GRAS-microparticle microarrays identify dendritic cell tolerogenic marker-inducing formulations.

Microarrays, miniaturized platforms used for high-content studies, provide potential advantages over traditional in vitro investigation in terms of time, cost, and parallel analyses. Recently, microarrays have been leveraged to investigate immune cell biology by providing a platform with which to systematically investigate the effects of various agents on a wide variety of cellular processes, including those giving rise to immune regulation for application toward curtailing autoimmunity. A specific embodiment incorporates dendritic cells cultured on microarrays containing biodegradable microparticles. Such an approach allows immune cell and microparticle co-localization and release of compounds on small, isolated populations of cells, enabling a quick, convenient method to quantify a variety of cellular responses in parallel. In this study, the microparticle microarray platform was utilized to investigate a small library of sixteen generally regarded as safe (GRAS) compounds (ascorbic acid, aspirin, capsaicin, celastrol, curcumin, epigallocatechin-3-gallate, ergosterol, hemin, hydrocortisone, indomethacin, menadione, naproxen, resveratrol, retinoic acid, α-tocopherol, vitamin D3) for their ability to induce suppressive phenotypes in murine dendritic cells. Two complementary tolerogenic index ranking systems were proposed to summarize dendritic cell responses and suggested several lead compounds (celastrol, ergosterol, vitamin D3) and two secondary compounds (hemin, capsaicin), which warrant further investigation for applications toward suppression and tolerance.

Dual-Sized Microparticle System for Generating Suppressive Dendritic Cells Prevents and Reverses Type 1 Diabetes in the Nonobese Diabetic Mouse Model.

Antigen specificity is a primary goal in developing curative therapies for autoimmune disease. Dendritic cells (DCs), as the most effective antigen presenting cells in the body, represent a key target to mediate restoration of antigen-specific immune regulation. Here, we describe an injectable, dual-sized microparticle (MP) approach that employs phagocytosable ∼1 µm and nonphagocytosable ∼30 µm MPs to deliver tolerance-promoting factors both intracellularly and extracellularly, as well as the type 1 diabetes autoantigen, insulin, to DCs for reprogramming of immune responses and remediation of autoimmunity. This poly(lactic-co-glycolic acid) (PLGA) MP system prevented diabetes onset in 60% of nonobese diabetic (NOD) mice when administered subcutaneously in 8 week old mice. Prevention of disease was dependent upon antigen inclusion and required encapsulation of factors in MPs. Moreover, administration of this "suppressive-vaccine" boosted pancreatic lymph node and splenic regulatory T cells (Tregs), upregulated PD-1 on CD4+ and CD8+ T cells, and reversed hyperglycemia for up to 100 days in recent-onset NOD mice. Our results demonstrate that a MP-based platform can reeducate the immune system in an antigen-specific manner, augment immunomodulation compared to soluble administration of drugs, and provide a promising alternative to systemic immunosuppression for autoimmunity.

Poly(2-propylacrylic acid)/poly(lactic-co-glycolic acid) blend microparticles as a targeted antigen delivery system to direct either CD4+ or CD8+ T cell activation.

Poly(lactic-co-glycolic acid) (PLGA) based microparticles (MPs) are widely investigated for their ability to load a range of molecules with high efficiency, including antigenic proteins, and release them in a controlled manner. Micron-sized PLGA MPs are readily phagocytosed by antigen presenting cells, and localized to endosomes. Due to low pH and digestive enzymes, encapsulated protein cargo is largely degraded and processed in endosomes for MHC-II loading and presentation to CD4+ T cells, with very little antigen delivered into the cytosol, limiting MHC-I antigenic loading and presentation to CD8+ T cells. In this work, PLGA was blended with poly(2-propylacrylic acid) (PPAA), a membrane destabilizing polymer, in order to incorporate an endosomal escape strategy into PLGA MPs as an easily fabricated platform with diverse loading capabilities, as a means to enable antigen presentation to CD8+ T cells. Ovalbumin (OVA)-loaded MPs were fabricated using a water-in-oil double emulsion with a 0% (PLGA only), 3 and 10% PPAA composition. MPs were subsequently determined to have an average diameter of 1 µm, with high loading and a release profile characteristic of PLGA. Bone marrow derived dendritic cells (DCs) were then incubated with MPs in order to evaluate localization, processing, and presentation of ovalbumin. Endosomal escape of OVA was observed only in DC groups treated with PPAA/PLGA blends, which promoted high levels of activation of CD8+ OVA-specific OT-I T cells, compared to DCs treated with OVA-loaded PLGA MPs which were unable activate CD8+ T cells. In contrast, DCs treated with OVA-loaded PLGA MPs promoted OVA-specific OT-II CD4+ T cell activation, whereas PPAA incorporation into the MP blend did not permit CD4+ T cell activation. These studies demonstrate PLGA MP blends containing PPAA are able to provide an endosomal escape strategy for encapsulated protein antigen, enabling the targeted delivery of antigen for tunable presentation and activation of either CD4+ or CD8+ T cells.

