Symposium SB02 : Multiscale Materials Engineering Within Biological Systems

Diatoms are single-cell algae that form a hard silica/organic composite with a honeycomb-like structure. The fascinating and sophisticated structure of these algae, which is responsible for the multifunctionality of these organisms, has attracted several scientists and engineers for various reasons. Multiple functions and properties (e.g., mechanical, chemical, and optical) are achieved through a combination of different factors, such as compositions, geometrical features, and hierarchy. A characteristic feature of the diatoms is the protective shell, which has an optimal strength-to-weight ratio, especially if compared to other biological materials, offering enough resistance to predators’ attacks and preventing sinking. These characteristics make the shell an interesting biomimetic model for the design of lightweight structural materials. Here we focus on one of the most relevant and interesting diatom species, the Coscinodiscus, that is a centric diatom with radial symmetry. Keeping the effective density constant, we use numerical modeling, experimental testing, and additive manufacturing study the effect of systematically varying the size of the relevant geometrical features on the overall bending stiffness. The overarching hypothesis of this work is that the morphological features, like pores and hierarchical layers, drive the mechanical response of diatoms exoskeleton and are designed by Nature to simultaneously fulfill different functions. We build a simplified FE-model of a diatom-inspired architecture and we study the mechanical behavior under three-point bending and compressive loading. We evaluate the effect of the hierarchical level and the effect of material distribution amongst the hierarchical level on the overall bending stiffness and resistance to buckling. The numerical simulations are validated through experimental tests, carried out on polymeric geometries fabricated using multi-material 3D-printing. The results of this study reveal how the natural geometry is optimized to simultaneously provide lightweight, bending stiffness, and structural integrity, limiting local buckling and providing different dissipation mechanisms to absorb energy, thus preventing catastrophic failure. Ongoing studies are carryout on the effect of the pore distribution on both the mechanical behavior, with particular attention to fracture, and on the fluid-dynamics properties. Ultimately, this research offers an innovative perspective in terms of design and fabrication of multifunctional diatom-inspired materials for diverse applications, from drug delivery to membrane filtering and solar cells. Moreover, given the large diatom diversity and the plethora of architectures, there is great potential of applying machine learning algorithm for the design of novel diatom-like topologies with a novel set of properties.

Over 335 million tons of plastic is produced globally every year and nearly 80% of it have accumulated in landfills and water-bodies. Contamination of non-biodegradable plastics and microplastics (<5 mm fragmented particles) are causing potentially irreversible damage to our ecosystems and global health, including that of humans. Herein, we report AquaPlastic, a new class of microbially produced biodegradable bioplastic that is water-processable. We genetically engineered E. coli to fabricate AquaPlastic with minimal processing steps. AquaPlastic is entirely aqua-processable and aqua-healed by the addition of water. Additionally, it can be aqua-welded to create robust three-dimensional architectures by using water as a glue. It also readily forms well-adhered coatings on a wide variety of surfaces and is resistant to strong acid/base and organic solvents. AquaPlastic films can be imprinted with surface patterns with topographical features as small as few tens of nanometers. These unique features of AquaPlastic are believed to inspire further exploration and the development of much-needed alternatives to conventional plastics.

Design of stimuli responsive and structurally versatile materials with finelly controlled combination of properties represent the key bottlenecks of nearly all biomedical technologies. The central challenge for the design and realization of biologically inspired materials with such demanding set of properties is harnessing the processes of self-organization involving molecular, nanoscale, and microscale components.
This challenge can be addressed using integration of several methods of self-assembly producing hierarchically structured composites.

Replication of load-bearing and functional nanocomposites will be described for three examples of bioinspired nanomaterials: nacre; enamel and cartilage. Nacre-like composites allow for multidimensional design of materials properties: toughness, stiffness, strength, transparency, ion transport, and biological response. These type of hierarchoical nanocomposites resulted in biommetic neuroprosthetic implants. Replicating tooth enamel, we recently learn that the mechanics and other properties of this material can be replicated combining out-of-plane nanoparticle assembly into columns and molecular-scale self assembly of polymers between them. These composites reveal remarkably high vibrational damping unusual for stiff materials that imparts them resilience to aging. Replication of cartilage gives example how to nature reconciles load-bearing and transport properties. Versatile cartilage-like nanocomposites based on aramid nanofibers (ANFs) will be discussed in details.

Cephalopods possess unrivaled camouflage and signaling abilities that are enabled by their sophisticated skin, which alters the texture and coloration of their skin to blend into their surroundings. The color-changing capabilities of cephalopods, in particular, are enabled by multiple dermal layers, which contain chromatophore pigment cells (as part of larger chromatophore organs) and different types of reflective cells called iridocytes and leucophores, which function synergistically to alter the appearance of the cephalopods for both communication and signaling. The optical functionality of these cells (and thus cephalopod skin) critically relies upon subcellular structures partially composed of a class of unusual structural proteins known as reflectins. Reflectins have been found to have a unique amino acid sequence, but very little secondary structure. We will highlight studies that have investigated reflectins’ structure-function relationships, within the context of cell-reflectin interactions. We will also discuss these proteins’ multi-faceted material properties, associated challenges, and future potential. Our findings hold relevance for the development of biomedical technologies based on and inspired by reflectins.

Out-of-equilibrium self-assembly of biomolecules is one of key processes to forming organisms in nature. Spatial and temporal activation energy switches the biochemical properties of biomolecules, and altering the interactions between biomolecules results in self-assembled structures and, eventually, the construction of a functional organism. To explain the self-assembly mechanism in nature, biophysicists have constructed a variety of theoretical and experimental models. These models assume no interactions between biomolecules in general because it is not trivial to design an interaction-controllable system; this basic assumption, however, is highly simplified and does not represent the reality of nature. We demonstrate our recent efforts to design a biomimetic interaction-controllable self-assembly system. M13 bacteriophage possesses a long fibrous shape with a helically-arranged protein surface, analogous to naturally existing basic building blocks such as collagen, cellulose, and chitin. As a result of amplification through bacterial infection, M13 bacteriophage is identical, homogenous, and can be easily genetically engineered to make M13 bacteriophage a promising biomaterial in designing a biomimetic system. In this study, we genetically engineer M13 bacteriophage to install a thermally responsive peptide (TRP) at the end of the major capsid protein p8 to tune interactions between biomolecules during self-assembly. TRP phage self-assembles into a highly ordered structure with increasing temperature that is counter-intuitively inverse thermal response than usual liquid crystals. The temperature dependent phage transition of TRP-phage in solution is investigated by a macroscopic turbidity measurement. The phage transition temperature is decreased with increasing TRP phage concentration and ionic strength of solution. The microscopic nucleation and growth of TRP phage’s helical, fibrous, and smectic structure is observed by polarized optical microscope. To evaluate the temperature dependent orderedness of the TRP phage structure, small angle x-ray scattering measurement of (100) peak is utilized. TRP phage in solution shows 7.8 nm interspacing at 42^oC, and this peak is decreased to 6.9 nm at 68^oC, while there is no peak from room temperature to 42^oC. From atomic force microscopy and He-ion microscopy, the final nanostructure is the helically assembled TRP. The role of individual amino acids, especially valines in different positions, is also investigated through the turbidity measurement by inducing point mutations.

Artificially photosynthetic systems aim to store solar energy and chemically reduce carbon dioxide. These systems have been developed in order to use light to drive processes for carbon fixation into biomass and/or liquid fuels. We have developed a hybrid-biological system that manages both genetically controlled generation of products along with the photoactivability of a semiconductor system. We show an increase in the production of ethanol, a common biofuel, through the electron transfer stimulated by biologically produced cadmium sulfide nanoparticles and light. This work provides a basis on which to improve the production of many metabolites and products through endogenously produced nanoparticles.

Collagen is the most abundant protein in humans and animals with more than twenty-five different variations. Among them, Collagen Type I (COL1) is the main component of bone and skin. Owing to its structural nature, previous studies have investigated different mechanical perspectives, including strength, elasticity, toughness and viscoelasticity.
An interesting avenue of research, still poorly understood, is the capability of collagen to transfer mechanical energy when exposed to impulsive displacement loads. This topic is particularly relevant due to the tremendous implications for developing new biomimetic and bioinspired materials to be employed in bioengineering. Specifically, the energy transfer applications in the auditory apparatus are most interesting since this organ possesses abundant collagen and its physiological role is specific to mechanical energy transmission from sound waves.
In this work, we study a collagen peptide (with GXY triplets) at the molecular level to understand the role of hydration in the material behavior under impulsive loads. We employ molecular dynamics simulations to investigate wave speeds and energy dissipation.
We prepare a (GPO)₂₀ collagen peptide with Triple-Helical collagen Building Script with a length of about 180 Å. Both dry (DS) and the wet (WS) structures are equilibrated via LAMMPS aiming at relaxing the topology and reaching a convergence of the potential energy and RMSD (Root-mean-square deviation).
To study the behavior of COL1 along and perpendicularly to the helix axis, wave transfer analyses are performed on both the DS and WS by fixing the peptide at one end and by applying two different impulses: longitudinal case (LC) and transversal case (TC).
As for the LC, we load the free end with an axial impulsive displacement of 10 Å. In contrast, for the TC, we perform a preliminary slow stretching of the triple helix at the free end (up to 10% of the tensile strain) before applying a transversal impulsive displacement of 10 Å.
Our results for the LC show higher wave speeds for the DS than the WS’s (3082 m/s vs. 2190 m/s) with correspondent Young’s Moduli of 8.05 GPa and 4.07 GPa.
We discover that the kinetic energy is markedly dissipated, similarly for the DS and WS, and it results annihilated before the travelling wave reaches the fixed edge. We estimate for both the DS and WS a relaxation time in the order of 100 ps.
Concerning the TC, instead, the material behavior is strongly affected by the pre-strain applied before the impulsive load. We compare the results with the vibrating string analytical model to study the relationship between the wave speeds at different tensile strains and the Young’s Moduli. Our results show a monotonically increase of the travelling wave speed with the tensile strain (up to 1400 m/s and 829 m/s for DS and WS, respectively). A direct comparison of the Young’s Modulus vs. strain curves with previous works confirms the validity of our approach. Concerning the dissipating phenomena, our results show a quasi-elastic propagation of the wave along the DS with a dissipation about five times smaller than the WS’s in the same loading conditions (i.e., relaxation time: 429 ps vs. 80 ps). We believe that, during transversal perturbations, water plays a keyrole in enhancing the kinetic energy dissipation, otherwise driven by only hydrogen bonds.
This study represents a first step in understanding collagenous material properties under transient loading conditions in view of applications related to bone physiology and replacement, and to the auditory apparatus where COL1 is abundant.
This work has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement COLLHEAR No 794614.

Drug delivery systems have been extensively investigated for the last decade, with research in the field of microparticles, nanoparticles, micellar, lipid and hydrogel vesicles [1-4]. Unlike traditional drug formulations which typically consist of a raw drug dispersed within a matrix, encapsulating reservoirs present the advantage of sustained drug release with high efficacy, in an attempt to alleviate the risks of toxic side effects and overcome the challenges of providing an optimised therapy both in space and time [1, 5]. Such challenges are the driving force behind the design of novel drug delivery systems aiming to revolutionise the way drugs exert their actions. One example is the modification of the the release behaviour of core-shell carriers for the combined administration of drugs, within a safe therapeutic window [1, 6].
This work presents a novel route to process polymer-based core-shell microparticles, composed by an outer shell of poly(vinyl alcohol) (PVA) encapsulating ciprofloxacin (CPx), and a core comprising a curcumin (CM) loaded poly(e-caprolactone) (PCL) particle. The obtained core-shell particles size was in the range of 1 – 6 μm, making them ideal candidates for inhalable drug delivery systems.
The release patterns for both CM and CPx from the simple PCL or PVA particles took less than 72 h to being fully released. The core-shell microparticles, on the other hand, presented a biphasic release pattern, showing that the CPx was released over the first 24 h followed by the release of the curcumin from the core PCL particle, being the last one fully released after 15 days. Thus, the polymer layers worked as a physical barrier, preventing drug-drug interaction and modulating the release behavior.
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy coupled with synchrotron infrared (IR) beam was used to obtain the chemical mapping of the drug distribution within the individual core-shell particles. This technique demonstrated that the PCL particles were inside of the PVA layer, and that there was a distinct and traceable distribution of the both drugs inside the particles. Finally, the bioactivity and compatibility of the formulated particles were confirmed by antimicrobial and viability assays.
These novel polymer-polymer core-shell systems proved to be biocompatible and able to deliver, in a controlled fashion, two different drugs without compromising their bioactivity, hence offering unique properties for multidrug therapies.

Acknowledgements
This research was undertaken on the Infrared Microspectroscopy (IRM) Beamline at the Australian Synchrotron, part of ANSTO, under the project number M14463. This work was supported by the strategic program UID/BIA/04050/2019 funded by Portuguese national funds through FCT I.P.

References
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Statement of Purpose: Cancer related deaths are the second leading cause of death with more than 10 million new cases each year. Conventional treatment often affect both healthy and cancerous cells, causing severe adverse side effects. Moreover, systemic delivery of chemotherapeutic drugs requires higher doses of drugs which lead to higher toxicity to healthy cells. Therefore, biomaterials have been used to enable localized and effective delivery of therapies to avoid side effects and reduce the effective dose. Biomaterials engineering was used to develop a variety of drug delivery vehicles for different chemotherapeutic drugs. We hypothesize that by incorporating single walled carbon nanotube (SWCNT)-liposome complexes in hydrogels, a drug delivery system capable of controlling timing and sequence of multiple drug deliveries can be developed. Utilizing near infrared (NIR) lasers, SWCNTs can be preferentially heated to disrupt liposomal bilayers, resulting in drug release. Since NIR absorbance spectra of SWCNTs is dictated by their (n,m)-chirality, e.g. distinct Gaussian profiles with ~15 nm full-width at half maximum (FWHM), multiple formulations of single-chirality SWCNT-liposomes can be developed for the multiplexed release of drugs triggered by different NIR lasers. Finally, these SWCNT-liposome complexes will be retained in 3D hydrogel structures, enabling the localized delivery of chemotherapeutic drugs.
Methods: Single-stranded DNA was used to wrap the SWCNTs and disperse them in aqueous solutions. For each dispersion, 1 mg of raw HiPco nanotubes was added to 2 mg of desalted ss(GT)₁₅ oligonucleotide with 1 mL of 100 mM NaCl. The mixtures were then ultrasonicated using a 1/8″ tapered microtip for 2 hours at 40% amplitude, with an average power output of 8 W, in a 0 °C temperature-controlled vial. After sonication, the dispersion was ultracentrifuged for 30 min at 250,000 xg and the top 80% of the supernatant was extracted. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were mixed at a 1:1 molar ratio in chloroform at 10 mM and rotary evaporated to form a thin lipid film. 10 kDa FITC-Dextran was dissolved in DI water at 1 mg/ml and used to hydrate the lipid film for 5 minutes. The solution was then vortexed and extruded through polycarbonate membranes with 200 nm pore size to form unilamellar positively charged liposomes. DNA-wrapped SWCNTs were mixed with the liposomes using two-barrel syringe and syringe pump to form SWCNT-liposome complexes. Different rations of SWCNT to liposome were used to minimize aggregation (measurable by polydispersity index (PDI)). These SWCNT-liposome complexes were further encapsulated into alginate hydrogels.Dynamic light scattering (DLS) was used to characterize the SWCNT-liposome structures. Confocal fluorescence microscopy was used to verify the homogenous distribution of SWCNT-liposome structures in the alginate hydrogel.
Results & Conclusions: We verified the fabrication of SWCNT-liposome complexes using dynamic light scattering (DLS) to measure the size and zeta potential of these structures before and after fabrication. Different ratios of SWCNT to lipid were used and the optimal ratio for minimizing aggregation was identified. Furthermore, cryo- transmission electron microscopy (Cryo-TEM) was used to visualize the structure of SWCNT-liposome complexes. Confocal fluorescence microscopy demonstrated the retention of model drug in the complexes over a two-week period. In summary, SWCNT-liposome complexes were fabricated, characterized and their retention in a 3D hydrogel scaffold was demonstrated. Furthermore, drug retention in these complexes over a 14-day period was verified and drug release vs. time was quantified. We believe that these complexes can be useful for controlling the drug release by using NIR lasers. More studies need to be conducted to analyze the effect of different NIR wavelengths on release profiles.

Over the last 20 years nanomedicine has drastically evolved with consequent approval of 51 FDA nanomedicines which are currently in the market. Most of these nanomedicines are applied intravenously, although oral administration propitiates comfort to patients, reduced infection risks and invasiveness, easy application, and low cost.¹ Therefore, one critical challenge that remains in the pharmaceutical industry is the development of nanomedicines that can be orally administered.
Intestine is likely the key barrier facing oral administration-based medicines. Thus, the interaction and transport of nanoparticles (NPs) across this barrier is of paramount relevance. Currently, intestinal barriers production resembling human tissues involves the co-cultivation of human colon carcinoma epithelial (CACO-2, non-mucus secretion) and human colorectal adenocarcinoma (HT-29, high mucus secretion) cells at 9:1 ratio, respectively. While this co-culture leads to production of intestinal epithelia that mimics the human intestine with higher reliability, few reports have assessed the passage of nanomedicines across non-ideal models of intestine^2-4 either dealing exclusively with CACO-2 cells or with non-optimized CACO-2:HT-29 ratios. Further drawbacks in these works include the use of NPs undergoing aggregation in biological environment and/or protein-free culture media,^5,6 which lead to intestinal transport results of low biological relevance. Herein, we quantify the transport of stable fluorescent silica nanoparticles (SiO₂NPs) in complex culture medium across an optimized model of intestinal epithelium (CACO-2:HT-29).
SiO₂NPs were synthesized from the mixture of rhodamine B isothiocyanate dye precursor, tetraethyl orthosilicate (TEOS) and ammonia catalyzer in ethanolic solution. A silica shell was produced around the pre-formed fluorescent cores and the resulting particles have approximately 90 nm-size according to electron microscopy. These particles remained stable for more than 24 h of incubation in DMEM supplemented with 10.0% fetal bovine serum enabling reliable analyses of the SiO₂NP transport across the intestinal epithelium since aggregates can prevent this passage and hide accurate quantitative results.
The artificial intestine was formed by co-cultivation of CACO-2 and HT-29 at 9:1 v/v ratio and confocal images confirmed the structure of the intestine. SiO₂NPs at 0.5 mg mL^–1 were added on the top of the transwell allowing NPs to interact with intestine. After 24 h, samples from top and bottom (after passing through intestine) compartments were monitored by fluorescence spectroscopy. Quantitatively, 19 ± 2% (n=3) of the SiO₂NPs crossed the intestine through paracellular and transcellular pathways, 44 ± 8% remained at the top of the transwell, whereas roughly 37% were retained either in the transwell polycarbonate membrane or inside intestine cells. Cell viability assays further revealed these SiO₂NPs are non-toxic to intestine cells.
In addition to high stability and low toxicity, the SiO₂NP transport assays were performed using optimized intestine model. The obtained results are important towards a more reliable comprehension on the behavior of SiO₂NPs when in contact with intestinal barriers, then contributing for new advances related to the development of oral nanomedicines.

1.Fattal, E. et al. Clin. Transl. Imaging 2, 77–87 (2014).
2.Ye, D. et al. Beilstein J. Nanotechnol. 8, 1396–1406 (2017).
3.Bannunah, A. M. et al. Mol. Pharm. 11, 4363–4373 (2014).
4.Simovic, S. et al. Nanomedicine Nanotechnology, Biol. Med. 11, 1169–1178 (2015).
5.Yang, D. et al. ACS Appl. Mater. Interfaces 11, 11443–11456 (2018).
6.Strugari, A. F. G. et al. Nanomaterials 9, 5 (2018).

Micro and nanomotors are entities extensively present in nature where they perform complicated tasks in a simple way, making their artificial development interesting as it holds promise in a wide range of applications in the biomedical context, food science or environmental monitoring.
Artificial micro and nanomotors are micro or nanoscale sized devices able to exhibit motion in response to different stimuli or power sources. These motors can either employ fuels to move ¹ or they can require external energy.² However, they still need to overcome some difficulties, in the former case often toxic compounds in high concentrations were used to propel motors with high thrust.
Biocompatible systems with high enough operational lifetimes and speeds to perform their envisioned tasks remain scarce.

We have designed a polymer-based system to address the latter challenge.³ It consists of micromotors formed by polymer-multilayers assembled through hydrogen bonds via the layer-by-layer technique. They exhibit pH-dependent disintegration that results in micro-motors motion, outperforming the Brownian randomization expected for swimmers of these sizes.

The mobility properties of these micromotors have been assessed experimentally and theoretically based on their trajectories and velocities with the aim to yield micromotors with high thrust. We demonstrated their self-propulsion and directed motion for steep pH gradients, while more shallow gradients resulted in random enhanced motion. Interestingly, velocities were predominantly increased for lower micromotors masses, and higher amounts of adsorbed polymer multilayers in their surfaces. All these characteristics as well as their shape and time related dependence have been summarized.
Micromotors capabilities were further evaluated biologically based on their toxicity and mucus penetration ability within cells. We demonstrated an improvement in cell uptake for micromotors compared with their uncoated counterparts.

These micromotors can exploit the existence of different pH regions in the human body. Their high thrust at short times and their biocompatibility make them suitable to cross biological barriers.


1. Schattling, P., Thingholm, B. & Städler, B. Enhanced Diffusion of Glucose-Fueled Janus Particles. Chemistry of Materials 27, 7412-7418 (2015).
2. Schattling, P.S., Ramos-Docampo, M.A., Salgueiriño, V. & Städler, B. Double-Fueled Janus Swimmers with Magnetotactic Behavior. ACS nano 11, 3973-3983 (2017).
3. Fernández-Medina, M., Qian, X., Hovorka, O. & Städler, B. Disintegrating polymer multilayers to jump-start colloidal micromotors. Nanoscale 11, 733-741 (2019).

Platelets perform a variety of functions during the wound healing process, including targeting to wound sites, binding fibrin and promoting clotting following injury. Following cessation of bleeding, platelets also release cytokines and antimicrobial peptides and reorganize the fibrin clot through a process known as clot retraction. Clot retraction significantly decreases clot size, alters clot organization and increases clot stiffness, thereby promoting ongoing wound repair. The fibrin clot acts as a provisional matrix for cellular infiltration and tissue remodeling during the inflammation, proliferation, and migration stages of wound healing, and platelet-mediated reorganization of the fibrin network after hemostasis may be an important factor in promoting cellular infiltration. In cases of traumatic injury or disease, platelets can become depleted or dysfunctional, impairing their ability to promote hemostasis and contribute to subsequent healing. To that end, we have developed a biomimetic platelet-like-particle (PLP) that mimics native platelets by homing to injury sites in vivo, augmenting clot formation at sites of injury, and recapitulating platelet clot retraction in a tunable process. We hypothesized that PLPs would stiffen clot matrices as a result of this ability to induce clot retraction. Increased matrix stiffness has been shown to promote cell migration. Therefore, we also hypothesized that PLPs would subsequently improve in vitro cell migration and in vivo wound healing responses when incorporated into fibrin matrices. In these studies, we evaluated the ability of PLPs to reorganize fibrin clots, increase clot stiffness, and enhance cell migration and healing outcomes in healthy healing models, as well as in combinatorial therapies, infection models, and a deficient healing (hemophilia) model. We evaluated our base PLP design and an antimicrobial PLP design. Finally, PLPs were also applied in combination with ultrasound in order to determine whether this combinatorial therapy could be used to minimize the time required to bring about clot structural changes, as well as minimize the therapeutic dosage of PLPs required for treatment. To create PLPs, ultralow crosslinked poly(N-isopropylacrylamide) microgels co-polymerized with acrylic acid were synthesized using precipitation polymerization and following purification, were conjugated to a fibrin-specific IgG antibody through EDC/NHS coupling. Antimicrobial PLPs were also created by incorporation of nanogold or nanosilver into the base PLP design. The effect of PLPs on clot structure and stiffness were evaluated using confocal microscopy, cryogenic scanning electron microscopy, and atomic force microscopy. The influence of PLP-mediated clot retraction on cell migration was evaluated within 3D in vitro models of wound healing in both normal and hemophilia-B like conditions of deficient healing. The ability of antimicrobial PLPs to decrease bacterial burden was evaluated in a colony forming unit assay using both Escherichia coli and Methicillin-resistant Staphylococcus aureus. Finally, the ability of PLPs to augment hemostasis following trauma and promote healing were evaluated in a mouse liver laceration model and a full-thickness dermal injury model, respectively. Incorporation of PLPs into fibrin matrices increased clot density, stiffness, fibroblast migration in vitro, and markers of wound healing in vivo. Both normal and antimicrobial PLPs significantly decreased blood loss in vivo. Antimicrobial PLPs decreased bacterial burden in vitro and in vivo. Finally, co-application of ultrasound and PLPs increased clot density, stiffness, cell migration, and wound healing markers, but at lower dosages and time scales relative to those required with PLPs alone. Overall, these results demonstrate that PLPs are beneficial for augmenting hemostasis and promoting healing.

One of the important trends of next-generation biomedical sciences is the development of new tools that can accurately image, identify, and execute desired missions in a selectively programed manner. Nanotechnology is among one of the essential platform tools for targeted imaging, therapy, and simultaneous monitoring of therapeutic efficacy. In this talk, I will discuss magnetic nanoparticles as a core platform material and tool for a variety of functionalities such as sensing, targeting and signaling of cells in a selective and efficient way. Their unique utilizations in highly accurate dual-modal MR imaging, therapeutic hyperthermia of cancer cells, controlled drug/gene delivery, and molecular level cell signaling and cell fate control will be discussed.

