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.

Increase of antibiotic resistance in pathogens has directly impacted healthcare industry. With only a few novel discoveries in the field of antibiotics since last two decades, often referred to as the discovery void, drug-resistance in pathogens has increased. Previously, infections that were easily treatable have now become fatal. Infections caused by antibiotic-resistant pathogens can occur anywhere, but, it is observed to take maximum effect in healthcare settings such as hospitals and nursing homes. The pathogens adhere to surfaces such as linens, counter tops, drapes monitory equipment, sanitary ware and so on. Moreover, patients in hospitals are easily affected due to decreased immunity. According to a report by Center for Disease Control and Prevention (CDC), 1 out of every 20 hospital patients is affected by nosocomial infections subsequently resulting in 100000 deaths in the United States of America. Out of these, 23000 deaths on an average are attributed to drug-resistant pathogens such as methicillin resistantStaphylococcus aureus andvancomycin-resistant Enterococcus faecium. In addition to this, $9.8 billion are invested in treatment of such infections. The conventional routes of treatments are proving to be inconsequential and a major part of tackling such infections is use of antibiotics. But, with the increase in drug-resistant strains it is only a matter of time when all the treatment methods fail. Although several methods such as chemical disinfectants, ionizing radiation, ultra-violet treatments are used in addition to antibiotics but, they are not effective due to various reasons. Chemical disinfectants and ionizing radiation are not FDA approved. Moreover, radiation techniques are cost intensive and ultra-violet radiation is harmful to healthy cells. So, alternative techniques must be investigated that can be utilized to prevent the loss due to hospital acquired infections (HAIs). Consequently, alternative techniques for surface disinfection must be investigated for their application as preventive techniques instead of looking for a cure. One such method that has recently come into light in the last two decades is photodynamic therapy (PDT). Commercial photosensitizers (PSs) such as Photofrin and Protoporphyrin IX have been used in Photodynamic therapy (PDT) for treating initial stages of skin cancer, acne and psoriasis. It involves three components: a photosensitizer, a visible light source and cytotoxic singlet oxygen. Initially, the PS is applied to the area affected by the cancer cells. After the PS is absorbed into the cells, the target area is illuminated by red colored light, thus, activating the PS. Activated PS can exchange energy with oxygen that diffuses through the cells, converting it into singlet oxygen (¹O₂).¹O₂being highly energetic readily reacts with various components in the cell, ultimately leading to cell death. The idea of this study is to incorporate such photosensitizers into polymeric matrices that can be applied as surfaces that provide non-specific preventive measures, rather than as specific cures.
In this study, 1% (w/w)ZnTMPyP of porphyrin class has been physically incorporated in an Olefinic Block Copolymer (OBC) via melt-pressing. Inactivation studies were performed on five bacterial strains (two Gram-positive and three Gram-negative at various illumination intensities). Furthermore, time based illumination studies were conducted at various illumination intensities withStaphylococcus aureus(5 - 80 mW/cm², 5 – 60 min illumination time). Both the gram-positive bacteria showed ~ 6 log units reduction (~ 99.9999% killing) at the end of 60 minutes whereas all the three gram-negative bacteria showed at least ~3 log units reduction (~ 99.9% killing). Antiviral efficacy of films was determined against Vesicular stomatitis virus (VSV) (~ 99.9998 % inactivation), Human adenovirus-5 (~ 99.9596 % inactivation) and Influenza A virus (~ 99.9500 % inactivation).

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.