Symposium SB09—Genetically-Encoded and Bioinspired Materials Science

Radiolabeled nanomaterials have gained tremendous interest over the last 2 decades, which can play diverse roles in imaging, image-guided drug delivery, as well as theranostics of a number of diseases such as cancer. Some recent examples of radiolabeled materials from our recent work will be briefly described in this talk.

Although chelator-based radiolabeling techniques (commonly used for labeling nanomaterials with radiometals such as ⁶⁴Cu/⁸⁹Zr) have been used for decades, concerns about the complexity of coordination chemistry, possible alteration of nanomaterial pharmacokinetics, and potential detachment of radioisotopes have driven the need for developing a simpler yet better technique for future radiolabeling.

The emerging area of intrinsically radiolabeled nanomaterials can take advantage of the unique physical and chemical properties of well-selected inorganic or organic nanomaterials for radiolabeling, and more importantly, offer an easier, faster, and more specific radiolabeling possibility to facilitate future clinical translation. Generally speaking, the four major categories of intrinsically radiolabeled nanomaterials include: 1) hot-plus-cold precursors, 2) specific trapping, 3) cation exchange, and 4) proton beam activation.

Representative examples of each category will be briefly illustrated in this talk, with the main focus on our own work that involves the radiolabeling of a variety of nanomaterials via “specific trapping”. The nanomaterials investigated in our laboratory include silica-based nanoparticles, carbon-based nanomaterials, DNA nanostructures, iron oxide nanoparticles, micelles, multifunctional/multimodal hybrid nanomaterials, nanovaccines, among others.

Nanotubes and nanostraws are increasingly used to transfect cells with better efficiencies and cell viabilities compared to existing transfection methods (liposomes, viral vectors and electroporation)^[1,2]. A long-standing question in the field concerns the state of the cell membrane on such hollow nanostructures. Here, in order to investigate the state of the cell membrane during and after transfection, we have performed video-rate live cell STED microscopy imaging of the cell membrane when interfaced with nanostraws. The time-lapse STED images reveal that the cell membrane opens entirely on top of nanostraws upon application of gentle electrical pulses, and that it recovers and seals 30-60 min after turning off the electrical field^[3]. We further demonstrate that we can achieve direct delivery of fluorescent nanodiamonds to the cytosol using nanostraws. The nanostraw delivery leads to efficient and rapid nanodiamond transport into cells compared to when incubating cells in nanodiamond-containing medium. Moreover, whereas all internalized nanodiamonds delivered by incubation end-up in lysosomes, a significant larger proportion of nanostraw-injected nanodiamonds are located in the cytosol, which opens up for using nanodiamonds as cellular probes
References:
[1] X. Xie, A. M. Xu, S. Leal-Ortiz, Y. Cao, C. C. Garner, N. A. Melosh, ACS Nano 2013, 7, 4351.
[2] L. Schmiderer, A. Subramaniam, K. Zemaitis, A. Bäckström, D. Yudovich, S. Soboleva, R. Galeev, C. N. Prinz, J. Larsson, M. Hjort, Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 21267.
[3] E. Hebisch, M. Hjort, D. Volpati, C. N. Prinz, Small 2021, 17, 2006421.

This talk presents our recent work in carbon-based nanosciences. I will first briefly review the group’s earlier work of carbon-based nanomaterials, and then focus on fluorescence biological imaging in the newly coined 1000-1700 nm NIR-II window to benefit from suppressed light scattering at long wavelengths. I will show in vivo NIR-II imaging with millimeter tissue depth, single-cell spatial resolution, and real-time temporal resolution using a wide range of nano-scale fluorescent/luminescent nanoprobes emitting > 1000 nm including carbon nanotubes, quantum dots, rare-earth down-conversion nanoparticles, and donor-acceptor organic molecules.
In the second part of the talk, I will briefly present his group’s work on the rechargeable Al battery, Na/Cl2, and Li/Cl2 batteries based on novel carbon materials.

To reduce the toxicity and side effects of drugs, targeted intravenous delivery of drugs by nano/microparticles has been developed. Yet, the efficacy of the treatment is greatly reduced by biological organ filters that remove these delivery particles from the bloodstream, which then requires higher drug doses to ensure efficacy but increase adverse side effects. To date, research has mainly focused on geometric and electrostatic properties of delivery particles to improve their efficacy. Still, they lack the necessary mechanical properties to avoid filtering. Since erythrocytes pass through biological filters, the approach will be to mimic their mechanical behavior in protein polymer-network (PPN) materials. Discovered nanomechanics of specific protein tertiary structures are related to the mechanical properties of erythrocytes. However, it is still unclear how to incorporate these proteins into the PPN materials that can properly translate protein nanomechanics.

Here, we developed genetically-controlled protein polymers to identify design rules that will allow translation of protein nanomechanics to the micro/macroscale materials. In this protein polymer design, protein cross-linkers genetically fuse at the ends of protein polymer chains, containing protein tertiary structures. In buffer solutions, functionalized protein polymers bind to each other via self-recognition and noncovalent interactions and/or covalent bonds, and consequently form PPN materials, specifically hydrogels. By controlling the characteristics of protein polymer chains (i.e., composition, structure, and symmetry) and modulating crosslinker types as well as performing mechanical testing, we identified the specific design rules for the chain and the crosslinker that will properly exhibit protein nanomechanics at the micro/macroscopic material level. This design discovery will culminate in a protein polymer platform that harnesses mechanical proteins with diverse tertiary structures into biomimetic mechanical materials, which we expect to advance a wide variety of healthcare applications, including but not limited to erythrocyte-mimicking PPN-based microparticles that can pass through biological filters in the human body.

Adoptive cell therapies for in vivo tissue engineering are an exciting prospect, but are often limited by poor pharmacokinetics and pharmacodynamics. This can manifest as accumulation of transplanted cells in tissue sinks (e.g., lungs and spleen), leading to the formation of emboli and a poor prognosis. Accordingly, reengineering cells to improve their efficacy is key for the next generation of advanced cell therapies. In practise, this can be achieved through the synthesis of hybrid materials comprising highly cooperative biological and synthetic parts, which can be used to attenuate cell-target tissue interactions and provide compatible extracellular matrix components. Accordingly, we describe an emerging research programme that spans the fields of synthetic biology, nanomedicine, and regenerative medicine. Here, artificial membrane binding protein (AMBP) chimeras are designed and synthesised using a two-step process: protein supercharging to generate a supercationic plasma membrane binding anchor motif, followed by the electrostatic assembly of a membrane active polymer surfactant corona. Significantly, the resulting constructs spontaneously self-assemble at the plasma membrane of stem cells, providing resistance to hypoxia during cartilage tissue engineering.¹ Moreover, the methodology can be readily adapted to display a modified thrombin on stem cell, giving rise to plasma membrane nucleated fibrin hydrogels,² utilised to produce bacterial adhesin fusion constructs that direct stem cells to the myocardium,³ or applied to increase adhesion of stem cells on damaged articular cartilage via peptide display.⁴

1. Armstrong, J. P. K.; Shakur, R.; Horne, J. P.; Dickinson, S. C.; Armstrong, C. T.; Lau, K.; Kadiwala, J.; Lowe, R.; Seddon, A.; Mann, S.; Anderson, J. L. R.; *Perriman, A. W.; *Hollander, A. P., Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue. Nature Communications 2015, 6.
2. Deller, R. C.; Richardson, T.; Richardson, R.; Bevan, L.; Zampetakis, I.; Scarpa, F.; *Perriman, A. W., Artificial cell membrane binding thrombin constructs drive in situ fibrin hydrogel formation. Nature Communications 2019, 10.
3. Xiao, W. J.; Green, T. I. P.; Liang, X. W.; Delint, R. C.; Perry, G.; Roberts, M. S.; Le Vay, K.; Back, C. R.; Ascione, R.; Wang, H. L.; Race, P. R.; *Perriman, A. W., Designer artificial membrane binding proteins to direct stem cells to the myocardium. Chemical Science 2019, 10 , 7610.
4. Delint, R.C., Day, G.J., Macalester, W.J., Kafienah, W., Xiao, W., and *Perriman, A.W., An artificial membrane binding protein-polymer surfactant nanocomplex facilitates stem cell adhesion to the cartilage extracellular matrix. Biomaterials, 2021, 276, 120996.

Cell membrane-coated nanoparticles are made by wrapping natural cellular membranes onto synthetic nanoparticle cores. They leverage cell membranes to mimic some cell-like functions for biointerfacing, making it possible to enable novel biomedical applications. The targeting ability of these cell-mimicking nanoparticles is often mediated by specific proteins that are expressed on the source cells, and this bestows the nanoparticles with specific interactions with various disease substrates. On top of the natural biointerfacing capabilities of cell membrane-coated nanoparticles, their traits can be further enhanced by introducing exogenous moieties onto the membrane surface. One way to achieve this is to genetically engineer source cells to express desirable protein receptors, followed by membrane collection to prepare the nanoparticles. Herein, I will report on the latest development of genetically engineered cell-mimicking nanoparticle formulations for direct tumor antigen presentation to promote anticancer immunity as well as for targeted drug delivery to inflamed lungs.

Natural cells are highly complex microreactors that can be regarded as the basic building blocks of life. They serve as inspiration for artificial cells (ACs) in which certain structural and functional features are mimicked with the goal of creating ACs that mimic life-like properties¹. The focus is often on a single key property that assists to support or interact with their natural role models, here mammalian cells. Functions can be implemented using a variety of building blocks, either natural or synthetic. Further, hydrogels are a relevant option to serve as a structural scaffold since it creates an ‘intracellular’ environment akin to the dense cytosolic environment found in mammalian cells.
In this project, two essential cellular functions are explored: Energy generation and cellular communication.
Energy generation is essential for creating a self-sustaining system that can work out of equilibrium and to facilitate a wide array of functions that requires energy. In mammalian cells, mitochondria are responsible for the chemically driven adenosine triphosphate (ATP) synthesis. Here, mitochondria are used as a natural ATP producing subunit in gelatin methacryloyl (GelMA)-based ACs². Specifically, hepatocytes are employed as donor cells to isolate mitochondria. The maintained inner membrane potential, respiration and ATP production is confirmed in buffer and upon encapsulation in GelMA disks of the isolated mitochondria. Additionally, mitochondria are co-encapsulated with the ATP dependent enzyme firefly luciferase in GelMA particles to demonstrate the utilization of mitochondrially produced ATP in a cell-sized carrier, exemplified by the bioluminescent decarboxylation of D-luciferin.
Communication is ubiquitous amongst multicellular assemblies and on the tissue level to moderate collective behaviour. Here, communication is explored between alginate-based ACs and HepG2 cells.³ First, a one-way signal transfer is established by equipping two different types of ACs with catalytic function by the use of metalloporphyrins as artificial enzymes that mimic cytochrome P450 enzyme (CYP450) activity. Metalloporphyrins are employed to catalyse a dealkylation and hydroxylation, exemplified by the conversion of resorufin ethyl ether to resorufin and coumarin to 7-hydroxycoumarin. The two fluorescent products diffuse into the neighbouring HepG2 cells, establishing a one way signal transfer as an important first step in communication.
Taken together, these efforts work toward expanding the enzymatic functions that can be implemented in ACs by supplying energy to the system, here as mitochondrially produced ATP, moving towards a metabolically active AC. Further, the interaction between alginate-based ACs and HepG2 cells is explored, in one approach by utilizing metalloporphyrins as enzyme mimics as a synthetic approach to achieve catalytically active ACs.
¹ X. Qian, I.N. Westensee et al. (2020) WIREs Nanomed Nanobiotechnol. 13: e1683
² I.N. Westensee, E. Brodszkij et al. (2021) Small 17:2007959.
³ X. Qian, I.N. Westensee et al. (2021) Angew. Chem. Int. Ed., DOI: 10.1002/ange.202104904.

During the last decade, there has been a surge in scientific breakthroughs related to hydrogel-based active systems with electrical, thermal, or pH-sensing capabilities [1], [2]. However, these hydrogels are subject to direct contact with stimuli, drastic environmental changes, or exhibit low biocompatibility. Light-responsive systems offer the advantage of remote activation, wavelength selectivity, and localized actuation.[3] Previously, azo-moieties have been used due to their photo-isomerizable properties, yet harnessing the molecular folding to create tough, photo-responsive hydrogels with appreciable actuation strain is still a significant challenge.[4]
Herein present the design and facile fabrication process of a hierarchical material based on embedded, physically crosslinked azo-nanoclusters within a polyurethane scaffold. Due to its viscous nature, the material is suitable for casting or 3D printing. Furthermore, their surface can be functionalized to host living cells, resulting in a light-responsive active hydrogel, which exhibits mechanical properties, like those of native biological tissues, and high biocompatibility.
Its toughness allows the gel to be strained up 400% and yields the biologically relevant range of 10-17% contractile strain upon light-switching. During the switchable ON state (λ = 405 ± 5 nm) these photochromic nanoclusters fold, changing the local dielectric environment. The isomerization increases the overall material’s hydrophobicity and leads to a decreased water retention capacity, contracting up to 17%. This process is fully reversible on-demand by irradiation, the OFF state (λ = 525 ± 5 nm), where the gels scavenge water from the environment and recover by swelling to its original dimensions.
We have demonstrated the application of this remotely controlled system within the frame of biohybrid soft robotics, where the hierarchical cooperation between the inert and living components contributed to its behavior as a self-reinforcing material. This reversible contraction process has been used to evaluate mechanotransduction cues within a skeletal myoblast cell line (C2C12). The hydrogel protects the hosted cells from blue light irradiation, triggering localized contraction and inducing cellular fusion to form myotubes.
The system evolves with every ON-OFF cycle, beginning as a cell-laden scaffold that strengthens as the individual cells become muscle.
[1] M. C. Koetting, J. T. Peters, S. D. Steichen, and N. A. Peppas, “Stimulus-responsive hydrogels: Theory, modern advances, and applications,” Materials Science and Engineering R: Reports, vol. 93. Elsevier, pp. 1–49, 01-Jul-2015.
[2] G. Gerlach, M. Guenther, and T. Hartling, “Hydrogel-Based Chemical and Biochemical Sensors - A Review and Tutorial Paper,” IEEE Sensors Journal, vol. 21, no. 11. Institute of Electrical and Electronics Engineers Inc., pp. 12798–12807, 01-Jun-2021.
[3] M. P. M. Dicker, “Light and Chemistry Applied to the Control of Smart Materials and Structures,” University of Bristol, 2016.
[4] Y. Hu et al., “Photoactuators for Direct Optical-to-Mechanical Energy Conversion: From Nanocomponent Assembly to Macroscopic Deformation,” Adv. Mater., vol. 28, no. 47, pp. 10548–10556, Dec. 2016.

Interfacing cell-based therapeutics with bioelectronic devices can enable sense and respond capabilities to regulate therapeutic dosing. Herein, we present an implantable cell-based drug factory platform that interfaces with a bioelectric device to enable closed-loop, inducible and traceable protein production for in situ therapeutic dose regulation. For this system we engineered a clinically relevant cell line (ARPE-19) to co-express hormones which regulate circadian rhythms and EGFP under the control of a light-responsive optogenetic system. Engineered cells produced physiologically relevant peptides (leptin, glucagon-like peptide-1 and adrenocorticotropic hormone) and regulated their production by 16-fold in response to light actuation. We encapsulated engineered cells in an immunomodulatory hydrogel to facilitate implantation and demonstrated that a millimeter-scale implantable device could sense the produced EGFP fluorescence. By genetically linking the production of EGFP to the therapeutic peptide we have shown that this fluorescence reading can be used to calculate the dose of peptide produced by the cell factories. In combining the sense and actuate capabilities, we can create a cell therapy-bioelectric hybrid device capable of tracking and adjusting therapeutic dosing in situ. This technology will enable therapeutic modulation of precisely regulated biological processes including the circadian rhythm.

Achieving real-time monitoring of neurotransmitters enables one to monitor cognitive performance, diagnose mental health issues, and build the correlation between neurotransmitters and cognitive state. A subset of neurotransmitters, monoamine neurotransmitters (e.g. dopamine, serotonin, epinephrine), are commonly targeted for electrochemical biosensors, as they are inherently redox active, and thus can be detected directly by an electrical pulse. However, sensitivity and selectivity remain key challenges towards the fabrication and deployment of these sensors in wearable platforms. Au and Carbon-based electrodes have previously shown to be highly effective electrode materials as they are both biocompatible and easily modified for sensor fabrication. However, the current state-of-the-art form of the aforementioned electrodes show poor resolution at low analyte concentrations. Here we show multiple strategies to enhance the sensing capabilities for the Au and Carbon-based surfaces which have been specifically tailored to directly target neurotransmitters such as Serotonin and Dopamine. We demonstrate that electrodeposited PSS (polystyrene sulfonate) alone, likely due to its negative charge and aromatic structure, has improved sensitivity compared to the previously tested PEDOT:PSS (3,4-ethylenedioxythiphene polystyrene sulfonate). We have electrodeposited various thicknesses of pure PSS atop of a Carbon-based electrode sensor allowing us to detect Serotonin concentrations as low as 10 nM, well below the physiological levels in both the blood and Interstitial fluid (ISF). Furthermore we show two specifically designed peptides with enhanced sp₂ stacking which regain selectivity to Serotonin with a detection limit as low as 10-20 nM, mimicking the hybridization observed on graphene/carbon electrodes with an exceeding sensitivity.