The vortex configuration of spins emerges in anisotropic magnetic nanomaterials with certain dimensions in the absence of an external magnetic field (MF). This configuration enables rapid control over a particle’s magnetization direction and magnitude. Nanoparticles supporting vortex spin configuration possess near zero net magnetization in the absence of MF, which affords greater colloidal stability in suspensions. This property makes magnetic vortex particles uniquely suited for applications in biological systems. Guided by micromagnetic simulations, we predict and experimentally demonstrate magnetic vortex states in an array of colloidally synthesized magnetite nanodiscs (MNDs) 100–250 nm in diameter. The vortex state in MNDs was measured directly via electron holography. Following phase transfer into physiological solutions, we applied these MNDs for remote control of activity of sensory neurons with weak (7–26 mT), slow-varying (1–5 Hz) MFs. The latency of this magnetomechanical neuronal excitation was correlated to the MND volume and the applied MF amplitude and frequency.
Consistent with MNDs geometry and magnetite chemistry, MNDs exhibit direction-dependent hysteresis loops, which results in their hysteretic heating in high-frequency (100s kHz) alternating MFs. We determined MNDs capability of heat dissipation with efficiency (specific loss power) up to 1000 W/g in MFs with frequencies of 75-150 kHz. This is on par with record reported efficiencies for to the isotropic magnetite nanoparticles, but observed at 10-50 times lower particle counts owing it to the MND volumes. This characteristic allows for targeted activation of heat-gated ion channels in neurons upon application of high-frequency alternating MFs.
With their large induced magnetic moments in slow-varying MFs and high heating efficiencies in alternating MFs, MNDs allow for multiplexed stimulation of neurons by selectively activating mechanoreceptors or thermoreceptors

Nanoscale materials show great potential in the biomedical field as they can serve as superior bioimaging contrast agents, diagnostic and therapeutic tools while a key element for the successful implementation of nanoscale materials in clinical applications is multi-functionality. However, the two main bottlenecks for the successful commercialization of such nanotechnologies, that are often neglected in studies, are scalability and reproducibility. Here, a few recent examples will be shown of how flame nanoparticle synthesis, a nanomanufacture process famous for its scalability and reproducibility, may be employed for the production of sophisticated nanoscale materials to tackle important medical challenges.
We focus on nanoparticle formation by flame spray pyrolysis, a highly versatile nanomanufacture process and advance the knowledge for synthesis of complex nanoparticles and their direct integration in multi-scale biomedical devices. We place specific emphasis in multifunctional and responsive nanoparticles that may be used either as transducer elements or as diagnostic probes to monitor biological processes.
We synthesize nanoparticles with high purity and controlled sizes as a basis for functional nanoparticles. We have demonstrated functionalization of luminescent nanoparticles with targeting proteins, whose receptors are overexpressed in cancer cells, and detected them by fluorescence cell imaging. We have also recently explored the potential of flame-made nanoparticles in H₂O₂biosensing, using enzyme-mimetic luminescent CeO₂:Eu³⁺nanoparticles that exhibit catalase-mimetic activity and decompose H₂O₂. We have also shown the potential of stimuli-responsive nanoparticles as both photothermal agents by near-IR irradiation as well as superparamagnetic nanoparticles for the enhanced triggered-drug-release from alginate beads by hyperthermia.
Flame aerosol reactors for nanoparticle synthesis are a powerful toolbox for the scalable and reproducible production of sophisticated nanoparticles with properties not easily attained by other nanomanufacture processes. Their systematic employment in biomedicine has the potential to open up several avenues for nano-enabled solutions to medical challenges.

Acknowledgements
This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC Grant agreement nr. 758705). Funding from the Karolinska Institutet Board of Research, the Swedish Research Council (2016-03471), the Jeansson Foundations (JS2016-0029) and the Åke Wiberg Foundation (M16-0098) is kindly acknowledged.

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A. Pratsinis, G. A. Kelesidis, S. Zuercher, F. Krumeich, S. Bolisetty, R. Mezzenga, J. C. Leroux & G. A. Sotiriou. ACS Nano 11, 12210-12218 (2017).
A. Spyrogianni, P. Tiefenboeck, F. H. L. Starsich, K. Keevend, F. Krumeich, I. K. Herrmann, J. C. Leroux & G. A. Sotiriou. AIChE J. 64, 2947-2957 (2018).
D. F. Henning, P. Merkl, C. Yun, F. Iovino, L. Xie, E. Mouzourakis, C. Moularas, Y. Deligiannakis, B. Henriques-Normark, K. Leifer & G. A. Sotiriou. Biosens. Bioelectron. 132, 286-293 (2019).

Cystic Fibrosis (CF) is an inherited disorder affecting more than 70000 people worldwide,
especially Caucasian populations with a carrier prevalence of 1/3000. Currently, it has no
cure but an early diagnosis remains a critical issue for an optimistic prognosis. Sweat
chloride test has been the gold standard to diagnose CF since the affected present higher
sweat chloride concentrations than healthy individuals (≥ 60 mM). In this work, a planar
electrochromic point-of-care device, based on WO 3 nanoparticles produced by microwave
assisted hydrothermal synthesis, was developed for CF low-cost diagnostic testing
especially in resource-limited environments. For electrodes patterning, a CO 2 laser
technology was used in a PET/ITO sheet. The device presents a design that allows the
NaCl-based electrolyte deposition, used as artificial sweat, only on time of usage directly
on the nanoparticles or in a paper pad. By applying an operating voltage of 3 V for 1 min,
the nanoparticles change their optical properties according to NaCl concentration,
presenting a blue colouration with different intensities for different NaCl concentrations.
The device is able to differentiate between a positive and negative diagnosis within few
minutes, obtaining an RGB channel ratio of 1.37±0.03 for 60 mM of NaCl, when imaged.

While the translocation of cargos across biological barriers is the key issue for therapeutic applications, cell-penetrating peptides (CPPs) have demonstrated to be highly useful for the transport of molecules and even nanomaterials into cells. Herein, we studied how the intracellular delivery of silica nanoparticles can be modulated after decoration with differently active CPPs. The latter were obtained after an Ala scan of the recently developed CPP sC18. Out of the 16 peptides obtained, two were selected for further studies: the first displayed a higher positive net charge and enhanced amphipathicity resulting in significantly higher internalization rates than sC18. The second one demonstrated reduced cellular uptake efficiencies, and served as a control. Attachment of these CPPs to silica nanoparticles of different sizes (50, 150 and 300 nm) was performed electrostatically to preserve the secondary alpha-helical structure of the peptides, which was confirmed by CD-spectrometry. Flow cytometry studies showed that all conjugates were efficiently internalized into HeLa cells revealing a particle size-dependent uptake. Moreover, similar to the free peptides, a peptide-dependent internalization could be observed according to the position of the alanine residue in the biomolecular sequence. These results demonstrate the huge potential of sequential fine-tuning of CPPs and provide more insights into their interaction with inorganic nanocarrier surfaces.

Traumatic brain injury (TBI) affects ~1.7 million Americans every year and is the largest cause of disability-adjusted life-years lost worldwide. Treatments available in the clinic remain palliative, with no strategies that address the long-term brain health of patients. There is therefore an urgent need for new therapeutics. One hallmark of TBI is vascular damage, which is observed through clinical imaging modalities such as magnetic resonance imaging and X-ray computed tomography. We are engineering nanometer scaled materials to exploit this vascular damage in order to gain access to the injured tissue for the delivery of both diagnostic and therapeutic payloads. In addition, we are interested in studying and developing strategies to target nanomaterials to specific cell-types and structures within the injured brain.
We first explore the time window in which vascular damage allows the infiltration of nanomaterials into the brain tissue, and determine that we can deliver material into the brain when delivered hours after injury. Furthermore, we can exploit this transient window of vascular disruption to deliver targeted nanomaterials that carry therapeutic nucleic acids with neuronal specificity. We next explore the size range of materials that can infiltrate the brain after injury and find that similar to the enhanced permeation and retention (EPR) effect observed for nanomaterials in cancer, there is an optimal size for materials to accumulate in the injured brain. We leverage this accumulation in the injured brain for the delivery of a diagnostic payload that generates signal in response to an enzyme that has elevated and deleterious activity in brain injuries. We observe increased activation of this nanosensor in the context of TBI compared to uninjured brains. Collectively, we are engineering nanomaterials that provide diagnostic and therapeutic capabilities to provide new medicines for TBI.

Bioengineered systems often employ physical cues arising from intrinsic properties of the substrate in order to mimic specific physiological conditions. In particular, materials with micro- and nanoscale topographies are known to be regulators of cell adhesion, morphology, migration, proliferation, and differentiation. Of these topographies, high-aspect-ratio nanostructures have emerged as versatile platforms for facilitating drug/biomolecule delivery, and intracellular sensing.^1,2,3
Here, we present a new approach for the microfabrication of high-aspect-ratio, nondegradable silicon nanoneedle arrays that can support the long-term culture of human mesenchymal stem cells (hMSCs).⁴ Through a combination of different etching processes, we were able to finely control the sharpness of the nanoneedles from 20 to 700 nm. This enabled us to investigate the influence of the nanoneedle tip diameter upon the phenotype of interfaced hMSCs. High-content, image-based profiling of 100,000 individual cells cultured on different nanoneedle arrays revealed that the nanoneedle tip diameter could be used to influence cell shape, nuclear size, and actin alignment in a controllable manner. In particular, the polarization of actin on sharp nanoneedles was found to be mediated by the Rho-GTPase pathway. Mechanoresponsive cell behavior, including altered expression of lamins, Yes-Associated Protein (YAP) target genes and focal adhesion genes, could also be tuned by altering the tip diameter. These results were correlated to increased nuclear membrane impingement by the sharp nanoneedles, as shown by volumetric, superresolution imaging using focused-ion beam scanning electron microscopy (FIB-SEM).
Taken together, these observations demonstrate the importance of nanostructures for controlling the cell interface and phenotype. Our method of fabricating high-aspect-ratio nanostructures will be broadly applicable to the design of nanotopographical interfaces for biomedical devices, such as bioelectrodes and diagnostic platforms.

¹ C. Chiappini, …, M. M. Stevens, ACS Nano 2015, 9, 5500; C. Chiappini, M. M. Stevens et al., Nat. Mater. 2015, 14, 532
² S. Gopal, C. Chiappini, …, M. M. Stevens, Adv. Mater. 2019, 31, 1806788
³ C. S. Hansel, S. W. Crowder, …, M. M. Stevens., ACS Nano 2019, 13, 2913
⁴ H. Seong,…, M. M. Stevens, Submitted

Ultra-small gold nanoclusters (AuNCs) have emerged as agile probes for in vivo imaging, as they exhibit exceptional tumour accumulation and efficient renal clearance properties. However, their intrinsic catalytic activity,¹ which can enable increased detection sensitivity, has yet to be explored for in vivo sensing. By exploiting the peroxidase-mimicking activity of AuNCs and the precise nanometer size filtration of the kidney, we designed multifunctional protease nanosensors that respond to disease microenvironments to produce a direct colorimetric urinary readout of disease state in less than 1 h.²

Our nanosensor is comprised of renal clearable catalytic AuNCs (< 2 nm) tethered to a larger protein carrier via peptide linkages, that is disassembled in response to dysregulated protease activity at the site of disease. We demonstrated that the peptide-templated AuNCs can be filtered through the kidneys and excreted into the urine with high efficiency and retain catalytic activity in complex physiological environments. To demonstrate the modularity of the system, we synthesized functionalized peptide substrates shown to be specifically cleaved by either the serine protease thrombin or the zinc-dependent matrix metalloproteinase 9 (MMP9), which play a critical role in cardiovascular disease or cancer, respectively. We demonstrated the response of our protease nanosensors both in vitro and in vivo, achieving sensitive disease detection with a rapid, colorimetric urinary readout using our MMP-responsive nanosensors. We monitored the catalytic activity of AuNCs in collected urine of a mouse model of colorectal cancer where tumour-bearing mice showed a 13-fold increase in colorimetric signal compared to healthy mice. Nanosensors were eliminated completely through hepatic and renal excretion within 4 weeks after injection with no evidence of toxicity.

Our system exhibited a dual amplification platform: leveraging both in vivo protease activity and inorganic catalytic activity of AuNCs to provide a visual readout of disease state directly in urine. With this method, we demonstrate that these AuNCs are small enough to be filtered efficiently through the kidneys and retain catalytic activity in cleared urine, thus providing a versatile disease detection platform that is compatible for deployment at the point-of-care (PoC). Our adaptable nanocatalyst amplification platform should be applicable in low-resource settings for rapid detection of a diverse range of diseases by exploiting their specific enzymatic signatures and will democratize access to advanced and sensitive diagnostics.

1. Loynachan, C. N. et al. Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultrabroad Dynamic Range. ACS Nano 12, 279–288 (2018).
2. Loynachan, C. N. et al. Renal clearable catalytic gold nanoclusters for in vivo disease monitoring. Nat. Nanotechnol. 14, 883–890 (2019).

Graphene quantum dots (GQDs) are 2~3 nm sized nanoparticles that have a hydrophobic 2D graphitic domains with hydrophilic oxygen-containing functional groups along the edges. The carboxyl groups on GQDs can be modified to amine groups through simple organic chemistry to be decorated with many different small molecules. In our recent study, the application of GQDs as a therapeutic agent has been demonstrated to alleviatIe Parkinson's disease by degrading pre-existing α-synuclein fibers as well as by preventing its fibrillization. It is also proven that GQDs have negligible long-term toxicity in animal models as they are excreted through urine in a few weeks (D. Kim et al., 2018, Nat. Nanotechnol., 13, 812-818). Thus, GQDs are gaining growing interests in the field of nanomedicine by virtue of such novel capability to prevent or remove undesirable aggregation of biomolecules that causes various diseases. In this talk, we will introduce our recent finding that GQDs treatment considerably decreases the accumulation of intracellular cholesterol through physical interactions both in vitro and in vivo, resulting in a therapeutic effect against impaired functions in Niemann-Pick type C disease (NPC). The GQDs are found to induce autophagy to restore compromised autophagic flux, which eventually reduces the atypical accumulation of autophagic vacuoles. Moreover, GQDs injection prevents the loss of Purkinje cells in the cerebellum with decreased microglial activation. The finding that GQDs alleviate impaired functions in NPC provides a promising potential for the treatment of NPC and related lysosomal storage disorders (LSDs). In addition, it will be shown that the aggregation of amyloid beta and tau protein filbrils related to Altzheimer's Disease has been successfully prevented by GQDs in vitro. We suppose that the thermodynamic equilibrium can be repositioned by adding GQDs as they interfere with aggregating molecules in terms of enthalpy and entropy, leading to the change the Gibbs free energy to block or reverse the fibrosis. We expect that this new thermodynamic approach to treat the central nervous system disease would be expanded to other fibrosis diseases in liver, lung, and kidney in the future.

The CRISPR/Cas9 system has been one of the most innovative tools for genome editing. For therapy, viruses expressing Cas9 and sgRNA have been used, but show limitations in safety due to off-target effects and difficulty in viral packaging. Recently, strategies for nonviral, in vivo delivery have been developed, however problems reside due to the need of using excess carrier amount which causes toxicity. To solve this problem, we have developed conjugate systems of the Cas9 ribonucleoprotein, to safely deliver the cargo in vivo. Cas9 from Streptococcus pyogens (SpCas9) was covalently conjugated with cationic polymers or lipids, to enhance delivery by increasing the net charge or hydrophobicity of the ribonucleoprotein. Mixing the Cas9 conjugates with sgRNA resulted in the formation of nano-sized complexes, which showed enhanced cellular uptake and genome editing efficiencies compared to the native complexes, demonstrated in various human cell lines and bacteria. To improve the versatility of Cas9 as a platform for chemical modification, we have also generated a bioorthogonal Cas9 protein by incorporation of unnatural amino acids. Incorporation of azidohomoalanine could increase the number of functionalities for further modification, while azidophenylalanine allowed site-specific conjugation. Our platform technologies present a versatile platform for engineering the Cas9 ribonucleoprotein using a chemical route, which can be used as a therapeutic for various human diseases, such as cancer, infections, and genetic disorders.

The modern healthcare and biomedical systems show a clear trend towards personalized, predictive, and preventive medicine. Development of the concept, commonly known as mobile health (mHealth), means that a huge shift in the paradigms of medical device architectures is to be expected in the near future thanks to the increased portability of medical devices as well as increase in number of specific mobile-based apps. An ideal wearable device should possess a set of important requirements, such as (i) low cost of fabrication, (ii) being conformable and compatible with human skin, and (iii) multifunctionality. The latter is of special importance if the goal is to build not just a single specific device, but to rather develop a technology and basis for scalable fabrication of devices that are capable to detect a plurality of vital signals (HR, EEG, ECG, hydration, galvanic response, etc.).
In order to develop the universal technology that meets all three requirements mentioned above, we propose to utilize graphene in combination with epidermal technology. The conventional epidermal biosensors are based on metal and silicon based thin films that are patterned into special structures for softness and stretchability and embedded into soft biocompatible polymers. The choice of two-dimensional materials is the most natural due to their ultra-thinness, allowing extreme flexibility, transparency, and conformability to almost any rough surface, including skin [1]. Graphene based passive electrodes have been successfully used to epidermal sensing of electrocardiograms (ECG), electromyogram (EOG), electroencephalogram (EEG), skin temperature, and skin hydration [1], [2]. It is important to emphasize that the research work is based on large-area CVD-grown graphene, allowing us to develop low-cost, wearable, and fully conformable to skin devices. Furthermore, large area fabrication gives an ultimate promise for future devices fully based on 2D materials to be available on market. In terms of possible applications, the proposed technology can be easily expanded towards other fields of healthcare biosensing, such as in vivo electrophysiology, UV exposure sensing, pressure sensing, or even towards building electronic skin, and prosthetics.

[1] S. Kabiri Ameri et al., “Graphene Electronic Tattoo Sensors,” ACS Nano, vol. 11, no. 8, pp. 7634–7641, Aug. 2017.
[2] S. K. Ameri et al., “Imperceptible electrooculography graphene sensor system for human–robot interface,” npj 2D Mater. Appl., vol. 2, no. 1, pp. 1–7, 2018.

One of the major challenges in deciphering the fundamental principles of cognition is the lack of appropriate tools for seamless interfacing with neurons across all their signaling modalities. Gaining holistic understanding of neural circuits and their control of behavior requires invention of neural probes that can simultaneously record and modulate electro-chemical activity of neurons while evoking minimal inflammatory response for periods ranging from minutes to years. Multifunctional fibers have recently emerged as a promising platform for integrating multiple functional elements to probe and control neural activity that also minimizes the foreign body response.

In my presentation, I will describe our efforts in further expanding the multifunctionality of polymer-based fiber probes by incorporating an electrochemical sensor that enables real-time tracking of neurotransmitter dynamics in behaving animals. This is achieved by introducing a carbon nanotube (CNT) based electrocatalytic electrode within the multifunctional fiber-based probes during their fabrication via thermal drawing process. The resulting devices can be implanted chronically and perform electrical recording and stimulation of neurons, light delivery through waveguides for optogenetics, drug and gene delivery via microfluidic channels, and voltammetry via the CNT electrodes for dynamic detection of dopamine. We envision that these multimodal, miniature, and mechanically compliant probes will facilitate understanding of the neurophysiological underpinnings of dopamine-dependent behaviors including reward, addiction, and motor control.

Nanoscale materials enable unique opportunities at the interface between the physical and life sciences, for example, by integrating nanoelectronic devices with cells and/or tissue to make possible bidirectional communication at the length scales relevant to biological function. In this presentation, I will overview a new paradigm for seamlessly merging electronic arrays with the brain and other key components of the nervous system in three-dimensions. First, I will discuss the design considerations of matching structural, mechanical and topological characteristics of neural probes and brain tissue, thus leading to mesh electronics systems that are immune-privileged and enable uniquely stable electrophysiology such that it is possible to track and stably record from the same single neurons and neural circuits on the time scale of at least year. Second, I will describe a selection of new opportunities using the mesh electronics paradigm, including (i) nonlinear lesion-free implantation in the retina and brain, (ii) development of new nanoelectronic devices for subcellular resolution recording, and (iii) and advances in interfacing that can enable scalable recording and stimulation of large numbers of neurons. I will conclude with discussion of opportunities and challenges pushing towards tools that can significantly advance fundamental neuroscience and electronic medicine in humans.
Selected References
(1) http://meshelectronics.org/
(2) T.-M. Fu, et al., Nat. Methods 13, 875-882 (2016).
(3) G. Hong, et al., Science 360, 1447-1451 (2018).
(4) X. Yang, et al., Nat. Mater. 18, 510–517 (2019).
(5) G. Hong & C.M. Lieber, Nat. Rev. Neurosci. 20, 330–345 (2019).

Mammalian brains consist of billions of neurons operating at millisecond time scales, which current recording techniques only capture a tiny fraction. Recent advances in CMOS device design have led to high-recording quality planar probes, with diminishing sizes to ameliorate the extent of tissue damage. Matching these powerful silicon electronics to the inherently three dimensional architecture of the brain has remained challenging however, as devices are constrained to the planar two dimensional surfaces required for silicon processing. Here we describe a chronic interface using arrays of microwires read out by CMOS-based devices with a low-tissue damage, and controllable, three dimensional distribution of recording sites. The core concept is using a bundle of insulated microwires mated to a large-scale CMOS microelectrode array, such as found in modern camera chips or displays. We show recent results on the mechanics and tissue damage from microwire insertion scales strongly with wire diameter. Microwires with <25µm diameters are shown to have minimal to no vascular disruption or bleeding, as opposed to more conventional 75 to 100 µm devices. These microwires are then arranged into bundles to control the spatial arrangement and three dimensional structure of the distal (neuronal) end, while providing a robust parallel contact plane on the proximal side which is interfaced to a planar pixel array. The modular nature of the design enables a wide array of microwire types and size to be mated to a variety of different CMOS chips, making the same fundamental platform scalable from a few hundred electrodes to tens of thousands. We thus link the rapid progress and power of commercial multiplexing, digitisation and data acquisition hardware together with a bio-compatible, flexible and sensitive neural interface array. We present recent massively parallel recording using mouse and rat models, showing both spiking activity from single neurons and local field potentials within both chronic and acute settings.

The interaction between the cell membrane and the measuring electrode is crucial for crucial for sensitive measurement of cell electric activities. We are interested in exploring nanotechnology and novel materials to improve the membrane-electrode coupling efficiency. Recently, we and other groups show that vertical nanopillars protruding from a flat surface support cell survival and can be used as subcellular sensors to probe biological processes in live cells. The nanopillar electrodes deform plasma membrane inwards and induce membrane curvature when the cell engulfs them, leading to a reduction of the membrane-electrode gap distance and a higher sealing resistance. As an electrode sensor, nanoelectrodes offer several advantages such as high sensitivity, subcellular spatial resolution, and precise control of the sensor geometry. Furthermore, we found that the high membrane curvature induced by nanoscale electrodes significantly affects the distribution of curvature-sensitive proteins and stimulates several cellular processes in live cells. Our studies show a strong interplay between biological cells and nanoscale topography, which is an essential consideration for future development of interfacing devices.

In this talk, I will discuss several projects related to engineering conductive materials and developing fabrication methods to allow electronics with effective electrical interfaces with biological systems, through tuning their electrical as well as mechanical properties. The end result is a soft electrical interface that has both low interfacial impedance as well as match mechanical properties with biological tissue. Several applications of such electronics will be presented.

Bioelectronic medicines targeted at the peripheral nervous system have the potential to address a wide variety of diseases, from diabetes to bladder dysfunction. The foundational concepts have existed for decades but implementation has been limited and fraught with persistent challenges, including lack in target specificity, nerve interface biofouling, and inability to acquire real time physiologic signals for conditional delivery of corrective stimuli, leading to excess and unnecessary stimulation. Here we present a set of materials, a treatment strategy and supporting technology platform that address many of these challenges, using bladder control as model system. Specifically, we report capabilities for continuous monitoring of bladder function using an ultralow modulus, stretchable strain gauge to measure dimensional changes, real-time data analytics to identify pathological behavior based on the resulting data, and automated, closed-loop optogenetic neuromodulation of bladder sensory afferents to normalize bladder function in the context of acute cystitis, with generic applicability to many other organ systems and conditions.

Miniature, wireless bioelectronic devices enable less invasive surgical implantation and the ability to target tiny nerves or brain areas. However, as these neural stimulators become smaller, we must engineer new ways to deliver power. Conventional power deliver relies on long wires to deliver power from an implanted battery or subcutaneous antenna. These leads can limit device placement and cause device failure due to lead breakage or infection. Conventional wireless power delivery through biological tissue is difficult when devices are miniaturized and placed deep in the body. Here we show that magnetic materials can effectively harvest energy from magnetic fields and power millimeter-sized bioelectronics. These materials show excellent power densities even as the devices are made small allowing them to be fully implanted and wirelessly powered. We demonstrate that these mm-sized wireless devices can be used to power different types of conventional stimulation electrodes when implanted in rabbits, pigs, and freely moving rats. Furthermore, these miniature electrical stimulators can be adapted to power many individually addressable stimulation channels while still maintaining a small overall device footprint.

