to mechanical impact has become increasingly relevant for accurate assessment of potential blunt injuries and understanding injury mechanisms. When exposed to a blast, ballistic, or impact, a biological system, e.g., human brain or skin, is rapidly accelerated, which results in an acceleration-induced pressure gradient. In order to study cell behavior under these threats, we have developed a new experimental method that applies a well-controlled mechanical impact to live cells cultured in a custom-built in vitro setup compatible with live cell microscopy. A drop tower system is utilized to apply the mechanical impact while concurrently visualizing each impact using high speed cameras. The impact amplitude is controlled by changing the vertical height of a movable mass, i.e., drop height, which is then accelerated toward the setup once released. Our experiments show that the maximum pressure in the in vitro setup is very sensitive to the height of cell culture media. As an example, we experimentally correlated drop heights with the onset of cavitation nucleation in different cell culture setups. We find that the critical drop height decreases by about 7 times when the height of cell culture media increases from 1 cm to 4 cm. This result indicates that lower drop heights induce the critical pressure (a material related property) with increasing cell media height and, as a result, implies that impact-induced pressure in biological systems depends on their size due to inertial effects. Another interesting experimental observation is that cells near cavitation bubbles uptake propidium iodide, typically not permeable to live cells, which we suspect is due to transient damage to the cell membrane. Our study shows that in addition to acceleration - which is the most commonly used criteria for blunt injury - size of biological systems, e.g., head size, should be appropriately considered for accurate assessment of potential injuries due to mechanical impact.

The present study delineates preparation, characterization, and application of calcium alginate (CA) - Gum tragacanth (GT) beads carrying phenolic compounds from B. alba plant for oral drug delivery. The CA- GT beads were prepared by ionic gelation method and its physicochemical characterization was carried out by SEM, EDAX and FTIR analysis. The swelling analysis and in vitro degradation assay revealed variation in the percentage of swelling and degradation rate respectively with the change in the GT concentrations in beads. Variation was prominent in compositions with higher GT concentrations.
A critical examination of the drug release profile revealed that the presence of GT in the formulation retarded the release rate of phenolic compounds. Extract loaded formulations were tested against osteosarcoma cells (MG-63). Cytotoxicity data, live dead staining, nuclear condensation-fragmentation, DNA fragmentation (by TUNEL assay) and cell migration study together confirmed the therapeutic potential of the CA-GT formulations. The above findings suggest that B. alba phenolic extract loaded CA- GT beads could be used as the natural therapeutic source for cancer treatment and an ideal source of nutraceuticals.

In general, the vitiligo is diagnosed by wood lamp test which is also known as ultraviolet (UV) light test. However, UV light in the wavelength under 400-nm is not visible to the human eyes and cameras. Thus, it is difficult to obtain accurate UV images for analyzing lesions of vitiligo patients. The light incident into the human skin has different penetration depth depending on the wavelength, which means the information of the reflected light differs according to the incident light wavelength. If there were any cameras or sensors that are capable of obtaining UV reflected images of the lesion, more deliberate information such as lesion size, depth, and shape could be obtained through a depth profile according to the penetration depth of human skin along the wavelength range.
Here, we developed a novel UV camera applying blue-light emitting quantum-dots (QD) to conventional Si CMOS image sensor (CIS). Synthesized zinc-blend-structure QD absorbs UV light below 400-nm wavelength and emits the 433-nm-wavelength blue-visible-light by energy-down-shift mechanism, having a quantum yield of 75.2%, full-width half maximum (FWHM) of 20.7 nm. The reflected light from the object passes through the QD filter before entering the photodiode of the Si CMOS image sensor. The QD filter completely passes the visible light and only absorbs UV light and QD emits the blue-visible-light. In other words, the QD filter converts UV light, which the conventional image sensors cannot detect, into the blue light so that the image sensor can detect it. This process presents that the image sensor receives a further increased blue pixel’s signal caused by UV intensity. The difference of blue pixels intensity between with and without QD filters can be considered as a detected UV intensity.
In this work, we diagnosed and treated lesions of 2 vitiligo patients through our new QD UV camera. We photographed the UV images of their hands and faces and analyzed them in cooperation with dermatologists. Based on the analysis, the patients were recommended for proper treatment and we tracked their treatment progress for 2 months. It was observed that the UV images of the vitiligo lesions showed lower UV pixel intensities than the intact normal skin, which means that the location, size and distribution of vitiligo lesions could be clearly identified through the UV image. The UV pixel intensities effectively showed information of epidermis and dermis where vitiligo lesions mainly exist. Melanocytes reflect and scatter ultraviolet rays to protect the skin from harmful UV rays. So, in UV images of normal skin, melanocytes reflect UV, resulting in high UV intensity. However, in the case of vitiligo lesions, there is less reflection of UV light due to the lack of melanocytes, resulting in low UV intensity. In addition, we can also estimate the extent of melanin pigment deficiency by this UV intensity analysis. The regions with relatively high UV pixel intensity and those with relatively low UV intensity were clearly distinguished in the vitiligo region. The relatively lower UV intensity means that the deficiency of melanocyte is relatively large, indicating that lesions are more severe. We confirmed that the novel QD UV camera can accurately discriminate the lesions of vitiligo from the intact region, and also can quantify the severity of the lesion. It is expected that the UV images taken by the QD UV camera will provide guidelines for the diagnosis and treatment of vitiligo. In addition, UV pixel analysis is also considered to be valid for several skin disorders that can be diagnosed by the UV light test such as tinea capitis, erythrasma, albinism, etc.
^*This work was financially supported by the Brain Korea 21 Plus Program in 2018.