Engineered Living Materials (ELMs) are rapidly emerging as an exciting new class of functional materials. At the intersection of synthetic biology and materials science, these systems can take advantage of the broad range of modalities now readily achievable with modern genetic engineering, including responsivity, adaptability, growth, and self-healing.¹ Moreover, when coupled with advanced fabrication approaches such as 3D bioprinting, it is possible to generate precise spatial localization of multiple engineered cell populations within robust, handleable structures.²
The degradation of organophosphorus compounds (OPCs) is one challenge that could benefit from ELM development. OPCs are a class of organic molecule that act as acetylcholinesterase inhibitors; their toxicity varies widely, from pesticides such as parathion to chemical warfare agents including sarin and VX. Bioremediation presents an excellent method for soft, sustainable decontamination of OPCs that, along with being applicable directly for patient decontamination or protection, may address the issues of dealing with the presence of persistent hydrophobic nerve agents. Effective containment of engineered cells within an ELM, preventing cell release or DNA transfer to the environment, would enable environmental application.^3,4
Accordingly, we have 3D bioprinted a microporous alginate ELM laden with E. coli expressing the phosphotriesterase (PTE) that hydrolyses OPCs. The material’s 3D printability enabled benchmarking of the kinetic performance of the ELM for OP degradation, along with the behaviour of the living microcolonies contained within the structures. Utilizing the 3D geometries afforded by extrusion printing this living material was incorporated into a flow reactor system whose OPC-degrading performance could be tuned through both the design of flow dynamics and by alteration of proliferation and enzyme expression.
References
1) Gilbert, C. & Ellis, T. Biological Engineered Living Materials: Growing Functional Materials with Genetically Programmable Properties. ACS Synth. Biol. 8, 1–15 (2019).
2) Saygili, E., Dogan-Gurbuz, A. A., Yesil-Celiktas, O. & Draz, M. S. 3D bioprinting: A powerful tool to leverage tissue engineering and microbial systems. Bioprinting 18, (2020).
3) Mukherjee, S. & Gupta, R. D. Organophosphorus Nerve Agents: Types, Toxicity, and Treatments. J. Toxicol. (2020).
4) Thakur, M., Medintz, I. L. & Walper, S. A. Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges. Front. Bioeng. 7, 289 (2019).

Myocardial infarction (MI) is the leading cause of mortality in developed countries, resulting in a major psychological and financial burden for society. Current treatments target rapid restoration of perfusion to limit damage to the heart, rather than promoting tissue regeneration and subsequent contractile function recovery. Regenerative therapies, including stem cells, tissue grafts and extracellular vesicles (EVs), surge as a promising alternative, however, their efficacy is severely limited by low long-term cell survival, and poor homing and engraftment to the myocardium. Different strategies to overcome these problems include genetic-modification or membrane surface modification of the transplanted cells, and by their incorporation onto soft biomimetic materials.¹

Inspired by the Artificial Membrane Binding Proteins (AMBPs) technology previously developed within our group,² we have designed a novel versatile system comprised of two bio-orthogonal reacting pairs. First, a surfactant-coated protein fusion that anchors to bilayer lipid membranes and, second, a protein fusion that reacts selectively with the former³ and contains another protein or peptide that enhances the properties of artificial extracellular vesicles (AEVs), which can be used as carriers of therapeutic payloads. After confirming the biophysical interaction between the reacting AMBPs in solution, we have demonstrated that the interaction between the reacting AMBPs also occurs when embedded on the membranes of in-house built AEVs via liposome extrusion. Stability studies with dynamic light scattering (DLS) revealed that vesicles in our AMBP-AEV system were intact for up to a week.

We then selected a cardiac-homing protein to be fused to the second counterpart of the system, obtaining approximately a 20-fold uptake increase for fibronectin-rich cell types statically, upon no detriment to cell viability. Further experiments under physiologically relevant shear stress (2 dynes) resulted in a 10-fold increase of the desired AMBP-AEV uptake and retention. Additionally, we demonstrated exclusive localization of the cardiac-homing AMBP-AEV system in zebrafish hearts, highlighting their potential as therapeutic in cardiac disease. The extrusion-based AEV synthesis facilitates their loading with a cargo, for which we employed mCherry, a red-fluorescent protein, as a payload inside the vesicles to demonstrate only AMBP-modified red AEVs were uptaken in vitro and in vivo.

In conclusion, we have shown cellular uptake of AMBP-AEVs and the possibility of directing these to a specific target such as the heart. We demonstrated our cardiac homing AMBP-AEV system to function under physiological conditions (2 dyne shear stress), which localised exclusively in zebrafish hearts. This constitutes a plug-and-play system that can be readily modified or repurposed depending on the desired outcome for regenerative therapies and can be incorporated in any bilayer membrane.


(1) Cruz-Samperio, R.; Jordan, M.; Perriman, A. Cell Augmentation Strategies for Cardiac Stem Cell Therapies. Stem Cells Transl. Med. 2021, 10 (6), 855–866. https://doi.org/10.1002/sctm.20-0489.
(2) Xiao, W.; Green, T. I. P.; Liang, X.; Delint, R. C.; Perry, G.; Roberts, M. S.; Le Vay, K.; Back, C. R.; Ascione, R.; Wang, H.; Race, P. R.; Perriman, A. W. Designer Artificial Membrane Binding Proteins to Direct Stem Cells to the Myocardium. Chem. Sci. 2019, 10 (32), 7610–7618. https://doi.org/10.1039/c9sc02650a.
(3) Hatlem, D.; Trunk, T.; Linke, D.; Leo, J. C. Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. Int. J. Mol. Sci. 2019, 20 (9). https://doi.org/10.3390/ijms20092129.

Silica shell formation on nanoparticles via reverse microemulsion is one of the most promising ways to obtain biocompatible surface states. Furthermore, the silica layer is easily functionalized with versatile functional groups, which provides researchers with a universal design platform. However, for magnetic nanoparticles (MNPs) composed of magnetite or tertiary ferrites, commonly used for biological applications including hyperthermia and modulation of cell signaling, it was reported that the silica layer degrades the magnetic properties. The interaction between the silica shell and ferrite core diminishes the magnetic anisotropy, and thus the specific loss power (SLP, heating efficientcy in W/g_[Metal]) decreases. High SLP is necessary for MNPs to work as effective mediators of magnetothermal modulation of cell signaling. Additionally, further functionalization of the silica layer tends to increase the shell thickness, further decreasing the magnetic anisotropy and lowering the SLP values.

To suppress the degradation of MNP properties, creating thin (one or few layers) silica shells offers a promising approach, and several groups have conducted parameter studies to create thinner shells on MNPs used as magnetic resonance imaging (MRI) contrast agents. However, the size of MNPs for modulation of cell signaling or hyperthermia is larger (20-25nm) than those used as MRI contrast agents (10-15 nm), which means the number of particles differs by an order of magnitude for the same mass of material. Since the number ratio between nanoparticles and reverse micelles is critical during microemulsion synthesis, the conditions to make thin silica shells must be reconsidered.

We have developed a robust protocol for forming and functionalizing thin silica shells on 20-25 nm MNPs, a size range that shows the highest SLPs among metal-oxide nanoparticles under mild magnetic field conditions (H×f < 5×10⁹ A/m･s). Additionally, the relationship between SLP and silica shell thickness has been investigated. Through the protocol development, the significance of the crystal phase of magnetic cores to obtain uniform shells was uncovered. The influence of further biologically-motivated functionalization with amine groups or thiol groups has also been evaluated.

Adoptive cell therapies (ACT) are rapidly emerging as an exciting new therapeutic modality in medical oncology, due to their novel mechanisms of action that potentially overcome drug resistance and produce durable remissions ^1–3; these therapies aim to redirect the patient's own immune system to selectively target cancer cells. While ACT have shown great success for the treatment of hematologic malignancies such as Acute Lymphoblastic Leukemias (ALL) or Non- Hodgkin Lymphomas, response rates among patients with solid cancers are less favorable. The lack of widespread treatments for solid tumors, which accounts for 85% of all tumor deaths, stems from the inability of immune cells to effectively home to the tumor site and penetrate the tumor microenvironment. Moreover, a hypoxic and suppressive tumor extracellular matrix can inhibit cytotoxic T-cells’ ability to expand, persist and mediate cytotoxicity through the presentation of immunosuppressive modulators. Accordingly, the aim of this research project is to develop artificial membrane-binding proteins (AMBP) to improve the performance of adoptive cell therapies in solid tumors. AMBPs are designer proteins that have been modified to spontaneously insert into the membrane of cells, without penetrating into the cytoplasm. This approach allows the rapid display of functional proteins or peptides on the surface of T-cells, which can provide homing, adhesion, immune regulation, and hypoxia resistance to the donor cell, without affecting their function or viability^4,5. In this work, an AMBP is designed, expressed, purified, and characterized using SDS-PAGE, matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) and several biophysical techniques such as Circular Dichroism, UV-Vis spectroscopy and Dynamic Light Scattering. In addition, to demonstrate the improved effectiveness of antigen-specific T-cells coated with AMBPs in controlling tumor growth, in vitro functional assays such as Cell Viability and Cytotoxicity, Proliferation and Activation and Killing assays, will be done.
References:
1. Brentjens, R. J. et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Science translational medicine 5, 177ra38 (2013).
2. Maude, S. L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. The New England journal of medicine 371, 1507–1517 (2014).
3. Besser, M. J. et al. Adoptive transfer of tumor-infiltrating lymphocytes in patients with metastatic melanoma: Intent-to-treat analysis and efficacy after failure to prior immunotherapies. Clinical Cancer Research 19, 4792–4800 (2013).
4. Xiao, W. et al. Designer artificial membrane binding proteins to direct stem cells to the myocardium. Chemical Science 10, 7610–7618 (2019).
5. Armstrong, J. P. K. et al. Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue. Nature Communications 6, (2015).

Adoptive cell therapies (ACT) intend to redirect the patient’s immune system to effectively recognize and target tumor cells. Although these therapies have shown significant success in the treatment of hematological malignancies, the response has not been as favorable in patients with solid tumors¹. This is mainly caused by the immunosuppressive nature of the tumor microenvironment (TME) which prevents immune cells from infiltrating, penetrating, and homing into the tumor site².

Current in vitro models to test ACT for solid tumors measure cytotoxicity, chemotaxis and suppression a 2-dimensional(2D) arrangement. However, therapeutics tested in 2D models fail to exhibit the same effects in vivo, due to their inability to accurately display the characteristics of the TME, such as 3-dimensional (3D) architecture, chemoresistance, hypoxia, cell heterogeneity and the presence of an extracellular matrix (ECM)³. For this reason, spheroids have been proposed as an alternative to model human solid tumors. Spheroid refers to an aggregate of cells that grow and adhere to each other, adopting a 3D sphere-like structure without the need of a substrate or foreign material. This type of model has greater accuracy and similarity to the TME in vivo by exhibiting a 3D architecture comprised of three distinct cell layers (necrotic quiescent and proliferative) with different gradients of nutrients, gas, waste and oxygen⁴. Efficiency in this models is also increased by allowing a higher throughput capacity and reducing the need to use animal models⁵.
While there are several methods to generate spheroids (i.e. suspension, polymeric chambers, microfluidics), bioprinting displays more advantages than other methods due to its tight spatial control over spheroid growth and initial cell location that allows it to fully capture cell-cell and cell-ECM interactions⁶. Here, we aim to develop a 3D bioprinted tumor spheroid model to accurately evaluate the performance of ACT.

For the proposed model, spheroids were bioprinted by embedding breast cancer cells within several bioink formulations resembling the components of the ECM and then extruded. Cell viability, spheroid size and number were monitored throughout a 14-day period post-printing using high-throughput image-based approaches. Human T-cells were bioprinted in the same bioink formulations and tested for initial cell viability, infiltration and motility through image and flow cytometry based approaches. In conclusion, 3D bioprinting of spheroids is a promising tool to generate high-throughput, flexible and reproducible models that can be used to accurately evaluate ACT versus solid tumors without animal sacrifice.

References
(1) Rohaan, M. W.; Wilgenhof, S.; Haanen, J. B. A. G. Adoptive Cellular Therapies: The Current Landscape. Virchows Arch. 2019, 474 (4), 449–461.
(2) Joyce, J. A.; Fearon, D. T. T Cell Exclusion, Immune Privilege, and the Tumor Microenvironment. Science (80-. ). 2015, 348 (6230), 74–79. https://doi.org/10.1017/CBO9781107415324.004.
(3) Kapalczynska, M.; Kolenda, T.; Przybyla, W.; Zajaczkowska, M.; Teresiak, A.; Filas, V.; Ibbs, M.; Blizniak, R.; Luczewski, L.; Lamperska, K. 2D and 3D Cell Cultures – a Comparison of Different Types of Cancer Cell Cultures. Arch. Med. Sci. 2018, 14 (4), 910–919. https://doi.org/10.5114/aoms.2016.63743.
(4) Carvalho, M. P.; Costa, E. C.; Miguel, S. P.; Correia, I. J. Tumor Spheroid Assembly on Hyaluronic Acid-Based Structures: A Review. Carbohydr. Polym. 2016, 150, 139–148. https://doi.org/10.1016/j.carbpol.2016.05.005.
(5) Bahcecioglu, G.; Basara, G.; Ellis, B. W.; Ren, X.; Zorlutuna, P. Breast Cancer Models: Engineering the Tumor Microenvironment. Acta Biomater. 2020, 106, 1–21. https://doi.org/10.1016/j.actbio.2020.02.006.
(6) Gopinathan, J.; Noh, I. Recent Trends in Bioinks for 3D Printing. Biomater. Res. 2018, 22 (1), 1–15. https://doi.org/10.1186/s40824-018-0122-1.

Mesenchymal stem cells (MSCs) have been widely studied during the last thirty years for their potential applications in tissue engineering and cell therapy. They are multipotential cells that produce bioactive factors for the proliferation of tissue specific stem cells, inhibition of apoptosis, angiogenesis signaling, and modulation of the immune response. However, there are still unmet challenges for their translation to the clinic, such as the gradual osteogenic differentiation in vitro caused by the stiffness of tissue culture plastic, and the low cell survival and engraftment in vivo caused by the lack of vascularization into scaffolds, ischemia, and poor removal of cell metabolic waste. A way to address these challenges is by developing scaffolds with suitable chemical and mechanical properties that both mimic the stem cell niche and promote extended survival of MSCs. In this study, we propose the utilization of multidomain peptides (MDPs) as a base for such scaffolds, evaluating different chemical functionalities and their effect on stem cell phenotype preservation, survival, maintenance of immunomodulatory properties, and stem cell proliferation and differentiation.
MDPs are self-assembling peptides with an ABA motif, where B represents an amphiphilic domain of alternating hydrophilic and hydrophobic amino acids, and A represents the terminal domains that control peptide self-assembly. In aqueous solutions, MDPs self-assemble into nanofibers to form compliant hydrogels with storage moduli between 20 and 1200 Pa. In the present study, MSCs were 3D encapsulated in O₅(SL)₆O₅ (a neutral MDP), K₂(SL)₆K₂ (a cationic MDP), and D₂(SL)₆D₂ (an anionic MDP) hydrogels, to evaluate the cell viability and cell density between one and five days post-encapsulation. While K₂(SL)₆K₂ proved to be highly cytotoxic to MSCs and D₂(SL)₆D₂ showed high cell viability with moderate proliferation, the results obtained from O₅(SL)₆O₅ show a high cell viability percentage with low cell proliferation rate throughout the experiment. This is indicative of cells that have entered quiescence, a metabolic state also known as cell cycle arrest that is known to enhance the survival of MCSs in ischemic environments and improving their engraftment in vivo. Stem cell phenotype and quiescence induction is being further characterized by quantifying the expression of CCNB2 (expected in proliferating cells) and CDNK1 (expected in quiescent cells), and the retention of differentiation potential by inducing osteogenesis and adipogenesis on MSCs pre and post encapsulation. The maintenance of immunomodulatory properties was evaluated via IL-6 secretion measurements using ELISA, done by encapsulating cells in MDP hydrogels for 1, 5, 10, 15 and 20 days before retrieving them, stimulating them with TNFα and IFNγ for 24 hours, and recovering the supernatant to measure IL-6 concentration. From these experiments, neither O₅(SL)₆O₅ and D₂(SL)₆D₂ showed a decrease in IL-6 production, indicating the maintenance of MSCs immunomodulatory properties.
Overall, our current data suggest that by encapsulating MSCs in some MDP hydrogels, cells enter a quiescent state during encapsulation that could enhance their stem cell phenotype and preserve their immunomodulatory properties. Such scaffolds would have potential as delivery vehicles for enhanced survival and engraftment of MSCs in vivo and have broad applications for facilitating the use of stem cells in medical research and the clinic.

Transected peripheral nerve injury (PNI) causes loss of sensory and motor functions in patients, resulting in restricted daily activities and poor quality of life. Despite the use of autografts and commercial nerve guidance conduits (NGCs) in treating PNI, their limitations give rise to further research in bioengineered, artificial NGCs. Previously, cationic multi-domain peptide (MDP) hydrogels have accelerated nerve regeneration in rat crushed injury models, as demonstrated by higher sciatic function indices and degrees of myelination in experimental groups. To study their neuroregenerative capabilities, cationic and anionic MDP hydrogels were loaded in poly(caprolactone) conduits to bridge 10 mm sciatic nerve defects. Different rates of axonal outgrowth were observed between MDPs-treated groups after 3 weeks. For 16 weeks implantation, anionic MDP groups showed earlier occurrence of flexion contractures, greater amplitude of compound muscle action potentials, and higher axonal density/ myelination percentage. These results indicate better nerve recovery and potential application of anionic MDPs in the clinical settings, which may lead to more effective NGC systems when combined with other bioactive modifications.