Soft electronic devices that can acquire vital signs from the human body represent an important trend for healthcare. Combined strategies of materials design and advanced microfabrication allow the integration of a variety of components and devices on a stretchable platform, resulting in functional systems with minimal constraints on the human body. In this presentation, I will demonstrate a wearable multichannel patch that can sense a collection of signals from the human skin in a wireless mode. Additionally, integrating high-performance ultrasonic transducers on the stretchable platform adds a new third dimension to the detection range of conventional soft electronics. Ultrasound waves can penetrate the skin and noninvasively capture dynamic events in deep tissues, such as blood pressure and blood flow waveforms in central arteries and veins. This stretchable platform holds profound implications for a wide range of applications in consumer electronics, sports medicine, defense, and clinical practices.

Demands for precise health information tracking techniques are increasing, especially for daily dietry requirements to prevent obesity, diabetes, etc. Many commercially available sensors that detect dynamic motions of the body lack accuracy, while novel strain sensors at the research level mostly lack the capability to analyze measurements in real life conditions. Here, we demonstrate a stretchable, patch-type calorie expenditure measurement system that integrates ultra-sensitive crack-based strain sensor and Bluetooth-enabled wireless communication circuit to offer both accurate measurements and practical diagnosis of motion. The crack-based strain gauge transformed into pop-up-shaped structure provides reliable measurements and broad range of strain (~100%). Combined with the stretchable analysis circuit, the skin attachable tool translates variation of knee flexion angle into calorie expenditure amount, using relative resistance change (R/R0) data from the flexible sensor. As signals from knee joint angular movement translates velocity and walking/running behavior, the total amount of calorie expenditure is accurately analyzed. Finally, theoretical, experimental and simulation analysis of signal stability, dynamic noises and calorie expenditure calculation obtained from the device during exercise are demonstrated. For further applications, we expect our devices to be used in broader range of dynamic motion of the body for diagnosis of abnormality and for rehabilitation.

An ex-vivo heart perfusion device preserves the donor heart in a warm beating state during transfer between extraction and implantation surgeries. One of the current challenges includes the use of rigid and noncompliant plastic tubes, which causes injuries to the heart at the junction between the tissue and the tube. The compliant and rapidly strain-stiffening mechanical property that generates a J-shaped stress-strain behavior, is necessary for producing the Windkessel effect, which ensures continuous flow of blood through the aorta. In this study, we mimic the J-shaped and anisotropic stress-strain behavior of human aorta in synthetic elastomers to replace the problematic noncompliant plastic tube.

Firstly, we measured the mechanical properties of human (n = 1) and porcine aorta (n = 14) to quantify the nonlinear and anisotropic behavior under uniaxial tensile stress from five different regions of the aorta. Here, the human and porcine aortas demonstrated the J-shaped strain-stiffening in uniaxial tensile testing and were stiffer in the longitudinal direction when compared to the circumferential direction. In terms of location dependence, we observed that the porcine aorta appears to be stiffer at the distal sections from the heart when compared proximal regions.

Secondly, fabric-reinforced elastomer composites were prepared by reinforcing silicone elastomers with embedded fabrics in trilayer geometry. The knitted structures of fabric provide strain-stiffening as well as anisotropic mechanical properties of the resulting composite in a deterministic manner. By optimizing the combination between different elastomers and fabrics, the resulting composites matched the J-shaped and anisotropic stress-strain behavior of natural human and porcine aorta. In order to find the optimal combination between the elastomeric matrix and the fabric reinforcement, the uniaxial tensile properties of various commercial elastomers and commercial fabrics/textiles were tested and the data were archived. Here, neat elastomer materials could not mimic the J-shape, nor the anisotropy. Among composite elastomers, knitted rayon/spandex fabric sandwiched by the two layers of Ecoflex 0050 was the best in mimicking both the strain-stiffening and anisotropy features of the aorta. Here, the knitted fabrics played a role of anisotropically crumped elastin and collagen, which enable low moduli at low strains and a rapid stiffening at high strains.

Thirdly, improved analytical constitutive models based on Gent’s and Mooney-Rivlin’s constitutive model (the elastomer matrix) combined with Holzapfel–Gasser–Ogden’s model (the stiffer fabrics) were developed to describe J-shaped behavior of the natural aortas and the fabric-reinforced composites. Overall, the elasticity of natural aorta at low strains is attributed to the elastin (Mooney-Rivlin and Gent) and stiffening of the curve at higher strains originates from collagen contribution and k₁ and k₂ parameters (Holzapfel). In the fabric-reinforced elastomers, the low elastic modulus at low strains comes from the elastomer matrix and the stiffening at higher strains comes from embedded fabric. Moreover, the values of the main parameters, namely µ, k₁ and k₂ for Gent’s model, and stress-like parameters C₁₀, C₀₁, and k₁, and k₂, and for Mooney-Rivlin’s were very similar between the natural aorta and our fabric-reinforced composites. These support the aorta-like behavior of the developed fabric-reinforced composites.

In a broad context, J-shape and anisotropy are general properties of many soft biological tissues. We anticipate that the suggested design criteria and our proposed analytical models can be helpful in designing biomaterials that mimic properties of complex soft biological tissues with synthetic materials. The material design concept can also be used in emerging engineering fields such as soft robotics and microfluidics, where the Windkessel effect can be useful in regulating the flow of fluids.

The enhanced design and fabrication of cardiovascular devices relies on technological advances in implantable materials. In this talk I will discuss the importance of tunable and optimized materials for realizing implantable cardiovascular implants for structural repair, active assistance and biological therapy. I will discuss representative devices, and their component materials, in each of these three areas, each addressing an identified shortcoming of existing technologies. In terms of structural repair devices, I will discuss a minimally invasive delivery system that uses a biodegradable, photo-activated adhesive for atraumatic repair of intracardiac defects. Moving to active assist devices I will discuss the modelling and design of a bioinspired soft active material technology that enabled the fabrication of a robotic direct cardiac compression device whose design mimics the orientation of the heart muscle. In vivo testing of this device has demonstrated that it is possible to improve cardiac output without the need for a blood-contacting approach in an acute heart failure animal model. Building on the platform of soft robotic approaches to enhance organ function, I will discuss pediatric cardiac assist devices and mechanical devices to enhance respiratory function. Lastly, to illustrate examples of enhanced biological therapy, I will discuss the use of biomaterials as vehicles for cell delivery and a targeted, refillable bio-implant for increasing retention of therapy in the heart, which enables repeated local administration of biological or pharmacological delivery, and some preliminary steps to combine these mechanical and biological therapies in order to improve delivery of drugs and modulate the host response.

An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. In this talk I will describe our recent work in the development of materials for bioelectronics including polymers and functionalised nanoneedles. I will also describe our new imaging technologies for monitoring and elucidating the cell-material interface.

Tissue-wide electrophysiology with single-cell and single-spike spatiotemporal resolution, and cell-type specificity is critical for heart and brain studies. In this talk, I will first discuss the creation of cyborg organoids: the three-dimensional (3D) assembly of soft, stretchable mesh nanoelectronics across the entire organoid by the cell-cell attraction forces from 2D-to-3D tissue reconfiguration during organogenesis. We demonstrate that stretchable mesh nanoelectronics can migrate with and grow into the initial 2D cell layers to form the 3D organoid structure with minimal impact on tissue growth and differentiation. The intimate contact between the dispersed nanoelectronics and cells enables us to chronically and systematically observe the evolution, propagation and synchronization of the bursting dynamics in human cardiac organoids through their entire organogenesis and maturation. Second, I will discuss a general concept of genetically-targeted functional assembly in tissue--in this case through a convergence of protein engineering and polymer chemistry that genetically instructs specific living neurons to guide chemical synthesis of conductive polymers onto the plasma membrane. Conductive polymers were assembled in vivo at genetically- and subcellularly-targeted locations per design specifications, and were demonstrated to achieve intended functionality in the form of newly-created electrical conduction pathways. Imaging, electrophysiology, and behavioral analyses confirmed that in vivo conductive polymer assembly preserved neuronal viability, remodeled cellular membrane properties, and elicited cell-type-specific behaviors in freely-moving animals. In the end, I will discuss the prospects for future advances in bioelectronics to overcome challenges in neuroscience and cardiology.

Organic semiconductors are widely applied in bioelectronic devices, such as organic electrochemical transistors (OECT) and ion pump for drug delivery.^1,2This is largely due to their mixed electronic/ionic conductivity, which enables strong coupling between these two charge carriers in the bulk of the material. While electronic charge carrier transport in these materials is rather well understood, the transport of ions has not been studied to equal depth.³NMR spectroscopy is a powerful nuclei-specific technique that can provide atomic structural information as well as quantitative and dynamic information of the disordered polymer system.⁴Here, we report on the interaction between the sodium ion and the poly(3,4-ethylenedioxythiophene) polymer doped with poly(styrene sulfonate) (PEDOT:PSS) using ²³Na NMR spectroscopy. The structure of PEDOT:PSS is heterogenous, and it mainly consists of PSS-rich region and PEDOT:PSS nano-domains (known as “pancakes” due to the non-sphere shape revealed by X-ray scattering measurement).⁵We found that Na⁺absorbed in the pancakes gives rise to unique NMR patterns due to the strong quadrupolar interaction between the Na⁺and the polymer. Thus, we are able to distinguish Na⁺in aqueous NaCl solution and Na⁺absorbed in the polymer. Then we selectively monitor the absorption and desorption of the Na⁺and Cl^-ions in the polymer when it is biased to different voltages, in order to elucidate and quantify the species within the polymer. Lastly, the Na⁺dynamics (self-diffusion coefficient and ion drift mobility) is probed by pulsed-field gradient NMR. The result suggests a fast Na⁺mobility in the hydrated polymer with notable anisotropy possibly due to the preferred orientation of the PEDOT:PSS nanodomains. This work demonstrates the application of in-situ NMR to probe ion-polymer interactions and to correlate the microstructure of the polymer to ion mobility, which improves our fundamental understanding of mixed conductors and our ability to optimise the performance of their devices.
Reference:
(1) Inal, S.; Rivnay, J.; Suiu, A. O.; Malliaras, G. G.; McCulloch, I. Acc. Chem. Res.2018, 51(6), 1368–1376.
(2) Rivnay, J.; Inal, S.; Salleo, A.; Owens, R. M.; Berggren, M.; Malliaras, G. G. Nature Reviews Materials. 2018.
(3) Stavrinidou, E.; Leleux, P.; Rajaona, H.; Khodagholy, D.; Rivnay, J.; Lindau, M.; Sanaur, S.; Malliaras, G. G. Adv. Mater.2013, 25(32), 4488–4493.
(4) Forse, A. C.; Griffin, J. M.; Merlet, C.; Carretero-Gonzalez, J.; Raji, A.-R. O.; Trease, N. M.; Grey, C. P. Nat. Energy2017, 2(February), 16216.
(5) Rivnay, J.; Inal, S.; Collins, B. A.; Sessolo, M.; Stavrinidou, E.; Strakosas, X.; Tassone, C.; Delongchamp, D. M.; Malliaras, G. G. Nat. Commun.2016, 7, 1–9.

3D cell cultures in biomimetic environments are finding numerous applications in drug discovery, regenerative medicine, among many other fields. 3D cultures often rely on scaffolds, typically made of polymeric materials, which support cell attachment and favour tissue development. These scaffolds possess certain structural and mechanical properties, such as porosity and stiffness, that mimic the extracellular matrix providing a similar microenvironment to that found in vivo. However, these scaffolds are typically passive, merely providing a templated support. We have been focusing on a synthetic biology approach where we are integrating additional functionality into cell culture scaffolds, by using electroactive scaffolds capable of monitoring cell growth and differentiation. Porous conducting polymer scaffolds made of PEDOT (poly(3,4-ethylenedioxythiophene): PSS were obtained by the freeze-drying method. This method allows the addition of different components to tune mechanical and biochemical properties (e.g., collagen), to enhance conducting properties (e.g., carbon nanotubes), thereby fine-tuning the chemicophysical properties of the scaffold to render the system suitable for a variety of biological applications. We demonstrate that these scaffolds support cell growth of a variety of cell and tissue types and that the electroactive properties of the scaffolds can be used to monitor cell growth. Current work is focusing on understanding the role of electrical stimuli on cells growing in the scaffolds, in addition to the typical mechanical and biochemical cues present.

Cardiovascular disease (CVD) is the most leading cause of mortality and morbidity in the USA and costs $300 billion per year. Myocardial infarction (MI or a heart attack) is currently one of the most frequent types of CVD in the world. Among diverse technologies for rebuilding the infarcted myocardium, cardiac patches can adequately and simultaneously meet the biochemical, electrical and mechanical demands of the native heart tissue to promote regeneration following MI. Here, we first engineered a gelatin-based porous scaffold by electrospinning technique. We then incorporated graphene nanofibers (GNFs) to the acellular porous matrices to fabricate a conductive electrospun composite scaffold. The gelatin-graphene fibrous composite scaffolds were engineered by dissolving 10% (w/v) of gelatin from porcine skin into various concentrations of GNFs in the range of 0 to 0.5 %(w/v) in hexafluoro-2-propanol (HFIP) and electrospinning of resulting solutions. The effects of GNFs on physical properties (such as mechanical properties, degradation, swelling, surface energy, etc.), electrical conductivity and in vitro cytocompatibility of the scaffolds were evaluated. The results revealed that by increasing the amount of GNFs from 0 to 0.5% there was no significant difference in elongation of the scaffolds, while the tensile modules was increased from 3.2 to 7.02 MPa. Moreover, incorporation of GNFs can bridge the electrically resistant pore walls of the matrices, to support and facilitate the internal electrical interactions between adjacent CMs. Co-cultures of primary cardiomyocytes isolated form neonatal rats and cardiac fibroblasts grown on gelatin-graphene patches exhibited remarkably better contractile profiles compared to pristine gelatin control, as demonstrated by over expression of the gap junction protein connexin 43. In the next step, some epicardial-secreted factors such as paracrine factors and proteins was incorporated into the conductive patches obtained from the previous stage, with the aim of promoting the myocardial regeneration. It is expected that the integration of conductive GNFs and epicardial factors within 3D scaffolds may improve the therapeutic value of current cardiac patches and will open new avenues for engineering cardiac tissues.

Advances in material science during the last decades resulted in a new class of nanocomposite materials through the combination of polymeric matrices and a large number of different nanoparticles. In this context, nylon 6,6 (N6) is one of the most important engineering plastics in biomedical applications and has been investigated as a bone tissue scaffold. Trisodium trimetaphosphate (STMP) can be used as potential candidates for dental applications due to the anticaries action of STMP when present in a biocompatible scaffold as N6. Therefore, the insertion effect of different STMP concentrations in a N6 polymeric matrix was evaluated and correlated to the physicochemical and microbiological properties of nanocomposites. STMP nanoparticles were prepared by mechanical milling for 48h. N6 and its nanocomposites were prepared by electrospinning technique, while, the N6/STMP nanocomposites were processed by adding 2.5, 5 and 10% w/w (TMP:N6). The milling processing reduced the particle size of the STMP powders without affecting its crystalline structure. Particle size was reduced of micrometric to nanometric scale after mechanical milling producing particles with ~70 nm and spherical morphology. The phase structure was analyzed by XPS technique and nuclear magnetic ressonance spectroscopies in solid state. ¹³C NMR all chemical shifts were well resolved and were assigned according to N6. XPS results demonstrated that phosphate groups were bound to C=O groups on N6 by covalent bonds, showing that, STMP was incorporated in N6. The morphology it was analyzed by Scanning Electron Microscopy (SEM) technique. SEM images showed the formation of nanofibers in N6 and its nanocomposites with ~150 nm of thickness for N6 and thickness higher for N6-STMP nanocomposite, showing the presence of STMP homogeneously distributed over the nanofibers. The thermal behavior was analyzed by TGA technique. Thermogravimetry analyses demonstrated improved thermal stability of N6-STMP nanocomposites with higher TMP concentration according to its barrier effect. The mechanical properties were evaluated, and N6-STMP -2.5% nanocomposite presented higher elastic modulus, elongation at rupture and tensile strength, presenting as a potential candidate in dentistry. Additionally, the concentration of the cariostatic agent solution and its microbial effect were positively observed, where the inhibition halos corresponding to Saforide® against S. mutans showed the largest inhibition zone, with 4.18 mm and the nanocomposites were not cytotoxic in the concentrations evaluated. These findings showed a new approach to add STMP nanoparticles by electrospinning in a polymeric matrix, forming stable nanofibers with potential application in dental biomaterials. With this methodology, it was possible with this methodology to insert the STMP nanoparticles in a polymeric matrix and increase the physicochemical properties of the nanocomposites formed.

Periodontitis is a chronic dental infection caused by a complex bacterial microbiota that can lead to the progressive destruction of periodontal tissue and loss of teeth. Antimicrobial agents are frequently administrated to prevent/treat bacterial infections in periodontal defects and the development of controlled release drug systems based on nanostructures appears as a promising alternative to enhance the efficiency of such treatment. Here we report the preparation of core-shell nanofibers via coaxial electrospinning by using chitosan as shell layer and poly (vinyl alcohol) (PVA) containing tetracycline hydrochloride (TH) as core layer as a potential system to treat periodontitis. Chitosan samples possessing different average degrees of deacetylation (DD = 82% and 93%) were prepared via ultrasound-assisted deacetylation reaction of β-chitin extracted from squid pens. PVA was chosen because it is a biocompatible, biodegradable and easily electrospinnable polymer, while TH, a broad-spectrum antibiotic presenting activity against both Gram-positive and Gram-negative bacteria, was chosen as model drug. The effects of degree of deacetylation of chitosan and the post-electrospinning genipin crosslinking on physicochemical and biological properties of resulting nonwovens were evaluated. Defect-free and geometrically uniform nanofibers with diameters in the range of 100−300 nm were obtained, and transmission electron microscopy (TEM) revealed the core-shell structures of the produced nanofibers. The mechanical properties and stability of nonwovens in aqueous medium were greatly improved by genipin-crosslinking. As a consequence, a sustained release of TH from these structures was possible for 14 days. The degradation rate and the release profile of TH in the presence of lysozyme can be controlled by properly selecting the chitosan to be used in the shell layer of nanofibers, as the degradation and TH release rate was higher the lower the average degree of acetylation of chitosan. Further in vitro antimicrobial activity demonstrated that the cross-linked nonwovens containing TH showed strong activity against bacterial strains associated with periodontal disease. Additionally, the nonwovens did not demonstrate cytotoxicity toward fibroblast (HDFn) cells, indicating the potential of this novel drug delivery platform for periodontitis treatment.
The authors thank the financial support from FAPESP (grant number: 2017/20973-4), CNPq, MCTI-SisNano, FINEP, and Embrapa AgroNano research network.

Controlled drug release responding to external stimulus is an important technique to focus area of drug eluting, and to reduce side effect of the drug at normal tissue. Previously, we modified double stranded DNA on gold nanorods that can be heated by near-infrared (NIR) light irradiation. After heating the gold nanorods by NIR irradiation, single stranded DNA was released [1]. Thus, we succeeded in constructing controlled release system responding to NIR irradiation. However, capacity of loading drugs (DNA) was limited on surface of the nanoparticles. Chu et al. prepared gold nanorods-coated PLGA nanoparticles and achieved controlled release of drugs from the nanoparticles responding to NIR [2]. Large amount drugs could be encapsulated in the PLGA as a payload, but disruption of PLGA by the photothermal effect of the gold nanorods was limited on their surface. Here, we prepared PLGA nanoparticles encapsulating drugs in the nanoparticles and the release efficiency was evaluated.
Gold nanorods were coated with disulfiram and dispersed in dichloromethane. The gold nanorods and PLGA were mixed in chloroform, and then added to 1% PVA aqueous solution with sonication. After washing resultant nanoparticles by water, gold nanorods-encapsulated in PLGA nanoparticles (GNR-PLGA NPs) were obtained. TEM observation revealed that some gold nanorods were encapsulated in one PLGA nanoparticles. Mean diameter of GNR-PLGA NPs was about 300 nm. When GNR-PLGA NPs dispersion in water were irradiated by continuous wave (CW) near-infrared laser, dispersion temperature increased and destruction of PLGA nanoparticles was observed. In case of pulsed near-infrared laser, the gold nanorods were converted to spherical form in PLGA nanoparticles by strong heating effect by the pulsed near-infrared laser; however, no temperature increase was observed. After changing the shape, the gold nanospheres have little absorption band at NIR region, therefore, they could not be heated anymore.
Next, drug release from the gold nanorods-encapsulated PLGA nanoparticles responding to the CW laser was examined. Curcumin as a model drug was encapsulated in the GNR-PLGA NPs. When the nanoparticles were irradiated by CW NIR laser, release of the curcumin was observed, while little release was observed when the laser had been turned off.
We constructed the controlled drug release system responding to NIR laser from GNR-PLGA NPs. Heating from inside of PLGA nanoparticles and destruction of the whole nanoparticles will be advantageous in high contrast on/off control of drugs release.

References
1. S. Yamashita et al., Bioorg. Med. Chem. 19 (2011) 2130–2135
2. C.-H. Chu et al., J. Mater. Chem. 20 (2010) 3260-3264

Capacitive biosensors, combined with inexpensive fabrication technologies and low cost materials, may provide simple, sensitive devices for detecting clinically relevant cardiovascular disease biomarkers. Herein, we report a novel platform for detecting the C reactive protein using low-cost ink-jetted interdigitated electrodes modified with biomass-based composite. First, a comb-like interdigitated silver electrode was inked on a Kapton sheet substrate by using an ink jetting Dimatix. Different parameters were tested: tension (20 to 30V), substrate heating temperature (273.15 to 333.15K), distance between nozzle and substrate (100 to 500mm) and number of printed layers (01-15) for obtaining good electrical characteristics. The electrodes were characterized by SEM and resistivity measurements. The best parameters were set 30V, 313.15K, 300mm and 01 layer. A simple synthesis was used to prepare the biomass carbon derived from bamboo and its composite with ZnO nanorods. The biomass carbon derived from bamboo was prepared by direct pyrolysis of the ground sample at 750C for 4 hours. For preparing its composite, a quantity of biomass carbon derived from bamboo was added a hexamethylenetetramine and zinc nitrate in the proportion 1:1 in a Polytetrafluoroethylene (PTFE) vessel. The PTFE vessel was placed in silicone bath. The solution was stirred and heated at 90°C for 2 h, aiming to promote the growth of ZnO NRs on the surface of the biomass carbon. The samples were characterized by FTIR, RAMAN, EDX and SEM microscopy. The biomass-base composite consisting of bamboo derived biomass carbon with a high degree of graphitization and ZnO nanorods were deposited on silver electrodes to aid in the immobilization of C reactive protein antibody. Immobilization of the anti-C reactive protein was proven by immunofluorescence confocal microscopy and FTIR. The capacitance was measured in a frequency range of 1 Hz to 1 MHz and diverse analyte concentrations. The modification of the electrode with biomass-based composite significantly improved the analytical performance of the immunosensor. The biosensor exhibited high reproducibility and relatively low limit of detection.

Gene therapy is a promising technology for treatment of human diseases due to the versatility and specificity in designing the drug according to the target. The CRISPR/Cas9 system has been greatly attractive as a gene therapeutic since the target gene can be reprogrammed with high efficiency and selectivity. Non-viral gene delivery methods that can effectively transport the carrier to a target site are important for addressing the safety concerns of viral delivery methods, due to their low toxicity. Herein, we used polyethyleneimine carbon dot nanoparticles (PEI-Cdots) to deliver the CRISPR plasmid efficiently to cancer cells. An all-in-one CRISPR plasmid expressing Cas9 and GAL4UAS-luciferase sgRNA was prepared and allowed to form a complex with the PEI-Cdots by electrostatic interaction. The complexes were then functionalized with hyaluronic acid (HA) to stabilize the complexes and specifically target CD44 overexpressed on tumor cells. HA-functionalized PEI-Cdot/plasmid complexes were characterized by dynamic light scattering and zeta potential measurements. Delivery of the HA-functionalized PEI-Cdot/plasmid complexes into tumor cells were investigated by using an all-in-one plasmid to CD44-overexpressing cancer cell lines. The cellular uptake of the complexes and subsequent knockout of the target reporter gene were determined by confocal microscopy.

Stents used for cardiovascular applications are composed of three main elements; a metal, polymer coating and the specific drug component. Nickel-based metals and polymer coatings currently used in the stent market have increased the recurrence of in-stent restenosis and stent failure due to inflammation. In this study, a Ti-8Mn alloy was used to fabricate a nanostructured surface that can be used for drug eluting stents to overcome the hypersensitivity of metals that are currently used in stent making as well as introducing a new built-in nano-drug reservoir instead of polymer coatings. Two different systems were studied: titanium dioxide nanotubes (NTs) and Ti-8Mn oxides NTs. The materials were characterized using field emission electron microscope (FESEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), roughness, wettability and surface energy measurements. Nanoindentation was used to evaluate the mechanical properties of the nanotubes as well as their stability. In-vitro cytotoxicity and cell proliferation assays were used to study the effect of the nanotubes on cell viability. Computational insights were also used to test the blood compatibility using band gap model analysis, comparing the band gap of the materials under investigation with that of the fibrinogen, in order to study the possibility of charge transfer that affects the blood clotting mechanism. In addition, the drug loading capacity of the materials was studied using acetyl salicylic acid as a drug model.