Trauma brain injury (TBI) is a problem in public health locally and globally. The consequences of this pathology are serious and its mortality rate is high. TBI severity is classified using the Glasgow coma scale. This scale assigns points to the patient’s eye opening, motor and verbal response based on a qualitative test. Although this method is part of several TBI protocols, its application is susceptible to errors associated to the specific physiological response of each person. Misclassification of the initial TBI severity will in turn impact the therapeutic management causing in many cases delays in proper treatment. In this research work, we are proposing a quantitative method to classify the severity of the TBI, using highly specific serum biomarkers in order to reduce misclassification as well as to provide a fast, easy to use tool for monitoring the patient's progress. An electrochemical immunosensor have been developed by means of a specific antibody against S100B protein. S100B is a calcium binding protein present in the central nervous system; its concentration increases within the initial hours of the TBI event, its value as TBI biomarker have been demonstrated before. The biosensing platform was created by electrochemical polymerization of a nanodiamond enhanced polyaniline matrix, this matrix was then functionalized with antiS100B by crosslinking reaction after APTMS-GA (siloxane 3-aminopropyltriethoxysilane - glutaraldehyde) initial surface modification. The amount of antibody was optimized for the specific surface active area using different depositing dilutions, the results showed that after 5ugmL no statistically significant increment in the electrochemical signal is observed. Response of the biosensor to the antigen was recorded using impedance spectroscopy experiments in 0.2 M KCl and 10 mM potassium ferrocyanide as supporting electrolyte. Our results showed the characteristic semicircle behavior at the high frequency range associated to the redox probe of the electrolyte; also, the Warburg line has been observed at the low frequency range.

Functional brain changes represent a significant social and economic burden that has increased considerably over the last decades. The development of new scientific capabilities that are able to analyze brain structure and connectivity has recently revitalized the neuroscience field. Biomaterial based ex vivo models of healthy and diseased brain tissue are among those tools that may help study the complexity of cerebral tissue to better diagnose, prevent, and treat brain diseases (1). We have established engineered brain tumor biomaterials based on methacrylamide-functionalized gelatin hydrogels, and used microfluidic-forming techniques to generate platforms that combine transitions of biophysical and biomolecular properties found in the glioblastoma tumor microenvironment, from the core to the tumor margins (2). Moreover, this biomaterial approach is able to monitor the response of patient-derived xenograft (PDX) cell populations with different molecular signatures; in particular, we have analyzed the amplified and the constitutively activated vIII mutant EGF receptor. We have also characterized PDX response to targeted inhibitors, such as erlotinib, a tyrosine kinase inhibitor that specifically blocks EGFR.

The ex vivo biomaterial platforms that recreate transitions in the native GBM tumor microenvironment have shown the capacity to describe cell response to therapeutic inhibitors and classify tumors response to local gradients in extracellular matrix properties (3, 4). This in vitro tumor model is able to analyze the relationship between the molecular weight of matrix-bound hyaluronic acid (HA) and the invasive phenotype of GBM tumor cells. We found that cell growth, motility, and proteomic responses of GBM cells within our platform were significantly altered by HA molecular weight. These results provide additional insights regarding the importance of extracellular microenvironment in the invasive potential of glioblastoma tumors (5). Recently, we demonstrated that repeated dosing of erlotinib promotes expression of PDGFRb in EGFR vIII mutant cells and that extracellular HA plays a favorable role in the inhibition of EGFR, through STAT3 deactivation (6).