Liquid crystal polymer networks (LCNs) and elastomers (LCEs) are gaining attention as bio-inspired materials for tissue engineering because of their parallels to biological architecture at multiple length scales. At the tissue level, dynamic epithelial monolayers can be modeled as active nematic liquid crystals and muscle tissues possess anisotropic material properties analogous to monodomain LCNs or LCEs. On the molecular scale, (pro)collagen fibers display liquid crystalline phases and cytoskeletal proteins within the cell can self-organize into nematic phases. Prior research has focused on 2-dimensional LCN substrates and rigid, porous scaffolds. These LCNs are stiff (E~MPa–GPa) compared to native tissues (E~Pa–kPa) and do not present the 3-dimensional (3D) cell-matrix interactions that cells normally experience in vivo. Meanwhile, poly(ethylene glycol) (PEG) based hydrogels have been used extensively as synthetic matrices for 3D cell culture.
We hypothesized that introducing PEG into LCE compositions would be a route to better match native tissue properties while retaining the anisotropy of liquid crystals. To this end, we detail an approach in which we oligomerize commercially available reactive liquid crystal mesogens and linear hydrophilic PEG spacers. Using base-catalyzed thiol-Michael addition followed by acrylate or thiol-ene photopolymerization, we retain and can dynamically adjust liquid crystalline character in the final hydrogel. X-ray studies, differential scanning calorimetry, and mechanical testing of bulk hydrogels (shear rheology, dynamic mechanical analysis) confirm the retention of the liquid crystalline phases in swollen gels. While PEG hydrogels require the use of adhesive peptide sequences to facilitate cell adhesion and proliferation, cells grew on LC-PEG hydrogels without these modifications. Furthermore, LC-PEG block copolymers were amenable to 3D extrusion printing. Hydrated oligomers were molecularly aligned after printing and retained alignment after secondary photo-crosslinking even when subject to repeated swelling and drying cycles. While cells can easily be cultured on the top of these printed scaffolds, cells can also be mixed with LC-PEG oligomers before printing, enabling 3D bioprinting into complex geometries.

Collagen I and hyaluronic acid (HA) are commonly utilized to form hydrogels that mimic the extracellular matrix for tissue engineering applications. Scaffold microstructure, component incorporation, and modulus govern molecular transport and provide biochemical and mechanical signals for cellular proliferation, differentiation, and migration. Despite their widespread use in tissue engineering, the physical and mechanical properties of these materials are variable across studies utilizing similar conditions. In our study, we developed an empirical model toward the formation of tunable, well-defined, and robust collagen-HA blended hydrogels (ColHA) for a wide range of tissue engineering applications.

Statistical modeling was utilized to fully characterize the design space of the collagen and HA blended hydrogels. In particular, we probed the contribution of parameters, such as HA concentration, HA molecular weight, and the polymerization temperature of collagen, to modulate materials properties, including scaffold modulus, microstructure, and functional assays such as molecular transport through the gels. Hydrogels were formulated with 4 mg/mL of collagen, and physiologically relevant HA concentrations (0, 0.5, 1, and 2 mg/mL) and molecular weights (50, 500, and 1500 kDa) were chosen to fully represent various in vivo tissues and processes. It is known that the entanglement point, or the concentration at which polymer chains form entangled networks, of 1500 kDa HA is around 1 mg/mL.¹ Thus, the molecular weights and concentrations chosen reflect HA behavior below, at, and above the entanglement point. Numerous matrix polymerization temperatures (15, 20, 25, and 37 °C) were investigated to capture the full range of fibril formation capabilities of collagen.

Robust fabrication protocols were developed and validated by five individuals reproducing the matrix and resulted in uniform component incorporation and microstructure. The collagen incorporation into the ColHA gels after 14 days is nearly 100%, and thus independent of polymerization temperature and HA presence in the gels. The HA incorporation appears to be directly related to the entanglement point of HA. The gels consisting of HA above entanglement point appear to have slower diffusion of HA out of the ColHA gels compared to those gels containing HA below the entanglement point. Polymerization temperature appears to have an effect on gel swelling as the percent mass change is smallest for gels polymerized at 37 °C and largest for gels polymerized at 15 and 20 °C. The changes in microstructure that arise from varying polymerization temperatures result in a range of mechanical properties. ColHA gels polymerized at 25 °C resulted in gels with the highest complex modulus (G*), which was three times higher than those of gels polymerized at 37 °C. A Transwell mass recovery assay was performed to assess how different matrix properties alter diffusion profiles. The Transwell assay highlighted the importance of electrostatic charge and viscous matrix effects for mass transport efficiency of various molecules through the matrices. Overall, the results of this study demonstrate that ColHA gels span a large design space that can be tailored for tissue engineering applications with specific mechanical properties, fibril microstructure, equilibrium HA concentration, and macromolecule transport profiles.


¹Morris, E. R., Rees, D. A., Welsh, E. J. Conformation and Dynamic Interactions in Hyaluronate Solutions. J. Mol. Biol. (1980) 138, 383-400

Collagen hydrogels are used extensively in literature for a wide range of applications. Collagen I hydrogels are the most prevalent type explored due to its availability and lower cost. However, in native tissue, multiple collagen types are often found to work in conjunction to create stable structural support for tissues and provide specific biological cues for cells. Thus, blended hydrogels have been studied, under physiological conditions, for various applications such as cartilage tissue engineering (I/II)¹ and for cardiac and vocal tissue engineering (I/III)² to more accurately recapitulate native tissue. The addition of collagen II and III for these applications resulted in improved biological results which highlights the importance of blended collagen hydrogels. The impact of polymerization temperature on the polymerization of collagen I hydrogels has been explored in literature and has been shown to have a profound impact on the microstructure and resulting mechanical and transport properties of the hydrogels.
This work extends the notion of exploring polymerization temperature to adjust the gelation of blended hydrogels to alter hydrogel microstructure and thereby tune the mechanical and transport properties of the gels. In this work, we studied the effect of various polymerization temperatures (25, 30, and 37 °C) on the polymerization kinetics of blended hydrogels (I/II and I/III) compared to collagen I hydrogels alone, and the corresponding impact on fundamental hydrogel properties. Through turbidity measurements and rheological time sweeps, we found that the addition of collagen II or III slows the polymerization time compared to pure collagen I hydrogels. Additionally, within each hydrogel type, increasing temperature reduced polymerization time. The differences in polymerization kinetics contributed to variable microstructures. Hydrogels polymerized at 37 °C appeared similar across the different polymerization conditions tested. These gels resulted in smaller, more compact fibrils. When polymerization temperatures were lowered to 30 °C, the blended hydrogels began to exhibit differences as compared to collagen I alone. Microstructure differences in the hydrogels led to variations in mechanical properties. Mechanical properties for all gel types were the highest for hydrogels polymerized at 25 °C and lower for both 30 and 37 °C. When exploring the transport properties, the addition of collagen II or III to collagen I altered the diffusion of dextran through the matrix as observed by Transwell macromolecular recovery studies. Finally, the degradation rates of the gels slowed under collagenase with the addition of collagen II or III.
¹Kilmer, C. E. et al. Collagen Type I and II Blend Hydrogel with Autologous Mesenchymal
Stem Cells as a Scaffold for Articular Cartilage Defect Repair. ACS Biomater. Sci. Eng. 6,
3464–3476 (2020).
²Roman, B. et al. A Model for Studying the Biomechanical Effects of Varying Ratios of Collagen Types I and III on Cardiomyocytes. Cardiovasc. Eng. Technol. (2021).

Multifunctional biomaterials which exhibit well-defined physicochemical properties and encode spatiotemporally controlled biological signals are emerging as next generation advanced cell culture systems. One of the most biologically potent and interesting molecules are self-assembling peptides. Minimalistic approaches can be used to find short peptide sequences with tendency for aggregation towards particular structures.¹ Indeed, by the choice of primary peptide sequence, functional materials with defined nanostructure, mechanics and biological responsiveness can be designed.² So-called β-sheet forming peptides are very attractive for the design of biomaterials, in particular hydrogels, emulgels/emulsions and microgels.^3-5 These materials are highly hydrated and built from amphiphilic or polymeric nanofibres that can mimic extracellular matrix (ECM),⁶ and have been shown to be biocompatible and tailorable in terms of their physicochemical and biological properties.
In this presentation we uncover the link between basic molecular interactions driving self-assembly of functional peptide-based biomaterials (hydrogels, emulgels)^{3, 5} and demonstrate their capabilities as platforms for a range of biomedical applications spanning tissue engineering, controlled drug delivery, controlled organoids growth as well as shed light on their new immunopharmacological potential when formulated with hyularonic acid. Chemical modifications of amino acids including edge-modifications and substitions will be discussed and their impact on generating biofunctional materials revealed.
These materials were used to produce human induced pluripotent stem cell (hiPSC)-derived kidney organoids, which have prospective applications ranging from basic disease modelling to personalised medicine. We demonstrate how the self-assembling peptide hydrogels with varied mechanical properties affect kidney organoids growth and provide fully controllable and physiologically relevant 3D growth environments for differentiation of cells, critically improving organoid reproducibility and maturation, as compared to the animal-derived Matrigel matrix. The resulting organoids contained complex structures comparable to those differentiated within the animal-derived matrix. Single-cell RNA sequencing of organoids compositional analysis revealed a larger proportion of nephron cell types within Transwell-derived organoids, while organoids grown in peptide-based hydrogels were enriched for stromal-associated cell populations. Notably, differentiation within a higher stiffness matrix generated podocytes with more mature gene expression profiles. As such, tuneable peptide-based platforms are effective minimally complex microenvironments for the selected differentiation of kidney organoids with distinct subpopulation dependent on the selected stiffness.
Acknowledgements
The authors acknowledge support from Science Foundation Ireland (16/IA/4584 and 13/IA/1840), from the Royal Society of Chemistry (M19-6613) and from Manchester BIOGEL. J.K.W. acknowledges European Union’s Horizon 2020 (H2020-MSCA-IF-2019) research and innovation programme under the Marie Sklodowska-Curie grant agreement 893099 — ImmunoBioInks.
References
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Hydrogel networks synthesized through efficient click reactions have become prevalent biomaterials to further our understanding into how cells interact with their surrounding environment¹. Intracellular hydrogelation is an emerging application for these materials and presents new opportunities for hydrated polymer networks. The intracellular microenvironment is highly crowded and is composed of complex biochemical networks that regulate the internal state of a cell. Through hydrogelation, intracellular macromolecular crowding can be achieved, reducing reaction rates on a cellular level, and creating gelation-induced quiescence. It is envisioned that this technique could expand routes for preservation, reducing the reliance on costly cryopreservation techniques and cold-chain transportation². Herein, we induce gelation in the cytosol of living cells with high molecular weight poly(ethylene glycol) (PEG) macromers through click reactions causing supraoptimal macromolecular crowding. PEG macromers were functionalized with either dibenzylcyclooctyne (DBCO), a ring strained alkyne, or a terminal azide to form networks through a spontaneous strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. For photocatalyzed intracellular gelation, PEG macromers were functionalized with norbornene or thiol reactive groups. With the additional of nitrobenzyl moieties (UV-degradable) into the polymer network, degradation of the network could be achieved in a spatiotemporal manner. Efficient transfection of PEG macromers into various cell lines and primary cells was achieved with lipofectamine as confirmed through flow cytometry (>85%) with minimal apoptosis. Intracellular PEG uptake was quantified and confirmed higher retention of the gelled network in comparison to individual PEG macromers. Using immunofluorescence, flow cytometry, and bioactivity assays, cellular PEG uptake, structure, and proliferation were assessed. The effects of intracellular hydrogelation included decreased DNA turnover, delayed new protein synthesis, and disrupted cytoskeletal organization. To track cell-cycle progression in real time, cells with a genetically encoded fluorescent sensor for Cyclin-Dependent Kinase 2 (CDK2) activity were implemented³. CDK2 activity builds up continuously in cells that are actively progressing through the cell cycle and turns off when cells enter gelation-induced quiescence. By tracking single cells in real time, intermitotic times were demonstrated to increase with gelation, and a reduction in cellular speed indicated a decrease in motility (30 μm/hr vs. 15 μm/hr). Fluorescent correlation spectroscopy (FCS) was employed to understand changes in the cytosol’s viscosity after intracellular crosslinking⁴. With this technique, it was demonstrated that hydrogelation significantly slowed diffusion inside the cytoplasm, doubling the viscosity of the cytoplasm on a nanometer length scale when PEG macromers were crosslinked. Wound healing assays demonstrated reduction in the coordinated response of cells to repair after intracellular gelation, suggesting potential relevance for this technique for wound preservation. With the addition of UV-degradable moieties into PEG macromers, the effects of intracellular gelation were reversed through degradation of the hydrogel network. Intracellular polymerization using bioorthorgonal click reactions offers new opportunities to further explore intracellular dynamics and the effects of intracellular polymerizations. It is envisioned that this high-throughput method will enable the expansion of biomaterial-based approaches to study a facile, low-cost method of preservation.

1) M. W. Tibbitt, K. S. Anseth, Sci. Transl. Med. 2012, 4, (160), 160ps24-160ps24.
2) L. J. Macdougall, et.al., ACS Biomater. Sci. Eng. 2021, 7, (9), 4282-4292.
3) S. L. Spencer, et.al., Cell. 2013, 155 (2), 369–383.
4) K. Kwapiszewska, et.al., J. Phys. Chem. Lett. 2020, 11, (16), 6914–6920.

Injectable hydrogels have great potential in healthcare, as they can locally deliver therapeutics to target tissue in a minimally invasive manner, overcoming the negative side-effects of systemic delivery. However, injectable delivery to mechanically active tissues, such as the heart, has been notoriously difficult, as the injected material is often pushed out of the tissue, leading to loss of the therapeutic cargo. Therefore, there is a pressing need to develop hand-injectable hydrogels that are biocompatible and mechanically stiff enough to be retained within mechanically active tissue.

To address this demand, our group hypothesized that a hydrogel crosslinked with dynamic covalent bonds; which are strong like primary bonds, yet dynamic like secondary bonds; would result in the desired mechanical properties. For clinical translation, the ideal material would be cell-adhesive, biodegradable, and fully chemically defined. Thus, we chose two recombinant biopolymers to comprise our gel system: (1) an engineered elastin-like protein (ELP) modified with hydrazines and (2) a hyaluronic acid (HA) modified with aldehydes. Upon mixing, the two components spontaneously form a network, which we term HELP gels, as the hydrazines react with the aldehydes to form dynamic covalent hydrazone bonds.

To investigate the role of molecular-level material design choices on the macroscopic mechanical properties, we synthesized a family of HELP gels with control over the HA molecular weight (20 - 100 kDa), the degree of HA-aldehyde modification (6-30%), and the kinetics of the dynamic hydrazone bond (a faster aldehyde moiety and a slower benzaldehyde moiety). The ELP component was kept constant with a molecular weight of 37 kDa and 14 hydrazines per molecule. These gels achieved plateau shear storage moduli (G’) ranging from 10 - 1,000 Pa. At the lowest HA molecular weight, gel stiffness was predominately dictated by the ratio of hydrazine to aldehyde groups, with a theoretical ratio of 1:1 predicted to have the highest stiffness. In general, as HA molecular weight was increased, this led to higher gel stiffness, presumably due to enhanced chain entanglements.

Injectability of each sample was assessed empirically following two criteria important for clinical translation: injectability with one hand post complete gelation through a 30-gauge syringe and the absence of burst injection, which can produce local tissue damage. We determined that the molecular weight of the HA had a direct impact on the injectability of the resulting hydrogel, with all gels containing HA of 60 kDa or less being easily injectable through a 30-gauge syringe. Further, the kinetics of the hydrazone bond was also found to impact injectability, with the 100-kDa, benzaldehyde-modified HELP system not being injectable, while the aldehyde-modified version was injectable. To demonstrate potential clinical translatability, we observed that some gel formulations were injectable through a 5-ft catheter, allowing for potential minimally-invasive delivery to different systems in the body.

In summary, we have demonstrated a reproducible synthetic strategy to produce injectable HELP gels with tunable molecular-level design parameters. Our analyses of gel mechanics and preclinical injectability show that both HA molecular weight and kinetics of the dynamic covalent hydrazone bond impact gel injectability. This biocompatible, catheter-injectable HELP gel has great potential to significantly improve the retention of therapeutic cargo within the beating heart, opening a door to direct prolonged delivery of pharmaceuticals and gene therapies.

Microporous annealed particle (MAP) scaffolds are materials composed of hydrogel microparticle (HMP) building blocks. Thus, rather than use polymers as the building block that form the hydrogel, we use particles. This makes MAP scaffolds granular materials, which open unique properties such as inner porosity, exterior porosity, injectability, and heterogeneity. We have found that these properties make MAP uniquely suited for applications in tissue regeneration applications. We have found that simple changes in the MAP composition can have dramatic changes in the immune response to the material and subsequent regenerative healing response. This talk will cover the concept of MAP, software that we have developed to understand MAP microstructure, and some of our findings that relate the immune response and regenerative healing.

