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.

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.

Displacement Affinity Release of Antibodies from Injectable Hydrogels.

Current methods to tune release rates of therapeutic antibodies (Abs) for local delivery are complex and routinely require bioconjugations that may reduce Ab bioactivity. To rapidly tune release profiles of bioactive Abs, we developed a biophysical interaction system within a neutravidin modified poly(carboxybetaine) hydrogel (pCB-NT) that tunes release rates of desthiobiotinylated Abs (D-Abs) using a constant hydrogel and D-Ab combination. Herein, we delivered desthiobiotinylated bevacizumab (D-Bv), a recombinant humanized monoclonal IgG1 Ab for antiangiogenic cancer therapies. D-Bv's high affinity for pCB-NT (KD 7.8 × 10-10 M; t1/2 ∼ 2 h) produces a slow D-Bv release rate (∼5 ng day-1) that is increased by the dissolution of hydrogel encapsulated biotin derivative pellets, which displaces D-Bv from pCB-NT binding sites. In contrast to traditional affinity systems, displacement affinity release of Abs (DARA) does not require Ab or hydrogel modifications for each unique release rate. D-Bv release rates were tuned by simply altering the total biotin derivative concentration; the effective first-order (keff) and mass per day release rates were tuned 25- and 8-fold, respectively. Local surface plasmon resonance (LSPR) and biolayer interferometry (BLI) confirmed the D-Bv binding affinity for the corresponding ligand and Fc receptor, demonstrating that the biophysical interaction system is amenable to anticancer Abs for receptor or cytokine blockade and immune cell recruitment to cancer cells.

Tunable degradation of low-fouling carboxybetaine-hyaluronic acid hydrogels for applications in cell encapsulation.

Low-fouling hydrogels with tunable degradation rates and biochemical environments have the potential to improve adoptive cell therapies for cancer immunotherapy and regenerative medicine. To this end, we developed in situ gelling hydrogels from low-fouling poly(carboxybetaine-co-maleimide) (pCBM) random copolymers and thiolated hyaluronic acid (HA-SH). pCBM-HA hydrogel enzymatic degradation rates were tuned 5 fold by altering pCBM composition (4, 11, and 16 maleimide mol%) and 2.3 fold by HA-SH concentration (1-2 wt%). pCBM-HA gels were low-fouling towards bovine serum albumin (BSA; adsorbed ∼20 µg cm-2) and resisted fibroblast adhesion. To control pCBM-HA bioactivity, the cell adhesive peptide CGRGDS was immobilized on pCBM to promote fibroblast adhesion (39% decrease in circularity), which increased metabolic activity by ∼50%. pCBM-HA modified with CGRGDS enhanced the metabolic activity of encapsulated T cells by ∼21% compared to gels without HA, indicating their potential for immunotherapies. Low-fouling pCBM-HA hydrogels provide a vehicle with tunable degradation rates and biochemical environments for encapsulation applications in cell adoptive therapies.

Controlled degradation of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) hydrogels.

Degradable low-fouling hydrogels are ideal vehicles for drug and cell delivery. For each application, hydrogel degradation rate must be re-optimized for maximum therapeutic benefit. We developed a method to rapidly and predictably tune degradation rates of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) (P(EG) x MA) hydrogels by modifying two interdependent variables: (1) base-catalysed crosslink degradation kinetics, dependent on crosslinker electronics (electron withdrawing groups (EWGs)); and, (2) polymer hydration, dependent on the molecular weight (M W) of poly(ethylene glycol) (PEG) pendant groups. By controlling PEG M W and EWG strength, P(EG) x MA hydrogels were tuned to degrade over 6 to 52 d. A 6-member P(EG) x MA copolymer library yielded slow and fast degrading low-fouling hydrogels suitable for short- and long-term delivery applications. The degradation mechanism was also applied to RGD-functionalized poly(carboxybetaine methacrylamide) (PCBMAA) hydrogels to achieve slow (∼50 d) and fast (∼13 d) degrading low-fouling, bioactive hydrogels.

