Investigation on Adaptability and Applicability of Polymer-Mediated Cell Surface Engineering by Ligation with Transglutaminase.

Polymer-mediated cell surface engineering can be a powerful tool to modify the cell's biological behavior, but a simple ligation strategy must be identified. This manuscript assessed the use of transglutamination as a versatile and adaptable approach for cell surface engineering in various cellular models relevant to biomedical applications. This enzymatic approach was evaluated for its feasibility and potential for conjugating polymers to diverse cell surfaces and its biological effects. Transglutaminase-mediated ligation was successfully performed at temperatures ranging from 4 to 37 °C in as quickly as 30 min, while maintaining biocompatibility and preserving cell viability. This approach was successfully applied to nine different cell surfaces (including adherent cells and suspension cells) by optimizing the enzyme source (guinea pig liver vs microbial), buffer compositions, and incubation conditions. Finally, polymer-mediated cell surface engineering using transglutaminase exhibited immunocamouflage abilities for endothelial cells, T cells, and red blood cells by preventing the recognition of cell surface proteins by antibodies. Employing transglutaminase in polymer-mediated cell surface engineering is a promising approach to maximize its application in cell therapy and other biomedical applications.

HyClear: A Novel Tissue Clearing Solution for One-Step Clearing of Microtissues.

3-D cell cultures are being increasingly used as in vitro models are capable of better mimicry of in vivo tissues, particularly in drug screenings where mass transfer limitations can affect the cancer biology and response to drugs. Three-dimensional microscopy techniques, such as confocal and multiphoton microscopy, have been used to elucidate data from 3-D cell cultures and whole organs, but their reach inside the 3-D tissues is restrained by the light scattering of the tissues, limiting their effective reach to 100-200 µm, which is simply not enough. Tissue clearing protocols, developed mostly for larger specimens usually involve multiple steps of viscous liquid submersion, and are not easily adaptable for much smaller spheroids and organoids. In this work, we have developed a novel tissue clearing solution tailored for small spheroids and organoids. Our tissue clearing protocol, called HyClear, uses a mixture of DMSO, HPG and urea to allow for one-step tissue clearing of spheroids and organoids, and is compatible with high-throughput screening studies due to its speed and simplicity. We have shown that our tissue clearing agent is superior to many of the commonly used tissue clearing agents and allows for elucidating better quality data from drug screening experiments.

Prevention of vascular-allograft rejection by protecting the endothelial glycocalyx with immunosuppressive polymers.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery.

An improved in vitro model for studying the structural and functional properties of the endothelial glycocalyx in arteries, capillaries and veins.

The endothelial glycocalyx is a dynamic structure integral to blood vessel hemodynamics and capable of tightly regulating a range of biological processes (ie, innate immunity, inflammation, and coagulation) through dynamic changes in its composition of the brush structure. Evaluating the specific roles of the endothelial glycocalyx under a range of pathophysiologic conditions has been a challenge in vitro as it is difficult to generate functional glycocalyces using commonly employed 2D cell culture models. We present a new multi-height microfluidic platform that promotes the growth of functional glycocalyces by eliciting unique shear stress forces over a continuous human umbilical vein endothelial cell monolayer at magnitudes that recapitulate the physical environment in arterial, capillary and venous regions of the vasculature. Following 72 hours of shear stress, unique glycocalyx structures formed within each region that were distinct from that observed in short (3 days) and long-term (21 days) static cell culture. The model demonstrated glycocalyx-specific properties that match the characteristics of the endothelium in arteries, capillaries and veins, with respect to surface protein expression, platelet adhesion, lymphocyte binding and nanoparticle uptake. With artery-to-capillary-to-vein transition on a continuous endothelial monolayer, this in vitro platform is an improved system over static cell culture for more effectively studying the role of the glycocalyx in endothelial biology and disease.

Shattering barriers toward clinically meaningful MSC therapies.

More than 1050 clinical trials are registered at FDA.gov that explore multipotent mesenchymal stromal cells (MSCs) for nearly every clinical application imaginable, including neurodegenerative and cardiac disorders, perianal fistulas, graft-versus-host disease, COVID-19, and cancer. Several companies have or are in the process of commercializing MSC-based therapies. However, most of the clinical-stage MSC therapies have been unable to meet primary efficacy end points. The innate therapeutic functions of MSCs administered to humans are not as robust as demonstrated in preclinical studies, and in general, the translation of cell-based therapy is impaired by a myriad of steps that introduce heterogeneity. In this review, we discuss the major clinical challenges with MSC therapies, the details of these challenges, and the potential bioengineering approaches that leverage the unique biology of MSCs to overcome the challenges and achieve more potent and versatile therapies.