Antibiotic-resistant bacteria present a global threat because they are increasingly difficult to treat. Therefore, it is highly important to develop advanced methods for the identification of antibiotic resistance gene in the virulent bacteria. Here, we report the development of novel nanoprobes for fluorescence in situ hybridization (FISH) and the application of the nanoprobe for detection of ampicillin-resistant Escherichia coli (E. coli). The nanoprobe was prepared by the modified sol–gel chemistry and consisted of fluorescent dye-loaded poly(d,l-lactide-co-glycolide) (PLGA) and silica nanoparticles. The synthesized nanoprobe showed strong fluorescent signals and pH stability even under natural light condition. For the double-identification of bacteria species and ampicillin-resistance with a single probe in situ, the nanoprobes were conjugated to the two kinds of biotinylated probe DNAs; one for E. coli-species specific gene and the other for a drug-resistant gene. By using the nanoprobe-DNA conjugants, we successfully detected the ampicillin-resistant E. coli through the FISH technique. We anticipate that the nanoprobe-based FISH method will be employed for the detection of various kinds of pathogenic bacteria, and diagnosis of the emergence of the infectious drug-resistance bacteria.

Solid lipid nanoparticles (SLNs) have a crystalline lipid core which is stabilised in solution by interfacial surfactants. They are considered favourable candidates for future drug delivery vehicles as they are capable of storing and release bioactive molecules. However, when stored over time it is thought that the lipids undergo polymorphic transitions which result in the premature expulsion of the drug molecules. To date, significant experimental studies have been conducted with the aim of investigating the physicochemical properties of SLNs, including their long-term stability, but as-of-yet, no molecular scale investigations have been reported on the behaviours that drive SLN formation and their subsequent polymorphic transitions. Using a combination of small angle neutron scattering (SANS) and all-atom molecular dynamics simulations (MD) we have generated a detailed, atomistic description of the internal structure of an SLN formed from the triglyceride, tripalmitin, and the Brij O10 surfactant. In addition to studying the SLN, we have performed further experiments and molecular-dynamic simulations on the formation of a triolein-based liquid lipid nanoparticle (LLN) which is stabilised by the same Brij O10 surfactant. LLNs are, like SLNs, of interest for their potential applications in drug delivery. This has allowed us to characterise the structure of the LLN in a similar manner to the SLN and to compare the two contrasting nanostructures in order to better understand the relationship between a nanoparticle’s internal structure and its role in drug delivery. As well as studying the structure and formation of the nanoparticles, we have conducted molecular dynamics simulations and experiments to characterise and compare the processes involved in the encapsulation and localisation of a steroidal drug, testosterone propionate, by both the SLN and the LLN.

Electrospun chitosan offers an attractive platform for biomedical applications such as wound healing, drug delivery etc. However, chitosan is an extremely difficult material to electrospin and there are few systematic reports of process optimisation for electrospinning chitosan. Graphene-based materials have potential applications in biomedical engineering, due to its large surface to volume ratio and superior mechanical properties¹ but can show poor biocompatibility². Studies have shown that the biocompatibility of graphene-based materials is improved with the addition of chitosan, which also serves to increase the drug loading capacity of the material ^3-5.

It is hypothesised that blending graphene oxide (GO) with chitosan (CS) and polyvinylpyrrolidone (PVP) would improve the biocompatibility of the graphene and increase its drug loading capacity, whilst also improving the processability of the CS. The research aimed to investigate if a CS / PVP / GO composite could be electrospun and to test the efficacy of this construct for drug release of an anti-cancer drug.

Solution and process parameters were optimised to perform electrospinning of CS/ PVP and these scaffolds were then characterized. PVP was fixed at 6 wt% concentration while CS concentration varied from 0 wt% to 4 wt%. A solvent system of trifluoroacetic acid and glacial acetic acid in a 9:1 ratio was used. SEM analysis showed that the sample containing 4% CS 6% PVP had a homogenous structure with a mean fibre diameter of 0.653μm. XPS analysis of the surface showed that there were no significant chemical shifts present as CS increased, possibly due to masking of the CS by PVP. Raman and FTIR analysis proved the existence of intermolecular interactions (via H-bonding) between PVP and CS molecules, which increases with the increase of CS content in PVP. This is shown through a meaningful downshift of the carbonyl band in Raman analysis (by nearly 13cm^-1) along with the linear increase of the ratio of integrated intensities of CH stretching bands (A₂₉₃₀/A₂₉₈₁) versus CS content in x%CS/6%PVP composites (with x = 0, 1, 2, 3, and 4). In FTIR spectra, the contribution of CS to CS/PVP scaffolds was also confirmed by the less dramatic downshift of a C=O band and by the linear increase of intensity of C-O stretching bands of chitosan at 1034 and 1076 cm^-1 with increase of the CS content.

GO was integrated into the system to construct GO/4%CS/6%PVP composites in a concentration range of 0, 0.1, 0.2 and 0.7 wt%. Cell viability testing was performed on A549 cells using a neutral red assay. There was no statistical difference in cell viability between constructs loaded with 0 wt%, 0.1 wt% and 0.2wt% GO. With this, a composition of 0.2%GO/4%CS/6%PVP was chosen for drug studies. The anti-cancer drug 5-Fluorouracil (5-Fu) was loaded in concentrations of 10, 5, 1 and 0.1mg/mL to construct electrospun 5-Fu/0.2%GO/4%CS/6%PVP composites. In this case, a significant decrease in cell viability between 10 mg/mL and 5 mg/ml drug loaded samples compared to control of cells only after 48 hours of exposure was detected. At the same time, no statistical difference was observed between control cells and 1mg/mL and 0.1mg/mL samples, showing the drug released was only significant at higher concentrations and had toxic effects on the cells.

The study concludes that electrospun constructs of 0.2%GO/4%CS/6%PVP were successfully fabricated and showed good biocompatibility; capable of releasing a bioactive drug to cells. Current research is aimed at optimising the drug release system and further assessing cell toxicity.

1 Pumera, M. Chemical Society Reviews 39, 4146-4157 (2010).
2 Liao, C. et al. Int J Mol Sci. 19(11), 3546 (2018)
3 Bao, H. et al. Small 7, 1569–1578. (2011).
4 Fan, H. et al. Biomacromolecules 11, 2345-2351 (2010).
5 Ardeshirzadeh, B. et al. Materials Science and Engineering: C 48, 384-390 (2015).

Breast cancer accounts for 11.6% of all cancer cases in both genders. Even though several diagnostic techniques have been developed, the mostly used are invasive, complex, time-consuming and can’t guarantee an early diagnosis, something that is key when it comes to the tumour treatment success rate. Exosomes are extracellular vesicles that carry biomolecules from tissues to the peripheral circulation, representing an emerging non-invasive source of markers for early cancer diagnosis. Current techniques for exosomes analysis are frequently complex, time consuming and expensive. Raman spectroscopy interest has risen lately due to its non-destructive analysis and little to no sample preparation, while having very low analyte concentration/volume, due to surface enhancement signal possibility (SERS). However, active SERS substrates are needed, and commercially available substrates come with a high cost and low shelf life. In this work, composites of commercial nata de coco to produce bacterial nanocellulose and in situ synthesised silver nanoparticles are tested as SERS substrates, with a low cost and green approach. Enhancement factors (EF) from 10⁴ to 10⁵ were obtained, detecting rhodamine 6G (R6G) concentrations as low as 10^-11 M. Exosome samples coming from MCF-10A (non-tumorigenic breast epithelium) and MDA-MB-231 (breast cancer) cell cultures lineages were tested on the synthesized substrates and the obtained Raman spectra were subjected to statistical Principal Component Analysis (PCA). Combining PCA with Raman intra and inter variability in exosomal samples, data grouping with 95% confidence was possible, serving as a low cost, green and label free diagnosis method, with promising applicability in clinical settings.

Tetracyclines (TCs) are a type of antibiotic that exhibits activity against most gram-positive and gram-negative bacteria. These antibiotics are often added at subtherapeutic levels to the feedstock to act as growth promoters. Since they can cause allergic and toxic reactions and also can lead to an increased antimicrobial resistance, several countries implemented a maximum level of residues for TCs of 0.1 µg/mL in milk, 0.2 µg/mL in cattle muscle, 0.6 µg/mL in cattle liver and 1.2 µg/mL in cattle kidney. The current available methods of TCs detection are based on inhibition tests but most of them are time consuming, expensive and inadequate for field analysis.
In the present work a colorimetric biosensor was developed using paper as platform with gold nanoparticles, for the detection of four types of TCs, presenting an alternative in the performance of point-of-care tests. The construction of the sensors was performed using Lab-on-Paper technology and is based on the synthesis of gold nanoparticles by reducing a gold salt precursor for which tetracyclines constitute the reducing agent itself. Different concentrations of tetracyclines were tested and analyzed using image software, allowing to a obtain linear calibration, that correlates the concentration of antibiotics in a range between 0.1 and 10 μg/mL with the color intensity of the gold nanoparticles.
Validation tests of the sensors developed with tetracycline (TC) in milk were also performed., confirming that it is possible to detect this type of antibiotic in pre-treated milk.

The CRISPR-Cas9 system is a very robust platform for genome engineering and has a great impact on applications of gene modulation in various organisms. For their delivery, viral vectors are commonly used which have been shown high efficiency and persistent expression of the edited gene. However, the risk of insertional mutagenesis, immunogenicity and off-target effects, disclosed in clinical trials, poses serious safety concerns. Alternatively, CRISPR-Cas9 can be delivered in the form of ribonucleoprotein (= RNP) to reduce these side effects. Most of the recently reported non-viral delivery methods of Cas9 RNP involve the non-covalent encapsulation in carrier materials, which show limitations due to the cytotoxicity by the need of treating at high doses because of the low packaging efficiency. In this study, we introduce a polymer-derivatized Cas9 by direct covalent modification of the protein with cationic polymer, for subsequent complexation with sgRNA and ssODN. Nano-sized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, in which the functionality of Cas9 endonuclease was well maintained. After characterization of the complex, the Cr-nanocomplex was treated to various types of mammalian cells, which showed enhanced cellular uptake and nuclear localization of both Cas9 and sgRNA compared to the native complex or lipofectamine control. Furthermore, we generated a fluorescent reporter cell line to determine HDR efficiency, through which the Cr-nanocomplex was shown to induce effective and precise genome editing, exhibited by phenotypic changes in the reporter cells. Treatment of the Cr-nanocomplex even at high doses did not cause any cytotoxicity problem, which has been a major hurdle for application of conventional lipid-based formulations. In conclusion, the covalent modification of Cas9 with a cationic polymer shows great potential for the application CRISPR complexes as therapeutics for treating a variety of diseases such as cancer and genetic disorders.

Minimally invasive microneedle (MN) patches have shown its potential for the rapid detection of biomarkers toward diagnostic point-of-care testing. However, achieving sufficient detection sensitivity for target molecules is a significant challenge. Here we present a highly sensitive detection platform using nanoporous MNs, enabling a rapid capturing of biomarkers present at sub-nanogram level. The uniform nanopore arrays on the MN surface was prepared by a controlled anodization process and then the nanoporous MNs were functionalized by the immobilization of biomarker-specific antibody to detect a target biomarker based on an immunoassay method. The bio-functionalized MN patch showed a rapid capture of estradiol (E2) known as a biomarker of preeclampsia following 1 min incubation time and exhibited a concentration-dependent change in fluorescence intensity over the E2 range of 0.5 to 1000 ng mL⁻¹ after treating fluorescent detection antibodies. Additionally, multiple biomarker detection using a single MN patch was investigated to improve the accuracy of preeclampsia diagnosis. The nanoporous MN platform can be employed to detect diverse biomarkers including metabolites and proteins and could be integrated into a handheld system for point-of-care clinical diagnostics.

Skin-mountable and wearable electronic devices have potential scientific and technological applications due to their facile integration, interaction with the human body and long-term monitoring capabilities ¹.
Up to now, efforts in the development of polymer-based strain sensors was performed with a synthetic elastomeric matrix like polydimethylsiloxane (PDMS) ¹, Ecoflex ², Dragon Skin ¹, or butadiene-styrene copolymers ³. These materials are not biodegradable, and their disposal after usage raises critical issues related to the increasing amount of electronic waste generated and its negative impact on the environment.
Polyglycerol sebacate (PGS) which is an elastomer obtained from the polycondensation of glycerol and sebacic acid, has tremendous potential for biomedical applications since it is both biodegradable and biocompatible.
In this work, we showed a novel route to manufacture sustainable porous piezoresistive sensors with an outstanding performance by adding multiwall carbon nanotubes (MWCNTs) to the PGS prepolymer, followed by the curing the elastomeric matrix at 120 °C, during 96 h under low pressure (100 mTorr). The sensors before biodegradation reveal high sensibility, with a Gauge Factor (GF) up to –9, very fast response (≤ 3 ms), negligible mechanical and electromechanical hysteresis, reliability and very long lifetime under cycling loading (> 1,200,000 cycles), and a differential pressure sensibility of 34 Pa. Due to their porous structure, they can detect low and high frequency vibrations (up to 300 Hz), small forces (200 mN) covering from the low detection limit of metallic strain gauges up to the large strains characteristic of elastomeric-based nanocomposites. Overall, these characteristics closely match the properties of the human fingertip and hence pave the way towards tactile and compliant sense elements embedded in prosthetic devices.
After biodegradation in a simulated body fluid (SBF) at physiologic conditions (37 C), a loss in the sample weight of 15% was observed after 8 weeks of incubation. While their GF or hysteresis behavior was not dramatically affected, a decrease in the nanocomposite density and an increase in the sample cross-linking was observed. Furthermore, their vibration detection limit decrease from 300 Hz for the as processed sample down to 50 Hz, after one week of incubation in the SBF solution. These sensors presented no detectable cytotoxic effects against normal human skin fibroblasts, revealing the great potential of these sensors for the fabrication of wearable electronics.
Overall, this work demonstrates prospective applications of novel sustainable soft resistive foam sensors that meet the future challenges of soft robotic systems^4,5.

Acknowledgments
This study is supported by ARC Centre of Excellence for Electromaterials (ACES) (Grant No. CE140100012).

References
(1) Amjadi, M.; Kyung, K.-U.; Park, I.; Sitti, M. Advanced Functional Materials 2016, 26 (11), 1678-1698.
(2) Mai, H.; Mutlu, R.; Tawk, C.; Alici, G.; Sencadas, V. Composites Science and Technology 2019, 173, 118-124.
(3) Sencadas, V.; Mutlu, R.; Alici, G. Sensors and Actuators A: Physical 2017, 266, 56-64.
(4) Alici, G. Biomaterials and Soft Materials, Vol.3, No.28, pp. 1557-1568,
(5) Tawk, C.; in het Panhuis M.; Spinks . G. M; Alici, G. Advanced Intelligent Systems, doi: 10.1002/aisy.201900002

Graphene quantum dots (GQDs) are 2~3 nm sized nanoparticles that have a hydrophobic 2D graphitic domains with hydrophilic oxygen-containing functional groups along the edges. The carboxyl groups on GQDs can be modified to amine groups through simple EDC/NHS chemistry to be conjugated with many different small molecules. Recently, the application of GQDs as a therapeutic agent has been demonstrated to alleviate Parkinson's disease by degrading pre-existing α-synuclein fibers as well as by preventing its fibrillization. It is also proven that GQDs have negligible long-term toxicity in animal models as they are excreted through urine in a few weeks (D. Kim et al., 2018, Nat. Nanotechnol., 13, 812-818).
On the other hand, kidney disease occurs due to the weakening of the filter function of a glomerulus, and while it can be cured by early diagnosis, a full recovery is almost impossible when CKD (Chronic Kidney Disease) proceeds. When the symptom gets severe, dialysis or even transplantation is required. About 15% of adults in the US are suffering from CKD, and the population has been steadily increasing (National Chronic Kidney Disease Fact Sheet, 2017). When a kidney is injured, it loses tubular cells and to fill in space, an excess amount of ECM (Extracellular Matrix) is synthesized, which results in fibrosis. EMT (Epithelial-Mesenchymal Transition) occurs during fibrosis, and myofibroblasts make more ECM proteins, causing the kidney to become stiff and lose its function. Renal fibrosis is a typical and inevitable histological symptom of CKD.
Herein, we develop a method to employ GQDs as a therapeutic agent to prevent or reduce renal fibrosis. GQDs was treated to human kidney cells at the concentration of 5~200 μg/ml and did not show significant cytotoxicity according to the MTT assay. rTGFβ was added to TECs (Tubular Epithelial Cells) to provoke fibrosis and measured whether GQDs can alleviate this process. After rTGFβ treatment, the level of fibronectin and collagen (I), which are major components of the ECM were increased. When GQDs was treated at 2~10 μg/ml, a decrease in the ECM proteins was observed from the immunofluorescence. The increase in E-cadherin confirmed GQDs' role in preventing cell death. Collagen (I) monomers were incubated for 24 hours with and without GQDs, and thicker collagen (I) fibers were formed when GQDs was treated. GQDs quenched the emission of photoluminescence of collagen (I), showing a physical interaction between the protein and particles.
A UUO (Unilateral Ureteral Obstruction) mouse model was used for generating AKD (Acute Kidney Disease), and GQDs were intravenously injected at the concentration of 20 mg/kg. The level of fibronectin and collagen (IV) were significantly reduced after GQDs treatment, while an increase in E-cadherin was observed. TGFβ, as a key profibrotic factor, is linked with diverse signaling pathways, and we focused on the TGFβ-Smad pathway. Smad2/3 promotes fibrosis by directly binding to the promoter region of collagen and triggers its production. Smad7 is an inhibitory regulator that suppresses TGFβ via negative feedback. GQDs were shown to decrease the level of Smad2/3, and increase Smad7, which correlates with the less formation of the fibrotic region as confirmed by histological staining. Overall, we demonstrate that GQDs can be used as a therapeutic agent for renal fibrosis, which is expected to provide an alternative route to treat CKD patients without pain.

Magnetic oxide (Fe₃O₄) nanoparticles have a wide range of medical applications, such as magnetic separation, contrasting agents for MRI scans, DNA detection, drug delivery, and magnetic hyperthermia. Water solubility is important for nanoparticles to be viable in biological applications. In this study, synthesis of Fe₃O₄ nanoparticles were synthesized under an inert N₂ atmosphere with ferric acetate, 1,2-hexadecanediol, phenyl ether, oleic acid, and oleylamine. And ligand exchange process via citric acid were performed to yield water soluble nanoparticles under basic environment (pH >7). These synthesized nanoparticles are characterized by UV-Vis-NIR spectroscopy, fluorescence spectroscopy, XRD, TEM, etc. are also heavily relevant in producing stable and uniform hybrid nanoparticles that lack physical impurities. By further optimizing synthesis conditions, such as altering growth temperatures and precursor reagent ratios, functional hybrid magnetic nanoparticles can be obtained for MRI and other multimodal biomedical applications. Correlations between fluorescent lifetime and sizes, compositions, shapes of hybrid nanoparticles have been studied further as well.

Drug-eluting stents (DES) are favored over bare metal stents due to the low risk of angiographic restenosis. To improve the adhesion strength of drug emitting polymer coatings, surface chemical treatments are often adopted. However, to optimize surface modification, it is important to understand the interface phenomenon and mechanical properties of polymer coatings in a greater detail. Here, a new methodology was adopted where, in situ confocal imaging in combination with nanoDMA and nano-scratch tests were used to determine the mechanical and adhesion strength of polymer coatings on stents that contains pharmacologic agents. Force modulations and real-time contact images are correlated to understand adhesion characteristics. Further, the methodology was extended to characterize the mechanical properties of biomimetic materials.

Artificial protein hydrogels have immense potential as functional biomaterials, incorporating the intrinsic mechanical and bioactive properties of proteins in the polymer network. However, current artificial protein designs are limited in their ability to translate protein nanomechanics to macroscale materials due to inefficient crosslinking density or weak crosslinkers. Chemical crosslinkers are strong but, in general, the lack of specificity or uncontrollable binding kinetics can lead to a spatially inhomogeneous crosslinking density within the polymer network. Physical crosslinkers have specific self-associations with temporary bonds, capable of rearranging in a timescale dependent on their dissociation constants to form a relatively homogeneous network. However, their low rupture strength limits possible applications due its effect on mechanical properties of the material. With many self-oligomerizing proteins that form quaternary structures available in nature, there is great potential for finding physical protein crosslinkers with high affinity and specificity. Streptavidin, well known for having one of the strongest protein-ligand interactions with biotin, is utilized in diverse biotechnology applications ranging from molecular identification to drug delivery. Four streptavidin monomers naturally self-associate with high affinity physical bonds to form a tetramer quaternary structure. Here, we used streptavidin as a strong and specific physical crosslinker to model its effect on the spatial homogeneity and mechanical strength of artificial protein hydrogels. In addition, biochemical methods and single-molecule studies revealed that biotin binding to streptavidin enhances the thermal and mechanical stability of the tetramer structure. We analyzed how the stabilizing effect of biotin on streptavidin tetramers translates from the single-molecule scale to macroscale hydrogel mechanical properties.

The rising prevalence of organ loss and failure due to acute and long term illnesses, and the lack of available therapies and organs for transplantation has been one of the most critical unmet needs in medicine worldwide. In order to meet this critical need in tissue, the field of tissue engineering has emerged over the last several decade with major efforts focused on the liver, lung, kidney, and heart. However, for any organ regeneration, consistent supply of blood to the targeted regeneration sites is considered the most essential criteria for the initiation as well as continuous regeneration. Most engineered tissues, particularly highly metabolic tissues or those with poor oxygen diffusivity, require an intrinsic microcirculation that reaches to within 100 – 200 m of every cell in the construct. This requirement has spurred the development of angiogenesis induction to create capillary beds. Such approaches are promising but face challenges including the long-term robustness and viability of the vessels and integration with larger vessels in the construct and in the host. As an alternative, this work presents biodegradable scaffolds containing bifurcated microchannels designed to replicate the fluid mechanics and transport properties of organ vasculature through 3D printing of glycerol-based polymers. Vasculatures of varying sizes ranging from aortic to micro-vasculatures are successfully printed and are proven robust for repeated flow. Intrinsic micropatterns from 3D printing are shown to facilitate cell adhesion for vasculature regeneration. This work shows potential in advancing the developments in microfluidics and microfabrication technology enabling the supply of nutrients and waste removal for regenerated tissue.

Valvular heart disease is one of the leading causes of death worldwide and is projected to increase because of our aging population and the lack of effective treatments. Current heart valve bioprostheses are engineered from chemically-fixed animal tissues and are therefore prone to calcification and structural degeneration over time. To overcome these limitations, tissue-engineered heart valves (TEHVs) based on cell-free polymeric scaffold materials have emerged as a promising replacement alternative, as they are designed to leverage the regenerative and self-remodeling capabilities of the host to regrow a living, native-like tissue. Such cell-free approaches can furthermore rely on predictable and low-complexity manufacturing methods, thus further addressing the cost and variability of cell-based TEHVs. However, despite recent advances in synthetic scaffold manufacturing, there remains a lack of automated and scalable platforms for the rapid production of biomimetic TEHVs. We previously designed a nanofiber rotary jet spinning process capable of rapidly fabricating (<15min) fibrous biohybrid heart valve scaffolds. In a proof-of-concept preclinical acute study, we confirmed TEHVs functionality up to 15 h in the pulmonary valve position of a sheep model. Here, we propose to utilize this spinning process to manufacture in a single-step biohybrid TEHVs comprising physiologically-shaped leaflets using a newly synthesized elastomeric polymer. We demonstrate that controlled material composition, mechanical properties, and biocompatibility of TEHVs are essential to promote in-vitro cellular infiltration, as well as sustain in-vitro valve functionality at physiological pulmonary conditions. Future in-vivo acute and chronic studies will demonstrate the feasibility to implant such newly designed TEHVs via transcatheter approaches in a preclinical animal model and confirm long-term functionality and remodeling.

Acknowledgments: The authors thank the Wyss Institute of Biologically Inspired Engineering at Harvard University and the Institute for Regenerative Medicine (IREM) at University of Zurich for their ongoing support throughout this project. The authors also thank Harvard MRSEC (NSF award number DMR-1420570), SEAS Scientific Instrument Shop and Harvard Center for Nanoscale Systems (NNIN member, NSF award number 1541959).

The extracellular matrix (ECM) is a macromolecular network existing within all tissues. It provides physical scaffolding for the cell while initiating crucial biochemical and biomechanical processes required for tissue morphogenesis, differentiation and homeostasis. Therefore, the study of the ECM is essential to understand the former processes.

Some cells present apical-basal polarity, where the ECM is mainly created on the basal side of the cell. More specifically, in the case of culturing cells with apical polarity, the ECM lies underneath the cells in direct contact to the substrate, which makes it difficult to access it for direct measurements.

In the last years, atomic force microscopy (AFM) was successfully used to study both cells and their ECM. However, when the ECM is synthetized on the basal part of the cell culture, lying between the substrate and the single or multi-layered cell culture, it becomes challenging to introduce the cantilever tip through the cell layer(s) to measure the ECM underneath the cells.

In order to overcome the above, we have developed a novel technique: culturing cells in a standing thin membrane with holes. First, cells are cultured on one side of the membrane and then, the structure is flipped upside-down to measure the ECM produced on the basal side of the cells and spread through the membrane holes. This technique demonstrates that AFM measurements can be performed on unaltered ECM, without any fixation, chemical or temperature treatment. Using this method, we have successfully studied the collagen matrix produced by osteoblasts.