Extrusion-based three-dimensional (3D) bioprinting is an emerging biofabrication technique in which bioinks composed of a mixture of cells and polymer are spatially patterned to create tissue-like constructs. While advances have been made to enable printing of more geometrically complex architectures, the technique remains limited by a lack of biomaterials suitable for both printing and subsequent cell culture. Since it is well-established that cell behaviors such as growth, migration, and differentiation are dependent on matrix properties, it is becoming increasingly important to tailor a bioink’s matrix cues to the cell type of interest in order to create more biomimetic and biofunctional structures. In particular, recent work has demonstrated the importance of a material’s viscoelastic, time-dependent properties in regulating cell behavior. Most extracellular matrices within the body are viscoelastic and stress-relaxing, which allows for dynamic remodeling of the matrix through cell-generated forces. However, few currently available bioinks are able to replicate such dynamic behavior. Here, we present a bioink crosslinked by dynamic covalent bonds which enable cellular remodeling within printed constructs, and demonstrate how tuning the kinetics of these dynamic bonds impacts bioink printability. To prepare these inks, we modified hyaluronan (HA) with aldehyde functional groups and a recombinant elastin-like protein (ELP) with hydrazine functional groups to create a hyaluronan elastin-like protein (HELP) matrix. When mixed, these two components form hydrazone bonds, which can break and reform at physiological conditions, creating a material that is stress-relaxing, but difficult to extrude. We modulate the printability of these inks by introducing two small molecules: a competitor and a catalyst. The competitor binds to aldehydes present on the functionalized HA, which disrupts crosslink formation and thus reduces the overall stiffness of the ink. The catalyst increases the rate of hydrazone bond exchange and thus increases the ink’s ability to shear-thin. Together, these two molecules enable the ink to be more readily extruded during printing. When printed into a gel support bath, these small molecules can then freely diffuse away from the printed structure while the ink remains in place, stiffening and stabilizing the final structure. We demonstrate that addition of the competitor reduced the stiffness of the HELP material up to three orders of magnitude in a dose-dependent manner. Addition of the catalyst was shown to improve the ink’s ability to shear-thin without changing the overall stiffness of the material. Sufficient diffusion of the competitor and catalyst out of the printed structure occurred within hours, allowing stabilized printed structures to be removed from the support bath and cultured for one week. Finally, we utilize these inks to print a 3D model of breast cancer invasion, and show that incorporating dynamic crosslinks into the ink increases growth factor-induced cell invasion as compared to a statically crosslinked control. Altogether, we present a dynamic bioink material in which we are able to modulate printability and long-term stability based on the addition of a small molecule competitor and catalyst to create 3D in vitro models of the tumor microenvironment that better recapitulate native physiology.

Introduction/Motivation: Organoids have emerged as the current state-of-the-art multicellular in vitro model for nearly all mammalian tissues and organs. As organoid culture methods have been refined, their utility has become unmatched in developmental biology and disease modelling, owing to their biomimetic cellular composition and 3D architecture, as well as their capacity to match tissue and organ-level functionality on the benchtop. Concurrent to advances in organoid culture, significant progress has been made in 3D imaging, which has enabled characterization of cell composition and fate with spatiotemporal precision. Traditionally, organoids have been imaged by embedding and sectioning or by removal from their culture microenvironment, but the intricacies of the 3D architecture, as well as the role of extracellular matrix (ECM), are lost with these methods. Noninvasive optical sectioning microscopy, such as confocal microscopy, has been used for in situ imaging, but signal attenuation is a persistent issue with confocal imaging techniques, thus requiring additional processing through optical clearing to achieve artifact-free imaging. Additionally, biomolecules smaller than the diffraction limit of light cannot be resolved. To address these numerous limitations, we have developed a method, termed photo-expansion microscopy (PhotoExM), to image organoids grown in natural and synthetic hydrogels. This method imparts a multi-factorial advancement in organoid imaging by overcoming signal attenuation, achieving nanoscale resolution, and facilitating in situ imaging of cell-matrix interactions.
Methods: Murine intestinal organoids were cultured in both natural hydrogels (Matrigel) and phototunable PEG hydrogels. Samples were immunostained using standard methods, then fluorophores were linked with acryloyl X to the expansion gel, which was generated by a thiol/acrylate mixed-mode photopolymerization. Unlinked proteins were then digested, the biomaterial was degraded or photo-transferred, and the photopolymerized expansion gel was immersed in diH₂O for expansion. Super-resolution, post-expansion imaging (PostExM) was performed by conventional confocal microscopy. To analyze cell-ECM interactions and ECM dynamics, we developed a custom Matlab script, where we could quantify (on a pixel-by-pixel basis) the thickness of the ECM, as well as integrin-ECM co-localization.
Results: Intestinal organoid growth and crypt formation was achieved in both Matrigel and synthetic, phototunable PEG hydrogels. Following fixation and immunolabeling, organoids were isotropically expanded ~4.3x, corresponding to single point resolution of ~120 nm with a conventional confocal microscope. Analysis of PreExM z-stacks revealed striking signal attenuation with ~50% reduction in fluorescent intensity at a depth of just 40 µm, much smaller than the size of many intestinal organoids. PostExM, signal attenuation was eliminated, facilitating imaging of large z-stacks without signal loss. Further, heterogeneities in cell-matrix interactions were imaged and quantified before and after crypt formation, revealing a potential role of differential cell-matrix interactions in organoid symmetry breaking. Finally, using phototunable synthetic hydrogels, we were able to assess the spatial organization of newly synthesized ECM proteins and cell-matrix interactions during organoid growth and crypt formation.
Conclusions: Intestinal organoids can be expanded in both Matrigel and synthetic, PEG-based hydrogels to achieve resolution at the nanoscale. The method presented here for PhotoExM also eliminates signal attenuation and can provide broad utility for super-resolution imaging of organoids derived from any tissue, while maintaining 3D architecture and ECM context.
Acknowledgements: NIH R01 DK120921, NSF RECODE 2033723

Toward more physiological disease models and efficacious therapies for heart failure, researchers aim to recreate the three-dimensional myocardial architecture in engineered tissues using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we report a novel bioprinting method to align heart tissue in arbitrary geometries at high cellular densities. First, we generated a bioink composed of cellularly aligned, contractile cardiac tissues, which we termed anisotropic organ building blocks (aOBBs). Next, the extrusion of this densely compacted bioink caused the aOBBs to orient parallel to the direction of the print path. To demonstrate our spatial control of cardiomyocyte alignment, we generated linear, spiral, and chevron patterned centimeter-scale sheets. In filaments suspended between elastomeric pillars, discrete aOBBs fused into electromechanically synchronous tissue. Cardiac maturation markers and functional values of force and conduction velocity increased overtime. These studies demonstrated the capacity to generate functional heart tissue with arbitrarily complex cellularly alignment at high cellular densities.

The extracellular matrix directs stem cell function through a complex choreography of biomacromolecular interactions in a tissue-dependent manner. Far from static, this hierarchical milieu of biochemical and biophysical cues presented within the native cellular niche is both spatially complex and ever changing. As these pericellular reconfigurations are vital for tissue morphogenesis, disease regulation, and healing, in vitro culture platforms that recapitulate such dynamic environmental phenomena would be invaluable for fundamental studies in stem cell biology, as well as in the eventual engineering of functional human tissue. In this talk, I will discuss some of our group’s recent successes in customizing the chemical and physical aspects of synthetic/recombinant cell culture platforms with user-defined spatiotemporal control through genetically encoded photochemistries. Results will highlight our ability to modulate intricate cellular behavior including stem cell differentiation, protein secretion, and cell-cell interactions in 4D.

An ability to switch a nano-structure on demand is important for enabling a dynamic control over the material properties. However, achieving this via highly specific triggering and with single nano-object precision presents a significant challenge. To tackle this problem, we developed method for assemble designed and switchable DNA-based “screen” composed of addressable pixels for the manipulation of nano-object arrays. We established approaches for creating the desired screen and their switching to exhibit different types of patterns from nano-objects. Our study shows that the DNA screen can display single to multiple types of nano-objects in discrete and reconfigurable mesoscale patterns that can be visualized by electron microscopy and atomic force microscopy. We have demonstrated that switching between different patterns can be realized predictably and by-design, and quantified efficiencies of the screen assembly and switching processes. We further showed that developed switchable screens allow for structural control of complex material systems composed from inorganic nanoparticles and proteins, as well as can be used for readout of molecular-level algorithmic operations.

Barnacles adhere permanently underwater through amyloid materials that are delivered as a liquid of free proteins, triggered to assemble, and set as a bulk adhesive in extreme seawater environments. More cosmopolitan than most other fouling organisms, barnacles are able to remain permanently adhered at frigid ocean depths, as well as on hot intertidal coasts. We have previously been successful in designing sequences that can mimic the natural glue chemistry and structure, however bridging the gap between natural sequences and materials of practical use still remains a challenge. Here, we mimic protein aggregation from the barnacle and use unmodified food proteins as model systems to fabricate adhesives by curing them at the adhesive joint. We use processing conditions to control protein assembly and identify the effect of amyloid structure on adhesive strength. Using thermal processing, we fabricate adhesives that approach the underwater lap shear strength of commercial marine and contemporary bioinspired chemistries. Though we find differences in adhesive behavior between the examined proteins, the presence of amyloids improves underwater performance for a given protein. As many proteins can form amyloids under the proper conditions, our results encourage further research into in situ self-assembled protein adhesives across proteins of varying chemistry. We show that commonplace amyloidogenic proteins can be delivered as a liquid, triggered to cure with chemistry or heat, and form strong underwater adhesives at the contact. The aggregation of commonplace proteins is therefore a viable pathway in creating a new class of strong underwater adhesives which, like the organisms that use them, can operate in extreme underwater conditions.

From DNA to proteins, from cells to plants, biomaterials demonstrate superior functions by harvesting environmental resources for adaptive assemblies and actuations. The movement, whether passive or active, are largely dependent on amplification by geometry and material anisotropy in response to environmental cues. Inspired by nature, we create soft polymer networks with tailored molecular chemistry and anisotropy at nanoscale. Together with geometric designs at micro- and macroscales, we pre-program and reprogram material’s dynamic and active responses. Further, by incorporating 1D and 2D nanomaterials (e.g. carbon nanotubes, graphene and gold nanorods) and chiral dopants, we demonstrate soft robots with tendon-like actuators, shape shifting into different curvatures, and inflatable 3D displays in response to heat, light, magnetic field, electric field and pressure.

This talk will present attempts towards designing and assembling a biocompatible artificial electric organ that may be implanted into living organisms to produce electrical power. The inspiration for this functional material stems from the electric organs of strongly electric fish such as the electric eel and the torpedo ray. The talk will demonstrate that stacking hydrogel compartments with alternating high and low salt concentration between appropriate hydrogel membranes makes it possible to power a microcontroller and small electrical devices. It will introduce first attempts to power artificial electric organs continuously by converting metabolic energy into electricity and it will discuss obstacles and possible future strategies for achieving this far-reaching goal.

For some commonly used chemotherapeutic cancer treatments, there is an issue that the substance is ineffective on dormant cells, and in some cases can itself induce cell dormancy. This leads to a less efficient treatment and a higher risk of cancer reoccurring [1]. As a step towards developing new, more efficient cancer treatments, it is of great importance to have a platform where it is possible to study the behavior and response of dormant cells to different treatments. Such a cell dormancy switch would enable the possibility to make cells enter the dormant cell cycle state in a controlled way, to further study the dormant cell phenotype.
Here, we use illuminated photovoltaic indium phosphide nanowires to induce dormancy in cancer cell lines. A549 lung cancer cells are seeded on the nanowire array, which is then illuminated. Fluorescence microscopy is used to investigate the proportion of active cells on the array. Nanowires without photovoltaic properties are used as control. A significant difference in proportion of active cells is seen when comparing cells grown on illuminated photovoltaic nanowire arrays, compared to controls [2].
The device is now tested on multiple cell types, both cancer cells and normal cells. Cells will be exposed to Cisplatin, which is a chemotherapy substance leading to an accumulation of cells in the sub G1 cell cycle stage and eventually inducing apoptosis [3], to assess the effect of the drug on nanowire-induced dormant cells. Variation of light source intensity and chemical environment will be done as a way of increase the efficiency of the developed cell dormancy tool.

[1] Gao et al, Onco Targets Ther., 2017, 10, 5219-28
[2] Olsson et al, Nanoscale, 2020, 12, 14237
[3] Velma et al, Biomarker Insights, 2016, 11, 113-121

Neural probe technologies link the nervous system and external system and help to reveal the neural mechanisms underlying psychological and neurological disorders. To extend the capability to investigate the complicated neural activity with multiple modalities, it is important to integrate independent functional components within miniaturized devices to maintain multifunctionality and minimize tissue damage. Using soft hydrogel substrates, here we developed a fabrication technique to miniaturize hydrogel-based soft multifunctional neural probes with tunable polymer nano-crystallization processes. By introducing in-situ crosslinking and controlling the growth of polymer nano-crystalline, we were able to trigger large volumetric shrinkage during hydrogel probe fabrication. By covalently anchoring polymer chains and the surface of individual components, hydrogel optical waveguides, elastic microfluidic channels, and soft electrodes can be integrated into a miniaturized device with a one-step crosslinking process and further be applied into optical stimulation and recording, pharmacological interventions, and electrical recording in mouse brains.

Interrogation and intervention of single-cell functional and transcriptional states in intact three-dimensional (3D) tissues across time and space are crucial to fields ranging from developmental biology to cardiology and neuroscience. Such multimodal methods require stable and continuous recording and controlling of individual cell electrical activity with high spatiotemporal resolution across 3D tissue, multiplexed profiling of a large number of genes in electrically recorded cells, and cross-modal computational data integration and analysis. In this talk, I will introduce 1) “tissue-like” electronics that possess tissue-like properties for long-term stable single-cell electrophysiology in tissues, organs, and behaving animals; 2) “cyborg organisms”, where stretchable multifunctional sensor arrays are embedded in 2D sheets of stem/progenitor cells and reconfigured through 2D-to-3D organogenesis, enabling 3D multimodal interrogation and intervention over the time course of development; 3) “in situ electro-sequencing” that integrates “tissue-like” electronics, spatial transcriptomics, and machine learning to chart cell electrophysiology and gene expression at single-cell resolution across time and space; and 4) genetically targeted functional assembly in tissue, where living neurons are genetically instructed to guide electrically functional components onto the plasma membrane for bidirectional remodeling of cellular membrane properties and modulation of cell-type-specific behaviors in freely-moving animals. Finally, I will discuss potential applications of these technologies in biology and medicine.

Amyloid-like nanoribbons (Amel NRs) of amelogenin protein and derived peptide analogues similar in ultramicrostructure to those found in vivo were recently discovered to promote formation of filamentous apatite crystals via an initial amorphous calcium phosphate (ACP) phase¹. Thus, Amel NRs provide a conceptually simple scaffold for enamel formation. To understand the impact of nanoribbon structure and chemistry on ACP nucleation and identify the physical mechanism of templating we applied in situ atomic force microscopy (AFM) combined with molecular dynamics (MD) simulations to a number of Amel NRs sequences². From the analysis of nucleation rates as a function of chemical potential, our results show that all sequences substantially reduce nucleation barriers by creating low-energy interfaces. Moreover, they dramatically enhance kinetic factors associated with ion binding. Furthermore, the MD simulations predict that the distribution of negatively charged residues along the nanoribbons present a potential match to the Ca-Ca distances of the multi-ion complexes that comprise ACP. However, these experiments were performed on graphite wherein Amel NRs with hydrophobic interfaces cover 100% of the exposed surface area, forming domains that orient randomly along the three equivalent crystallographic directions of graphite. To extend the knowledge from these studies towards templating substrates on which Amel NRs can be patterned for tissue engineering of synthetic bone or tooth, we employed block copolymer (BCP) lamellae with alternating hydrophilic and hydrophobic stripes. We previously demonstrated that thin-films of BCPs, which self-assemble to produce topographically and chemically tunable patterns, can direct localized binding and self-assembly of proteins such as S-layers and collagen fibers³. Moreover, dimensions of Amel NRs are comparable to stripes of BCP patterns and BCP assembly can be tuned to fabricate three-dimensional structures. Therefore, we investigated whether Amel NRs can template ACP or apatite crystals specifically on hydrophobic stripes. Which, without further functionalization, remain inert during mineralization. Topographical and chemical surface characterization of the BCP and BCP coated with Amel NRs by AFM shows that Amel NRs do indeed bind predominantly to the hydrophobic polystyrene (PS) stripes while retaining the β-sheet conformation. In situ AFM observations of subsequent mineralization in either supersaturated solutions or solutions containing a so called “polymer-induced liquid precursor” (PILP) of calcium phosphate demonstrate that nucleation is localized at the PS-Amel NR stripes. Moreover, mineralization potency is sequence specific, following the order observed in the previous results for Amel NRs immobilized on graphite. Furthermore, mineralization using PILP solutions shows that PS-Amel NR stripes can template smooth, filament-like mineral without large grain boundaries, as seen in studies of calcium carbonate nucleation on self-assembled monolayers. Taken together, the results indicate that 1) Amel NRs template calcium phosphate, 2) the functionality of Amel NRs is retained on BCPs, and 3) bottom-up fabrication of filamentous mineral on substrates can be achieved through a combination of low surface energy, high nucleation rates and discrete nucleation sites.
1. Bai, Y. et al. Proc. Natl. Acad. Sci. U.S.A. 117, <a href="tel:19201–19208">19201–19208</a> (2020).
2. Akkineni, S. et al. Res. Sq. (2021) doi:10.21203/rs.3.rs-265667/v1.
3. Stel, B. et al. ACS Nano 13, <a href="tel:4018–4027">4018–4027</a> (2019).

Protein self-assembly is a highly efficient bottom-up process for building organized tissues, including fibrils, membranes, and 3D composites. In the last two decades, engineered proteins have become versatile building blocks for designing new supramolecular materials that mimic natural biomolecular materials for applications in biomedicine, energy, and the environment, due to their monodispersity, atomically tunable interactions, and structural diversity. Although protein self-assembly at solid-liquid interfaces offers opportunities for developing hybrid materials that respond to environmental cues and external triggers, the mechanisms and controlling parameters are largely unexplored when compared to those at work in bulk solution. In this project, we designed β-cyclodextrin and azobenzene modified protein building block l-rhamnulose-1-phosphate aldolase (^CDRhuA and ^AzoRhuA) with predicted host-guest interactions and photo-response via azobenzene photoisomerization. This synthetic strategy results in multiple physiochemically distinct, precisely patterned 2D protein polymorphs. Furthermore, each of ^CDRhuA and ^AzoRhuA also self-assembles individually into two distinct 2D protein polymorphs. To determine the structures, follow the assembly pathway, and delineate the mechanisms underlying the transitions between polymorphs, we used in-situ atomic force microscopy (AFM) to observe the assembly and transition processes of the protein polymorphs. The new level of understanding achieved through these observations provides opportunities for precise control over protein polymorph selection modified through interactions with inorganic interfaces. Moreover, these unique 2D assemblies exhibit high surface-to-volume ratios and porosities, with matter and surface-charge densities toggled between different states in a coherent manner. Thus, they offer potential for applications in adaptive biomedical devices, as well as energy storage and conversion.