Oncotically Driven Control over Glycocalyx Dimension for Cell Surface Engineering and Protein Binding in the Longitudinal Direction.

Here we present a simple technique for re-directing reactions on the cell surface to the outermost region of the glycocalyx. Macromolecular crowding with inert polymers was utilized to reversibly alter the accessibility of glycocalyx proteoglycans toward cell-surface reactive probes allowing for reactivity control in the longitudinal direction ('z'-direction) on the glycocalyx. Studies in HUVECs demonstrated an oncotically driven collapse of the glycocalyx brush structure in the presence of crowders as the mechanism responsible for re-directing reactivity. This phenomenon is consistent across a variety of macromolecular agents including polymers, protein markers and antibodies which all displayed enhanced binding to the outermost surface of multiple cell types. We then demonstrated the biological significance of the technique by increasing the camouflage of red blood cell surface antigens via a crowding-enhanced attachment of voluminous polymers to the exterior of the glycocalyx. The accessibility to Rhesus D (RhD) and CD47 proteins on the cell surface was significantly decreased in crowding-assisted polymer grafting in comparison to non-crowded conditions. This strategy is expected to generate new tools for controlled glycocalyx engineering, probing the glycocalyx structure and function, and improving the development of cell based therapies.

Surface Engineering for Cell-Based Therapies: Techniques for Manipulating Mammalian Cell Surfaces.

The introduction of cell-based therapies has provided new and unique strategies to treat many diseases and disorders including the recent approval of CAR-T cell therapy for the leukemia. Cell surface engineering is a methodology in which the cell surface is tailored to modulate cellular function and interactions. In addition to genetic engineering of cell surface proteins, a wide array of robust, innovative and elegant approaches have been developed to selectively target the cell surface. In this review, we will introduce the leading strategies currently used in cell surface engineering including broadly reactive chemical ligations and physical associations as well as more controlled approaches as demonstrated in genetic, enzymatic and metabolic engineering. Prominent applications of these strategies for cell-based therapies will be highlighted including targeted cell death, control over stem cell fate, immunoevasion, blood transfusion and the delivery of cells to target tissues. Advances will be focused specifically on cells which are the most promising in generating cell-based therapeutics including red blood cells, white blood cells (lymphocytes, macrophages), stem cells (multipotent and pluripotent), islet cells, cancer cells, and endothelial cells.

Hyperbranched polyglycerols: recent advances in synthesis, biocompatibility and biomedical applications.

In the pursuit of dendrimer alternatives, hyperbranched polymers have found increasing interest from academia and industry in a broad range of fields due to their topological and synthetic advantages. Hyperbranched polyglycerol (HPG), as the name implies, is a hyperbranched polymer with about 50-65% dendrimeric structure. Due to its ease in synthesis, globular nature, versatility in terms of functionalization, and superb biocompatibility profiles HPG provides a promising class of materials suitable for numerous applications in nanomedicine and biomedical technologies. The structural features of HPG can be easily tailored by adopting different synthetic methodologies. In this review, we briefly explore the synthesis of HPGs starting from the traditional Lewis acid based approaches to recent advances including the development of high MW HPGs, biodegradable HPGs, co-block HPGs and sustainable or 'green' HPG synthesis. The robust history of HPG biocompatibility is extensively reviewed giving examples of both in vitro and in vivo models. In particular, HPG showed very minimal polymer accumulation in vital organs after intravenous injection compared to other polymers widely used for various biomedical applications. HPG is well tolerated in mice and rats, and has been found to be non-immunogenic to date. Due to its demonstrated safety profile and multifunctionality, HPG has been extensively studied for different biomedical applications including as macromolecular therapeutics, multivalent inhibitors/scavengers, in controlled drug delivery systems, in organ preservation, dialysis and cell surface engineering, as imaging agents and theranostics, in the development of anti-fouling surfaces and proteomics reagents. We highlight these applications along with its advantages. Finally, we conclude by providing a future prospective of HPG as one of the promising PEG alternatives with a great potential to enter clinical trials in the near future.