Two-photon lithography (2PP) is a method of creating three-dimensional micro-structures on photosensitive materials. One of the key capabilities associated with 2PP is the ability to create sub-micrometer resolution materials with intricate shapes and features that are impossible to create using other fabrication techniques. Here we demonstrate a 2PP based approach to create a wide range of 3D structures and geometries to efficiently screen phagocyte cell responses on a range material chemistries (polymers).
Phagocytic cells (including monocytes, macrophages and neutrophils) play a key role in the response to invading pathogens and particulate matter as well as large foreign body implants such as medical devices. Upon encountering such materials in the body these cells will activate and attempt to engulf the foreign material. The physical dimensions and shape of these structures has been identified to play a key role in the response of these immune cells to engulf and encapsulate foreign materials. Therefore the aim of this study is to develop a high throughput screening approach to efficiently study the effect of surface structure size, geometry and complexity on phagocyte interactions and responses.
Initial data shows that complex biocompatible structures down to the micron level can be created which differentially drive the attachment of human monocytes. Up to 90 different chemistries with different micron-size complex structures can be tested in one batch to investigate the combinatorial effect of 3D geometry, shape, chemistry and architecture on cell fate. Furthermore, cells appear to completely remodel there morphology and cytoskeleton as part of this complex interaction with the 3D structures. Further studies are ongoing to understand the effect of these different geometries and chemistries on other cell processes including; differentiation, cellular polarisation and cytoskeletal changes. This fabrication method is fast and economical making it an ideal tool for developing large screening platforms of polymer surfaces and complex surface structures.

Elucidating genetic mechanisms of brain diseases requires tractable in vitro models of the human brain. Three-dimensional (3D) neural tissues are compelling systems to investigate brain diseases, but it has not been shown before how encapsulating materials of these tissues impact the transcriptome of neurons and how these changes relate to the human brain. Understanding how biomaterials and 3D culturing parameters affect the RNA-signature of in vitro neural tissues can help to develop model systems better approximating the gene expression profiles of the human brain. Herein, we developed 3D human neural tissues using cells directly derived from human embryonic stem cells and characterized how scaffolding materials and wide variety of 3D culturing conditions impact their transcriptome in comparison to that of the human brain. We demonstrate that altering crosslinking density of composite hydrogels of alginate and basement membrane matrix tunes the transcriptomic correlations to particular regions and stages of the developing human brain. Single-cell sequencing revealed that our 3D tissue system transcriptionally recapitulates cell types in the human brain. Finally, we show that our biomaterial-templated 3D tissue system is compatible with CRISPR gene editing and delivery tools to interrogate disease-associated genes. This study interfacing biomaterials engineering with genomics will support the development of more effective models of neurological diseases.

Tissue engineering is a rapidly growing field during the last decade. Cells within an artificial tissue need structural support and guidance for growth. For this purpose, we fabricate polymeric bio-compatible scaffolds by multi-photon lithography (MPL).
In MPL, a femtosecond-pulsed laser focused into a photosensitive resin solution initializes polymerization solely within the focal volume of the laser beam. Hence, sub-micrometer resolution can be achieved in three dimensions. Recently lateral and axial resolution of MPL of below 200nm and around 500 nm have been demonstrated respectively. Hence, its flexible additive manufacturing performance makes MPL a well suited technique for 3D-structuring of biocompatible materials for tissue scaffolds.

The challenge herein is the development of a photoresist that is biocompatible, mechanically stable and can be structured high writing speed. Herein we demonstrate a 2D and 3D biocompatible scaffolds structured onto cell culture membranes, which can be combined with microfluidics. For biocompatibility testing the scaffolds are seeded with cells. In order to promote cell adhesion, we developed strategies to functionalize the scaffolds with biomolecules like antibodies, DNA-linkers or RGD-peptides. This 3D structured cell scaffold within a microfluidic device are seeded with human endothelial cells models of a blood vessel wall. In the future molecular processes like transportation of bio-microparticles or macromolecules will be addressed with our platform.

Cell instructive biomaterials interact directly with biological systems, modifying their behavior by fine-tuning the crosstalk at cellular level. Cell spreading, polarization and cellular mechanical tension are only few examples of interface dynamics consequences. Great consideration has been given to the role of the cell membrane and the surface tension induced by the topography of the material in contact with it. In this context, many efforts have been focused in the last decade to characterize the cell-membrane interface, at the relevant scale. Beside the standard optical acquisition that allows imaging with major limitations in resolution (~ 100 nm), electron microscopy based-acquisition exceeds that limit. However, high resolution electron microscopy procedures require long specimen preparation processes as well as the imaging and processing of hundreds of specimen sections. Here, we present an advanced microscopy method (scanning electron microscopy/focused ion beam) based on ultra-thin resin plastificization which uniquely allows the visualization of the interface between cells and materials with 5-10 nm resolution (1,2,3). This technique allows for the visualization of a region of interest where the cell is in contact with the biomaterial underneath. The use of focused ion beam allows for etching through a variety of materials. In fact, here we will present relevant adhesion process of cells in contact with organic and inorganic materials, pseudo 3D materials (vertical nanostructures) and 3D scaffolds. Our results could give new insights in designing new efficient 3D structures for tissue engineering purpose and their interaction and effect on cellular ultrastructures at the cell-biomaterial interface.

References
(1) Li X., Matino L., Zhang W., Klausen L., McGuire A.F., Lubrano C., Zhao W., Santoro F., Cui B., A nanostructure plaform for live-cell manipulation of membrane curvature. Nature Protocols, 2019
(2) Santoro, F., Zhao, W., Joubert, L.-M., Duan, L., Schnitker, J., van de Burgt, Y., Lou, H.-Y., Liu, B., Salleo, A., Cui, L., Cui Y., Cui B., Revealing the Cell–Material Interface with Nanometer Resolution by Focused Ion Beam/Scanning Electron Microscopy, ASC Nano, 2017.
(3) Zhao, W., Hanson, L., Lou, H.-Y., Akamatsu, M., Chowdary, P.D., Santoro, F., Marks, J.R., Grassart, A., Drubin, D.G., Cui, Y., Cui B., Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nature Nanotechnology, 2017.
(4) Iandolo D., Pennacchio F. A., Mollo V., Rossi D., Dannhauser D., Cui B., Owens R. M., Santoro F., Electron Microscopy for 3D Scaffolds–Cell Biointerface Characterization, Advanced Biosystems, 2018.

Diabetic foot ulcers (DFU) are chronic non-healing wounds that often lead to lower leg amputations¹. The gold standard therapy includes surgical debridement and topical antibiotic treatment. Omnigraft (a tissue engineered collagen-glycosaminoglycan (GAG) scaffold) has shown partial success in clinical trials, motivating the use of extracellular matrix based scaffolds as possible solutions.
The Garlick lab has demonstrated that fibroblasts (“Pre-iPSF”) that have been cycled through iPS reprogramming and re-differentiated into fibroblasts (“Post-iPSF”) produce a foetal-like matrix compared to that produced by the parent Pre-iPSF cells². Building on this, our current project aims to first produce sufficient matrix from post-iPSF cells in order to next develop tissue engineered scaffolds from this matrix for the treatment of DFUs. Specifically, we have (1) analysed matrix components in 2D matrices from Pre- and Post-iPSF cells; (2) explored factors (e.g., Ascorbic Acid (AA) and Macromolecular Crowders (MMC)) that improve and scale-up production; (3) fabricated 3D porous scaffolds utilizing the 2D matrix; and finally (4) tested the biological function of the scaffold.
Human pre- and post-iPSF cells were seeded at 16,000 (low) and 64,000 (high) cells/cm², with the addition of increasing concentrations (0, 10 and 100 μg/mL) of Ascorbic Acid (AA) and/or a macromolecular crowder (Ficoll 70 + Ficoll 400). Gene expression and protein production was analysed after one and three weeks by RT-PCR and western blots for the presence of key structural genes and proteins (Collagens 1, 3, 4 (COL1, COL3, COL4) , Fibronectin (FN1), Laminins 1 and 15 (LAMA1 and LAMA5).
Our results demonstrated that post-iPSF cells produced an increased ratio of COL3/COL1 and increased production of fibronectin and LAMA5 compared to pre-iPSF cells, confirming the formation of a matrix with foetal characteristics, known to promote wound healing.
High AA concentration and high cell density enhanced matrix production from both pre and post-iPSF cells, with a 3 fold increase compared to controls. These culture conditions also increased COL1, COL3, COL4, FN1, and LAMA5 production in post-iPSF cells compared to pre-iPSF cells (p<0.05). Moreover, the amount of GAGs was increased in the post-iPSF cells while the overall amount of collagenous proteins was unchanged.
Macromolecular crowders are known to enhance matrix production by aiding in increased collagen deposition. Accordingly, addition of Ficoll in combination with high concentration of AA significantly increased matrix production and collagen deposition in both pre- and post-iPSF groups after three weeks. Moreover, RT-PCR analysis of different collagens (Col1, 3 and 4) revealed that Ficoll did not significantly affect their expression. Together, these results suggest that high density, high AA concentrations and the addition of Ficoll increased matrix production in pre- and post-iPSF cells without affecting its composition.
In order to fabricate a functional scaffold, we blended equal amounts of the pre- or post-iPSF derived matrix with additional Collagen 1 and freeze-dried the resulting slurry following previously established protocols. The scaffolds had a porous micro-architecture, held their shape when rehydrated and successfully promoted fibroblast migration and proliferation. Ongoing work is focused on assessing the functional capabilities of post-iPSF scaffolds in vascularization and wound healing assays, with the hypothesis that the foetal-like matrix will promote tissue repair. Together, this study has shown that matrix production from post-iPSF cells can be scaled up with optimized cell density, AA concentration and with the use of MMC to successfully produce sufficient amounts of matrix for fabricating porous free-dried scaffolds towards the treatment of DFUs.
¹ Kearney & Pandit, 2016 Tissue Engineering (PMID: 26671466)
² Yulia Shamis, 2013 PLOS (PMID: 24386271)
Funding : HORIZON 2020, ERC starting grant #758064

Periodontal disease, periodontitis, is a progressive destruction of periodontium including the gingival tissues, cementum, periodontal ligaments and alveolar bone. It is caused by infections, trauma, orthodontic tooth movement as well as certain systemic and genetic diseases. If left untreated, periodontitis leads to wide range of health problems from early tooth loss to severe systemic infections. Current clinical settings focus on stabilizing diseased tissues by removal of local debridement and conditioning with demineralizing agents. While these treatments generally have a positive effect on healing, they often lead to colonization of epithelial cells resulting in the formation of long junctional epithelium that prevents the regeneration of periodontium. A concerted effort in the past 20+ years has developed guided tissue regeneration strategies using bone grafts, cell sheets, tissue scaffolds, and growth factors. Although partially effective, these approaches have not yet led to predictable outcomes, mainly due to their inability to restore the structure and function of cementum and cementum-periodontal ligament (PDL) interface. Enabling remineralization of dentin lesion and promoting the regeneration of acellular cementum by progressive mineralization of the PDL fibers while preventing the downgrowth of gingival epithelial cells are requisites of connective tissue regeneration. In this study, the goal has been to adapt machine learning (ML) tools and high-throughput (HTP) screening methods into functional peptide design platform, first, to derive a set of peptides from periodontium related-proteins to form acellular cementum-like hybrid peptide-mineral complex, and then use these peptides with cell attachment/signaling functions to interface with PDL cells to guide cell attachment, proliferation and differentiation leading to generation of acellular cementum (aC) and aC-PDL junction. Using Amelogenin, the key protein in enamel mineralization, we have reported the development of set of Amelogenin-Derived Peptides (ADPs) with mineralization and hydroxyapatite (HAp) binding properties. In particular, using ADP5, we had previously demonstrated the formation of structurally and functionally integrated mineral layer on dentin and enamel tissues with mechanical properties similar to that of cementum. In addition, attachment and proliferation of PDL cells on cemento-mimetic layer has been demonstrated. Towards regenerating the acellular cementum (aC) and aC-PDL junction, the next steps included identification of protein-derived peptides from cell with signaling functions and incorporate them into the newly formed layer in hetero-functional form chimerized with ADP’s as heterofunctional biomolecular constructs that enable to form hybrid peptide-mineral tissue interface facilitating periodontal tissue regeneration. The preferential attachment and differentiation of PDL cells induced by the hybrid peptide-mineral tissue interface within a mixed culture including gingival epithelial cells, are demonstrated. The outcomes of this study provides a practical and effective approach towards the regeneration of periodontal connective tissues. This work is supported by UW-School of Dentistry Spencer Funds.

The use of non-covalent self-assembly to construct materials has become a prominent strategy in material science offering practical routes for the construction of increasingly functional materials for a variety of applications ranging from electronic to biotechnology. A variety of molecular building blocks can be used for this purpose, one such block that has attracted considerable attention in the last 20 years is de-novo designed peptides. Peptides offer a number of advantages to the material scientists. The library of 20 natural amino acids offers the ability to play with the intrinsic properties of the peptide such as structure, hydrophobicity, charge and functionality allowing the design of materials with a wide range of properties. Synthetic peptides are chemically fully defined and easy to purify through standard processes. Being build form natural amino acids they result usually in low toxicity and low immune response when used in-vivo and can be degraded and metabolised by the body. Self-assembling peptide-based hydrogels in particular have encountered increasing interest in the recent years as scaffolds for 3D cell culture or for controlled drug delivery. One of the main challenges is the fine control of the mechanical properties of these materials. The bulk properties of hydrogels not only depend on the intrinsic properties of the fibers but also on the network topology formed. In this work we show how fiber−fiber interactions can be manipulated by design to control the final hydrogel network topology and therefore control the final properties of the material. This was achieved by exploiting the design features of β-sheet forming peptides based on hydrophobic and hydrophilic residue alternation and exploiting the ability of the arginine’s guanidine side group to interact with itself and with other amino acid side groups. By designing peptides based on phenylalanine, glutamic acid, lysine, and arginine, we have investigated how fiber association and bundling affect the dynamic shear modulus of hydrogels and how it can be controlled by design [Biomacromolecules, 18, 826−834 (2017)]. Subsequently the fine tuning of the mechanical properties allowed us to design a family of functional hydrogels able to direct cellular behaviour from neurite sprouting of neuronal cells for nerve repair [Advanced Healthcare Materials under review 2019] and co-culture of oesophageal cells for Barrett’s oesophagus treatment [Advanced Functional Materials, 27, 1702424 (2017)] to direct differentiation of mesenchyme stem cells into osteoblasts for bone repair [Journal of Tissue Engineering, 7, 2041731416649789 (2016)].

Nanophotonic materials promise non-invasive, high-fidelity sensing and imaging of biological processes, provided they can exhibit biocompatibility, longevity in dynamic, aqueous environments, and improved targeting capability. Here we present our research developing nanophotonic imaging tools for bacterial identification, cellular mapping, and inter-cellular force visualization. First, we combine Raman spectroscopy and deep learning to accurately classify bacteria by both species and antibiotic resistance in a single step. We design a convolutional neural network (CNN) for spectral data and train it to identify 30 of the most common bacterial strains from single-cell Raman spectra, achieving antibiotic treatment identification accuracies exceeding 99% and species identification accuracies similar to leading mass spectrometry identification techniques. Our combined Raman-CNN system represents a proof-of-concept for rapid, culture-free identification of bacterial isolates and antibiotic resistance. Second, we describe a new technique for high-resolution vibrational spectroscopy in the TEM: electron-and-light-induced stimulated Raman scattering (ELISR). Unlike conventional stimulated Raman measurements, our technique uses a laser source as the pump and the electron beam as the broadband Stokes excitation. A small plasmonic nanoparticle amplifies the local Raman signature, with the spatial resolution determined by the electron beam spot size and the nanoparticle size. We show how this technique can enable molecular mapping at the nanoscale, en-route to an “atlas” of cellular receptors. Finally, we introduce a new class of in vivo optical probes to monitor biological forces with high spatial and temporal resolution. Our design is based on upconverting nanoparticles that, when excited in the near-infrared, emit light of a different color and intensity in response to nano-to-microNewton forces. The nanoparticles are sub-30nm in size, do not bleach or photoblink, and can enable deep tissue imaging with minimal tissue autofluorescence. We present the design, synthesis, and characterization of these nanoparticles both in vitro and in vivo, focusing on the forces generated by the roundworm C. elegans as it feeds and digests its bacterial food. Chronic cytotoxicity assays are used to confirm biocompatibility. Our force measurements are coupled with electrical measurements of muscle contractions in both wild-type and mutant animals, providing insight into the interplay between mechanical, electrical, and chemical signaling in vivo.

Near infrared (NIR) emitting quantum dots (QDs) have long held promise as probes of biological systems due to their brightness, deeper penetration depth, and narrow emission profiles, allowing the tagging and multiplexing of many targets simultaneously. However, after several decades of research, there has been limited translation of QD technology to meet biomedical problems. Two key barriers have hindered their use in vivo: the accumulation and persistence of QDs in essential organs over months to years, and the use of toxic materials (e.g. lead, arsenic, cadmium) in traditional QD compositions.

To address the latter barrier, copper indium sulfide (CIS) QDs have emerged in the last decade as a non-toxic alternative to traditional QDs.¹ However, all in vivo studies thus far have been confounded by the presence of a zinc sulfide (ZnS) shell,^2,3 which traps the core CIS material by preventing degradation and causing accumulation in vital organs. Additionally, the ZnS shell can act as a mask that hides the toxicity of the core material: in hepatocytes, CdSe cores under irradiation cause clear dose-dependent toxicity, while the same QDs with a ZnS shell cause no such toxicity.⁴ This bioaccumulation and masking greatly limits potential for clinical translation: of the ~50 FDA approved nanomedicines in the clinic today, all are biodegradable and clear from vital organs quickly.

For the first time, we assess the biodistribution and toxicity of unshelled CIS in a murine model at 1-day, 7-day, and 1-month time points. We show that bare CIS QDs breakdown quickly, with >75% of the initial dose being cleared by 1 month. Surprisingly, we also demonstrate a significant toxic response to these QDs as measured by organ weight, blood chemistry, and histology, in contrast to previous literature on this system. Specifically, we find that CIS particles induce severe hepatotoxicity and splenotoxicity. We also find that CIS particles alloyed with zinc ions before injections demonstrated significant, but lower, toxicity compared to bare CIS, while also degrading slower in several organs, suggesting that the release rate of ions correlates with increasing toxicity. Finally, we explore a new copper based semiconductor material that fully degrades in under a month without significant toxicity.

Overall, our data suggests a shift in perspective in interfacing QD platforms with biological systems. Firstly, QD cores and shell materials must be tested in vivo separately to truly assess the biocompatibility of the materials. Secondly, intentionally designing QDs to break down into biocompatible ions may be the key to future translation of QD platforms for biomedical challenges.


1. McHugh, K. J. et al. Biocompatible Semiconductor Quantum Dots as Cancer Imaging Agents. Adv. Mater. 30, 1–18 (2018).
2. Pons, T. et al. Cadmium-Free CuInS 2 / ZnS Quantum Dots for Sentinel Lymph Node Imaging with Reduced Toxicity. ACS Nano 4, 2531–2538 (2010).
3. Trapiella-Alfonso, L. et al. Clickable-Zwitterionic Copolymer Capped-Quantum Dots for in Vivo Fluorescence Tumor Imaging. ACS Appl. Mater. Interfaces 10, 17107–17116 (2018).
4. Derfus, A. M., Chan, W. C. W. & Bhatia, S. N. Probing the Cytotoxicity of Semiconductor Quantum Dots. Nano Lett. 4, 11–18 (2004).

Label-free platform diagnostic technologies hold great promise, capitalising upon their independence of target-specific binding structures and the associated burden of their discovery, complex conjugation and production procedures. Among a number of targeting-free sensing technologies, label-free surface-enhanced Raman spectroscopy (SERS) has attracted considerable attention, enabling direct profiling of physicochemical properties of endogenous biomolecules. Despite the promise of sensitive fingerprinting, reliable label-free SERS sensors are infrequently realized for biological samples due in part to the challenges of highly overlapped signatures in complex environments. Here, we present an artificial-nose inspired approach using an array of differently functionalised plasmonic surfaces to achieve increased output-data dimensionality in a label- and wash-free regime. Supported by molecular dynamics simulation, we propose that each self-assembled monolayer can provide a different physicochemical interface that promotes a diverse range of molecular interactions, resulting in modulated SERS signatures. As an artificial-nose-like sensing approach, the value of the increased information was illustrated using cell lysates where we achieved reliable improvements in mean discriminatory accuracy towards 100% with each additional surface functionality. The versatile, label-free artificial-nose based approach lays the groundwork for a broad range of biomedical applications where complex signatures of differing pathologies could be established through unguided compositional fingerprinting.

Light offers attractive ways to visualize and modulate biological systems. However, due to limited light penetration in tissues, many applications require solutions to deliver and generate light deep in the tissue. Here we present some of those solutions using biomaterial waveguides, bioluminescence energy transfer, and intracellular lasers.

Holistic study of neural dynamics in behaving subjects often demands simultaneous recording of neural activity across multiple organs in the nervous system. However, currently available techniques for the chronic recording of activity of specific neuronal ensembles are unsuitable for mobile regions of the nervous system such as the brain stem or spinal cord due to the rigidity of the probes.
Here we present a stretchable optical photometry platform that allows chronic recording of calcium signals as a proxy for neural activity across multiple regions of the nervous system in freely moving rodents during behavioral assays. We achieved simultaneous photometric readout from genetically identifiable neuronal populations in mice correlated to social interactions over a period of more than 6 months. Furthermore, we integrated microfluidic channels within these stretchable probes to realize chemical perturbation concomitant with optical recording. In targeted regions of the nervous system with high mobility, the stretchable fiber photometry platform provides an extended time window to record neural dynamics underlying complex behavioral phenotypes. We anticipate that the stretchable photometry probes will facilitate investigation of neural circuits across central and peripheral nervous systems in freely moving subjects.

In recent years, advances in imaging probes, microscopy techniques and bioinformatics image analysis have markedly expanded the imaging toolbox available to probe biological systems. Apart from conventional phenotypic studies, complex biological systems are increasingly investigated in vivo with improved accuracy in time and space and more detailed quantitative analyses down to the single-cell level (reviewed in¹). To get more insight into the elaborate chemical and mechanical dynamics that underlie development and disease progression, my laboratory addresses the growing imaging needs of the biological community by developing assays², imaging technologies^3-5, and reagents^6,7 for carrying out imaging with i) high spatiotemporal resolution at the single-cell level and with ii) sensitivities down to individual proteins. Such newly introduced and future imaging tools can then be used as a means of performing qualitative and quantitative imaging in order to mechanistically dissect development, disease progression, and tissue regeneration in vivo.

1 Pantazis, P. & Supatto, W. Advances in whole-embryo imaging: a quantitative transition is underway. Nat Rev Mol Cell Biol 15, 327-339, doi:10.1038/nrm3786 (2014).
2 Plachta, N., Bollenbach, T., Pease, S., Fraser, S. E. & Pantazis, P. Oct4 kinetics predict cell lineage patterning in the early mammalian embryo. Nat Cell Biol 13, 117-123, doi:10.1038/ncb2154 (2011).
3 Welling, M. et al. Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins. eLife 8, doi:10.7554/eLife.44491 (2019).
4 Dempsey, W. P. et al. In vivo single-cell labeling by confined primed conversion. Nat Methods 12, 645-648, doi:10.1038/nmeth.3405 (2015).
5 Mohr, M. A. & Pantazis, P. Primed Conversion: The New Kid on the Block for Photoconversion. Chemistry 24, 8268-8274, doi:10.1002/chem.201705651 (2018).
6 Pantazis, P., Maloney, J., Wu, D. & Fraser, S. E. Second harmonic generating (SHG) nanoprobes for in vivo imaging. Proc Natl Acad Sci U S A 107, 14535-14540, doi:10.1073/pnas.1004748107 (2010).
7 Mohr, M. A. et al. Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion. Angewandte Chemie (International ed. in English) 56, 11628-11633, doi:10.1002/anie.201706121 (2017).

An increasing number of commercial skincare products are being manufactured with engineered nanomaterials (ENMs), prompting a need to fully understand how ENMs in these products interact with their major biodistribution entry route: dermal barriers. Although animal studies show that certain nanomaterials can cross the skin barrier, physiological differences between human and animal skin, such as the lack of sweat glands, limit the translational validity of these results. Current optical microscopy methods have limited capabilities to visualize ENMs within human skin tissues due to the high amount of background light scattering caused by the dense, ubiquitous extracellular matrix (ECM) of the skin. We hypothesized that organic solvent-based tissue clearing (“immunolabeling-enabled three-dimensional imaging of solvent-cleared organs”, or “iDISCO”) would reduce background light scattering from the skin’s ECM to sufficiently improve imaging contrast both for 2D mapping of unlabeled metal oxide ENMs and also 3D mapping of fluorescent nanoparticles. Here, we demonstrate successful mapping of the 2D distribution of label-free TiO₂ and ZnO nanoparticles in cleared skin sections using correlated signals from darkfield, brightfield, and confocal microscopy, and micro-spectroscopy. Specifically, hyperspectral microscopy and Raman spectroscopy confirm the identity of label-free ENMs which we mapped within human skin sections. We also present measurements of the 3D distribution of fluorescently labeled Ag nanoparticles in cleared skin biopsies with wounded epidermal layers using light sheet fluorescence microscopy. Overall, our results represent a novel strategy for quantitatively mapping ENM distributions in cleared ex vivo human skin tissue models using multiple image modalities. By improving imaging contrast, we affirm label-free 2D ENM tracking and 3D ENM mapping as promising capabilities for nanotoxicology investigations.