Amyloid fibrils are one of the hallmarks of Alzheimer's disease (AD), although a causative link between plaque-forming amyloid fibrils and AD pathology remains to be clarified. This study demonstrates, for the first time for a naturally occurring amyloid, that fibrils comprising the 42-residue Amyloid-β peptides (Aβ42) exhibit significant catalytic properties. Aβ42 fibrils catalyzed the hydrolysis of the model ester, para-nitrophenyl acetate (pNPA), and of acetylthiocholine, a surrogate for the neurotransmitter acetylcholine. Aβ42 fibrils also catalyzed the oxidation of the prominent neurotransmitters, dopamine and adrenaline. Importantly, the catalytic activity was specifically exhibited by mature Aβ42 fibrils, as opposed to the peptide monomers, or oligomeric Aβ42, the putative neurotoxic species. Importantly, maximal catalytic activity was exerted by the full-length Aβ42 fibrils, whereas fibrillar assemblies comprising Aβ42 subdomains exhibited significantly lower catalytic activity. The catalytic activity of Aβ fibrils may be implicated in pathophysiology pathways associated with the generation of AD amyloid plaques

The dynamic nature of living organisms strongly relies on the transduction of mechanical forces into chemical signals, which is based on the conformational changes in proteins or macromolecular architectures to sense weak forces. Here we develop a modular chemomechanical system inspired by those in nature in which weak forces on hydrogels analytically control programmable chemical reaction networks. This system consists of two molecular modules: 1) tunable DNA force sensors and 2) cofactors that transduce force detection into different outputs. The DNA force sensors detect reversible hydrogel deformations without affecting a hydrogel’s mechanical moduli. During reversible deformations of a hydrogel, the hydrogel network could transfer force to DNA force sensors to dehybridize them, exposing a cryptic domain and thus reacting with different DNA complexes, i.e., cofactors to induce different downstream responses. With a DNA fluorophore-quencher reporter complex as cofactor, the DNA force sensor activation is detectable via fluorescence. Their output levels are precise and repeatable when either homogeneous or local forces are applied. With a partial transcriptional template as cofactor, the downstream DNA circuits can transduce, amplify, and broadcast force inputs. We anticipate that our programmable chemomechanical systems can serve as a general strategy for soft autonomous devices which respond to force across space and time.

The assembly and functionality of many biomolecules are coupled to a complex and dynamic solution microenvironment. For example, the effective nature and range of electrostatic forces that mediate interactions between charged objects are determined by (among other factors) the concentrations, types, and distributions of electrolyte ions present in solution. There is a growing body of evidence that the manifestations of electrostatic forces in concentrated solutions can be both counterintuitive and technically useful; thus, studies which help characterize such exotic behavior have the prospect to not only allow for more-informed design of new materials applied in such conditions, but also to open up new degrees of functionality in currently employed synthetic biomaterials. Consider nanoparticles functionalized with DNA, which are often selected for diagnostic or therapeutic applications owing to their capacity for highly selective Watson-Crick base-pairing; such nanoparticles are also highly charged, and thus can serve as responsive probes for changes in the strength and spatial extent of electrostatic interactions in various ionic conditions. In this work, we demonstrate via solution small-angle X-ray scattering (SAXS) that such particles, functionalized with non-base-pairing oligonucleotide sequences, assemble into colloidal crystals in the presence of the biologically relevant Ca²⁺ cation. Our measurements reveal that raising the solution ionic strength induces phase transitions from face-centered cubic (FCC), to body-centered cubic (BCC), to amorphous structures. Furthermore, the nearest-neighbor distances in the lattices begin to increase above a threshold salt concentration, suggesting that an increase in screening length drives the expansion of the spherical DNA brushes; this anomalous lattice expansion is coincident with the appearance of short-ranged correlations between ions in the bulk solution, as revealed by in situ wide-angle X-ray scattering. These initial results demonstrate how DNA-based nanotechnologies can be made to actuate in response to a very wide range of ionic conditions. To explore the generality of these observations, we investigate assembly induced via the addition of other divalent cations and observe similar general behavior, but distinct sequences of structures, suggesting an ion-specific component to these interactions. The influence of other solution parameters which suggest further avenues for manipulating DNA, such as temperature and dielectric constant, will be discussed. In demonstrating how interactions between DNA continue to evolve past the regime where classical theory predicts electrostatics to become negligible, this work suggests that it is possible to confer additional degrees of functionality based on electrostatic forces to other materials employed at, or far above, biological salt concentrations.

Tissue engineering offers great promise as a therapy for damaged tissues, a replacement for whole organs, or a platform for drug screening; however, many synthetic biomaterial scaffolds do not replicate the complexity of the natural extracellular matrix, especially with regard to sequence-specific bioactivity and hierarchical structure. Hence, our work aims to bridge this gap using a bioinspired polymer in synthetic hydrogel scaffolds. Specifically, peptoids are non-natural polymers composed of N-substituted glycines, which preserve the monomer level structure and hierarchical chain order of biopolymers. Using synthetic peptoid crosslinkers, we achieved control over two key properties of hydrogels: 1) bulk mechanics and 2) enzymatic degradability. We controlled hydrogel mechanics by varying peptoid chain stiffness, in a fashion reminiscent of semiflexible biopolymers. Specifically, helical peptoids increased the shear moduli of hydrogels due to increased chain stiffness as compared to non-helical peptoids, while keeping all other hydrogel parameters fixed. These changes in mechanics impacted the amount of secreted factors from encapsulated stem cells, such as indoleamine 2,3-dioxygenase (IDO). Furthermore, we examined the ability of peptoids to tune hydrogel degradability via proteolysis. We substituted peptoids into key sites of proteolytically degradable substrates, enabling a tailored material response to matrix metalloproteinases secreted by cells. Overall, our results suggest that sequence control of synthetic peptoids may provide effective strategies for expanding the functionality of biomaterial scaffolds for tissue engineering, particularly with respect to mechanics and degradation in complex biological environments.

Natural proteins display critical structural and bioactive properties that have evolved in nature for millions of years. However, depending on the specific protein, there may be useful functions, such as mechanical toughness, while other critical features may be more limiting, such as cell compatibility or a broader range of mechanical properties.
Naturally spun silks supply important fibers for a range of established applications, and in their re-liquified, reconstituted form they also provide increasingly valuable raw materials for the manufacture of a growing range of novel and useful products. Such reconstituted Silk Fibroins (RSF) are now a widespread technology platform. However, they are rather different not only in degree but also in kind to the unprocessed, unspun Native Silk Fibroins (NSF) which are found in the silk gland of the animal prior to spinning. Our research focus on characterization of structural, biophysical and biochemical properties of different types of fiber-forming protein materials including amyloid-based and native silk-based molecular complexes to unveil structure-function relationships, with emphasis on studies related to self-assembled biomaterials. We aim to achieve deep understanding of distinctive properties of fiber-forming protein constructs from atomic to macromolecular levels and molecular pathways from pathological entities to diverse functions to enable expand the concept from soluble proteins to insoluble amyloids, from inhibition to functionalization, and from drugs to new materials.

The formation of liquid condensates has been recently implicated as a mechanism of control in biological systems. While non-specific interactions between subunits are considered to be major drivers in the formation of natural condensates, molecules with programmable specific interactions are of particular interest to engineers and materials scientists. The toolkit of DNA nanotechnology has recently provided a framework to design and build subunits for liquid-liquid phase separation (LLPS) using DNA as a programmable biological polymer. Using DNA nanotechnology we are exploring the mechanisms and design rules by which condensates can be controlled through chemical reactions.
Here, we demonstrate, by theory and experiment, how modification of the structure of DNA nanostar subunits affects their macroscopic condensation kinetics. By changing the various domains of the DNA monomer we achieve a predictable growth profile of the condensates. We conceive tunable and reversible dynamic control of condensate formation and dissolution by utilizing DNA strand displacement reactions that deactivate or reactivate the DNA monomer interaction domains. By changing DNA monomer design and sequence makeup, we explore the tunability of these reactions under different conditions. We quantify the temporal evolution of different designs of DNA condensates via fluorescence microscopy and report population-level kinetics of phase separation and condensate growth. We also develop a mean field theory of condensates under the impact of chemicals which disrupt their ability to phase separate, and show how the impact of said chemicals leads to phase diagrams for the system which have similar features to the standard temperature control. We show how this control is dependent on various factors under our direction, such as the concentrations of the chemicals involved, the valency of the nanostars and the size of the nanostars. Our results provide a versatile DNA-based toolkit for designing and controlling macroscopic condensates through sequence design methods. This toolset may be useful to build synthetic membraneless organelles whose formation can be temporally controlled via chemical reactions.

Protein corona formation and opsonization of nanoparticles is one of the major concerns that disturb the targeting receptor recognition. Coating the surface of nanoparticles with polyethylene glycol (PEG) is widely demonstrated as an anti-opsonization strategy to reduce such protein corona formation for improving specific delivery to targeted cells. However, excessive surface passivation with PEG can lead to strong inhibition of cellular uptake and less efficient binding to target receptors, resulting in reduced potential of targeted delivery. To improve the specific cell targeting while reducing the protein corona formation, the secondary packaging of the nanoparticles with shorter PEG chains, making the targeting ligands densely stretched out. In particular, we present the tailored surface functionalization of the porous nanoparticles that require the stealth shealing onto the open-pore region. Since the distance between neighboring PEG chains determines the structural conformation of the grafted PEG molecules, well-controlled PEG combination can efficiently resist the adsorption of serum proteins onto the pores. The balanced PEG conformation with secondary packaging of shorter PEG chains could be a promising anti-opsonization and active targeting strategy for efficient intracellular delivery of the nanoparticles.

Tumors are a complex mixture of cancer cells, non-cancer cells, extracellular matrix, and other stimuli. To maximize clinical translation of in vitro models, it is critical to recapitulate the high degree of complexity found in vivo. This work presents heterogenous in vitro high grade serous ovarian cancer (HGSOC) models that recapitulate key features of the HGSOC tumor microenvironment (TME).
HGSOC is characterized by high rates of chemoresistance development and recurrence due to the presence of cancer stem cells (CSCs) which are inherently chemoresistant and have the capacity to repopulate the entire tumor. The non-cancer cells in the TME, such as mesenchymal stem cells (MSCs), endothelial cells, and immune cells help to maintain CSCs and influence the classification of HGSOC molecular subtypes, which have variable clinical prognoses. However, it is unclear exactly how CSCs and the other TME cells collectively promote chemoresistance and poor outcomes.
To better understand this phenomenon, practical in vitro model systems that more closely represent in vivo microenvironments are needed. We hypothesize that development of more physiologically relevant in vitro models will contribute novel insights into TME-mediated CSC regulation and development of chemoresistance in HGSOC.
In this work, we developed a tumoroid culture system that enabled culture of patient-derived tumor cells with controlled ratios of MSCs, endothelial cells, and immune cells to study how non-cancer cells in the TME drive CSC maintenance and chemoresistance. We found composition-specific changes in CSC marker expression, increased tumorigenic potential, and increased chemoresistance in tumoroids with evidence of epithelial-to-mesenchymal transition (EMT), altered CSC phenotypes, a malignant matrisome signature, and a mesenchymal subtype molecular signature. Together, this indicates that the non-cancer cells in the TME contribute to the development of advanced, chemoresistant disease. Overall, the model presented in this work can be used to better understand the role of CSCs and the TME in chemoresistance and clinical outcomes. These personalized models could ultimately lead to the development of novel therapies, enhanced clinical management, and improved clinical outcomes.

This presentation will describe the development of small molecule tools that can directly modulate the bundling of neuronal actin filaments in vitro and in vivo. Actin is a globular protein that forms linear polymeric microfilament in eukaryotic cells, and is involved in essential cellular functions such as muscle contraction, cell motility, cell adhesion, and cell shape. In neurons, this cytoskeletal protein is also involved in the formation of gap and synaptic junctions that are essential for intercellular communication responsible for processes such as nerve propagation and memory formation. We will discuss the examination of an actin bundling protein that can be modulated by small molecule inhibitors and agonists. Recent results will be presented to highlight the mechanistic details of how these molecules influence activity on actin bundling. We will also present results from in vivo and organotypic brain slice culture studies that highlight the effects of actin bundling modulation on synapse formation and memory and learning.

The majority of dynamic biological phenomena, from signaling cascades to structural rearrangement, involve protein-based assemblies that respond to external stimuli. Such natural protein-based machines and materials have inspired extensive efforts in protein design and engineering. Light represents a particularly attractive stimulus to control protein self-assembly as it is non-invasive, spatially and temporally controllable and does not rely on diffusive processes like chemical stimuli. However, to the best of our knowledge, there are no designed protein assemblies with a built-in light trigger, and most proteins that are engineered to be light-responsive involve fusion to a naturally light-sensitive protein domain. One of the overarching goals of our research program is to develop chemical strategies to design protein assemblies using chemically tunable interactions. Here, we set out to exploit light-controllable, supramolecular host-guest interactions to mediate the formation of artificial protein assemblies, with the ultimate goal of developing novel functional, responsive and self-healing biomaterials. Toward this end, we used the well-characterized β-cyclodextrin (β-CD) as a molecular host and azobenzene as the molecular guest whose UV-visible light-triggered trans-cis isomerization effects the association and dissociation of the host-guest complex. A C₄ symmetric, tetrameric protein, RhuA, was site-specifically labeled with β-CD or azobenzene to yield two complementary building blocks ^CDRhuA and ^AzoRhuA. We have observed that ^CDRhuA and ^AzoRhuA co-assemble into well-defined 1D nanotubes in solution, which were characterized extensively by electron microscopy, X-ray crystallography and atomic force microscopy. Importantly, the formation of the ^CDRhuA-^AzoRhuA co-assemblies was reversible through irradiation with UV or visible light.

Peptides are a major class of therapeutics with lower manufacturing cost and greater stability over antibodies and higher binding affinity and specificity than small molecules. However, the efficacy of peptide drugs is limited by poor stability in plasma, fast clearance and low cell permeability. Using the non-covalent intermolecular interactions, supramolecular peptide chemistry offers the possibility of partitioning peptides into compartments within nanostructures, preventing aggregation or degradation of the peptides in aqueous environment. In this talk, we will describe the co-assembly of lipidated β-sheet-forming peptides, known as peptide amphiphiles (PAs) with soluble short peptides. We have investigated the use of hydrated β-sheet motifs within the PA nanostructures to create supramolecular copolymers with a series of complementary peptides. The integration of cryogenic transmission electron microscopy, circular dichroism spectroscopy and synchrotron X-ray scattering allowed us to characterize the variations of PA nanostructures as well as internal order upon peptide incorporation at different concentrations. The location and mobility of the peptides within the PA nanostructures were probed by transmission Fourier transform infrared spectroscopy and fluorescence anisotropy, respectively. Both kinetic and equilibrated states of these peptide-PA co-assemblies were also characterized, indicating that the metastability of the co-assembly could serve as a potential peptide release mechanism for drug delivery. To demonstrate the potential therapeutic functionality of the peptide–PA supramolecular copolymers, a short peptide as β-amyloid (Aβ) inhibitor for Alzheimer’s disease was co-assembled with the PA molecules, and the efficacy of reducing Aβ neurotoxicity in vitro was evaluated using target-based assays and cell-based assays on primary cortical neurons.

Obesity is an alarmingly common and serious disease. Adipose tissue stores excess calories as triacylglycerol (TAG) and cholesterol ester in lipid droplets (LDs) and mobilizes them as needed. How adipose tissue regulates fat packing in the different depots—white versus brown—during obesity remains poorly understood. We show that adipose LDs are liquid-crystalline, packing TAG in a disordered core and a multilamellar crystalline shell. During obesity, the number of TAG lamellae increases in a manner that is adipose depot-specific. Additionally, collagen packs randomly in white fat forming a permissive environment for LD expansion, but is highly-oriented in brown fat. Finally, during obesity, there is reduced levels of chenodeoxycholic acid (CDCA), a bile-acid (BA). Loss of the BA receptor, Farnesoid X receptor (FXR) leads to enlarged adipocytes. In fact, CDCA, a major ligand of FXR, promotes LD breakdown. These findings implicate that BA profiles, diet, and tissue niche dictate LD remodeling.

The squid sucker ring teeth, used in tandem with the suckers on the squid’s tentacles to capture prey, is comprised of mainly suckerin, a protein material. Squid suckerin is a promising biopolymer for wound dressings due to its biocompatibility, antibacterial properties, and its ability to form gels/films of varying mechanical properties. However, issues such as low recombinant solubility and the requirement of harsh photochemical means for gel/film formation hinders suckerin’s suitability for encapsulation and delivery of stem cell-secretome, which is a bioactive yet sensitive therapeutic shown to overcome persistent wound inflammation and accelerate wound healing. Via bioinspired design, we developed a novel squid suckerin-spider silk fusion protein hydrogel, where the fusion of key spider silk sequences conferred high solubility and heat-induced gelation properties that enabled encapsulation of secretome without denaturation. The protein hydrogel is capable of long-term delivery of secretome via protein-protein interaction. Additionally, due to its modular design, we incorporated cell adhesion peptides to promote biocompatibility of the hydrogel. In our results, we demonstrate the secretome-loaded hydrogel potential for clinical use via accelerated healing of excisional chronic wounds in vivo.

