Biomaterials are being developed as therapeutics for spinal cord injury (SCI) that can stabilize and bridge acute lesions and mediate the delivery of transgenes, providing a localized and sustained reservoir of regenerative factors. For clinical use, direct injection of biomaterial scaffolds is preferred to enable conformation to unique lesions and minimize tissue damage. While an interconnected network of cell-sized macropores is necessary for rapid host cell infiltration into—and thus integration of host tissue with—implanted scaffolds, injectable biomaterials have generally suffered from a lack of control over the macrostructure. As genetic vectors have short lifetimes in vivo , rapid host cell infiltration into scaffolds is a prerequisite for efficient biomaterial-mediated delivery of transgenes. We present scaffolds that can be injected and assembled in situ from hyaluronic acid (HA)-based, spherical microparticles to form scaffolds with a network of macropores (∼10 μ m). The results demonstrate that addition of regularly sized macropores to traditional hydrogel scaffolds, which have nanopores (∼10 nm), significantly increases the expression of locally delivered transgene to the spinal cord after a thoracic injury. Maximal cell and axon infiltration into scaffolds was observed in scaffolds with more regularly sized macropores. The delivery of lentiviral vectors encoding the brain-derived neurotrophic factor (BDNF), but not neurotrophin-3, from these scaffolds further increased total numbers and myelination of infiltrating axons. Modest improvements to the hindlimb function were observed with BDNF delivery. The results demonstrate the utility of macroporous and injectable HA scaffolds as a platform for localized gene therapies after SCI.

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Immune‐mediated hypersensitivities such as autoimmunity, allergy, and allogeneic graft rejection are treated with therapeutics that suppress the immune system, and the lack of specificity is associated with significant side effects. The delivery of disease‐relevant antigens (Ags) by carrier systems such as poly(lactide‐co‐glycolide) nanoparticles (PLG‐Ag) and carbodiimide (ECDI)‐fixed splenocytes (SP‐Ag) has demonstrated Ag‐specific tolerance induction in model systems of these diseases. Despite therapeutic outcomes by both platforms, tolerance is conferred with different efficacy. This investigation evaluated Ag loading and total particle dose of PLG‐Ag on Ag presentation in a coculture system of dendritic cells (DCs) and Ag‐restricted T cells, with SP‐Ag employed as a control. CD25 expression was observed in nearly all T cells even at low concentrations of PLG‐Ag, indicating efficient presentation of Ag by dendritic cells. However, the secretion of IL‐2, Th1, and Th2 cytokines (IFNγ and IL‐4, respectively) varied depending on PLG‐Ag concentration and Ag loading. Concentration escalation of soluble Ag resulted in an increase in IL‐2 and IFNγ and a decrease in IL‐4. Treatment with PLG‐Ag followed a similar trend but with lower levels of IL‐2 and IFNγ secreted. Transcriptional Activity CEll ARrays (TRACER) were employed to measure the real‐time transcription factor (TF) activity in Ag‐presenting DCs. The kinetics and magnitude of TF activity was dependent on the Ag delivery method, concentration, and Ag loading. Ag positively regulated IRF1 activity and, as carriers, NPs and ECDI‐treated SP negatively regulated this signaling. The effect of Ag loading and dose on tolerance induction were corroborated in vivo using the delayed‐type hypersensitivity (DTH) and experimental autoimmune encephalomyelitis (EAE) mouse models where a threshold of 8 μg/mg Ag loading and 0.5 mg PLG‐Ag dose were required for tolerance. Together, the effect of Ag loading and dosing on in vitro and in vivo immune regulation provide useful insights for translating Ag‐carrier systems for the clinical treatment of immune disorders.

Food allergy is a growing health concern worldwide. Current allergen-specific immunotherapy (AIT) approaches require frequent dosing over extended periods of time and may induce anaphylaxis due to allergen-effector cell interactions. A critical need remains to develop novel approaches that refine AIT for the treatment of food allergies. Previous studies show that poly(lactide-co-glycolide) (PLG) nanoscale particles (NP) effectively suppress Th1- and Th17-driven immune pathologies. However, their ability to suppress the distinct Th2-polarized immune responses driving food allergy are unknown. Herein, we describe the safety and efficacy of NPs containing encapsulated peanut allergen in desensitizing murine models of peanut allergy. Peanut extract encapsulation allowed for the safe intravenous delivery of allergen relative to non-encapsulated approaches. Application of 2–3 doses, without the need for dose escalation, was sufficient to achieve prophylactic and therapeutic efficacy, which correlated with suppression of Th2-mediated disease and reduced mast cell degranulation. Efficacy was associated with strong reductions in a broad panel of Th1, Th2, and Th17 cytokines. These results demonstrate the ability of PLG NPs to suppress allergen-specific immune responses to induce a more tolerogenic phenotype, conferring protection from intragastric allergen challenge. These promising studies represent a step forward in the development of improved immunotherapies for food allergy.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue-and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.