Many drug delivery strategies demand the need for the adsorption and transport of charged, therapeutic biomolecules. These requirements can be met by nanoparticles (NPs) encompassing multiple molecular species that endow electrostatically-induced interfacial binding of specific biomolecules. We are interested in understanding the role of the architecture and composition of the molecular species on the morphological characteristics of the NPs. We study multicomponent NPs encompassing phospholipids and amphiphiles bearing hyper-branched polyelectrolytes (namely, polyamidoamine (PAMAM) dendrons) via the Molecular Dynamics simulation technique used in conjunction with a coarse-grained force field. We examine the impact of dendron generation and relative concentration on the mechanisms and processes dictating the morphology of NPs. Furthermore, we examine the theory underlying the organization and conformation of the hyper-branched polyelectrolytes.

The development of scaffolded DNA nanotechnology has enabled the self-assembly of various 2D and 3D DNA nanostructures with nanometer scale precision. The basic principle of DNA origami is the sequence programmability of multiple DNA strands and uniqueness between them. It uses a long scaffold DNA as a template, and hundreds of staple strands programmed to hybridize with the specific binding locations of the scaffold. By utilizing its excellence in shape design with high precision, a number of design methods based on the lattice-packing or lattice-free rules have been established. By contrast, a method for controlling the mechanical stiffness of the DNA origami structures has been rarely investigated, although the importance and the demand of it has been increased for their various biological application.
Here we provide two design approaches to effectively control the mechanical stiffness of DNA origami nanostructures. The first method uses stiffness-tunable modules consist of up to 11 staple strands. By revising the staple connectivity within the selected module, the location, stiffness, and included angle of hinges can be controlled precisely. Therefore, it enables the construction of dozens of single- or multiple-hinge structures with the minimized replacement of staple strands. The second method uses multiple engineered defects, consist of one to five-nucleotide (nt)-long single-stranded segments as stiffness design components. Systematic spatial distribution of these local mechanical defects with controlled lengths and positions can weaken the stiffness of the entire structure up to 70% while preserving overall structural integrity. Since our methods shown here are based on the basic principle of scaffolded DNA origami, it is anticipated that they are compatible with existing lattice-based or algorithmic routing shape design rules, as well as commonly used design program (caDNAno) and computational shape prediction platform. Therefore, our module-based stiffness design approaches can be widely adopted to biological application such as intracellular delivery carriers and templates of functional nanomaterials for therapeutics.
This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (NRF-2017M3D1A1039422 and NRF-2019R1A2C4069541).
References
Rothemund PWK. Folding DNA to create nanoscale shapes and patterns. Nature 2006, 440(7082): 297-302.
Lee C, Lee JY, Kim D-N. Polymorphic design of DNA origami structures through mechanical control of modular components. Nature Communications 2017, 8: 2067.
Kim D-N, Kilchherr F, Dietz H, Bathe M. Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructures. Nucleic Acids Research 2012, 40(7): 2862-2868.

The encapsulation of hydrophobic drugs at the hydrophobic core of a micelle gives superior properties in drug delivery, such as drug biocompatibility, better solubility and longer circulation times. Pluronic micelles are one of the widely used polymeric materials in drug encapsulation processes. In this work, we employ coarse-grained Dissipative Particle Dynamics simulations with the hydrogen bonds added explicitly to study the drug encapsulation property, structure and interactions of Pluronic L64/Ibuprofen combinations at different mixing proportions. The coarse-grained simulations reveal that the computed total drug encapsulation efficiency is around 80%, where the hydrophobic drug Ibuprofen is mainly kept in the hydrophobic core of the micelle. As for the micelle structure, the simulations show a decrease in the micelle size upon encapsulation of the drug in line with the experimental literature. The computed Radial Distribution Functions point out that the micelle shrinkage can be caused by an increased local packing of the hydrophobic-hydrophilic units around each other, and the absence of water molecules inside the micelles when there are drug molecules present in the system. Overall, the coarse-grained DPD simulations predict the structural and drug encapsulation properties of a polymeric system consistent with the experiments, whereby bringing new insights to its molecular understanding in terms of micelle shrinkage upon inclusion of Ibuprofen. The results confirm the promising role of the simulation procedure reported in this work to study the drug encapsulation, molecular structure and interactions of polymeric micelles used as drug delivery materials.

Calcium phosphate (CaP) nanoparticles are preferred in many applications owing to their excellent biocompatibility, bioactivity and chemical affinity towards biological molecules. Stoichiometric or non-stoichiometric hydroxyapatite (HA or CDHA), β-Tricalcium phosphate (β-TCP) and biphasic calcium phosphates (BCP, mixtures of HA and β-TCP in a variety of ratios) are the most widely used CaP compounds. Non-stoichiometric Ca-deficient hydroxyapatite (CDHA) is the main inorganic component of hard tissues and its synthetic forms are extensively utilized in a large spectrum of bio-applications ranging from hard tissue repairment and scaffolds to targeted drug delivery and gene therapy. Among all CaP compounds, β-tricalcium phosphate (β-TCP) exhibits better biodegradability hence it can be absorbed better and aid in the generation of new hard tissue or effective release of the nanotherapeutic molecules and/or drugs in the targeted tissue. β-TCP is also a forthcoming structure enabling highly effective osteointegration. In synthetic wet-chemical processes, β-TCP cannot be directly precipitated but only be transformed from CDHA at relatively high temperatures (>900°C) where it apparently gets stabilized irreversibly upon cooling. Low-temperature sintering of β-TCP, on the other hand, is desired for use as a biocompatible coating on alloy-based load bearing implant structures inside the body, acting as the osteointegration agent between the host tissue and the implant. In this work, we report on a procedure to obtain β-TCP phase from CDHA at temperatures as low as 720°C where the transformation already starts. Low calcination temperatures are favored in order to obtain pure β-TCP phase with finer morphology in adapt to natural skeletal tissue. However, whether or not adjusting pH can be an effective means to allow so has partly remained elusive. We, therefore, decided to systematically explore the range of pH that we thought would allow the low-temperature transformation of CDHA to β-TCP. A full transformation of CDHA to β-TCP at 750°C in under 3 hours from Ca⁺⁺ and PO₄^3- precursor solutions prepared under a pH of 5.5 was observed. The lower temperatures and the shorter sintering time reported herein allow for a fine nanostructured morphology along with high crystallinity, a sought outcome for effective osteointegration. The effect of synthesized β-TCP particles on the cell viability of human osteoblast-like cell line MG-63 was also assesed by using MTT colorimetric assay for verifying the biocompability of the obtained nanomaterials. We finally note that such a substantial lowering of the sintering temperature, when used as a coating on metallic implants, also aids in minimizing the high-temperature corrosion of the substrate alloy as most metals are prone to oxidation at elevated temperatures in addition to changing the intended fatigue-resistant microstructure.

In this study, the metallothionein gene of Candia Albicans was assembled by PCR, inserted into pUC19 vector, and further transformed into E coli DH5α cells. The capacity of these recombinant E coli DH5α cells to synthesize silver nanoparticles was tested. Our preliminary data obtained by UV-Vis spectrophotometer and SEM analysis suggested that the metallothionein gene transformed E coli DH5α cells were able to synthesize silver nanoparticles earlier and faster than DH5α cells transformed with pUC19 vector. The composition and morphology of the nanoparticles will be further characterized using Fourier-transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDS), and high-resolution transmission electron microscope (HRTEM). The capacity of metallothionein gene transformed bacteria to produce other metallic nanoparticles will be tested. The details of bio-synthesis of nanoparticles and the analyzed results on the bio-synthesized metallic nanoparticles will be demonstrated and reported.

Structural DNA nanotechnology has proposed a bottom-up approach to assemble various complex structures with the nanoscale resolution based on complementary self-assembly principles. DNA origami method is used to design higher order structures by programming complementary base sequences of DNA strands and the inter-helical connectivity between DNA strands. Accordingly, in order to precisely design and analyze DNA nanostructures, it is important to characterize and model the material properties of DNA structural motifs such as base-pair steps, which is the smallest unit of DNA helix, and Holliday-junctions connecting helices. Here, we first quantitatively investigated the sequence-dependent characteristics of DNA structural motifs at the base-sequence level using the molecular dynamics simulation. The atomic fluctuation of structural motifs consisting of base-pairs was reduced to intrinsic configuration and covariance matrices, providing the elastic properties as mechanical rigidities and coupling coefficients. A significant difference in material properties was observed with respect to the base sequences, suggesting that consideration and modeling of the sequence-dependent properties may play a crucial role in designing and analysis of the DNA origami structures. Furthermore, we developed a finite element model that can consider the base level properties of structural motifs driven by molecular dynamics simulation. It was confirmed that the proposed model was in good agreement with the previous experimental or simulation results, suggesting detailed and accurate insights into complex and highly deformable structures. Our study demonstrates the importance of characterizing material properties of DNA by atomic simulations, and their proper modeling can contribute greatly to the mechanical analysis of DNA nanostructures. This multiscale strategy developed for DNA origami nanostructures could potentially be applied to various biomolecule system, indicating its generalizability.

Acknowledgment
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1A2C4069541 and NRF-2017M3D1A1039422).

In this study, Mo₃Se₃^– single-chain atomic crystals (SCACs) with atomically small chain diameters of ∼0.6 nm, large surface areas, and mechanical flexibility were synthesized and investigated as an extracellular matrix (ECM)-mimicking scaffold material for tissue engineering applications. The proliferation of L-929 and MC3T3-E1 cell lines increased up to 268.4 ± 24.4% and 396.2 ± 8.1%, respectively, after 48 h of culturing with Mo₃Se₃^– SCACs. More importantly, this extremely high proliferation was observed when the cells were treated with 200 μg mL^–1 of Mo₃Se₃^– SCACs, which is above the cytotoxic concentration of most nanomaterials reported earlier. An ECM-mimicking scaffold film prepared by coating Mo₃Se₃^– SCACs on a glass substrate enabled the cells to adhere to the surface in a highly stretched manner at the initial stage of cell adhesion. Most cells cultured on the ECM-mimicking scaffold film remained alive; in contrast, a substantial number of cells cultured on glass substrates without the Mo₃Se₃^– SCAC coating did not survive. This work not only proves the exceptional biocompatible and bioactive characteristics of the Mo₃Se₃^– SCACs but also suggests that, as an ECM-mimicking scaffold material, Mo₃Se₃^– SCACs can overcome several critical limitations of most other nanomaterials.

Silver nanoparticles (Ag NPs) have unique optical, electrical, and thermal properties and are being incorporated into products that range from photovoltaics to biological and chemical sensors. The new generation of silver nanoparticles brings potential applications for antimicrobial coatings, biomedical devices, molecular diagnostics, photonic devices between others. The production of silver nanoparticles has been increasing worldwide in the nanotechnology industry due to the variety of applications mentioned and are very likely to reach aquatic ecosystems damaging them. Due to their small size and high surface area to volume ratio of NPs, they can strongly interact with life cells and cause damage to tested animals. Based on the mentioned previously, it is necessary to evaluate the silver nanoparticle nanotoxicity in aquatic ecosystems to prevent possible ingestion or transfer to humans. Also, the research will benefit aquatic systems due to less pollution around aquatic organisms. The objectives of this research included: i) production and characterization of stable silver nanoparticles in water, ii) characterizing the optical properties by UV-Vis spectroscopy, morphology by HR-TEM, crystalline structure by X-Ray Diffraction (XRD) and the nature of the surface by Infrared spectroscopy. Additionally, Electron Diffraction and Energy Dispersive X-Ray analyses were also evaluated for Ag NPs. iii) Optimize the synthesis of silver nanoparticles by changing the molar ratio of silver/citrate and, iv) evaluate the toxicity of silver nanoparticles in aquatic organisms, i.e Artemia salina. Results obtained evidenced that AgNPs showed absorption peaks in a range of 410 nm and 440 nm. These peaks are due to the phenomenon called surface plasmon resonance (SPR) that are responsible for a variety of phenomena, including nanoscale optical focusing, negative refraction, and surface-enhanced Raman scattering. Also, the concentration of nanoparticles was dependent of the reaction time. HR-TEM measurements evidenced the spherical form of the nanoparticles and its small size at around 12-14 nm and it was confirmed by XRD. In addition, Electron Diffraction analyses suggested the composition of the nanoparticle, which contained only Ag⁰. The toxicity assays were evaluated using different concentration of Ag NPs and a control test. The effect and cytotoxicity of these nanoparticles were studied in the nauplii state of Artemia salina. During the toxicity assay, it was demonstrated that the Artemia salina was able to uptake silver nanoparticles and stored it into the gastrointestinal tract.

The precise manipulation, localization, and assembly of biological and bioinspired molecules into organized structures have greatly promoted material science and bionanotechnology. Further technological innovation calls for new patternable soft materials with the long-sought qualities of environmental tolerance and functional flexibility. Here, we report a Patterned Amyloid Material (PAM) platform for producing hierarchically ordered structures that integrate these material attributes. This platform, combining soft lithography with generic amyloid monomer inks (consisting of genetically engineered biofilm proteins dissolved in hexafluoroisopropanol), along with methanol-assisted curing, enables the spatially controlled deposition and in situ reassembly of amyloid monomers. The resulting patterned structures exhibit spectacular chemical and thermal stability and mechanical robustness under harsh conditions. The PAMs can be programmed for a vast array of multi-level functionalities, including anchoring nanoparticles, enabling diverse fluorescent protein arrays, and serving as self-supporting porous sheets for cellular growth. This PAM platform will not only drive innovation in biomanufacturing but also broaden the applications of patterned soft architectures in optics, electronics, biocatalysis, analytical regents, cell engineering, medicine, and other areas.

Leishmaniasis has been classified as one of the most neglected tropical diseases, causing 50 thousand deaths and 1.5 to 2 million new cases every year, according to the World Health Organization. This disease, promoted by protozoan parasites of the genus Leishmania, has a high incidence affecting 89 countries worldwide. Nowadays, current treatment strategies still rely on the antifungal agent amphotericin B (AmB) but are rather inadequate due to the high prevalence of the disease within low-income population of sub-developed regions, the intracellular location of the parasite and the emergence of parasite resistance. Thus, other strategies have been pursued to improve the therapeutic efficacy and to reduce the toxicity of AmB such as the use of biocompatible polysaccharides as carriers. In this work, a simple and inexpensive production process using hyaluronic acid (HA, 50 kDa) was used in order to develop water-soluble hyaluronic acid-amphotericin B nanocomplex (HA-AmB). HA is the main ligand of CD44 receptor, thus being favorably internalized by macrophages that overexpress this receptor upon infection. Therefore, HA arises as a suitable polysaccharide to target the AmB delivery to the leishmania-infected macrophages.
The nanocomplex, obtained by simply processing the mixture of the polysaccharide with the drug in a nanospray dryer (HA-AmB SD), was characterized in terms of size/zeta potential (DLS) and morphology (SEM and Cryo-SEM). Furthermore, an HPLC-MS detection method was optimized and used to determine the AmB content in the nanocomplex. Also, to ascertain the interaction between AmB and the HA, FTIR, DSC and PXRD analysis were performed. Cytotoxic and hemolytic effects were assessed on different cell lines through the resazurin test and in dog’s blood, respectively. Anti-leishmanial activity was assessed in vitro in axenic cultures of Leishmania by resazurin and in infected bone marrow-derived macrophages (BMMΦ) stained with different fluorescent probes using high-content microscopy.
Our results shown that the produced material has a spherical morphology in aqueous solution with a mean hydrodynamic diameter of 318.4 ± 34.7 nm and low polydispersity (0.239 ± 0.02). Moreover, this material that presents an AmB content of 13.56 ± 3.49 %, has a good colloidal stability due to the highly negative surface charge (-39.45 ± 1.12 mV). DSC and PXRD analysis strongly suggested the formation of an amorphous inclusion complex between AmB and the complex polysaccharide chain networks, explaining the high solubility of the drug in water. The in vitro assays showed that compared to free-AmB, the nanocomplex had significantly less cytotoxicity against BMMΦ and HEK293T cell lines, significant less hemolytic effect and inhibited the infection in the Leishmania-infected BMMΦ. Exploratory in vivo assays are being conducted in mice. In conclusion, this work has shown that the hyaluronic acid-AmB nanocomplex is a promising system for the treatment of Leishmaniasis, possessing similar effects to the free-AmB against Leishmania-infected macrophages and Leishmania axenic cultures, with reduced cytotoxicity. Given the affordability, simplicity, low-toxicity and facile scale up of the developed formulation, the hyaluronic acid-AmB nanocomplex may represent an alternative to the expensive nanoformulations available.


The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for supporting this study under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ricardo Silva-Carvalho also acknowledges FCT for the PhD scholarship SFRH/BD/118880/2016.

RNA delivery into deep tissues with dense extracellular matrix (ECM) has been challenging. For example, cartilage is a major barrier for RNA and drug delivery due to its avascular structure, low cell density and strong negative surface charge. Cartilage ECM is comprised of collagens, proteoglycans, and various other noncollagneous proteins with a spacing of 20nm. Conventional nanoparticles are usually spherical with a diameter larger than 50-60nm (after cargo loading). Therefore, they presented limited success for RNA delivery into cartilage. Here, we developed Janus base nanotubes (JBNTs, self-assembled nanotubes inspired from DNA base pairs) to assemble with small RNAs to form nano-rod delivery vehicles (termed as “Nanopieces”). Nanopieces have a diameter of ~20nm (smallest delivery vehicles after cargo loading) and a length of ~100nm. They present a novel breakthrough in ECM penetration due to the reduced size and adjustable characteristics to encourage ECM and intracellular penetration.

JBNTs are comprised of self-assembled supramolecular structures which are further broken down into guanine and cytosine DNA base pairs. The hollow channels formed by these nanotubes are ideal for drug loading and the six-member rosette comprised of hydrogen bonding is essential for its low cytotoxicity profile. By controlled assembly between our nucleic acid cargos (in this case siRNA) with JBNT solution, long segmented Nanopieces are synthesized. They can be further separated via a regulated sonication process. Nanopiece with formulations were assembled and their material properties were studied using DLS and Zeta potential measurements. Their morphological characteristics and distributions were analyzed by TEM (transmission electron microscopy) imaging. The RNA delivery abilities of Nanopieces with different formulations were determined in vitro using human chondrocytes. Nanopiece binding and ECM penetration was also studied in vitro using fluorescence and confocal microscopy. Results of this study provide in-depth characterizations of these Nanopieces along with evaluating their abilities of RNA delivery. Our data also determines how Nanopieces bind with and penetrate into cell ECM to understand their delivery mechanism.

Stem cells, which are defined as a progenitor cell having differentiation potential and self- renewal property, have been used in clinical and biomedical application as a therapeutic method for various diseases; particularly, adipose-derived stem cells (ADSC) have shown promise for regenerating tissues and organs damaged by injury and diseases. However, since the non-human derived molecules can induce the immune response ex vivo, xenogeneic proteins should be eliminated from the clinical application of stem cells [1,2]. Although researchers attempt the development of non-serum media to replace the fetal bovine serum (FBS) containing media, the non-serum media has their limitations including the lower cell activity and adhesion rates.
In order to overcome those limitations, nano-materials coated tissue culture polystyrene (TCPS) plates, which are commercially available, are suggested as a novel lab-ware to improve cell adhesion on culture plate. However, we suggest a compatible alternative, the single-walled carbon nanotube (SWCNT), which is a seamless cylinder comprised of a graphene sheet with diameter of the order of a nanometer and has no toxicity when it is properly functionalized and treated to mammalian cells [3].
The aim of this study is 1) the fabrication and characterization of SWCNT film based scaffolds having an optimal property for ADSC culture in non-serum media and 2) the analysis and evaluation of ADSC adhesion mechanism on the SWCNT film based scaffolds in this culture condition.

References
[1] Mochizuki, M. et al. Stem Cell Research & Therapy 9:25 (2018).
[2] Naskou, M. C. et al. Stem Cell Research & Therapy 9:75 (2018).
[3] Francis, A. P. et al. Toxicology Industrial Health 34, 200-210 (2018).

Surface chemistry plays an important role in regulating cellular behavior, in vitro. Recent studies showed that functional groups on biomaterial surfaces regulate cellular adhesion, migration, proliferation and differentiation. Self-assembled molecules (SAMs) form organized structures and with these molecules, desired surface properties can be easily generated. In the present study, we aimed to prepare polydimethylsiloxane (PDMS) substrates in natural myocardium-like stiffness range and investigate the effect of their surface modifications with SAMs, having two functional end groups (-CH₃ and –NH₂) and different wettability properties, on cardiac differentiation of murine induced pluripotent stem cells (mIPS). PDMS (Sylgard 184) substrates were prepared with different ratios of silicone elastomer base and curing agent (10:1 – 70:1) and spin coated on glass slides. Young’s moduli of substrates were characterized with nanoindentation. Next, -OH were created on substrate surfaces by using oxygen plasma treatment for 1 min, followed by dipping into 1% concentrations of either 3-Aminopropoyl triethoxy silane (APTES) for -NH₂ end groups or Trimethoxy (octadecyl) silane for –CH₃ end groups. Characterizations of these modified substrates were done by water contact angle measurements (WCA) and X-Ray ray photoelectron spectroscopy analysis (XPS). Cell viability (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT) and Western Blot analysis were performed. According to nanoindentation results with different silicone elastomer base crosslinker concentrations of PDMS, the ratio of 50:1 was found to be in natural myocardium-like stiffness range with a Young’s modulus of 26.42 ± 6 kPa. In low resolution XPS survey spectra of all unmodified and modified PDMS, four characteristic peaks were found as O1s, C1s, Si2s and Si2p at 533, 286, 155 and 104 eV, respectively. -CH₃ functionalization of PDMS substrates lead to a remarkable increase in the carbon peak, due to high carbon content of the molecule, whereas N1s peak appeared after modifications with APTES at 401 eV. WCA analysis showed hydrophobic nature of native and –CH₃ modified PDMS (104.1±5 and 112.7±5, respectively) and hydrophilic properties of –NH₂ modified PDMS (61.9±4). MTT analysis of mIPS cells on these substrates showed no statistical difference between SAMs modified PDMS. Both modified substrates showed higher viability when compared to native PDMS (p<0.05). Western blot analysis results were given in Fig.1 on day 12 of differentiation. Similar to viability analysis, both functional end groups enhanced cardiac differentiation significantly, when compared to native PDMS. Cardiac marker Troponin-T expressions were higher in hydrophilic -NH₂ groups. Conventional SAMs modified PDMS substrates in myocardium-like stiffness range were confirmed to be nontoxic to mIPS cells and also these modifications enhanced cardiac differentiation of mIPS cells on PDMS substrates.

We fabricated an artificial oral mucosa using a fish scale-collagen scaffold for clinical applications of oral mucosa defects. Fish derived collagen is appropriate for human use due to zoonotic-free biomaterial. The intrinsic wavy papillary structure of in vivo oral connective tissues was patterned to the collagen scaffold by soft lithography. The soft lithography mold was fabricated from an initial mold fabricated via Si anisotropic/isotropic etching process with the error less than 5%. The histology of artificial oral mucosa with the fish scale-collagen patterned by soft lithography (150 µm in height and 300 µm in pitch) showed a differentiated stratified epithelial layer, similar to the native oral mucosa. Soft lithography can be applied to fabricate a negative molds to create the micropattern mimicking the intrinsic wavy papillary structure of oral mucosa, suggesting a useful technique for developing the proposed biomimetic artificial oral mucosa.

As a remedy of oral mucosa defects, transplantation of the oral mucosa substitutes has been clinically applied. Although collagen scaffolds derived from animals have been proposed, the use of animal derived-collagen may cause infectious diseases by foreign contaminants. Instead, attention has been paid on fish scale-collagen because there is no risk of disease transmission from fish to human ^[1]. In addition, previous studies on a skin epithelial regeneration revealed that undulating microstructure of the scaffold enables to create the specific microenvironment for epithelial cells. ^[2] However, microstructuring of the fish scale-collagen has not been reported. In this study, we applied soft lithography to fabricate an artificial oral mucosa using a fish scale-collagen mimicking the topographical structure between oral mucosa epithelium and the underlying the connective tissue. Our proposed artificial oral mucosa with the micropatterned fish scale-collagen facilitates the regeneration of oral mucosa epithelium in vitro.

The microstructure was designed to obtain the wavy papilla structure of oral connective tissues. ^[3] A polydimethylsiloxane (PDMS) was used as a material for soft lithography mold. First, a Si initial mold was prepared via anisotropic deep-reactive ion etching with a photoresist mask. Subsequently, Si isotropic wet etching was performed to fabricate the wavy structure. The PDMS was molded after applying a release agent on the initial mold by dip coating. Second, a solution of fish scale-collagen was poured and structured by soft lithography with the PDMS mold. Finally, the artificial oral mucosa was constructed by culturing oral keratinocytes on the micropatterned fish scale-collagen tissue.

Results of scanning electron microscope observation showed that the initial mold was successfully patterned with the error less than 5% from the designed value. This result indicates the anisotropic/isotropic etching process enables to fabricate the undulating microstructure similar to oral connective tissue. Furthermore, it was confirmed the PDMS mold was replicated from the initial mold with the error less than 5%. Moreover, our histological examination of the artificial oral tissue revealed the formation of the stratified epithelial cell layer on top of the micropatterned collagen scaffold. The papillary structure with 150 µm in height and 300 µm in pitch was successfully fabricated. The histology of artificial oral mucosa was similar to an in vivo oral mucosa tissue. Consequently, soft lithography can be a useful technique for developing the proposed biomimetic artificial oral mucosa to create micropatterns mimicking the intrinsic wavy papillary structure of oral mucosa.