Bone healing complications resulting from non-unions and delayed non-unions are continuing to burden the clinical sector, therefore, there is a growing demand for advancing bone regeneration research. During one of the many, complex, steps in wound healing, inflammatory cells present at the site release a plethora of signalling factors that recruit nearby mesenchymal stem cells (MSCs). Upon binding of these factors to transmembrane receptors on MSCs, cell signalling cascades are engaged, resulting in the initiation of processes such as differentiation. MSCs also have the capacity to secrete paracrine factors in the instance of tissue injury, contributing to the repair response. Due to these important attributes, MSCs and growth factors are extensively researched in tissue engineering to reconstruct or repair mesenchymal tissues. A particular growth factor, namely Bone Morphogenetic Protein 2 (BMP2) is critical for efficient bone formation and mineralisation, as it has the ability to induce osteoblast differentiation in MSCs. So far, the administration of growth factors, such as BMP2, as therapeutics, has primarily focused on bolus or systemic intravenous injections. However, these proteins have a short half-life and tend to diffuse away from their target tissues. To tackle this, we have explored a way in which the growth factor could be produced on-site to minimise the problematic administration of BMP2. It involves the embedment of a Cell-Free Protein Synthesis (CFPS) system into 3D hydrogel networks, giving rise to biologically active, protein-producing bioink.

Firstly, a collection of growth factor-reporter fusion constructs was designed and selected for the highest yielding by E.coli CFPS. The efficiency of protein synthesis was further optimised by varying the input DNA concentrations, temperature and CFPS buffer additives. Secondly, an investigation was launched into the interactions and effect on MSCs with CFPS-made fusion protein and native BMP2. The phosphorylation of downstream BMP2 signalling molecules detected by Western Blotting was a good indicator that the CFPS-made fusion and the native BMP2 were comparable in their receptor-binding activity. Flow cytometry was also conducted to confirm the interaction of the fusion protein with MSCs. Furthermore, an upregulation of osteogenic markers, calcium deposition and enhanced alkaline phosphatase activity were observed, indicating that the fusion protein is capable of MSCs differentiation towards the osteogenic lineage. Thirdly, CFPS was incorporated into a series of hydrogel networks, where the increasing fluorescence of the fusion protein produced in-gel was successfully detected. Eventually, we aim to develop this CFPS system into an injectable bioink capable of spatio-temporal osteogenesis stimulation, which would pose as a powerful technique in tissue engineering research.

Supramolecular hydrogels have shown promise as biomaterials for sustained drug delivery. Peptide-based hydrogels have garnered special interest because they can form rapidly under physiological conditions and are inherently biocompatible.¹ Synthetic peptides can be easily customized to impart desired functionality, but a major impediment to the widespread adoption of these materials is the high cost of bulk manufacturing.² Modified phenylalanine (Phe) derivatives are a privileged class of low molecular weight (LMW) supramolecular gelator that can be produced inexpensively on a large-scale, making them promising potential replacements for peptide gelators. Recently, we demonstrated that Phe derivatives modified at both termini (Fmoc-Phe-DAP) form hydrogels suitable for in vivo sustained drug release.³ The hydrogel, loaded with a nonsteroidal anti-inflammatory drug, acted as a reservoir for localized and sustained release in a mouse model to mitigate pain for nearly two weeks. To further understand the interactions between small molecules and the hydrogel network, we characterized the release of cationic, neutral, and anionic cargo from both cationic and anionic supramolecular hydrogels.⁴ Cargo that was neutral or of the same charge as the gelator were released at similar rates, but cargo in hydrogels with complementary charge was highly retained. These results highlight the importance of considering electrostatic interactions between the cargo and LMW gelator when designing supramolecular hydrogel systems for drug delivery.
References:
1. Caliari, S. R. and Burdick, J. A. Nat. Methods 2016, 13, 405–414.
2. Sis, M. J. and Webber M. J. Trends Pharmacol. Sci. 2019, 40, 747–762.
3. Raymond, D. L.; Abraham, B. L.; Fujita, T.; Watrous, M. J.; Toriki, E. S.; Takano, T.; Nilsson, B. L. ACS Appl. Bio Mater. 2019, 2, 2116–2124.
4. Abraham, B. L.; Toriki, E. S.; Tucker, N. J.; Nilsson, B. L. J. Mater. Chem. B 2020, 8, 6366–6377.

Oxidized form of graphene such as graphene oxide (GO) attracts lots of interests in metal ion sensing, cell imaging, and adsorption of protein enzyme. GO is known to preferentially bind single-stranded nucleic acids with fluorescence quenching near the GO surface and thus has been recognized as a promising carrier for gene delivery. However, GO has low dispersibility in cell culture media and has shown significant cytotoxicity in cells. To improve the solubility in cell culture media and cytotoxicity of GO, I prepared nano-sized GO (nGO) and modified the nGO surface with polyethylene glycol (PEG), which is often used as a biocompatible coating for surface modification of nanomaterials.1 In the present study, I used PEGylated graphene oxide (PEG-nGO) as biocompatible carriers of antisense peptide nucleic acid (PNA) oligonucleotides for gene delivery. GO was cracked into nGO by tip sonication. For PEGylation, –OH groups on the nGO surface were converted to –COOH groups via conjugation of acetic acid moieties. Carboxylated nGO (HOOC-nGO) was conjugated with 6-arm PEG-amine by EDC coupling. The characterization of PEG-nGO were measured via atomic force microscopy, transmission electron microscopy, and dynamic light scattering spectrophotometer. To observe the interactions between PNA and PEG-nGO, I used fluorescein-labeled PNA. Cellular uptake and distribution of PNA or PEG-nGO were examined by flow cytometry and confocal microscopy. PEG-nGO showed lower cytotoxicity and higher solubility than GO. PEG-nGO were taken up by cells and more effectively delivered PNA than free PNA or PNA/nGO complexes. PEG-nGO readily adsorbed single-stranded PNA on the surface. Moreover, the adsorbed PNA were readily desorbed from the PEG-nGO surface by adding complementary RNA or under low pH conditions, which are similar to the cellular environments in endosomes and lysosomes. PEG-nGO were taken up by lung cancer cells and more effectively delivered PNA than free PNA or PNA/nGO complexes. Cellular uptake of PEG-nGO was mediated via endocytosis, whereas free PNA was not observed to be delivered via endocytosis. As illustrated in schematic illustration, PNAs adsorbed onto the PEG-nGO surface are released under acidic conditions of endosomes/lysosomes and eventually diffuse to the cytosol through endosomal escape, while PEG-nGO appears to be trapped in the endosomes/lysosomes. Gene knockdown of green fluorescent protein and EGFR was performed to validate the effectiveness of target gene knockdown via PEG-nGO-mediated PNA delivery. I suggest that PEG-nGO is a biocompatible carrier useful for PNA delivery into cells and serves as a promising gene delivery tool.

Both the stiffness and stress relaxation of the extracellular matrix affect cell behavior. To study mechanotransduction in environments that change over space or time, 4D cell culture scaffolds are required that enable user-defined changes in mechanical properties. As an external stimulus, light provides high-resolution spatiotemporal control. Our lab has developed stress-relaxing poly(ethylene glycol) hydrogels in which stiffness can be reversibly controlled using irradiation with two different wavelengths of visible light. By manipulating the equilibrium of a dynamic boronic ester bond via the conformation of an adjacent photoswitch, we observed up to 2 kPa reversible changes in shear modulus (G’) independent of the rate of stress relaxation. Unfortunately, this boronate ester hydrogel is readily dissolved in the presence of excess water or cell culture media. To address the instability of these hydrogels, we introduced a fraction of covalent cross-links, which enhances the stability under cell culture conditions while maintaining the reversible photoresponse. Moreover, to better mimic the fibrillarity of the natural extracellular matrix and introduce adhesion sites, we formed a semi-interpenetrating network with collagen I. In this talk, I will discuss the development of this 4D hydrogel platform and its application to study reversible mechanotransduction in mesenchymal stem cells.

New hierarchical hydrogel architectures have been pioneered to create multi-layered soft hydrogels in a concentric fashion as providing fundamental insights at the molecular level. Yet the strategies might not be standardized because different design considerations are required due to a wide range of chemical processes involved. At small scales, the fabrication of micro-size architectures requires higher reaction rates than that of macroscopic objects, which causes agglomeration between gels in a reaction bath. Due to the agglomeration issue, there has been an absence of reliably scale-down models for mass production. Only a few methods have been introduced to produce a single multi-layered capsule with diameters between 4 and 20 mm. Even with predictive mechanisms in various hydrogels, challenges exist in both mass production and scaling-down of the size of hydrogel architectures, and multi-layered techniques for cell encapsulation studies are even sparse.
Soft hydrogel-based modalities have been designed to study cell-cell communication systems including bacterial quorum-sensing by confining genetically engineered microorganisms, which allows the diffusion of solutes across the hydrogel matrix. The communication modes typically use chemical signals mediated by intra/extracellular messenger molecules which not only governs the basic activity of cells but also regulates their physiological functions in a milieu. Despite numerous efforts, technical obstacles to create confined architectures for cell communications has remained elusive. Above all, an effective platform for bacterial confinement should be to retain functionally engineered cells inside the hydrogel architectures without disruption of the structures. Herein, microbial colonization occurs ensuring cell longevity and production of designed signaling molecules for quorum sensing through diffusive biological gels. However, to the best of our knowledge, most of the previous strategies failed to demonstrate an effective platform to confine bacterial communities effectively. In some cases, to constrain cell leakage, cytotoxic processes and/or chemicals such as acrylamide polymerization were utilized to form a size-exclusive barrier, which is not suitable for bacterial biocontainment. Even if bacterial cells could be contained and are able to survive in adverse conditions, chemical toxicity and cell damage are inevitable. Unfavorable procedures lead to rapid cell death within two days in such confined environments, impeding the potential to study the basic dynamics of cellular responses and interactions between microbial communities.
To address the challenges, we have designed and developed a biocompatible confinement model utilizing alginate-based hierarchical architectures. Importantly, our methodology can be used to develop hydrogel architectures that can be reliably scale-down and controlled for mass production. The confinement technology conceived has excellent permeable properties to allow diffusion of signaling molecules and essential nutrients, including oxygen, and byproducts, to maintain their physiological function for a prolonged period. Numerous small ecological systems in a hydrogel environment can be generated to serve as independent microbial modulator with direct or indirect activation of a response regulator by stimuli, including signaling molecules. Specifically, we demonstrate three representative models (i.e., bead-to-bead, stratified communication and bacteria-mammalian cell interaction system) based on hydrogel modulators by entrapping specific microorganisms. This platform will have significant impact in intra/multicellular communications by evaluating interspecies’ signaling in a highly specific manner.

Three-dimensional culture of cells, allowing cells to interact with their surroundings in all dimensions, provides a more in vivo like microenvironment than traditional two dimensional cell culture. Organoids and artificial tissues or organs made by three dimensional cell culture are promising in both in vivo transplantation and in vitro experiments such as disease modeling and drug screening. However, existing three dimensional culture methods are always dependent on specific substrates or equipment, such as round-bottom culture plate, cell spinner and cell rotator. In addition, cell spheroids with uniform and controlled size are still difficult to make while are highly required in various applications to ensure the reliable and reproducible results.
In this study, we developed a universal coating for three dimensional cell culture. The coating was made with chitosan-gallol (CHI-G), and both somatic cells and stem cells automatically self-assemble into spheroids on it. CHI-G coating could be universally coated onto any materials in any shapes simply by immersing and shaking in the CHI-G solution for 48 hours. As a result, any materials coated with CHI-G coating could be substrates for spheroid culturing. We coated CHI-G onto the surface of regular two-dimensional cell culture platform (eg. cell culture plate with flat bottom and glassware for confocal) and inner-surface of three dimensional materials (eg. porous scaffolds and silicon tubes), and cell spheroids were cultured inside. Interestingly, cell spheroids cultured on substrates with different shapes could be applied in different scenarios. Porous scaffolds with spheroids cultured and imbedded inside is promising to be used as tissue engineering scaffolds. Silicon tubes with CHI-G coated on the inner-surface could be used as the device for continuous generation of spheroids. With the continuous injection of cell suspension solution and cell culture medium into the CHI-G coated silicon tube, spheroids could be generated and collected at the another end of the long silicon tube. The size of cell spheroids generated in silicon tubes is much more uniform than those generated on the two dimensional culture plate, which is mainly because of the restriction of round shape in tube. Furthermore, by changing the cell density of the cell suspension solution injected, spheroids with controlled size ranging from 60 um - 300 um could be generated, thus realizing fabricating spheroids with controlled size in a continuous and high-throughput way.

Many of the more than 3000 antimicrobial peptides (AMPs) that have been identified are considered promising alternatives to existing antibiotic, antiviral, and antifungal drugs to treat antimicrobial resistant infections. However, the clinical implementation of AMPs remains limited by their low in vivo efficacy, compared to in vitro activity, due to their vulnerability to protease/enzyme degradation and renal filtration. AMPs that are tethered to biomaterial surfaces are resistant to the filtration barrier by increasing the size of the material system and to the protease degradation due to physicochemical properties of macromolecular tethers. Yet, tethering AMPs typically comes at the cost of reduced potency, and challenges for conventional AMP-incorporated materials include ensuring the consistent synthesis of each material component, reproducible conjugation efficiency with high yields for reliable experimental results, and the flexibility to precisely modulate design parameters. Overcoming these challenges will jumpstart investigations of AMP-incorporated biomaterials to establish more informed design principles for advanced AMP therapeutics.

Here, we developed an AMP-incorporated material platform that simplifies manufacturing for high reproducibility and enables a systematic investigation of design parameters to potentially permit the utilization of many unique AMPs with improved potency. Our approach is an artificial protein platform, comprised of an AMP, a protein tether, and a material-forming protein as a modifiable biomaterial scaffold for various clinical applications. We biosynthesized the artificial proteins in high yields with reproducibility and fabricated strength-controllable, cytocompatible hydrogels and temperature-responsive, self-assembled micelles. We also validated developed materials to inhibit bacterial growth. Since the biosynthesis of artificial proteins is reproducible, and scalable, we expect that there is significant potential of the AMP-incorporated materials for clinical translation. Furthermore, the results will lead future studies to develop an artificial protein-based biomaterial platform for diverse AMPs, as well as a combinatorial approach that mixes several different AMPs into one material as an effective wide-reaching therapeutic strategy to treat a broad range of microbial infections.

Cornea, the outermost layer of the eye, is a transparent tissue composed of highly ordered collagen fibrils. Cornea provides pressure resistance and protection. Corneal cells residing in different corneal layers have unique properties to constitute this transparent, permeable, and durable tissue. Endothelial cells located on Descement’s membrane define fluid entrance to the cornea. Keratocytes give the unique structure of stroma and play an important role in light transmission. Epithelial cells constitute a protective barrier and regulate water, gas, and nutrient transport in and out of stroma. External chemical/mechanical damage, aging and diseases can directly affect these cells and thereby cause visual disturbances. Damaged cornea can be replaced through corneal transplantation, however only a limited number of patients can receive corneas due to limited tissue availability. A bioengineered corneal substitute can be a substantial solution for transplantable tissue shortage. An ideal bioengineered cornea is expected to be optically transparent, biocompatible, water and glucose permeable with mechanically stable. Various biomaterials including decellularized tissues, natural polymers, and peptide amphiphiles have been found investigated for corneal applications.
Our aim is to sustain the growth of multiple cell types in three layers as co-cultures on peptide scaffolds to mimic natural cornea. Briefly, peptide hydrogel constituting the corneal stroma and polymer membrane being home to the epithelial and endothelial layers will be constructed. We initially isolated primary human corneal cells. Epithelial, stromal and limbal cells were characterized by immunocytochemistry and gene expression profiling. Keratocyte cells were immortalized and confirmed exhibiting cell specific phenotype until passage 15. Limbal cells were either immortalized or differentiated into distinct cells with corneal keratocyte or epithelial characteristics by using conditioning media. Endothelial cells proliferate only in initial culture and couldn’t be immortalized.
Peptide hydrogels with different bioactive sequences to provide cell adhesion and anti-angiogenic properties were formed within serum free culture media. Refractive indexes of all hydrogels were found to be around 1,33, which is slightly lower than natural cornea making them suitable candidates for corneal tissue engineering. For all hydrogel formulations, storage and loss modulus were measured lower than elastic modulus of natural cornea. MAX8:MAX8-IKSAV and MAX8:MAX8-HRH exhibit higher mechanical strength relative to MAX8:MAX8-RGD. The hydrogels showed advanced biocompatibility to promote cell adhesion and proliferation for corneal keratocytes. Hydrogels’ transparency was decreased from 95% to only 85% following a two-week culture period.
Elastomer membranes were prepared by curing pre-polymer at 120^oC under vacuum for 12-48 hours. Two methods were followed for membrane casting: (1) Dr. Blade and (2) polyHIPE. Membranes prepared by Dr. Blade exhibit amenable optical transmission, mechanical durability, and cellular biocompatibility for corneal epithelial cells. However, this membrane was lack of glucose permeability. Membranes prepared by polyHIPE method produced porous structures and exhibit glucose permeability with a diffusion coefficient of 3.4 x 10^-4 cm²/s. Decreasing the thickness of this membrane from 3 mm to 0.3 mm, we expect to decrease diffusion rate further.
This work is funded by 2232 International Fellowship for Outstanding Researchers Program of TUBITAK (Grant number 118C371). The funder had no role in study design, data collection or analysis, the decision to publish, or the preparation of this abstract.