In addition to tumor tissue samples, these gelatin-based platforms permitted the survival and growth of the large nucleus gigantocellularis (NGC) neurons, considered as the 'master cells' for initiating CNS arousal. The better understanding of NGC neurons might help to understand particular characteristics of certain brain disorders (7). Until now, the lack of a physiologically relevant microenvironment for in vitro culture has been associated with unusual cell morphologies and cell death. We showed that NGC neurons were able to survive and differentiate in the presence of HA, and without the assistance of growth factors (8). In this context, 3D in vitro cultures of NGC neurons may allow the analysis of the extracellular matrix contributions and growth factors to their survival, growth and development. Due to the success of these platforms to recreate cerebral tissue peculiarities, we have also cultured neural stem cells within gelatin – HA hydrogels and we are currently working on the development of neurodegenerative diseases models.

References
(1) Pedron S and Harley BAC (2018) Editorial: Biomaterials for brain therapy and repair. Front. Mater. 5:67.
(2) Pedron, S, Becka, E, Harley, BA (2015). Adv. Mater., 27: 1567-1572.
(3) Pedron S, Hanselman JS, Schroeder MA, Sarkaria JN, Harley BAC (2017). Adv. Healthcare Mater., 6, 1700529.
(4) Pedron, S, Polishetty, H, Sarkaria, JN, Harley, B et al. (2017). MRS Commun., 7(3), 442-449.
(5) Chen J-WE, Pedron S, Sarkaria JN, Harley BA et al. (2018). Front. Mater. 5:39.
(6) Pedron S, Wolter GL, Sarkaria JN, Harley BA et al. (2019). Neuro-oncology. Under review.
(7) Inna Tabansky, Yupu Liang, Joel N. H. Stern, Donald W. Pfaff et al. (2018). PNAS, 115 (29) E6900-E6909.
(8) Magarinos AM, Pedron S, Pfaff DW, Harley B et al. Front. Mater., 28 June 2018

Electronic devices are one of the most promising candidates for chemical and biological sensor platforms because of its high sensitivity, and application to Internet of Things (IoT). However, a major challenge to developing such sensor systems is the use of battery power owing to its limited life-time, inconvenience of recharging, and insufficient battery level to operate the integrated sensor system.
In this study, we suggest self-powered biosensors by integration of n-IGZO/p-Si photodetectors and enzyme-based colorimetric reactions, which are operated by light sources in daily life environment such as fluorescent light and sunlight. A colorimetric reaction was performed at a polydimethylsiloxane vessel located at the upper side of the IGZO/Si photodetector, which is physically apart from the photodetectors. Photocurrent changes of the photodetectors are induced by the colorimetric reaction depending on target concentration, which enables quantitative analysis. The self-powered biosensors showed high sensitivity towards glucose level in real human samples without matrix effect due to physical separation of the photodetectors and colorimetric reaction part. This sensor platform could pave the way for highly sensitive portable IoT biosensors operating without batteries.

Recently the interest in bio-degradable Magnesium alloys as an alternative implant material has increased. Since no surgery is needed to remove the inserted implant, the risk of bacterial infection and the medical burden is decreased. In particular, anti-adhesion is one of the prerequisite properties in medical applications to avoid bacterial colonization on the implant. In this study, biocompatible Mg-Zn-Ca alloys are synthesized by squeeze casting and hydrophobic nano-SiO₂ particles are coated on the alloy surface. In the process, a porous SiO₂ layer is formed and its condition controlled via dip-coating. The human pathogen most frequently found on implant materials, Staphylococcus Aureus (S. Aureus), is prepared through the pour-plating method to evaluate the anti-adhesive properties and the bacteria on the surface are counted by scanning electron microscopy. The result shows that hydrophobically treated SiO₂ particle layers significantly mitigate the bacterial attachment compared to a bare Mg surface and a SiO₂ particle layer.

Introduction: Extracellular matrix (ECM) alterations accompany the onset and progression of many diseases. In this work, we sought to develop tissue engineering-based approaches to mimic the disease environment in order to perform prospective analyses that probe the mechanistic role of the ECM in disease etiology. Our specific focus was on calcific aortic valve disease (CAVD), whose pathogenesis is poorly understood, but is known to involve excessive glycosaminoglycan (GAG) accumulation early in the disease process. We implemented two different strategies to create tissue-engineered platforms that mimic elements of early CAVD: 1) construction of engineered environments using traditional “bottom-up” methods, which enable tuning and control of scaffold parameters but fail to capture the complexity of the native tissue. 2) construction of a top-down organ culture platform that retains native complexity, but lacks the ability to precisely control tissue composition.

