Controlled swelling of biomaterial devices for improved antifouling polymer coatings.

Nonspecific interactions between cells and implantable elastomers often leads to failure modes for devices such as catheters, cosmetic and reconstructive implants, and sensors. To reduce these interactions, device surfaces can be coated with hydrophilic polymers, where greater polymer density enhances antifouling properties. Although graft-from coating techniques result in higher density polymer films and lower fouling in controlled settings, simpler graft-to methods show similar results on complex implanted devices, despite limited density. To address the need for improved graft-to methods, we developed Graft then shrink (GtS) where elastomeric materials are temporarily swollen during polymer grafting. Herein, we demonstrate a graft-to based method for poly(oligo(ethylene glycol) methyl ether methacrylate) (pOEGMA) on swollen silicone, GtS, that enhances grafted polymer content and fouling resistance. Total grafted polymer content of pOEGMA on toluene swollen silicone increased over ~ 13 × compared to non-swollen controls, dependent on the degree of silicone swelling. Increases in total grafted polymer within the top 200 µm of the material led to bacterial and mammalian cell adhesion reductions of 75% and 91% respectively, compared to Shrink then Graft (StG) antifouling polymer coated controls. GtS allows for the simple 3D coating of swellable elastomers (e.g., silicone medical devices) with improved antifouling pOEGMA coatings.

Tunable Multivalent Platform for Immune Recruitment to Lower Antigen Expressing Cancers.

Chemical immunotherapeutic strategies including Antibody Recruiting Molecules (ARMs - bivalent small molecules containing an antibody-binding domain (ABD) and a target-binding domain (TBD)) direct immune-mediated clearance of diseased cells. Anti-cancer ARM function relies on high tumor antigen valency, limiting function against lower antigen expressing tumors. To address this limitation, we report a tunable multivalent immune recruitment (MIR) platform to amplify/stabilize antibody recruitment to cells with lower antigen valencies. An initial set of polymeric ARMs (pARMs) were synthesized and screened to evaluate ABD/TBD copy number, ratio, and steric occlusion on specific immune induction. Most pARMs demonstrated simultaneous high avidity binding to anti-dinitrophenyl antibodies and prostate-specific membrane antigens on prostate cancer. Optimized pARMs mediated enhanced anti-cancer immune function against lower antigen expressing target cells compared to an analogous ARM.

Real-time evaluation of a hydrogel delivery vehicle for cancer immunotherapeutics within embedded spheroid cultures.

Many protein immunotherapeutics are hindered by transport barriers that prevent the obtainment of minimum effective concentrations (MECs) in solid tumors. Local delivery vehicles with tunable release (infusion) rates for immunotherapeutics are being developed to achieve local and sustained release. To expedite their discovery and translation, in vitro models can identify promising delivery vehicles and immunotherapies that benefit from sustained release by evaluating cancer spheroid killing in real-time. Using displacement affinity release (DAR) within a hydrogel, we tuned the release of a CD133 targeting dual antigen T cell engager (DATE) without the need for further DATE or hydrogel modifications, yielding an injectable vehicle that acts as a tunable infusion pump. To quantify bioactivity benefits, a 3D embedded cancer spheroid model was developed for the evaluation of sustained protein release and combination therapies on T cell mediated spheroid killing. Using automated brightfield and fluorescent microscopy, the size of red fluorescent protein (iRFP670) expressing spheroids were tracked to quantify spheroid growth or killing over time as a function of controlled delivery. We demonstrate that sustained DATE release enhanced T cell mediated killing of embedded glioblastoma spheroids at longer timepoints, killing was further enhanced with the addition of anti-PD1 antibody (αPD1). The multi-cellular embedded spheroid model with automated microscopy demonstrated the benefit of extended bispecific release on T cell mediated killing, which will expedite the identification and translation of delivery vehicles such as DAR for immunotherapeutics.

Graft-Then-Shrink: Simultaneous Generation of Antifouling Polymeric Interfaces and Localized Surface Plasmon Resonance Biosensors.