A cell-based microarray to investigate combinatorial effects of microparticle-encapsulated adjuvants on dendritic cell activation.

Experimental vaccine adjuvants are being designed to target specific toll-like receptors (TLRs) alone or in combination, expressed by antigen presenting cells, notably dendritic cells (DCs). There is a need for high-content screening (HCS) platforms to explore how DC activation is affected by adjuvant combinations. Presented is a cell-based microarray approach, "immunoarray", exposing DCs to a large number of adjuvant combinations. Microparticles encapsulating TLR ligands are printed onto arrays in a range of doses for each ligand, in all possible dose combinations. Dendritic cells are then co-localized with physisorbed microparticles on the immunoarray, adherent to isolated islands surrounded by a non-fouling background, and DC activation is quantified. Delivery of individual TLR ligands was capable of eliciting high levels of specific DC activation markers. For example, either TLR9 ligand, CpG, or TLR3 ligand, poly I:C, was capable of inducing among the highest 10% expression levels of CD86. In contrast, MHC-II expression in response to TLR4 agonist MPLA was among the highest, whereas either MPLA or poly I:C, was capable of producing among the highest levels of CCR7 expression, as well as inflammatory cytokine IL-12. However, in order to produce robust responses across all activation markers, adjuvant combinations were required, and combinations were more represented among the high responders. The immunoarray also enables investigation of interactions between adjuvants, and each TLR ligand suggested antagonism to other ligands, for various markers. Altogether, this work demonstrates feasibility of the immunoarray platform to screen microparticle-encapsulated adjuvant combinations for the development of improved and personalized vaccines.

A combination hydrogel microparticle-based vaccine prevents type 1 diabetes in non-obese diabetic mice.

Targeted delivery of self-antigens to the immune system in a mode that stimulates a tolerance-inducing pathway has proven difficult. To address this hurdle, we developed a vaccine based-approach comprised of two synthetic controlled-release biomaterials, poly(lactide-co-glycolide; PLGA) microparticles (MPs) encapsulating denatured insulin (key self-antigen in type 1 diabetes; T1D), and PuraMatrix(TM) peptide hydrogel containing granulocyte macrophage colony-stimulating factor (GM-CSF) and CpG ODN1826 (CpG), which were included as vaccine adjuvants to recruit and activate immune cells. Although CpG is normally considered pro-inflammatory, it also has anti-inflammatory effects, including enhancing IL-10 production. Three subcutaneous administrations of this hydrogel (GM-CSF/CpG)/insulin-MP vaccine protected 40% of NOD mice from T1D. In contrast, all control mice became diabetic. In vitro studies indicate CpG stimulation increased IL-10 production, as a potential mechanism. Multiple subcutaneous injections of the insulin containing formulation resulted in formation of granulomas, which resolved by 28 weeks. Histological analysis of these granulomas indicated infiltration of a diverse cadre of immune cells, with characteristics reminiscent of a tertiary lymphoid organ, suggesting the creation of a microenvironment to recruit and educate immune cells. These results demonstrate the feasibility of this injectable hydrogel/MP based vaccine system to prevent T1D.

Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells.

A new paradigm in oncology establishes a spectrum of tumorigenic potential across the heterogeneous phenotypes within a tumor. The cancer stem cell hypothesis postulates that a minute fraction of cells within a tumor, termed cancer stem cells (CSCs), have a tumor-initiating capacity that propels tumor growth. An application of this discovery is to target this critical cell population using chemotherapy; however, the process of isolating these cells is arduous, and the rarity of CSCs makes it difficult to test potential drug candidates in a robust fashion, particularly for individual patients. To address the challenge of screening drug libraries on patient-derived populations of rare cells, such as CSCs, we have developed a drug-eluting microarray, a miniaturized platform onto which a minimal quantity of cells can adhere and be exposed to unique treatment conditions. Hundreds of drug-loaded polymer islands acting as drug depots colocalized with adherent cells are surrounded by a nonfouling background, creating isolated culture environments on a solid substrate. Significant results can be obtained by testing <6% of the cells required for a typical 96-well plate. Reliability was demonstrated by an average coefficient of variation of 14% between all of the microarrays and 13% between identical conditions within a single microarray. Using the drug-eluting array, colorectal CSCs isolated from two patients exhibited unique responses to drug combinations when cultured on the drug-eluting microarray, highlighting the potential as a prognostic tool to identify personalized chemotherapeutic regimens targeting CSCs.