Methods: To create scaffolds via the bottom-up approach, we copolymerized methacrylated gelatin with either methacrylated hyaluronic acid (HA) or chondroitin sulfate (CS) at concentrations intended to replicate the GAG enrichment found in early CAVD (1x GAG = healthy valve; 4x = diseased) and seeded them with valvular interstitial cells (VICs). To create top-down scaffolds, we transduced scaffolds to overexpress hyaluronic acid synthase-3, an enzyme responsible for HA synthesis. Porcine aortic valve leaflets were treated with feline immunodefficiency virus (FIV) containing a vector for either GFP (FIV.GFP) or human hyaluronic acid synthase-3 (FIV.HAS3) and then cultured in vitro for up to 7 days. Cultures were also treated with low density lipoprotein (LDL) or oxidized LDL (oxLDL), which mimic CAVD risk factors, and analyzed for overall ECM composition, compressive modulus, cell phenotype, and cytokine/growth factor production.

Results: Analysis of CS-enriched scaffolds demonstrated that pathological CS enrichment alone was not sufficient to promote a change in VIC phenotype or secretion of inflammatory factors. Instead, the main function of increased CS appeared to be sequestration of oxLDL. Sequestered oxLDL increased the secretion of multiple inflammatory cytokines (e.g., IL-6, IL-8, PDGF-BB, MCP-1) by VICs, thereby implicating oxLDL as a potent factor in regulating pathological VIC behavior. Moreover, a positive feedback loop was uncovered where oxLDL increased CS production, which further increased oxLDL retention. These findings suggest that increased CS in early CAVD plays a central role in regulating the onset of inflammation.

Meanwhile, a different role for HA enrichment was demonstrated in both bottom-up and top-down scaffolds. ECM-edited valve organ cultures were generated to achieve a 4-fold enrichment in HA content, with the level of HA enrichment regulated by the amount of virus administered. This result demonstrated the ability to tailor the ECM composition of intact, 3D tissues, thereby providing a novel disease-mimicking culture platform. In both culture approaches, HA enrichment served to stimulate vascular endothelial growth factor (VEGF) production by the VICs, but HA was not involved in LDL/oxLDL sequestration or subsequent inflammatory activity. Instead, HA-induced VEGF production was sufficient to stimulate the proliferation and tubulogenesis of endothelial progenitor cells, suggesting that increased HA in early CAVD may promote the pathological angiogenesis that is a hallmark of this disease.

Conclusions: We developed complementary tissue-engineered platforms to mimic disease pathogenesis, including a novel approach to use intact organ cultures as semi-tunable biomaterials. These platforms were applied to reveal new information about CAVD pathogenesis. The applications of these approaches extend beyond valvular disease, as there are many pathologies which involve selective enrichment of specific ECM components.

Molecularly targeted nanoparticles that produce X-ray excited optical luminescence, also known as radioluminescence, enable unique multimodal imaging capabilities not available with current clinical technologies. These functional nanomaterials, often comprised of lanthanide-doped nanophosphors, have also been evaluated as energy mediators for deep tissue photodynamic therapy (PDT) and other X-ray stimulated therapies. As both imaging probes and therapeutics, the application of radioluminescent nanoparticles (RLNP) is limited by their photophysical properties. Here we present the synthesis and characterization of lanthanide-doped nanoparticles with core-shell structure, incorporation of high-Z elements, and their surface modification with biocompatible polymers to improve functional properties for biological applications. In this radioluminescent platform, optical emission can be tuned by selection of dopants, such as Eu³⁺ and Tb³⁺. Utilizing a thermal degradation of organometallic precursors, uniform cubic (~11 nm) nanocyrstals were obtained and observed by transmission electron microscopy (TEM). To evaluate the radioluminescence of these nanophosphors a suspension of the particles was irradiated at 130 kV while an emission spectrum was obtained by a spectrophotometer. Emission was improved significantly through protection from surface quenching of luminescent centers. In addition, surface modification with a layer-by-layer approach was developed and cell specific uptake was evaluated using folic acid as a model targeting agent. Based on these results, we discuss the feasibility of X-ray luminescence computed tomography (XLCT) and X-ray excited PDT.

Cancer-associated fibroblasts (CAFs) are one of the major types of stromal cells within the tumor microenvironment that