Development of injectable/implantable sensors that can monitor different biomarkers related to cognitive and physiological performance has gained significant focus in recent years. We report on the initial research related to the development of implantable hydrogel-based sensor for continuous assessment and autonomous monitoring of biomarkers associated with physical/cognitive performance. Several biocompatible hydrogel formulations based on synthetic and natural polymers were selected to investigate the effect of the polymer matrix on the function of the structure switching sensor probes in a hydrogel environment. We optimized the hydrogel formulations to yield biocompatible and transparent network, with optimal mesh size structures and gelation time transitions for sensing. The synthetic hydrogel network was based on hyperbranched polyethylene imine (PEI) polymer network crosslinked with polyethylene glycol (PEG) linkers and natural hydrogel was prepared during enzymatic crosslinking of reconstituted silk fibroin (SF) protein. Both formulations can be processed at physiological conditions in aqueous buffers without the need for organic solvents, and hence can be considered for biocompatible processes. The optimization of sensor function in both hydrogel formulations was done using several fluorogenic DNA and RNA-based sensors including a model fluorogenic DNA aptamer responsive to crystal violet, a spinach RNA sensor for adenosine metabolite and a forced intercalation (FIT) DNA aptamer responsive to dehydroepiandrosterone sulfate (DHEAS) hormone. For all these sensors to be functional and produce a fluorescent signal, the matrix should allow a structure switching mechanism to take place and allow the formation of the aptamer binding pocket. We demonstrate that hydrogels with optimal mesh size network and hydrophilicity can ideally support the diffusion of analytes and the switching of aptamer conformations in DNA-based sensors, leading to generation of the signal within 10-20 min depending on the polymer volume and concentration of sensor probes. While DNA-based sensors were successfully activated in hydrogels, RNA-based sensors function require further optimization. This optimization is necessary to understand the mechanism of sensor activation with long RNA sequences that are composed of several structurally important parts such as ligand-binding pocket, connecting module, stabilizing scaffold and fluorogenic aptamer.

Recapitulating the functional architecture of myocardium is one of the main challenges in regenerating cardiac tissues and disease modeling. Current tissue engineering methods primarily rely on synthetic polymeric materials to fabricate nanofibers and micropatterned templates to mimic the anisotropic cardiac conduction. However, these non-biological substrates lack the functional advantage rendered by the use of native extracellular matrix proteins. In this work, we showcase a highly anisotropic, fibrillar extracellular matrix solely composed of fibronectin (FN) spanning several millimeters in length which is achieved through a fluidically-induced polymerization across two surfaces. Using this engineered biomimetic FN matrix, we test the hypothesis that the topographical alignment of regenerated cardiomyocytes is essential for the proper hierarchical organization and the development of cardiac electrophysiological functions. Human pluripotent stem cell-derived atrial-like cardiomyocytes (hPSC-ACMs) cultured on anisotropic FN matrices adapt the physiological orientation and display significant sarcomeric organization with synchronous contractility when compared to those cultured on FN matrix without anisotropic alignment. Electrophysiological readouts of intracellular calcium transient reveal that the anisotropic FN matrices promote better intercellular calcium handling and yield greater maturity in the cardiac tissues than the less-anisotropic counterparts. Notably, the cardiac tissues regenerated on the anisotropic FN matrix (CTAFs) exhibited higher average conduction velocities than the cardiac tissues on the less-anisotropic FN matrix (CTLFs). Likewise, the upstroke velocity of calcium transient was significantly higher in CTAFs, indicating greater intercellular connectivity of cardiomyocytes through gap junctions and an increase in calcium handling functionalities in CTAFs. The regulated alignment of fibrillar fibronectin matrix achieved in this work unlocks a new possibility of generating cardiac tissue transplants with anisotropic functionality for regenerative medicine and disease modeling.

The extracellular matrix (ECM) is a complex web of interwoven proteins and glycans that influence cell fate through biochemical and biophysical cues, where desmoplastic ECM signatures demarcate the progression of metastatic breast cancer. Accumulation of ECM glycan hyaluronan (HA) in breast tumor stroma has been correlated with poor patient prognosis. While this clinical correlation is well established, the functional role stromal HA serves in metastatic disease progression remains unclear but is suspected to be underpinned by differential HA molecular weight (Mw).

Fibronectin (Fn) is the bedrock of many native tissues, existing as a fibrous protein network that sequesters cell signaling factors and other ECM macromolecules and has been implicated in diseased tissue remodeling [1]. Given the complexity of native tissue-genesis, controlling the presentation of multivariant ECM components such as HA, Fn and collagen (COL) remains incredibly challenging using cell-based approaches. Engineered materials can be leveraged to resolve these outstanding ECM-related biological questions, but current platforms fail to utilize native proteins and glycans. Hence, it remains an outstanding challenge to recapitulate fibrillar ECM morphology in a scalable, three-dimensional (3D) platform with defined multi-component heterogeneity utilizing native materials.

Our lab recently pioneered an in vitro technique enabling the creation of 3D fibrillar Fn networks suspended across hyper-porous polymer scaffolds on the millimeter length scale by mimicking Fn fibrillogenesis that can be controlled to define fibril architecture [2,3]. This is achieved by shearing a native Fn solution across a polymeric scaffold at the solution/air interface, which promotes the formation of robust 3D fibrillar protein networks, herein referred to as engineered ECMs (EECMs). We showed Fn EECMs enrich for tumorigenic stem-like tumor cells [2] making them an ideal candidate for the creation of defined tumor niches to model the complexity of stromal ECM dynamics during metastatic disease progression.

In this work, we demonstrate the creation of defined multi-component fibrillar engineered ECMs comprised of native Fn, COL-I and HA. This was achieved by taking advantage of Fn’s binding activity, assembly modalities and developing novel bio-conjugation strategies. We show that Fn-based EECMs embody characteristics of fibrillar cell secreted tissue including conformationally active assembled, insoluble fibrillar structures with ECM component co-assembly. We further demonstrate remarkable control over the co-presentation of high and low Mw HA in porous, 3D fibrillar constructs for the first time in an engineered system using native materials. We confirmed Fn-HA EECMs retain critical biological characteristics compared to tumor associated cell secreted matrices. Finally, rationally designed Fn-HA EECMs enabled the study of HA’s role in influencing metastatic breast tumors. Conformationally active Fn increased metastatic tumor cell potential which may be enhanced or perturbed by HA in a Mw dependent manner.

Tumor-mimetic EECM marks a substantial advancement in the field of naturally derived material engineering where the findings presented here underscore the broader utility of native material systems. With further work, EECMs can be leveraged to dissect ECM-derived epigenetic regulation of the cellular milieu in the tumor microenvironment with potential application in personalized cancer treatment.

[1] Zollinger, AJ & Smith, ML DOI: 10.1016/j.matbio.2016.07.011
[2] Jordahl, S, Neale DB, Lahann, J et al. DOI: 10.1002/adma.201904580
[3] Neale, DB, Lahann, J et al. DOI: 10.1002/sstr.202000137

In cancer treatment, drugs can be categorized into two types, small molecules and biologicals. Therefore, it leaves a gap in the size of the druggable therapeutic targets. While next-genetration peptides fall in this size range can potentially be developed into new class of drugs. However, preclinical peptide therapeutics generally have low in vivo efficacy due to poor cell permeability and high proteolysis sensitivity. By attaching hydrophobic moieties to therapeutic peptides, the conjugated peptidic amphiphilic compounds can self-assemble into micelles with some physicochemical properties analogous to proteins. Because of the high chemical versatility of peptides, such a design of protein analogous micelle system can be highly modular to achieve the cell permeability, targeting capability, and combined delivery of multiple therapeutics at the same time. In this work, therapeutic peptides targeting intracellular p53 pathway and BCL-2 family proteins are chemically modified as peptide amphiphiles to formulate into protein analogous micelles. In vitro mechanism of action and cellular trafficking processes were investigated in wild type p53 DLBCL cell lines, providing guidelines for next generation micelle design.

Introduction
Vascular composite Allograft (VCA) transplantation (Tx) involves the Tx of a functional unit of multiple tissues, such as skin, muscle, tendon, nerve, and bone. VCA, such as hand transplants, have tremendous potential to restore normal form and function (body integrity and sensory-motor ability) for amputees (such as wounded warriors) lacking conventional reconstructive or prosthetic options. Despite encouraging patient and graft survival, the widespread application of the life-enhancing benefits of VCA has been hampered by the toxicity and complications associated with high-dose, multi-drug immunosuppression, which are highly toxic and cause kidney and heart failure. We have recently developed a platform called Sustained-release, Microparticle-based, Anti-Rejection Therapy through Enhancement of Regulatory T-cells (S.M.A.R.T.E.R.) Platform for VCA Immunomodulation that takes advantage of the body’s own regulatory functions instead of suppressing the entire immune system. Indeed, these formulations mimic a strategy used by tumors to avoid immune rejection. We have shown that these systems can effectively recruit Tregs toward the site of injection in vivo, with these recruited Tregs demonstrating significant promise for promoting local immunological homeostasis. Here, we propose this S.M.A.R.T.E.R. platform in the context of VCA for the establishment of tolerance of transplants.

Methods
The S.M.A.R.T.E.R. platform consists of safe, biodegradable polymer (poly(lactic-co-gylcolic)acid) microspheres that are engineered to either sustain a gradient of a Treg-recruiting chemokine (CCL22) or release a combination of three Treg inducing factors (TGF-beta, IL2, rapamycin) at the local site of injection. Formulations were fabricated using a double emulsion method and characterized for size, morphology, drug loading, release, and bioactivity. Blank formulations (unloaded polymer microspheres) were prepared as a control. Formulations were applied subcutaneously to rat full hind limb allografts (Brown Norway to White Lewis) at both the time of transplant and 3 weeks post-transplant. ALS and FK-506 were applied at the time of transplantation, with FK-506 being removed at day 21. Rats were monitored for signs of rejection out to 300 days post-transplant. In addition, both brown Norway (donor antigen) and Wistar Furth (3^rd party) skin transplantation was performed on long term survivors to probe for systemic dominant tolerance.

Results
ALS and FK-506 immunosuppression removal resulted in 100% graft rejection in all untreated and blank carrier groups. Rats treated with the S.M.A.R.T.E.R. platform showed over 90% long term survival (post 300 days). White Lewis survivors treated with the S.M.A.R.T.E.R. platform accepted Brown Norway skin transplants with no systemic immunosuppression, but robustly rejected skin transplants from Wistar Furth rats.

Conclusion/Impact
The proposed S.M.A.R.T.E.R. platform is a powerful strategy in inducing survival and systemic dominant tolerance in the most difficult and immunogenic transplantation model (VCA) and would represent a way to avoid the life-long systemic immunosuppression that currently follows VCA procedures.

Introduction
The cryogels, a type of macroporous scaffolds, are referred hydrogels formed through the controlled freeze-thawing or drying of a polymer solution. They recently have attracted much attention in the various biomedical fields due to their unique interconnected macroporous structure, enhanced mechanical stability, and high absorption ability. Cryogels provide innovative avenues for tissue regeneration and engineering, exhibiting unique properties. Recently, many researchers have developed cryogels as a dynamic scaffold that incorporates various bioactive molecules to stimulate biological activity for enhanced tissue regeneration. Among these various factors, molecular oxygen (O₂) plays an essential role as a signaling molecule in wound healing. In particular, it is demonstrated that hyperbaric oxygen enhances wound healing and tissue regeneration through temporally increased intracellular reactive oxygen species (ROS) of surrounding tissues. Herein, we design a new type of O₂-releasing cryogels scaffold composed of thiolated-gelatin and calcium peroxide (CaO₂) for enhanced wound healing.
Experimental Methods
We first synthesized thiolated-gelatin (GtnSH) polymer through conjugation of Traut’s reagent into the gelatin backbone. The cryogels are fabricated by CaO₂-mediated disulfide bond followed by lyophilization. The porosity and macroporous structure of cryogels was confirmed by the gravimetric method and scanning electron microscope (SEM, JSM-7800F). In vitro degradation test using collagenase (type II) was conducted to examine proteolytic degradation of the cryogels. We monitored dissolved O₂ (DO) levels using a commercially available O₂ sensor and a non-invasive sensor (Microx-4, Presens). For in vitro cytocompatibility test, we cultured human dermal fibroblasts (HDFs) with cryogels swelled and cell viability was analyzed by WST-1 assay and live/dead assay. To confirm tissue compatibility of the cryogels, we subcutaneously implanted cryogels. We conducted a wound closing test using a critical defect model and analyzed it through immunohistochemistry to demonstrate the effect of oxygen and porosity of cryogels on wound healing. To evaluate the hemostatic efficacy of cryogels, we performed a mouse liver injury model.
Results and Discussion
The O₂-releasing cryogels with the macroporous structure were prepared via CaO₂-mediated crosslinking followed by lyophilization. The cryogels showed improved resistance to proteolytic degradation due to increased crystallinity compared to hydrogels of the same composition. The gelatin-based cryogels showed high porosity by varying CaO₂ concentrations due to the generation of oxygen bubbles. The O₂ release behavior of cryogels in aqueous media is controlled depending on CaO₂ concentrations (8.4 to 15.2 mg/L O₂ level), and hyperoxia level was retained for up to 14 hours in vitro. We demonstrated that our cryogels showed good cytocompatibility on HDFs and tissue compatibility in major organs. Furthermore, we found that our O₂-generating cryogels enhanced wound closing rate and vascular recruitment through temporary acute hyperoxic conditions and porosity of cryogels. We also demonstrated that our bioactive cryogels had a hemostatic capacity due to their inherent high absorption capability and released calcium ions from cryogels compared to control (without treatment).
Conclusion
We developed a new type of O₂-releasing scaffold formed by CaO₂-mediated cross-linking. The macroporous cryogels showed controllable physicochemical properties depending on CaO₂ concentration. The biocompatible O₂-releasing scaffolds as bioactive wound dressing promoted wound closure as well as vascular recruitment. We suggest that our O₂-releasing cryogels have great potential as a promising wound dressing material for facilitating wound healing.

Our body maintains homeostasis through three network systems: blood vessels, lymph vessels, and nerves. Among them, blood vessels and nerves interact with each other, especially when morphogenesis occurs during development. In this study, we fabricated a microdevice to test neuro-vascular interactions with neurovascular organoids consisting of neuronal stem cells and vascular endothelial cells. We used fatal rat cerebral-derived neural stem cells and human umbilical cord-derived vascular endothelial cells to generate neurovascular organoids. Reciprocal interactions of each cell types on angiogenic and neuronal gene expression were evaluated by RT-PCR and fluorescent immunostaining. Interestingly, in the co-culture of neural stem cells and vascular endothelial cells, the neurogenesis and angiogenesis potential increased with the increase in the ratio of vascular endothelial cells. Furthermore, considering the length of axons in the central nervous system is several mm, we fabricated microdevices with a few mm-long microfluidic channel using photolithography. When two organoids were placed at both ends of the microchannel, axons and vascular sprougin spontaneously grew from each of them, and finally the two organoids were connected by axon bundle tissues including vascular endothelial cell networks. These results show that when the direction of axon and vascular extension from the organoids is spatially controlled, formation of neuro-vascular interconnections in neural axon bundles can be partially replicated in vitro. This neurovascular organoid will be a useful tool for understanding neural-vascular interactions during neurogenesis and testing drugs.

Introduction: Hair loss is caused by aging, drugs, and diseases, and greatly affects people's quality of life. One of the current treatments for hair loss is hair transplantation, but there is a critical limitation that only an insufficient number of hairs are sometime available for this treatment due to the progression of the alopecia. Thus, hair regenerative medicine has emerged as a novel approach, in which hair follicle stem cells are grown in culture and are used to prepare tissue grafts. Previous studies demonstrated that de novo hair follicles can be generated by transplanting hair follicle stem cells into nude mice. We have previously demonstrated that hair follicle germ (HFG)-like aggregates prepared using hair follicle stem cells efficiently generated de novo hair follicles in the back skin of nude mice¹⁾. Although the fabricated HFGs had high hair regeneration ability, considering that HFGs in vivo are surrounded by extracellular matrices, further improvement of hair regeneration ability of HFGs may be possible by replicating such in vivo extracellular matrix microenvironments. In this study, we focused on collagen, which is abundant in the skin, and fabricated HFGs containing high-density collagen and cells by contracting collagen microgels. Then, using a bioprinter, we scaled-up this approach for preparing a large number of HFGs automatically.
Methods: Two types of hair follicle stem cells, epithelial and mesenchymal cells, were isolated from the mouse embryonic skin, and were then suspended in 2.4 mg/mL of collagen gel solution, respectively. Collagen drops were placed adjacent to each other using an electromotive pipette or bioprinter to prepare collagen-containing HFGs. Changes in the microgels diameter and cell distribution in HFGs were observed during 3 days of culture. To investigate the relationship between spontaneous contraction of microgels and hair regeneration ability, a myosin II ATPase inhibitor, blebbistatin, was added to the culture medium. Collagen-containing HFGs with/without blebbistatin after 3 days of culture and no collagen-containing HFGs which were prepared by our previous approach¹⁾ were transplanted into shallow stab wounds prepared on the back of nude mice. The number of hairs generated per transplanted site was evaluated after 3 weeks of transplantation.
Results: The collagen-containing HFGs showed spontaneous contraction by cell attraction forces during 3 days of culture. The long-side diameter of collagen drops reduced from 3.2 mm to less than 0.8 mm after 3 days of culture, wherein the cell density and collagen were enriched 12 times and higher. Interestingly, the contraction was significantly inhibited in the presence of blebbistatin, and no hairs were regenerated post transplantation. However, collagen-containing HFGs without blebbistatin regenerated hair follicles and shafts post transplantation more efficiently compared to the HFGs without collagen gel. This suggested that the spontaneous contraction of cell-encapsulated microgels provided a more suitable environment for the improvement of hair regeneration ability of HFGs. Using a bioprinter, more than 1,000 HFGs were prepared within 12 minutes automatically, and the preparation efficiency and the uniformity of the collagen microgels were improved 20-fold and 2-fold, respectively.
Conclusions: We demonstrated that HFGs can be prepared through spontaneous contraction of cell-suspended collagen microgels in the culture. The cell- and collagen-dense microenvironments were suitable for the improvement of trichogenic functions. The preparation of the HFGs were automated using a bioprinter and thus scalable for preparation of a large number of HFGs. This approach may provide a promising strategy for advancing hair regenerative medicine.
References: 1) T. Kageyama et al., Biomaterials, 154, 2018