Improved Efficacy of Antibody Cancer Immunotherapeutics through Local and Sustained Delivery.

Antibodies are a growing class of cancer immunotherapeutics that facilitate immune-cell-mediated killing of tumors. However, the efficacy and safety of immunotherapeutics are limited by transport barriers and poor tumor uptake, which lead to high systemic concentrations and potentially fatal side effects. To increase tumor antibody immunotherapeutic concentrations while decreasing systemic concentrations, local delivery vehicles for sustained antibody release are being developed. The focus of this review is to define the material properties required for implantable controlled antibody delivery and highlight the controlled-release strategies that are applicable to antibody immunotherapeutics.

Influence of Hydrophobic Cross-Linkers on Carboxybetaine Copolymer Stimuli Response and Hydrogel Biological Properties.

Poly(carboxybetaine) (pCB) hydrogels do not elicit a foreign body response due to their low-fouling properties, making them ideal implantable materials for in vivo drug and cell delivery. Current reported pCB hydrogels are cross-linked using cytotoxic UV-initiated radical polymerization limiting clinical and in vivo translation. For clinical translation, we require in situ and biorthogonal cross-linking of pCB hydrogels that are both low-fouling and low-swelling to limit nonspecific interactions and minimize tissue damage, respectively. To this end, we synthesized carboxybetaine (CB) random copolymers (molecular weight (MW): ∼7-33 kDa; D: 1.1-1.36) containing azide (pCB-azide) or strained alkyne (Dibenzocyclooctyne (DBCO); pCB-DBCO) that rapidly cross-link upon mixing. Unlike CB homopolymers and other CB copolymers studied, high DBCO content pCB-DBCO30 (30% DBCO mole fraction) is thermoresponsive with a upper critical solution temperature (UCST; cloud point of ∼20 °C at 50 g/L) in water due to electrostatic associations. Due to the antipolyelectrolyte effect, pCB-DBCO30 is salt-responsive and is soluble even at low temperatures in 5 M NaCl, which prevents zwitterion electrostatic associations. pCB-azide and pCB-DBCO with 0.05 to 0.16 cross-linker mole fractions rapidly formed 10 wt % hydrogels upon mixing that were low-swelling (increase of ∼10% in wet weight) while remaining low-fouling to proteins (∼10-20 µg cm-2) and cells, making them suitable for in vivo applications. pCB-X31 hydrogels composed of pCB-azide32 and pCB-DBCO30 formed opaque gels in water and physiological conditions that shrunk to ∼70% of their original wet weight due to pCB-DBCO30's greater hydrophobicity and interchain electrostatic interactions, which promotes nonspecific protein adsorption (∼35 µg cm-2) and cell binding. Once formed, the electrostatic interactions in pCB-X31 hydrogels are not fully reversible with heat or salt. Although, pCB-X31 hydrogels are transparent when initially prepared in 5 M NaCl. This is the first demonstration of a thermo- and salt-responsive CB copolymer that can tune hydrogel protein and cell fouling properties.

Hydrogels with reversible chemical environments for in vitro cell culture.

Methods to reversibly control the chemical environment of hydrogels have application in three-dimensional cell culture to study cell proliferation, migration and differentiation in environments more representative of in vivo environments. Herein, we have developed a method to temporally control the chemical environment of agarose hydrogels through non-covalent attachment of peptide motifs. Streptavidin-GRGDS conjugates were immobilized in desthiobiotin-modified agarose hydrogels through the desthiobiotin-streptavidin interaction (KD 10-11 M). Streptavidin-GRGDS was then displaced from the gel by the addition of biotin, which has a higher affinity for streptavidin (KD 10-15 M). This process was repeated to sequentially and simultaneously immobilize different biomolecules and model compounds in hydrogels over the course of several hours to weeks. The influence of dynamic chemical environments on cellular activity was demonstrated by monitoring HUVEC tube formation for 30 h.