[1] Zoonoses and communicable diseases common to man and animals, 3rd Edition, Scientific and Technical Publication; 2003.
[2]Priyalakshmi Viswanathan, et al., Integr. Biol., 2016, 8, pp.21-29.
[3] Michiko Terada, et al, Journal of Biomedical Materials Research, 2012, 7, pp.1792-1802.

The developing field of tissue regeneration depends on the smart design of biomaterial scaffolds that mimic the surface and topological features of the extracellular matrix and as well as support the formation of new viable tissue. However, there are an excessive variety of scaffolds and some of them improve biochemical properties but lack from mechanical strength. Nowadays, fibrous scaffolds have demonstrated excellent potential due they mimic the in vivo physiology, where cells grow and interact according to the spatial and mechanical conditions provided. Here, we prepared a scaffold that incorporated bioglass nanoparticles into the polymeric matrix of electrospun nanofibers. Bioglass nanoparticles were loaded and studied at different concentrations, to induce osteogenic differentiation of human fetal osteoblastic cells (hFOB). Further, we produced a bio-adhesive scaffold, based on mussel-inspired polydopamine (PDA) coating of bioglass-loaded nanofibers. This scaffold not only enhances cell adhesion but also improve its mechanical strength. The chemical composition and morphology of these hybrid composite scaffolds were characterized using Fourier-Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM), respectively. SEM micrographs showed highly interconnected pores, a suitable characteristic for cell penetration and showed the PDA coating transition as a function of time. After the fabrication, mechanical tests were performed, and the properties of the scaffolds were evaluated. Immunostaining assays were conducted to study cell adhesion and differentiation of hFOB cells. Our results indicate that our bio-adhesive and bioactive composite could support tissue growth and is proposed as a scaffold for bone tissue regeneration.

Tryptophan and tyrosine, aromatic peptides are widely known for their redox properties and roles in neurotransmitter synthesis. Dityrosine cross-links are present in several proteins and these bonds play a crucial role in stabilization of proteins, also redox-active properties of tyrosine and tryptophan facilitate charge hopping to allow long-distance electron transportation in proteins. Self-assembly is the organization of molecules into ordered structures to form various structures depending on different conditions. Self-assembling peptide-based nanostructures are attractive due to their chemical versatility, biological recognition abilities, tunable mechanical strength and biodegradability. In this study, we have synthesized nanotubes composed of tryptophan-tyrosine and dityrosine (YY) peptides, using solution phase self-assembly and plasma enhanced chemical vapor deposition. These were characterized using various chemical, mechanical, morphological, and thermal characterization techniques such as FTIR, RAMAN, UV-VIS spectroscopy, and Liquid chromatography mass spectroscopy, nuclear magnetic resonance, powder X-ray diffraction and circular dichroism, and nanoindentation, and scanning electron microscopy (SEM), and transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and thermogravimetric analysis, respectively. In addition, Self-assembly of aromatic dipeptides involves complex non-covalent interactions and understanding these forces allows manipulation of material properties at a molecular scale. A cost-effective way of computational studies and simulations can be employed to elucidate the forces involved. Quantum chemical computational methods were used to study the lowest possible conformations and energy decomposition of the tyrosine based self-assembled nanostructures.
There is an increasing need for biocompatible neuronal cell proliferation and regeneration scaffolds as regeneration of the neurite structures is slow and complex, which results in permanent nerve cell damage. Our hypothesis was by facilitating a scaffold made of monomer units that were biocompatible and contributed to upregulation of neuro-transmitters such as dopamine we could help in neuronal regeneration. After the analysis of physicochemical properties of these peptide nanostructures, we studied the biological interactions and influence these scaffolds had on rat adrenal pheochromocytoma cells (PC-12), human bone marrow neuroblasts and neural progenitor cells of Parkinson’s disease model. To test the same, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity studies, morphological cellular interaction imaging through SEM and confocal imaging, dopamine-enzyme linked immunosorbent assay, immunostaining for neuronal marker, and real-time polymerase chain reaction (q-PCR) for gene expression were carried out after the growth of above-mentioned neuronal cells on the synthesized peptide bioscaffolds.

The human skin prevents our bodies against an entry of various microbes. Once the skin is damaged, its infection can occur via anaerobic and aerobic bacteria. The necessary skin wound dressing must fulfil some properties, such as to create a sufficiently moist environment, to prevent further infection, absorb the wound fluids and exudates, to decrease the wound skin necrosis and to prevent the skin against external harsh conditions. Bandages should be elastic, non-antigenic and biocompatible materials. To further increase their healing functions, they are loaded with antimicrobial, antibacterial and anti-inflammatory agents (1, 2). However, these compounds are also present in some biopolymers, such as chitosan and oxycellulose, which are used in the form of nanofibers for wound covers (2, 3).
Biopolymeric nanofibers possess in general a high porosity, large specific surface area, variable pore size, excellent breathability and also biocompatibility (1). The most common technique to produce nanofibers so far has been electrospinning (4). This technique enables synthesis of various nanofibers with diameters in the range of dozens to hundreds of nm. However, it possesses several drawbacks, such low production rate, sensitivity on temperature and humidity, incomplete utilization of spun solutions. In addition, electrospun fibers often have residual electrostatic charge, which renders their handling difficult. More recent spinning method, known as centrifugal spinning (5), uses only centrifugal force, which eliminates drawbacks of electrospinning. The centrifugal spinning also broadens the pool of fibers to allow spinning of even non-conductive materials (polymers).
In this work, deacetylated natural polysaccharide Gum Karaya (GK) was used as the main material for the centrifugal spinning. This biocompatible and biodegradable polysaccharide can be prepared into 3D foams of fibrous materials with great prospects in regenerative medicine (6). Fiber blends of Gum Karaya with other biodegradable polymers, such as poly(ethylene oxide) (PEO) and poly(vinyl alcohol) (PVA), were also investigated.
Obtained fibers from aqueous blended from GK/PVA and GK/PEO were characterized by various means (Fourier-transformed infrared spectroscopy, scanning electron microscopy and swelling behaviour). Most importantly, they were characterized for biomedical effect using antibacterial testing and commercial skin analyzer. We show that the newly prepared fibers represent very promising material for the skin wound dressing.
The work was supported by Technology Agency of the Czech Republic (project TJ02000329), Ministry of Education, Youth and Sports of the Czech Republic (project LQ1601).

REFERENCE
1. Zahedi P., Rezaien I., Ranaei-Siadat S.-O., Jafari S.-H., Supaphol P. A review on wound dressing with an emphasis on electrospun nanofibrous polymeric bandages. Polymers Advanced Technologies 2009, 21: 77-95.
2. Fouda M. M. G, Wittke R., Knittle D. and Schollmeyer E. Use of chitosan/polyamine biopolymers based cotton as a model system to prepare antimicrobial wound dressing International Journal of Diabetes Mellitus 2009, 1: 61-64.
3. Svachova V., Vojtova L., Pavlinak D., Vojtek L., Sedlakova V., Hyrsl P., Alberti M., Jaros J., Hampl A., Jancar J. Novel electrospun gelatin/oxycellulose nanofibers as a suitable platform for lung disease modelling. Materials Science Engineering C 2016, 67: 493-501.
4. Wendorff J. H., Agarwal S., Greiner A.: Electrospinning: Materials, Processing, and Applications, Wiley-VCH Verlag GmbH & Co., Germany 2012.
5. Hromadko L., Koudelkova E., Bulanek R., Macak J. M. SiO₂ Fibers by Centrifugal Spinning with Excellent Textural Properties and Water Adsorption Performance. ACS Omega 2017, 2: 5052-5059.
6. Padil V. T. V., Senan C., Waclavek S. and Cernik M. Electrospun fibers based on Arabica, karaya and kondagogu gums. International Jounral of Biological macromolecules 2016, 91: 299-309.

While engineered nanomaterials (ENMs) have emerged as important components of various consumer products, human exposure to ENMs has been associated with negative health outcomes, such as cardiovascular diseases. To better understand and quantify the pathophysiological effects of exposure to different ENMs, there is a pressing need to develop in vitro models that faithfully recapitulate the native form and function of cells and tissues. In particular, two platforms developed for screening ENM effects in the cardiovascular space will be presented: 1) micropatterned cell pairs as a minimalistic model of the endothelial barrier; and 2) fiber-based cardiac microphysiological devices for contractile stress measurements. These models allow for a multi-scale assessment of the effects of nano-bio interactions at different stages of ENM biodistribution—from the translocation of ENMs across barrier tissues such as the endothelium, to their delivery towards target tissues such as the myocardium. Geometrically-controlled endothelial cell pair microtissues were produced via protein micropatterning, which allows for a systematic assessment of ENM-induced changes in multiple cellular-level parameters related to vascular barrier integrity. These measurements included changes in cellular morphology, junction protein expression, intercellular gap formation and cytoskeletal network organization upon ENM exposure. On the other hand, our “chip-based” platform can be used for measuring changes in tissue-level cardiac function, such as contractile stress and beat rate, during ENM exposure. Aligned polydopamine (PDA)/polycaprolactone (PCL) nanofibers were used as a tissue scaffold for this device in order to mimic the 3D architecture of cardiac microenvironments under physiological conditions. An instrumented version of this platform with embedded strain sensors will also be presented, which provides a way to continuously and non-invasively monitor the effects of ENM on cardiac tissue contractility at different time points. Together, these next generation in vitro and analytical testing platforms provide physiologically relevant 2D- and 3D-models of ENM exposure routes towards a more comprehensive evaluation of microvascular and cardiac response profiles to different nanomaterials.

Engineered nanomaterials (ENMs) are being increasingly used in a variety of products due to their unique physicochemical characteristics. However, the potential hazards that ENMs pose to human health are still not fully understood. These materials are delivered to target tissues by following several biodistribution routes across multiple biological tissue barriers, with different microenvironments that vary from one another. Of particular interest is their impact on the vascular endothelium, which is an important selective barrier that regulates the exchange of materials between the blood and tissues throughout the body. Current studies of ENM toxicity towards the endothelium are conducted using endothelial cell monolayers, which are low throughput and not particularly amenable to quantitative image analysis of changes in local structural features upon ENM exposure. To address this issue, we have used a micropatterning technique to isolate endothelial cell pairs as a reductionist model of the vascular barrier. Cell pairs, or two cells with a shared junction, serve as the basic functional repeating unit of a continuous biological tissue such as the vascular endothelium. The micropatterned cell pair in vitro model enables both higher throughput ENM toxicity studies, as well as the measurement of a series of quantitative parameters, such as changes in cellular structure, junction protein expression, and cytoskeletal network reorganization (e.g., transition from predominantly cortical actin to 24 stress fiber formation), which are all relevant to barrier function. We used the cell pair assay to evaluate the dose-dependent changes in induced by a library of ENMs (Au, Ag, TiO₂, ZnO, CuO, Fe₂O₃, SiO₂, Al₂O₃, and 2 nanocellulose polymorphs) from 10 to 100 μg/mL exposure dosage. The summative ENM-induced changes in these endothelial structural features were then assessed for their correspondence to effects on cellular viability and tissue-level barrier function. We found that endothelial exposure to some of these materials, such as Ag and TiO₂, induce changes with negative implications on barrier function, including increased formation of actin stress fibers, reduced junction protein expression, and increased intercellular gap formation. Here, a similarity index scoring method is also implemented to quantitatively compare the extent by which a cell pair deviates from the unexposed “healthy” phenotype after an acute 24-hour ENM exposure. Collectively, these results demonstrate that our cell pair assay represents a viable new method for standardizing ENM endothelial toxicity screening.

Paper fluidics is based on patterning hydrophilic paper with channels bounded by hydrophobic barriers. Fluids move along channels by capillarity. Several methods are available for patterning paper, with different costs/resolutions. Paper patterning for microfluidics also used the embossing technique to design open-channel microfluidic devices, fabricated by compressing the sheet of paper with the help of 3D plastic printing moulds.
These approaches are adopted in this work to develop a multiwell-microplate paper-based microfluidic, aiming the creation of organs-on-chip, combining complexity and miniaturization. Bacterial Cellulose (BC) represents a source of highly pure and biocompatible cellulose, with huge technological potential in many fields - biomedical, composites, textiles, food and cosmetics textiles - but currently still rather underexploited [Gama et al, 2916; Klemm et al, 2018]. This work describes a novel approach towards the development of a nanostructured and multifunctional cellulose-based device for the continuous culture of animal cells and tissues. A multilayered system of modified BC (hydrophobized and electroconductive) was used to assemble the skin-on-chip, a microfluidic platform, using the lab-on-paper technology intended to mimic vascularization, with controlled flow, to introduce external stimuli, such as electrical or mechanical, and to support multicellular growth.
This chip serves a multifactorial purpose, aiming the control of each part that make up the overall complex 3D system, including dynamic control of physical, chemical and gaseous gradients, ensure mimetic vascularization, introduce favourable stimuli and co-culture of skin cells. This model sustains cell growth and allow real time and in a high throughput manner to assess cellular phenomena, such as cell-cell crosstalk, paracrine factor exchange, ECM production, as well as tissue homeostasis in the presence of chemical, mechanical, electrical and biological stimuli, and also kinetics of substance delivery on/through the skin.
BC hydrophobization was achieved using a new strategy for the surface modification of BC through the combination of oxygen plasma deposition and silanization with trichloromethyl silane. The combined use of the two techniques modifies both the surface roughness and energy and therefore maximizes the hydrophobic effect obtained. These modified membranes were characterized by SEM, water contact angle measurements, FTIR-ATR and XPS, and its cytotoxic potential was investigated using both indirect and direct contact studies with cells. Importantly, this surface modification revealed no short-term cytotoxic effects on L929 and hDNFs cells. This material was used for the construction of a BC-based well plate for cell culture, which can be supplied continuously with culture medium in long term studies (ranging from days to weeks), using a two-layered 3D full-thickness skin equivalent consisting of an epidermal and a dermal tissue layer, cultivated in alginate scaffolds, that can be maintained and studied as a skin surrogate on the SkinChip [Maia et al, 2014].

The authors thank funding through the project “SkinShip - Dispositivo de microfluídica inovador baseado em celulose capaz de suportar a modelação 3D de pele”, with reference PTDC/BBB-BIO/1889/2014 supported by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Programa Operacional Competitividade e Internacionalização - COMPETE 2020, do Programa Operacional Regional de Lisboa e por Fundos Nacionais através da FCT - Fundação para a Ciência.

REFERENCES
Gama et al, Bacterial Nanocellulose: From Biotechnology to Bio-Economy, Elsevier, 2016. ISBN: 9780444634580
Klemm et al (2018) Nanocellulose as a natural source for groundbreaking applications in materials science: Today’s state, Materials Today, 21(7), 720-748
Maia et al (2014) Matrix-driven formation of mesenchymal stem cell-extracellular matrix microtissues on soft alginate hydrogels. Acta Biomater.10(7):3197-208.

Understanding the triggers of stem cell differentiation furthers the field of tissue engineering and contributes to the applications for implantation and bioprinting. Substrate mechanics and topography effects have been widely studied as factors to trigger stem cell differentiation. In our previous studies, we have shown that monodispersed polybutadiene (PB) is able to form biocompatible thin film with adjustable substrate mechanics depending on film thickness, to which the cells can adhere without additional coatings. A threshold was determined at 2.3 MPa when large amounts of biomineralized deposits were observed after 28 days on substrates with mechanics higher than this value. [1] In this study, we explored the understanding and analyzing how dental pulp stem cells (DPSC) respond to both surface moduli and topography effects in nanoscale. In order to achieve this nanopattern, a mixture of Polybutadiene (PB) and Polystyrene (PS) was used in a 3 to 1 ratio for spin cast. Due to the repulsion between two polymers, phase separated nanopattern was created with mechanics and topography differences on different domains, where PB formed flat and soft film with rounded domain of hard PS forming spikes of ~ 100 nm height. DPSC were cultured onto this substrate and were monitored over 28 days. Through the analysis of the images from the confocal microscopy and SEM at first week, DPSC grew slower on pattern substrate compared to the cells cultured on the pure PB substrate. Furthermore, DPSC appeared to be skinny and elongated, seeming to avoid attaching the spikes of PS on pattern substrates. It suggests that DPSC preferred to attach on PB rather than on PS. The cell moduli were also tested at first week through Atomic Force Microscopy with SMFM mode, where we found three different groups of cell moduli instead of two different moduli for the substrates, indicating that DPSC behaved more complicated in response to external triggers. On day 28, biomineralization were observed by SEM and Raman microscopy, where we found fibers templated mineralized deposits on the substrates. RT-PCR was also performed to determine differentiation pathway with selected markers: Alkaline Phosphatase(ALP), Runt-related transcription factor(Runx), and late markers, Dentin Sialophosphoprotein(DSPP) and Osteocalcin(OCN).
This research explored the combination of substrate mechanics and topography effects for DPSC differentiation, which is important for applying printed scaffolds as dentin/tooth regenerative biomaterials, which surface is rough and the mechanics is not homogeneous.

[1] V. Jurukovski, M. Rafailovich, M. Simon, A. Bherwani, C.-C. Chan, Citation: Entangled Polymer Surface Confinement, an Alternative Method to Control Stem Cell Differentiation in the Absence of Chemical Mediators. Annals of Materials Science & Engineering, 2014.

The extracellular matrix (ECM) environment of tissues permits 3D cell growth in a complex fibrous protein architecture with delicate mechanical properties. Existing biomaterial fabrication techniques struggle to simultaneously attain: micro/nano-scale fibril feature resolution, low fibre stiffness and the 3D organisation crucially provided by the ECM without comprising cell motility. This work utilises 3D printing and low voltage electrospinning patterning synergistically to address these conflicting engineering challenges and act as a minimalist guide for self-directed 3D cell growth. Low voltage electrospinning patterning was adapted as a sequential process on a modified 3D printer. Applied voltage and 3D printed geometry can modulate the suspended behaviour of hydrogel fibres that span between 3D printed support pillars, a parametric study characterised threshold conditions and established a predictive model for patterning suspended fibres. Applications in 2D fibre patterning and 3D cell culture on suspended fibres were explored, including the creation of in vitro glomerulus memberane, and fibre guidance of glioblastoma cell aggregate outgrowth.

In the field of tissue engineering, design and fabrication of precisely patterned, highly porous scaffolds/matrixes are required to guide overall shape of tissue growth and replacement. Although Rapid Prototyping fabrication techniques have been used to fabricate the scaffolds with desired design characteristics, controlling the interior architecture of the scaffolds has been a challenge due to CAD constrains. Moreover, large thick tissue scaffolds have reported limited success primarily due to the inability of cells to survive deep within the scaffold. Without access to adequate nutrients, cells placed deep within the tissue construct die out, leading to non-uniform tissue regeneration. This study aims to overcome these design and fabrication limitations. In this work, research has been expanded to design of scaffolds which have inbuilt micro scale fluidic networks. In this procedure, inbuilt channels serve as material delivery paths to provide oxygen and nutrients for the cells. First of all, negative of a cylindrical shape with a single channel was designed with AutoDesk Inventor and printed with a 3D printer to be used as a mold. Then, 3D printed mold was filled out with Poly(ethylene glycol) diacrylate (PEGDA) which is a photo-curable solution to fabricate the cylindrical hydrogel. Once PEGDA was exposed to UV light with the wavelength of 365nm, polymerization completed in about 3 minutes. After that, the same procedure was repeated for cylinders with two and three channels respectively. Then, their mechanical characterization tests were done to compare the compressive strengths of the scaffolds that has different internal architectures. Our preliminary results indicate that, 3D printing and polymerization techniques can be used together to control the interior architectures as well as the compressive strengths of scaffolds.

Severe wounds result in the formation of stiff and fibrotic scars. During embryogenesis, fetal wounds have however the capacity to restore skin tissues to their native scarless configuration. Although the underlying mechanisms of this process are still incompletely understood, several spatiotemporal differences have been observed in fetal and postnatal wounds that provide unique insight for regenerative scaffolds design. Biomaterials that attempt to recapitulate the biophysical and biochemical properties of fetal skin have accordingly emerged as promising pro-regenerative strategies. The extracellular matrix (ECM) protein fibronectin (Fn) in particular is believed to play a crucial role in directing this regenerative phenotype. Accordingly, Fn has been implicated in numerous wound healing studies, yet remains untested in its fibrillar conformation as found in fetal skin. Here, we show that high extensional and shear strain rates in a nanofiber manufacturing system, termed rotary jet spinning (RJS), can drive high throughput Fn fibrillogenesis, thus producing fibrous scaffolds that are used to promote wound healing. Förster resonance energy transfer first confirmed the fibrillar conformation of Fn attained using RJS. Next, when tested on a full-thickness wound mouse model, Fn nanofiber dressings not only accelerated wound closure, but also improved tissue restoration, recovering dermal and epidermal structures and promoting regeneration of skin appendages and adipose tissue. Together, these results suggest that bioprotein nanofiber fabrication via RJS could set a new paradigm for enhancing wound healing and may thus find use in a variety of regenerative medicine applications.

Grant acknowledgement: The authors thanks the Wyss Institute of Biologically Inspired Engineering, John A. Paulson School of Engineering and Applied Sciences, Harvard MRSEC (NSF award number DMR-1420570), SEAS Scientific Instrument Shop and Harvard Center for Nanoscale Systems (NNIN member, NSF award number 1541959), the Harvard Medical School Department of Neurobiology Imaging Facility supported in part by the Neural Imaging Center (NINDS P30 Core Center grant #NS072030) and the Rodent Histopathology Core at Dana-Farber/Harvard Cancer Center (NIH award number NIH 5 P30 CA06516).

Bioprocessing applications that derive meat products from animal cell cultures require food-safe culture substrates that support volumetric expansion and maturation of adherent muscle cells. This is important, because meat consists of muscle, fat, and connective tissues proportioned according to tissue source, each containing a diverse array of nutrients produced by constituent cells. Cell types composing meat can be cultured in vitro but production scale-up is limited by the anchorage dependence of these cells, which require attachment to culture substrates for survival, proliferation, and maturation. This requirement is especially stringent for muscle maturation, where alignment of densely packed muscle fibers is observed. For this reason, controlling cell phenotypes in volumetric cultures is a key challenge for adherent cell bioprocessing, including emerging strategies for meat production. Here we demonstrate scalable production of microfibrous gelatin that supports cultured adherent muscle cells derived from cow and rabbit. As gelatin is a natural component of meat, resulting from collagen denaturation during processing and cooking, our extruded gelatin microfibers recapitulated structural and biochemical features of natural muscle tissues. Using immersion rotary jet spinning, a dry-jet wet-spinning process, we produced gelatin fibers at high rates and, depending on process conditions, we tuned fiber diameters to values comparable to natural collagen fibers. To inhibit fiber degradation during cell culture, we crosslinked them either chemically or by co-spinning gelatin with a microbial crosslinking enzyme. To produce meat analogs, we cultured bovine aortic smooth muscle cells and rabbit skeletal muscle myoblasts in gelatin fiber scaffolds, then used immunohistochemical staining to verify that both cell types attached to gelatin fibers and proliferated in scaffold volumes. Short-length gelatin fibers promoted cell aggregation, whereas long fibers promoted aligned muscle tissue formation. Histology, scanning electron microscopy, and mechanical testing demonstrated that cultured muscle lacked the mature contractile architecture observed in natural muscle but recapitulated some of the structural and mechanical features measured in meat products. Our results demonstrated that gelatin fibers provide a suitable scaffold to study muscle cell aggregation or formation of 3D aligned tissues. The general nature of cell adhesion to gelatin, and its recognition as a safe edible material, suggest these scaffolds can support a variety of adherent cell types with utility for food bioprocessing. With further research and development, we believe that muscle bioprocessing and tissue engineering will play increasingly important roles in food science.

Grant acknowledgments: This work was sponsored by the John A. Paulson School of Engineering and Applied Sciences at Harvard University, the Wyss Institute for Biologically Inspired Engineering at Harvard University, Harvard Materials Research Science and Engineering Center grant DMR-1420570, and the TomKat Foundation.

Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Inspired by the fibrous extra-cellular matrix of myocardial heart muscle, we produced ellipsoidal 3D ventricle chambers based on blended polycaprolactone (PCL):gelatin fibers with average fiber diameter ~500 nm and circumferential fiber alignment achieved by collecting fibers on a rotating mandrel. Ventricle scaffold porosity enabled cardiomyocyte infiltration and fiber alignment controlled cardiomyocyte shape and tissue alignment. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles had a diastolic chamber volume of 0.5 mL (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle). We measured tissue coverage and alignment, calcium-transient propagation, and pressure-volume loops in the presence or absence of test compounds. Proof-of-concept structural arrhythmia disease modelling demonstrated that stable, pinned spiral waves could be generated by inflicting geometrically controlled injuries. Moreover, we describe a bioreactor for modular assembly of tissue engineered ventricles with optional valves and ventricular assist, providing a path towards inline automated experiments. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings, suggesting future use in pre-clinical cardiology where patient-derived models are preferred to animal models.


Grant acknowledgments: This work was sponsored by the John A. Paulson School of Engineering and Applied Sciences at Harvard University, the Wyss Institute for Biologically Inspired Engineering at Harvard University, Harvard Materials Research Science and Engineering Center grant DMR-1420570, Defense Threat Reduction Agency (DTRA) subcontract #312659 from Los Alamos National Laboratory under a prime DTRA contract no. DE-AC52-06NA25396, and the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers UH3TR000522 and 1-UG3-HL-141798-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was supported in part by the US Army Research Laboratory and the US Army Research Office under Contract No. W911NF-12-2-0036. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office, Army Research Laboratory, or the US government. The US government is authorized to reproduce and distribute reprints for government purposes notwithstanding any copyright notation hereon. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. 1541959. CNS is part of Harvard University.