Antifouling polymer coatings that are simple to manufacture are crucial for the performance of medical devices such as biosensors. "Grafting-to", a simple technique where presynthesized polymers are immobilized onto surfaces, is commonly employed but suffers from nonideal polymer packing leading to increased biofouling. Herein, we present a material prepared via the grafting-to method with improved antifouling surface properties and intrinsic localized surface plasmon resonance (LSPR) sensor capabilities. A new substrate shrinking fabrication method, Graft-then-Shrink, improved the antifouling properties of polymer-coated Au surfaces by altering graft-to polymer packing while simultaneously generating wrinkled Au structures for LSPR biosensing. Thiol-terminated, antifouling, hydrophilic polymers were grafted to Au-coated prestressed polystyrene (PS) followed by shrinking upon heating above the PS glass transition temperature. Interestingly, the polymer molecular weight and hydration influenced Au wrinkling patterns. Compared to Shrink-then-Graft controls, where polymers are immobilized post shrinking, Graft-then-Shrink increased the polymer content by 76% in defined footprints and improved the antifouling properties as demonstrated by 84 and 72% reduction in macrophage adhesion and protein adsorption, respectively. Wrinkled Au LSPR sensors had sensitivities of ∼200-1000 Δλ/ΔRIU, comparing favorably to commercial LSPR sensors, and detected biotin-avidin and desthiobiotin-avidin complexation in a concentration-dependent manner using a standard plate reader and a 96-well format.

Modulating the Thermoresponse of Polymer-Protein Conjugates with Hydrogels for Controlled Release.

Sustained release is being explored to increase plasma and tissue residence times of polymer-protein therapeutics for improved efficacy. Recently, poly(oligo(ethylene glycol) methyl ether methacrylate) (PEGMA) polymers have been established as potential PEG alternatives to further decrease immunogenicity and introduce responsive or sieving properties. We developed a drug delivery system that locally depresses the lower critical solution temperature (LCST) of PEGMA-protein conjugates within zwitterionic hydrogels for controlled release. Inside the hydrogel the conjugates partially aggregate through PEGMA-PEGMA chain interactions to limit their release rates, whereas conjugates outside of the hydrogel are completely solubilized. Release can therefore be tuned by altering hydrogel components and the PEGMA's temperature sensitivity without the need for traditional controlled release mechanisms such as particle encapsulation or affinity interactions. Combining local LCST depression technology and degradable zwitterionic hydrogels, complete release of the conjugate was achieved over 13 days.

Controlling Experimental Parameters to Improve Characterization of Biomaterial Fouling.

Uncontrolled protein adsorption and cell binding to biomaterial surfaces may lead to degradation, implant failure, infection, and deleterious inflammatory and immune responses. The accurate characterization of biofouling is therefore crucial for the optimization of biomaterials and devices that interface with complex biological environments composed of macromolecules, fluids, and cells. Currently, a diverse array of experimental conditions and characterization techniques are utilized, making it difficult to compare reported fouling values between similar or different biomaterials. This review aims to help scientists and engineers appreciate current limitations and conduct fouling experiments to facilitate the comparison of reported values and expedite the development of low-fouling materials. Recent advancements in the understanding of protein-interface interactions and fouling variability due to experiment conditions will be highlighted to discuss protein adsorption and cell adhesion and activation on biomaterial surfaces.

Molecular Mechanism for the Suppression of Alpha Synuclein Membrane Toxicity by an Unconventional Extracellular Chaperone.

Alpha synuclein (αS) oligomers are a key component of Lewy bodies implicated in Parkinson's disease (PD). Although primarily intracellular, extracellular αS exocytosed from neurons also contributes to PD pathogenesis through a prion-like transmission mechanism. Here, we show at progressive degrees of resolution that the most abundantly expressed extracellular protein, human serum albumin (HSA), inhibits αS oligomer (αSn) toxicity through a three-pronged mechanism. First, endogenous HSA targets αSn with sub-µM affinity via solvent-exposed hydrophobic sites, breaking the catalytic cycle that promotes αS self-association. Second, HSA remodels αS oligomers and high-MW fibrils into chimeric intermediates with reduced toxicity. Third, HSA unexpectedly suppresses membrane interactions with the N-terminal and central αS regions. Overall, our findings suggest that the extracellular proteostasis network may regulate αS cell-to-cell transmission not only by reducing the populations of membrane-binding competent αS oligomers but possibly also by shielding the membrane interface from residual toxic species.

The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.