Electrogenetics, which uses electric fields to control gene expression and cell function, has applications ranging from production and delivery of bioproducts and biomanufacturing of functional materials. We plan to design and synthesize next generation of electronic biomaterials by combining electrogenetics with synthetic and systems biology. We have found that deep in the ocean or underground, where there is no oxygen, Geobacter bacteria “breathe” by projecting tiny protein filaments called "nanowires" into the soil, to dispose of excess electrons resulting from the conversion of nutrients to energy (Cell 2019).
Using electric fields, we have induced the overexpression of cytochrome OmcZ nanowires that show 1000-fold more electron conductivity than the OmcS nanowires important in natural soil environments (Nature Chem.Bio. 2020). Nanowire-based electrogenetics platform responds rapidly to environmental changes to adapt to signals and stresses for stimuli-responsive and pH-responsive systems. In contrast to optogenetics, which requires specifically designed devices and whose signals are attenuated in high-density cells, these nanowires efficiently control bacterial growth, adhesion and communication.
I will present our recent studies revealed a surprise that these protein nanowires have a core of metal-containing molecules called hemes. Previously it was not suspected. Using high-resolution cryo-electron microscopy, we were able to see the nanowire’s atomic structure and discover that hemes line up to create a continuous path along which electrons travel. I will present our recent experimental and computational studies to identify the nanowire composition, structure and mechanism of nanowire assembly and high electron conductivity by measuring their DC and THz conductivity as a function of nanowire length, temperature, frequency, pH and heme stacking.
These studies solve a longstanding mystery of how microbial protein nanowires move electrons over micrometer distances and enable the bacteria to perform environmentally important functions such as cleaning up radioactive sites, generating biofuels and electricity (Nature Nano. 2011), or exchange electrons with other bacterial species to share nutrients and resources (Science 2010). This electron exchange process is important in microbial communities found in diverse environments. Nanowires confer novel properties to microbial consortia such as living transistors (Nature Nano. 2011) and living supercapacitors (ChemPhysChem 2012)
We have further found that the nanowires are translocated to the bacterial surface using unique heterodimeric pili that likely serve as a piston to secrete cytochromes, rather than functioning as nanowires as previously thought (Nature 2021.) These previously hidden bacterial hairs serve as a molecular switch for controlling the release of cytochrome nanowires. As all major phyla of prokaryotes use systems similar to these pili, this nanowire translocation machinery may have a widespread effect in identifying the evolution and prevalence of diverse electron-transferring microorganisms and in determining nanowire assembly architecture for designing synthetic protein nanowires.
I will also present our studies on bioinspired synthetic protein nanowires and new methodology to set standards for reporting protein conductivity and underlying mechanisms. Although most protein conductivity studies remain focused on electrons, protons play a very important role, not only in energy generation, but also in the electronic conductivity of proteins. Through measurements of the intrinsic electron transfer rate, we recently showed that both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine, regulate the hole transport over micrometers in amyloids through a proton rocking mechanism (PNAS 2021). I will present strategies to couple electron and proton transfer for the development of electronically conductive protein-based biomaterials.

Our laboratory is focused on the design of materials inspired by functional supramolecular systems in the plantea and animalia kingdoms. Three functional hallmarks of such systems are the photosynthetic machinery of green plants, the harvesting of mechanical energy from muscles, and the extracellular matrix that mediates signaling pathways in mammalian cells. All three use soft materials functionally optimized by evolutionary events. In this lecture I will describe bio-inspired synthetic hydrogels inspired by green leaves that are based on light harvesting supramolecular polymers. These systems can be designed to produce hydrogen, reduce carbon dioxide, or synthesize hydrogen peroxide which are all useful fuels in sustainable energy systems. The lecture will also describe life-like robotic materials that effectively transduce energy into mechanical actuation and could eventually participate in energy saving and smart technological systems. The third topic will be highly dynamic supramolecular polymers that mimic extracellular matrices and effectively signal cells to promote regenerative signaling pathways. This particular work will highlight the use of biomaterials for the “holy grail” of regenerative medicine, the repair of the brain and the spinal cord.

The structural complexity of composite biomaterials and biomineralized particles arises from the hierarchical ordering of inorganic building blocks over multiple scales. While empirical observations of complex nanoassemblies are abundant, physicochemical mechanisms leading to their geometrical complexity are still puzzling, especially for non-uniformly sized components. These mechanisms are discussed in this talk taking an example of hierarchically organized particles with twisted spikes and other morphologies from polydisperse Au-Cys nanoplatelets [1]. The complexity of these supraparticles is higher than biological counterparts or other complex particles as enumerated by graph theory (GT). Complexity Index (CI) and other GT parameters are applied to a variety of different nanoscale materials to assess their structural organization. As the result of this analysis, we determined that intricate organization Au-Cys supraparticles emerges from competing chirality-dependent assembly restrictions that render assembly pathways primarily dependent on nanoparticle symmetry rather than size. These findings open a pathway to a large family of colloids with complex architectures and unusual chiroptical and chemical properties.
The design principles for complex chiral nanoassemblies have been extended to engineer drug discovery platforms for Alzheimer syndrome,[3] materials for chiral photonics,[5] biomimetic composites for energy and robotics [2,4] catalysis[6] and antiviral vaccines.[7]

References
[1] W. Jiang, Z.-B. et al, Emergence of Complexity in Hierarchically Organized Chiral Particles, Science, 2020, 368, 6491, 642-648.
[2] Wang, M.; Vecchio, D.; et al Biomorphic Structural Batteries for Robotics. Sci. Robot. 2020, 5 (45), eaba1912.
[3] Jun Lu, et al, Enhanced optical asymmetry in supramolecular chiroplasmonic assemblies with long-range order, Science, 2021, 371, 6536, 1368
[4] D. Vecchio et al, Structural Analysis of Nanoscale Network Materials Using Graph Theory, ACS Nano 2021, 15, 8, 12847–12859.
[5] L. Ohnoutek, et al, Third Harmonic Mie Scattering From Semiconductor Nanohelices, Nature Photonics, 2021, conditionally accepted
[6] L. Tang et al. Self-Assembly Mechanism of Complex Corrugated Particles" JACS, 2021 accepted.
[7] L. Xu, et al Enantiomer-Dependent Immunological Response to Chiral Nanoparticles, 2022, conditionally accepted.

An effective resolution of inflammation contributes to favorable tissue regenerative therapeutic outcomes. However, fine coordination of local immunomodulation in a timely manner is limited owing to the lack of control strategies against disease dynamics. This study describes an inflammation-responsive hydrogel (IFRep gel) as an effective therapeutic strategy for on-demand epigenetic modulation against disease dynamics in wound healing. The IFRep gel is designed to control drug release by cathepsins according to the state of inflammation for active disease treatment. The gel loaded with an inhibitor of the epigenetic reader BRD4 regulates the translocation of nuclear factor erythroid 2 to the nucleus, thereby promoting antioxidant gene expression to reverse the inflammatory macrophage state in vitro. In addition, on-demand BRD bromodomain inhibition using the responsive hydrogel accelerates wound healing by controlling early inflammatory phase and keratinocyte activation in vivo. Collectively, these data suggest the clinical utility of the IFRep gel as a promising strategy for the desired therapeutic outcomes in inflammation-associated diseases.

De novo self-assembling peptides have been widely exploited as bioinspired platform for the development of extracellular matrices that mimic natural mammalian cells microenvironmental niches.¹ Setting the molecular design rules for assembling peptides is crucial for the meticulous control of scaffold material properties over the length scale, to enable tailoring systems that can mimic the nanoarchitectural and micro environment for different cell types. We have recently adopted a minimalistic design approach for the development of ultrashort ionic-complementary constrained peptides (UICPs), rationally designed to assemble into thermodynamically stable β-sheet nanofibers with cell scaffolding potential.² While we have previously shown the importance of aromatic stacking for the assembly and packing of UICP nanofibres,² in this talk we will unravel the role played by charge interaction in tuning the molecular assembly, nanostructure and mechanical properties of charge co-complementary binary UICP mixtures.
Our UICP binary mixtures are composed of a cationic peptide and an anionic counterpart, in which the charged residues are alternating around hydrophobic phenylglycine (Phg) and distributed in a pattern conferring a charge co-complementarity of the two sequences. Co-assembly into extended antiparallel β-sheet nanofibres, nanoribbons and twisted braids-like nanostructures was achieved upon mixing the charge co-complementary peptides and confirmed by FTIR, TEM and SAXS. Above critical gelation concentrations (~2-5 w/v%), these nanostructures spontaneously formed entangled networks and bulk hydrogels, as evidenced by the inverted vial test, oscillatory rheology and SEM. A full control over molecular co-assembly, size and morphology of nanostructures, as well as hydrogel stiffness, was possible via modulation of molar ratios of the binary components, the pH, and the overall peptide concentration. In contrast, mixtures of charge non-complementary sequences failed to co-assemble into any β-sheet-rich nanostructures, implying the importance of charge interactions for stabilizing nanostructure formation.
Our suggested ‘Mix & Gel’ approach is specifically designed to facilitate re-suspension of cell pellets in one of the peptide solutions. Upon mixing with the ionic co-complementary partner peptide solution, spontaneous formation of homogenous 3D nanofibrous network occurs, encapsulating cells, with superior homogeneity compared to traditionally used cell-hydrogel encapsulation techniques. This technique tackles the common issue encountered when mixing cells with pre-formulated hydrogels such as impairment of cell viability upon repeated pipetting and difficulty of de-aggregating cells from pellets in the viscous hydrogel matrix. In addition, molar ratios of binary mixtures, pH and total peptide concentration can provide divergent hydrogels properties to suit the requirements of distinct cell phenotypes.
Acknowledgements
The authors would like to thank Rachel Armitage at De Montfort University for her assistance with SEM experiments and Natalie Allcock at University of Leicester for helping with TEM imaging. This work was funded by the Newton-Mosharafa fund awarded to A.K. and M.E. and the Egyptian Government missions’ sector scholarship awarded to M.S. The authors are also grateful to Diamond Light Source for awarding beam times (SM28806 and SM28287) to this project and to the staff, in particular, Andy Smith on beamline I22 and Charlotte J.C. Edwards-Gayle for their support with the SAXS experiments.
References:
[1] Ding X., Zhao H., et al., 'Synthetic peptide hydrogels as 3D scaffolds for tissue engineering', Advanced Drug Delivery Reviews. 2020; 160:78–104.
[2] Wychowaniec J., Patel R., et al. 'Aromatic stacking facilitated self-assembly of ultra-short ionic complementary peptide sequence: β-sheet nanofibres with remarkable gelation and interfacial properties' Biomacromolecules 2020; 21: 2670-2680.

DNA nanotechnology has increasingly been used as a platform to scaffold materials based on its unmatched ability to create structure in a designed, desired format. Our group has focused on the organization of materials using 3D DNA scaffolds, ranging from individual DNA origami to mesoscale lattices. A particular source of interest across multiple disciplines has been the organization of enzymatic cascades, whereby enzymes can be arranged according to their associated substrates and intermediate exchange pathways. Yet while the concept of a nanoscale factory has been appealing, colocalization of enzymes on charged scaffolds introduces complex considerations arising from scaffold effects on the local reaction environment. The contribution of different effects makes it difficult to distill leading causes of activity changes associated with scaffolded enzymatic cascades. In working towards functional assemblies that present higher activity and guided reaction pathways, we can also shed light on questions of mechanistic causes for higher enzymatic activity on DNA scaffolds. Traditionally, this work has utilized 2D DNA scaffolds, but we utilize 3D wireframe DNA origami as a basis for our designs. Our group has organized enzymatic systems on multiple organizational scales in three dimensions, and has gained insight into the contribution of enzyme spacing, arrangement, and location on DNA scaffolds. We show that rather than being strongly dependent on specific spacing and DNA sequence makeup, enzyme activity is more strongly dependent on the nanoscale environment, providing experimental support that mesoscale organizations can yield significant enhancement in activity without direct substrate channeling between enzymes.

Introduction
Myofibroblasts are specialized cells that play a key role during the inflammatory phase of normal wound healing. They deposit extracellular matrix (ECM) components to restore the mechanical properties of the skin and physically contract to encourage wound closure. However, dysregulation of myofibroblast activity can lead to development of chronic inflammatory conditions such as delayed healing¹. To date, many studies have explored how material cues, including physicochemical and biological signals, influence cell behaviors such as differentiation, reprogramming, and proliferation². These findings raise the possibility of reprogramming gene expression of inflamed tissues via biomaterial contact to promote regeneration. In this study, we use a bio-inspired quasi-3D ex vivo skin dermis model³ that is comprised of human dermal (myo)fibroblasts seeded on top of a silk/collagen substrate to evaluate the influence of various material cues on gene expression that may delay or reverse pathological development.
Materials and Methods
Collagen and silk fibroin was crosslinked using HRP-mediated catalysis at 37°C³. To characterize the silk/collagen matrix, Texas Red^TM-X Succinimidyl Ester was conjugated to amine groups present on collagen. To compare cell morphology in 2D and in quasi-3D conditions, immunofluorescence imaging was performed with Hoechst 33342 dye and an anti-vimentin antibody to visualize nuclei and cytoskeletal vimentin, respectively.
Results and Discussion
It is now well appreciated that cells are sensitive to the geometry they are cultured in. Several cell types exhibit a flattened morphology in 2D culture, which leads to changes in chromatin topology that in turn, alters gene expression⁴. We address these issues using a quasi-3D model that allows for physiological recapitulation of the extracellular matrix to conserve typical baseline gene expression, as well as preservation of mechanical integrity, offered by collagen and silk components, respectively³. Originally, we elected to seed the cells within a 3D silk/collagen gel, but the dimensions were such that the majority of encapsulated cells would not be in contact with the material. To reduce the thickness of the model, we sought to dip glass slides in the reactive solution in a serial manner to obtain a thin coating of gel. However, only the first layer successfully adhered to the slides. We propose depositing the solution directly on top of the glass slides is optimal to achieve a homogeneous coating that preserves elongated cell morphology observed in vivo.
Conclusions
This skin model enables a biologically relevant yet accessible platform that more accurately recapitulates the mechanics of the 3D in vivo microenvironment for the purpose of identifying biomaterials that have the potential to delay the onset of and/or reverse pathological inflammatory phenotypes.
References
1. Darby, I. A., Laverdet, B., Bonté, F. & Desmoulière, A. Fibroblasts and myofibroblasts in wound healing. Clin. Cosmet. Investig. Dermatol. 7, 301–311 (2014).
2. Crowder, S. W., Leonardo, V., Whittaker, T., Papathanasiou, P. & Stevens, M. M. Material Cues as Potent Regulators of Epigenetics and Stem Cell Function. Cell Stem Cell 18, 39–52 (2016).
3. Vidal, S. E. L. et al. 3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components. Biomaterials 198, 194–203 (2019).
4. Smithmyer, M. E., Cassel, S. E. & Kloxin, A. M. Bridging 2D and 3D culture: Probing impact of extracellular environment on fibroblast activation in layered hydrogels. AIChE J. 65, e16837 (2019).
Acknowledgements
This work was supported by the Department of Bioengineering at Imperial College London.

Cells sense the physical environment at both the nano- and micro-scale. Specifically, cells interact with the surrounding via formation of focal adhesions, with integrins acting as a link between the ECM and the cytoskeleton. Several studies have shown that substrate topography, stiffness, and ligand type and density control these interactions, which determine reorganization of the cytoskeleton, and changes in cell and nuclear morphology, ultimately affecting chromosomal structure, gene expression, and cell behavior[1,2]. For these reasons, the ability to replicate the microenvironment of biological tissues creates unique biomedical possibilities for stem cell applications. Current fabrication methods are limited by either the control on feature size and shape, or by the throughput and size of the replicas.
Here we report a method to study the synergistic effect of the substrate topography, stiffness, and surface chemistry on the cell-surface interactions. This remains challenging due to the limitations of conventional nanofabrication methods in terms of costs, resolution, control of geometry and size in x-y-z (e.g. impossibility to fabricate complex 3D nano-structures), and choice of materials (e.g., materials with tunable chemistry and stiffness). We introduced a novel platform that combines thermal scanning probe lithography (tSPL)[2] with innovative methodologies for the low-cost and high-throughput nanofabrication of large area quasi-3D bone tissue replicas with high fidelity, sub-15 nm lateral precision, and sub-2 nm vertical resolution. This bio-tSPL platform [4] features a biocompatible polymer resist that withstands multiple cell culture cycles and can be patterned and chemically activated [5] allowing the reuse of the replicas, further decreasing costs and fabrication times. Moreover, we modulated the mechanical properties of this resist in order to study and improve cell-surface adhesion in acqueous solutions.
The achieved level of time and cost-effectiveness, as well as the cell compatibility of the replicas, make bio-tSPL a promising platform to produce tissue-mimetic replicas to study stem cell-tissue microenvironment interactions, test drugs, and ultimately harness the regenerative capacity of stem cells and tissues for biomedical applications.
[1] H. Donnelly, M. J. Dalby, M. Salmeron-Sanchez, P. E. Sweeten, Nanomed-Nanotechnol 2018, 14, 2455.
[2] M. M. Stevens,J. H. George, Science 2005, 310, 1135
[3] R. Szoszkiewicz, T. Okada, S. C. Jones, T. D. Li, W. P. King, S. R. Marder, E. Riedo, Nano Lett. 2007, 7, 1064
[4] X. Liu, A. Zanut, M. Sladkova-Faure, L. Xie, M. Weck, X. Zheng, E. Riedo, and G. M. de Peppo. Adv. Func. Mat 2021, 31, 2008662
[5] Liu, X. Y. et al. Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control. Acs Appl Mater Inter 11, 41780-41790