Through self-assembly of multiple DNA strands with rationally programmed sequences, DNA nanotechnology enabled us to construct various two- or three-dimensional structures at nanoscale with intricate structural shapes such as bending and twist [1, 2]. These structural features have proved their utility in broad fields including optical metamaterials [3], liposome synthesis [4], drug delivery [5], and synthesis of chiral colloidal liquid crystal [6]. The structural features could be achieved by applying engineered mechanical perturbations induced by geometrical mismatches neighboring helices at inter-helix junctions [2]. Contrary to well-developed techniques for curvature control, however, the resolution of twist rate still remains unsatisfactory lagging the development of optimized twisted structures for target functionality.
Here, we present a mechanical perturbation programming approach for fine control of twisted DNA origami nanostructures [7]. To this end, first, we program mechanical perturbation distributions through just determining the location of inserted or deleted base-pairs. This method can make different twist rates without change in the number of insertion or deletion used. For demonstration, a fine adjustment of twist angle in six-helix bundle was achieved with mean increment of 1.77 degree per a 21-base-pair-long unit block. Also, we found that different twist mechanism occurred depending on engineered mechanical perturbation through molecular dynamic simulations. Second, we locally relaxed the strain energy due to mechanical perturbations by introducing short unpaired nucleotides, which can broaden the range of achievable twist rate varying their length or density.
Our approach is expected to contribute to expanding design space of DNA nanostructures with high structural accuracy and accelerating utilization of various benefits derived from structural twist into the DNA nanostructures. Employing our method, for example, one can design plasmonic nanostructures with tailored optical response or macroscopic soft materials with controlled structural and physical properties and resolve unintended distortions of hierarchically assembled DNA nanostructures.

Acknowledgement
This work was supported by the National Research Foundation of Korea (NRF) grand funded by the Korea government (MSIP) (NRF-2019R1A2C4069541 and NRF-2017M3D1A1039422).

References
1. P. W. K. Rothemund, Nature 440, 297 (2006)
2. H. Dietz, et al., Science 325, 725 (2009).
3. MJ. Urban, et al., Journal of the American Chemical Society 138, 5495 (2016)
4. Y. Yang, et al., Nature Chemistry 8, 476 (2016)
5. Y-X. Xzao, et al., ACS Nano 6, 8684 (2012)
6. M. Siavashpouri, et al., Nature Materials 16, 849 (2017)
7. Y-J. Kim, C. Lee, J. G. Lee and D-N. Kim, ACS Nano, published online (2019)

Predictively designing peptides for executing materials science and technology relevant functionalities offers great potential to custom design materials systems with desired physicochemical properties at ambient conditions. While the functionality displayed by the biomolecules, such as peptides and proteins, are dependent on their conformation, traditional bioinformatics tools have largely used sequences of letters and implicit assumption of their physicochemical and structural features. Although heuristically interpretable, upon utilizing letter-based encodings in machine learning algorithms for predictively designing functional sequences for engineering applications, implicit physicochemical and structural features are unseen by the data-driven machine agents. Furthermore, transferring learned models from sequence-based molecules to non-sequence based conjugated biomolecules such as peptidolipids, peptidoglycans, peptide-nucleic acids, and even simple sugars and small molecules becomes impossible. Here we explore and demonstrate structure-based encodings inspired from valence shell electron pair repulsion structures (VSEPR), functional groups, and signal-processing to train machine learning algorithms that learn latent features which are transferable to other biomolecules of interest. Our results demonstrate clear progress over state-of-the-art similarity matrix approaches, as well as convolutional neural network approaches which are based on strings of letters. Fingerprints developed herein are peptide length invariant and are generalizable to heterogeneous molecules. We demonstrate the capabilities of VSEPRnet in predicting peptide-epitopes as well as peptidolipids binding to major histocompatibility proteins as well as solid binding peptides with binding and surface assembly characteristics. Such models will be applicable in predictively designing drugs, antimicrobial coatings, chimeric constructs and for sensing applications in disease diagnostics. The research is supported by NSF-DMREF program through the grant DMR-1629071 as part of the Materials Genome Initiative.

Structural DNA nanotechnology is the only synthetic materials paradigm that offers the ability to position molecules in nearly arbitrary 3D spatial patterns on the 1 to 100 nanometer scale. While the basic rules for this synthetic materials paradigm have largely been in place for over a decade, only now are computational and synthetic procedures reaching a tipping point at which broad adoption of this materials paradigm by the biological and materials science communities is enabled. For this reason, the next decade should witness an increasing number of researchers engaged in the application of this technology to explore its unique potential in diverse scientific domains. Here, I will begin by presenting advanced computational procedures that now offer the fully automated, top-down geometric design of nearly arbitrary 2D and 3D nanoscale assemblies using DNA. I will also review single-stranded DNA synthesis strategies needed to produce the raw material needed for the fabrication of programmed DNA assemblies, including bacterial, enzymatic, and solid-state approaches. I will then present the application of structural DNA nanotechnology to control the spatial organization of HIV antigens that reveals fundamental aspects of immune cell activation, with potential implications for novel vaccine development.

DNA nanotechnology allows to create programmable and precisely controlled molecular and nanoscale architectures due to the specific Watson-Crick base-pairing and molecular plasticity. In particular, the superior control over DNA origami structures has opened new venues for biomedical applications such as biosensing and drug delivery. However, protecting the integrity of DNA origami structures in complex biological fluids has remained a major challenge. Here, we demonstrate that hybrid peptoids can decorate 3D DNA origamis and provide an effective protection under different ionic and bio-active conditions. We have investigated two types of hybrid peptoid architectures, “brush” and “block” that were built from positively-charged moieties and oligo-ethylene glycol. We find that the brush-like peptoid design exhibits the best protection of DNA origamis against the damaging factors. Our detailed simulation study reveals the crucial role of electrostatic interaction between peptoids and the DNA backbone for the observed protection effect. Moreover, our systematic study shows that the interactions depend on the peptoid sequence, and that the oligo-ethylene glycol motifs contribute to the rigidity of the duplex DNA. Thus, the architecture of these hybrid peptoids can be optimized for desirable “coating” of DNA origamis. We have applied the developed strategy for creating peptoid-coated origamis that reduce a release of anti-cancer drug, doxorubicin and slow down trypsin digestion of proteins encapsulated in the nanostructure. As a proof of concept, we further show that alkyne-modified peptoids can be conjugated with fluorophores and antibodies. This new approach offers a functional and physiologically stable DNA origami-based for targeted biomedical applications.

Keywords: DNA nanostructure, DNA origami, peptoid, electrostatic interaction, molecular protection, molecular coating, drug release

The ultimate goal of nanotechnology is to build structures on the 10-100 nm length scale, with control of matter down to the atomic level. In recent years, DNA has emerged as a powerful molecular building block for the construction of nanostructure materials due to the specificity of Watson-Crick pairing. However, DNA nanostructures are limited by the physical and chemical properties of oligonucleotides, which can be a hindrance for biological applications, or for attaining higher resolution placement of synthetic functionality. Proteins and peptides, by contrast, are the natural “language” of biology, possess a greater diversity of chemical groups, and can position materials sub-nanometer precision. In this work, we present the novel integration of two self-assembling polypeptide motifs with DNA nanostructures: 1) the coiled-coil, and 2) hetero-trimeric proteins. We have synthesized peptide- and protein-DNA conjugates with high site-specificity, allowing us to integrate these components with self-assembled DNA structures with high precision. DNA tiles, wireframe cages, and DNA origami were modified in specific locations with one of two heterodimeric coiled coil peptides that bind with sub-nanomolar affinity. The coiled coil interaction drove the hierarchical assembly of these components into dimers and one-dimensional nanofibers, providing an orthogonal assembly “code” to DNA hybridization. In addition, we will demonstrate the assembly of hierarchically-ordered 3D cages comprised of trimeric protein vertices with tunable DNA arms linking them. These materials are the first to our knowledge to integrate synthetic, self-assembling peptide/protein-DNA hybrids with well-established DNA motifs, and demonstrate the potential of protein-DNA nanotechnology.

Control over the assembly structure and energetics of biomolecules is essential for the fabrication of micron-scale, higher order, functional systems, as required for a variety of chemical and biosensing applications. Promising candidates are solid-binding peptides selected for specific inorganic solids by directed evolution techniques. Specifically, these dodecapeptides have been shown to spontaneously form long-range ordered assemblies on two-dimensional solid surfaces, such as graphene. The conformation of the solid-binding peptide is dynamic in solution and depends on the solution conditions, such as pH, temperature, and salt concentration. As self-assembly of biomolecules relies heavily on the molecular conformation of the monomer, we show that it is possible to direct the formation of the equilibrium assembly structure by controlling the labile nature of the peptides in solution via environmental preconditioning. This environmental selection process entails the molecular tailoring of the aqueous peptides’ conformational states towards specific assembly structures at the solid interface. Based on structural kinetic analyses, molecular dynamics simulations and a scanning probe energetic analysis (dubbed Intrinsic Friction Analysis), our results demonstrate that (i) peptide-graphite binding can be tailored via thermal selection of specific conformational states and that the same binding state can be reached for even altered amino-acid sequences, and, (ii) the resulting assembly structure is specific to the most prominent peptide solution conformations. That the thermally selected solution conformation persists upon adsorption will be illustrated with a rational redesigned peptide sequence. Based on these findings, environmental processing of biomolecules may allow for the bottom-up fabrication of active bio-nano interfaces for multifarious applications from bioelectronic devices, biosensors, biomolecular fuel cells, to logic switches. The research was supported by NSF-DMREF program through the grant DMR-1629071 as part of the Materials Genome Initiative.

Designing the new sugar-derived poly(D-glucose carbonate)s (PGC) is motivated by a need to develop sustainable materials in response to a long-term environmental impact of traditional petroleum-based polymers. A recent work on the self-assembly of glucose-based molecules demonstrates they can assemble into various nanostructures including micelles, nanofibers and vesicles with tunable size and surface charge. Motivated by their potential for building blocks for functional nanostructures, in this work, PGC amphiphilic block copolymers (BCP) with targeted block compositions, chain lengths and side chain chemistries (cationic and anionic) are synthesized by organocatalyzed ROP and assembled into various nanostructures for an in-depth characterization. Their assembly behavior in water is investigated by developing CREASE, an algorithm that reverse-engineers assembled micelle structures based on calculated micelle features (e.g., aggregation number, core size, corona chain structure) from the experimental small-angle neutron scattering (SANS) intensity profile. We further explore the assembly behavior of PGCs using a range of solvent environment (e.g., solvent compositions and pH variation). We find that the PGC BCPs display a unique chain packing that is dependent on the degree of side chain ionization and can transition from micellar to fibrous nanostructures. These findings allow us to discover a variety of robust nanostructures that can be achieved by non-traditional polymers and their potential to be used in various scientific disciplines.

The area of peptide-based materials has seen a recent surge in interest due to its wide range of potential applications including targeted drug delivery, cancer treatment, tissue engineering, nanoelectronics and batteries. The 20 amino acids, the building blocks of peptides, yields an enormous molecular parameter space which can be used to design peptides with a wide variety of sequences. A fundamental understanding of the impact of peptide sequence on the structure-activity relation of these biological materials will accelerate their development and use, thereby advancing diverse disciplines.
We use a hybrid computational approach, which integrates molecular simulations with analytical techniques, advanced sampling and computing tools, to study and develop new biological materials with desired sequence-structure-activity properties in a more time- and cost-efficient way. The biological materials are formed via the self-assembly of amphiphilic ultrashort peptides encompassing aromatic amino acids (namely, diphenylalanine and phenylalanine-asparagine-phenylalanine). The extended spatiotemporal scales connecting molecules to materials is addressed via the use of coarse-grained representations of the molecular species (S. Mushnoori, et al Organic & Biomolecular Chemistry 2018, 16, 2499). The dynamics underlying the self-assembly of these peptides is resolved via the classical Molecular Dynamics simulation method.

With inspiration from proteins, we investigate synthetic random heteropolymers through molecular dynamics simulations. We explore the behavior of single macromolecular chains and can relate the impact that monomer chemistry and sequence have on their morphologies to protein folding. Examination of globule formation and the resulting conformations illuminate how the chains may interact and enable random heteropolymer multi-functionality which has been experimentally shown. By emulating the heterogeneity seen on the surfaces of some biological proteins, hydrophobic and hydrophilic polymer segments create a rich energy landscape, allowing for a variety of dynamic conformations and enabling its unique behavior.

There is great potential for genome sequencing to enhance patient care through improved diagnostic sensitivity and more precise therapeutic targeting [1]. To maximize this potential,
DNA-sequencing technologies need to be optimized and adapted to clinical requirements. Nano-channel analysis is one of the emerging strategies for non-optical DNA sequencing [2-5]. Each constituent nucleobase is identified via measuring the transverse tunneling current as a single DNA molecule approaches measuring electrodes. Accuracy and throughput are still limited by insufficient control over DNA elongation and transport dynamics. We employ a coarse-grained molecular model, as a robust framework for the simulation of biopolymer dynamics. Our computational model serves as an essential tool for the optimal design of nano-channel platforms offering high-resolution DNA analysis.

The model implementation is targeted towards predicting the degree of linearization of confined DNA molecules, and simulating the DNA uncoiling and entering the nano-channel. Nano-confinement opens the possibility of linearizing much longer DNA molecules that is currently possible. To uniformly stretch DNA chains, the dimensions of nano-fluidic structures should be smaller than the persistence length. (e.g, about 50nm for double stranded DNA in dilute solutions) [6, 7]. Channel size reduction introduces added challenges of DNA loading and fabrication cost. Mediated by channel characteristics and buffer concentration we investigate strategies of micro- and nano-confinement to improve DNA stretching.

In addition to the geometric aspects of sequencing DNA under confinement, controlling DNA electrophoresis is essential to acquiring a clear signal from the sensing device. The frequency of DNA treading can be increased by applying larger voltages. However, this approach can result in DNA transport velocity exceeding the sensor’s sampling rate. Our multiscale approach, combining continuum and coarse-grained models, is informative of the driving forces interplay at various scales. This allows for new design concepts to be numerically studied for feasibility. One strategy to facilitate the transition from a high to a low entropy state consists of introducing structures that provide multiple pathways of increasing confinement for the DNA to unravel [8]. Our systematic study reveals how to design the geometry and applied bias to realize shallow free energy paths for the loading of DNA molecules.

References:
[1] E. A. Ashley, Nature Reviews Genetics, 2016, 17, 507.
[2] N. Douville, D. Huh and S. Takayama, Analytical and Bioanalytical Chemistry, 2008, 391, 2395–2409.
[3] Y. Kim, K. S. Kim, K. L. Kounovsky, R. Chang, G. Y. Jung, J. J. dePablo, K. Jo and D. C. Schwartz, Lab Chip, 2011, 11, 1721–1729.
[4] M. Tsutsui, Y. He, M. Furuhashi, S. Rahong, M. Taniguchi and T. Kawai, Scientific Reports, 2012, 2, 394.
[5] J. Zhou, Y.Wang, L. D. Menard, S. Panyukov, M. Rubinstein and J. M. Ramsey, Nature Communications, 2017, 8, 807.
[6] T. Odijk, Physical Review E, 2008, 77, 060901(R).
[7] L. Dai, C. B. Renner and P. S. Doyle, Advances in Colloid and Interface Science, 2016, 232, 80-100.
[8] H. Cao, J. O. Tegenfeldt, R. H. Austin and S. Y. Chou, Applied Physics Letters, 2002, 81, 3058–3060.

Bio-composite materials with optimal mechanical and structural properties and capability of cell differentiation are crucial for tissue engineering. Synthetic collagen proteins with lengths of approximately 10 nanometers, along with natural spider silk proteins provide an opportunity for development of an optimal biomaterial for scaffolding applications in tissue engineering. This combines unique mechanical, structural and biological properties of two of the nature’s best polymer proteins. In the present work, we study the binding capability of these proteins at a molecular level via molecular dynamics modeling. Spider silk and synthetic collagen protein molecular models in biophysical saline conditions under standard pressure and temperature are investigated to understand if natural binding occurs between the two without any other external factors. An initial minimum separation of 10 angstroms between the proteins was used. Binding was observed between the two proteins throughout the dynamic simulation. Molecular dynamics simulations are performed for a minimum of 100 nanoseconds for all systems. The radius of gyration and minimum distance between the proteins shows a decreasing separation between the two proteins until a stable distance of 2.5 nanometers and 0.2 nanometers respectively, is achieved. Binding is further identified by binding observed between the proteins via formation of strong and stable hydrogen bonds. A hydrogen bond between collagen Proline-31 and silk Serine-96 was observed to be the most stable and frequent bond between the single collagen and silk system. Results clearly indicate a self assembly behaviour of these two systems illustrating their potential as a biomaterial for tissue engineering.

Bio-based and biocompatible antimicrobial materials have many advantages in term of environmental protection and sustainable development, comparing to common inorganic and organic antimicrobial materials. It has been found previously that fabrics made from bio-based PLA/PHBV blend filament fibers exhibit broad-spectrum bactericidal effects, with no addition of any antimicrobial agents. After extracting and analyzing the antibacterial components, here we first discover that Poly (3-hydroxybutyric acid) (PHB) oligomers with several degrees of polymerization have potent antibacterial and antifungal properties.

Subsequently, two preparation methods for PHB oligomers are described as extraction from biologically fermented PHB polymers and one-step ring-opening polymerization of β-butyrolactone. The topological application of the synthesized PHB oligomer imparts perdurable antibacterial properties to the non-antibacterial fabric.

Furthermore, to test its potential in medical applications, skin sensitization and infected wounds cure of the mice treated with PHB oligomer were observed. The antimicrobial mechanism of the synthesized PHB was revealed as the leakage of intracellular content, inhibition of protein activity and the change of transmembrane potential.

Finally, the antibacterial properties of homologues of PHB oligomer, including polylactic acid (PLA) oligomer and polyglycolic acid (PGA) oligomer, were also discovered incidentally, which has brought a wider map for the exploration of antibacterial materials.

Keywords: Poly (3-hydroxybutyric acid), PLA, PGA, oligomer, antimicrobial material, extraction, synthesis, medical applications, antimicrobial mechanism.

ACKNOWLEDGMENT
The work has been supported by Innovation and Technology Commission of SAR Government of Hong Kong (Grant No. ITP/039/16TP).

Zinc oxide (ZnO) particles possess unique semiconductive, photocatalytic, and antimicrobial properties and they have found wide applications in drug delivery, tissue engineering, and bioimaging. Morphology plays a major role in modulating the properties and activities of ZnO particles; however, gap in knowledge exists in evaluating the effect of capping agents on ZnO growth kinetics. In this contribution, we reported a facile water-based chemical precipitation method to prepare ZnO particles with distinct architectures (i.e. ZnO bowties, flowers, and nests) using zinc nitrate, urea, and polyvinylpyrrolidone (PVP) as building blocks [1]. Our detailed studies uncovered that the preferential adsorption of PVP onto different ZnO facets at different polymer concentration controlled the growth directions of ZnO and hence resulted in varied ZnO morphologies. Serendipitously, we discovered that ZnO particles have enzyme-like activities and demonstrated their ability to decompose exogenous and endogenous prodrugs to produce NO at physiological condition (pH 7.4, 37 °C). We also showed that physiologically relevant NO levels can be realized by varying the concentration of ZnO or NO prodrugs. In addition, we observed that ZnO particles preserved their catalytic capacity even after 6 months when suspended in PBS, which is beneficial for long-term biological applications. NO is a versatile player that participates in nearly every physiological system, such as cardiovascular, immune, central nervous system, and outflow physiology. However, the short half-life of NO in human tissues (approx. 5 s) and its limited diffusion radius (40-200 µm) have hampered the full potential of this molecule [2]. Our findings overcome the aforementioned challenges of NO delivery via utilizing an endogenous NO donor (GSNO) and an enzyme mimic (ZnO). Based on the infinite nature of endogenous NO precursors, NO delivery is therefore “limitless”, and NO can be generated throughout the lifetime of the enzyme mimic.

References
[1] Yang, T.; Oliver, S.; Chen, Y.; Boyer, C; Chandrawati, R. J Colloid Interface Sci 2019, (546) 43-52.
[2] Yang, T.; Zelikin, A.N.; Chandrawati, R. Adv Sci 2018, 5 (6), 1701043.

Infection of medical implants is a serious ongoing problem worldwide, caused by bacterial adhesion and subsequent biofilm formation on the implant interface. To prevent such infection, we designed a set of catechol-functionalized cationic peptide antibacterial hydrogels that were inspired by the lysine- and DOPA-rich mussel foot protein-5. These advanced supramolecular gels are designed to coat implants providing a barrier to surface colonization and can also be injected during surgery directly to the tissue implantation site to inhibit surgical site infection. Furthermore, utilizing the gels lysine rich amino acid composition and the redox-activity of catecholic residues, these gels kill bacteria by two distinctive mechanisms: via a direct contact mechanism between the polycationic gel and the bacterial cell surface and by DOPA-mediated production of hydrogen peroxide (H₂O₂), a known antibacterial agent. We demonstrated that these gels exhibit high bactericidal activity against clinically isolated gram-positive bacteria, including the notorious multidrug resistant bacteria, MRSA. We further showed how amino acid composition and peptide sequence can modify the amount of generated H₂O₂, and consequently alter the antibacterial activity of the gel. Moreover, we characterized the ability of the gels to act as adhesives at the implant-tissue interface by utilizing lap-shear tensile strain tests.
Collectively, these results indicate that DOPA-containing hydrogels hold promise as antibacterial adhesives, suitable for implantation at the tissue-implant interface.

Antimicrobial peptides are promising substitutes for conventional antibiotics with increasing bacterial resistance. Our previous work studied the relationship between self-assembly and antimicrobial activity using a designer peptide derived from a human salivary protein. This 13-amino acid amphiphilic and cationic peptide, named GL13K, can self-assemble to supramolecular nanofibrils in alkaline solutions. In our previous work, the coating of GL13K nanofibrils onto biomedical devices (e.g., Ti implant) and natural tissues (e.g., dentin) modified the surface hydrophobicity and showed a long-lasting antimicrobial activity. The amount of adsorbed GL13K was highly dependent on the surface chemistry and solution conditions, which eventually affected the activity of the coating. For example, the etched Ti adsorbed a much higher amount of GL13K than untreated Ti; the adsorption of GL13K predominated in the mineral-rich region on dentin. Separating and quantifying the effects on peptide adsorption of substrate hydrophobicity, charge, polarity, and roughness is a difficult, but critical task to design and control coating manufacturing and (bio)functionality. We hypothesized that surface negative charge is the main property favoring adsorption of self-assembled GL13K peptides.
In this work, we used quartz crystal microbalance with dissipation monitoring (QCM-D) to study in situ adsorption of GL13K self-assembled nanofibrils. The surface chemistry was controlled by coating Au QCM-D sensors with self-assembled monolayers (SAMs) with different terminal groups, including –COOH, –NH₂, and –CH₃. The SAM-coated sensors with polar terminal groups (i.e., –COOH and –NH₂) were more hydrophilic than the one with a nonpolar terminal group (i.e., –CH₃). It was observed that a significantly higher amount of GL13K adsorbed on the sensor with –COOH terminal group compared to the one with –CH₃. In the alkaline solution where GL13K self-assembled to nanofibrils, GL13K had negligible adsorption on the sensor with –NH₂ terminal group because –NH₂ was deprotonated. Notably, adsorption of GL13K was largely favored on the same sensor once it was protonated to –NH₃⁺ in lower pH. Self-assembled nanofibrils were found on sensors with deprotonated –COO^- and protonated –NH₃⁺ by atomic force microscopy (AFM). This indicated that polarity, not charge, was the critical factor determining the adsorption of these amphiphilic peptide self-assembled nanofibrils. Thus, we rejected our initial hypothesis.
With a better understanding of the adsorption behavior of GL13K, it is easier to control the coating of antimicrobial peptides on natural tissues. As a proof of concept, hydroxyapatite, the main inorganic component in tooth and bone, was also coated on sensors. Since hydroxyapatite is a very polar and hydrophilic material, GL13K adsorbed extensively on the sensor surface observed by QCM-D and AFM. The findings in this study could lead to better designs of coating amphiphilic peptides with different functionalities on biomedical devices and natural tissues.

The discovery of penicillin in 1940 is one of the most important medical inventions in reducing human morbidity and mortality but the intensive use of all kinds of antibiotics since then has led to an increase in multi-drug resistant bacteria and shows the necessity for alternative treatment options against bacterial infections. Alternatively to antibiotics, the antimicrobial properties of metals have been used for thousands of years, e.g. vessels made of copper or silver for water disinfection and food preservation. In this work hollow mesoporous silica capsules (HMSC) were synthesized using a hard iron oxide template which was coated with a silica shell through cross-condensation reactions. Afterwards the iron oxide core was removed by acidic leaching. Metallic nanoparticles such as Cu and Ag were incorporated into HMSC through the reduction of their metal salts (Ag@HMSC and Cu@HMSC). The formation of as-prepared rattle-type metal-silica particles was proven by TEM as well as XPS and EDX measurements. To determine the controlled leaching of metal ions under physiological conditions at 37°C, free Ag and Cu ions were complexed with dithizon to allow for their visualization and quantification via UV-Vis analyses at 454 nm and 543 nm. Moreover, INT assays revealed that both particle types exhibit a strong antimicrobial effect against gram-positive (B. subtilis) as well as gram-negative bacteria (E. coli), demonstrating their promising potential as antibiotic alternative in the future.