Adhesion of Blood Clots Can Be Enhanced When Copolymerized with a Macromer That Is Crosslinked by Coagulation Factor XIIIa.

The adhesion of blood clots to blood vessels, such as through the adhesion of fibrin, is essential in hemostasis. While numerous strategies for initiating clot formation and preventing clot lysis are being developed to create improved hemostatic agents, strategies for enhancing clot adhesion have not been widely explored. Here, we show that adhesion of blood clots can be increased by adding a previously characterized synthetic polymer that is crosslinked by coagulation factor XIIIa during clotting. Addition of the polymer to normal plasma increased the adhesive strength of clots by 2-fold. It also recovered the adhesive strength of nonadhesive fibrinogen-deficient whole blood clots from <0.06 kPa to 1.9 ± 0.14 kPa, which is similar to the adhesive strength of a fibrinogen-rich clot (1.8 ± 0.64 kPa). The polymer also enabled plasma clots to remain adhered under fibrinolytic conditions. By demonstrating that the adhesive strength of clots can be increased with a synthetic material, this provides a potential strategy for creating advanced hemostatic materials, such as treatments for fibrinogen deficiency in trauma-induced coagulopathy.

Toward Infection-Resistant Surfaces: Achieving High Antimicrobial Peptide Potency by Modulating the Functionality of Polymer Brush and Peptide.

Bacterial infection associated with indwelling medical devices and implants is a major clinical issue, and the prevention or treatment of such infections is challenging. Antimicrobial coatings offer a significant step toward addressing this important clinical problem. Antimicrobial coatings based on tethered antimicrobial peptides (AMPs) on hydrophilic polymer brushes have been shown to be one of the most promising strategies to avoid bacterial colonization and have demonstrated broad spectrum activity. Optimal combinations of the functionality of the polymer-brush-tethered AMPs are essential to maintaining long-term AMP activity on the surface. However, there is limited knowledge currently available on this topic. Here we report the development of potent antimicrobial coatings on implant surfaces by elucidating the roles of polymer brush chemistry and peptide structure on the overall antimicrobial activity of the coatings. We screened several combinations of polymer brush coatings and AMPs constructed on nanoparticles, titanium surfaces, and quartz slides on their antimicrobial activity and bacterial adhesion against Gram-positive and Gram-negative bacteria. Highly efficient killing of planktonic bacteria by the antimicrobial coatings on nanoparticle surfaces, as well as potent killing of adhered bacteria in the case of coatings on titanium surfaces, was observed. Remarkably, the antimicrobial activity of AMP-conjugated brush coatings demonstrated a clear dependence on the polymer brush chemistry and peptide structure, and optimization of these parameters is critical to achieving infection-resistant surfaces. By analyzing the interaction of polymer-brush-tethered AMPs with model lipid membranes using circular dichroism spectroscopy, we determined that the polymer brush chemistry has an influence on the extent of secondary structure change of tethered peptides before and after interaction with biomembranes. The peptide structure also has an influence on the density of conjugated peptides on polymer brush coatings and the resultant wettability of the coatings, and both of these factors contributed to the antimicrobial activity and bacterial adhesion of the coatings. Overall, this work highlights the importance of optimizing the functionality of the polymer brush to achieve infection-resistant surfaces and presents important insight into the design criteria for the selection of polymers and AMPs toward the development of potent antimicrobial coating on implants.

Bioorthogonal phase-directed copper-catalyzed azide-alkyne cycloaddition (PDCuAAC) coupling of selectively cross-linked superoxide dismutase dimers produces a fully active bis-dimer.

Superoxide dismutase (SOD) is a 32 kDa dimeric enzyme that actively removes a toxic oxygen species within red cells. The acellular protein itself does not survive circulation as it is filtered through the kidney. Conjugating the protein to another SOD should increase the size of the dual protein above the threshold for filtration by the kidney, making the material a potential therapeutic in circulation. Site-selective chemical cross-linking of SOD introduces a bioorthogonal azide group on the cross-link so that two SODs react efficiently with a bis-alkyne through phase-directed copper-catalyzed azide-alkyne cycloaddition (PDCuAAC). The modification has a negligible effect on the catalytic activity of the constituent proteins. Consistent with the retained activity, circular dichroism (CD) spectroscopy indicates that the secondary structures of the proteins are similar to that of the native protein.