Intra-articular delivery of an indoleamine 2,3-dioxygenase galectin-3 fusion protein for osteoarthritis treatment in male Lewis rats.

OBJECTIVE: Osteoarthritis (OA) is driven by low-grade inflammation, and controlling local inflammation may offer symptomatic relief. Here, we developed an indoleamine 2,3-dioxygenase and galectin-3 fusion protein (IDO-Gal3), where IDO increases the production of local anti-inflammatory metabolites and Gal3 binds carbohydrates to extend IDO's joint residence time. In this study, we evaluated IDO-Gal3's ability to alter OA-associated inflammation and pain-related behaviors in a rat model of established knee OA. METHODS: Joint residence was first evaluated with an analog Gal3 fusion protein (NanoLuc™ and Gal3, NL-Gal3) that produces luminescence from furimazine. OA was induced in male Lewis rats via a medial collateral ligament and medial meniscus transection (MCLT + MMT). At 8 weeks, NL or NL-Gal3 were injected intra-articularly (n = 8 per group), and bioluminescence was tracked for 4 weeks. Next, IDO-Gal3s's ability to modulate OA pain and inflammation was assessed. Again, OA was induced via MCLT + MMT in male Lewis rats, with IDO-Gal3 or saline injected into OA-affected knees at 8 weeks post-surgery (n = 7 per group). Gait and tactile sensitivity were then assessed weekly. At 12 weeks, intra-articular levels of IL6, CCL2, and CTXII were assessed. RESULTS: The Gal3 fusion increased joint residence in OA and contralateral knees (p < 0.0001). In OA-affected animals, both saline and IDO-Gal3 improved tactile sensitivity (p = 0.008), but IDO-Gal3 also increased walking velocities (p ≤ 0.033) and improved vertical ground reaction forces (p ≤ 0.04). Finally, IDO-Gal3 decreased intra-articular IL6 levels within the OA-affected joint (p = 0.0025). CONCLUSION: Intra-articular IDO-Gal3 delivery provided long-term modulation of joint inflammation and pain-related behaviors in rats with established OA.

Suppression of local inflammation via galectin-anchored indoleamine 2,3-dioxygenase.

The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 (Gal3). Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions.

Intra-Articular Delivery of an Indoleamine 2,3-Dioxygenase Galectin-3 Fusion Protein for Osteoarthritis Treatment in Male Lewis Rats.

Objective : Controlling joint inflammation can improve osteoarthritis (OA) symptoms; however, current treatments often fail to provide long-term effects. We have developed an indoleamine 2,3-dioxygenase and galectin-3 fusion protein (IDO-Gal3). IDO converts tryptophan to kynurenines, directing the local environment toward an anti-inflammatory state; Gal3 binds carbohydrates and extends IDO's joint residence time. In this study, we evaluated IDO-Gal3's ability to alter OA-associated inflammation and pain-related behaviors in a rat model of established knee OA. Methods : Joint residence was first evaluated with an analog Gal3 fusion protein (NanoLuc™ and Gal3, NL-Gal3) that produces luminescence from furimazine. OA was induced in male Lewis rats via a medial collateral ligament and medial meniscus transection (MCLT+MMT). At 8 weeks, NL or NL-Gal3 were injected intra-articularly (n=8 per group), and bioluminescence was tracked for 4 weeks. Next, IDO-Gal3's ability to modulate OA pain and inflammation was assessed. Again, OA was induced via MCLT+MMT in male Lewis rats, with IDO-Gal3 or saline injected into OA-affected knees at 8 weeks post-surgery (n=7 per group). Gait and tactile sensitivity were then assessed weekly. At 12 weeks, intra-articular levels of IL6, CCL2, and CTXII were assessed. Results : The Gal3 fusion increased joint residence in OA and contralateral knees (p<0.0001). In OA-affected animals, IDO-Gal3 improved tactile sensitivity (p=0.002), increased walking velocities (p≤0.033), and improved vertical ground reaction forces (p≤0.04). Finally, IDO-Gal3 decreased intra-articular IL6 levels within the OA-affected joint (p=0.0025). Conclusion : Intra-articular IDO-Gal3 delivery provided long-term modulation of joint inflammation and pain-related behaviors in rats with established OA.

Bcl11b sustains multipotency and restricts effector programs of intestinal-resident memory CD8+ T cells.

The networks of transcription factors (TFs) that control intestinal-resident memory CD8+ T (TRM) cells, including multipotency and effector programs, are poorly understood. In this work, we investigated the role of the TF Bcl11b in TRM cells during infection with Listeria monocytogenes using mice with post-activation, conditional deletion of Bcl11b in CD8+ T cells. Conditional deletion of Bcl11b resulted in increased numbers of intestinal TRM cells and their precursors as well as decreased splenic effector and circulating memory cells and precursors. Loss of circulating memory cells was in part due to increased intestinal homing of Bcl11b-/- circulating precursors, with no major alterations in their programs. Bcl11b-/- TRM cells had altered transcriptional programs, with diminished expression of multipotent/multifunctional (MP/MF) program genes, including Tcf7, and up-regulation of the effector program genes, including Prdm1. Bcl11b also limits the expression of Ahr, another TF with a role in intestinal CD8+ TRM cell differentiation. Deregulation of TRM programs translated into a poor recall response despite TRM cell accumulation in the intestine. Reduced expression of MP/MF program genes in Bcl11b-/- TRM cells was linked to decreased chromatin accessibility and a reduction in activating histone marks at these loci. In contrast, the effector program genes displayed increased activating epigenetic status. These findings demonstrate that Bcl11b is a frontrunner in the tissue residency program of intestinal memory cells upstream of Tcf1 and Blimp1, promoting multipotency and restricting the effector program.

Design principles of microparticle size and immunomodulatory factor formulation dictate antigen-specific amelioration of multiple sclerosis in a mouse model.

Antigen-specific therapies allow for modulation of the immune system in a disease relevant context without systemic immune suppression. These therapies are especially valuable in autoimmune diseases such as multiple sclerosis (MS), where autoreactive T cells destroy myelin sheath. This work shows that an antigen-specific dual-sized microparticle (dMP) system can effectively halt and reverse disease progression in a mouse model of MS. Current MS treatments leave patients immunocompromised, but the dMP formulation spares the immune system as mice can successfully clear a Listeria Monocytogenes infection. Furthermore, we highlight design principles for particle based immunotherapies including the importance of delivering factors specific for immune cell recruitment (GM-CSF or SDF-1), differentiation (GM-CSF or FLT3L) and suppression (TGF-ß or VD3) in conjunction with disease relevant antigen, as the entire formulation is required for maximum efficacy. Lastly, the dMP scheme relies on formulating phagocytosable and non-phagocytosable MP sizes to direct payload to target either cell surface receptors or intracellular targets, as the reverse sized dMP formulation failed to reverse paralysis. We also challenge the design principles of the dMP system showing that the size of the MPs impact efficacy and that GM-CSF plays two distinct roles and that both of these must be replaced to match the primary effect of the dMP system. Overall, this work shows the versatile nature of the dMP system and expands the knowledge in particle science by emphasizing design tenets to guide the next generation of particle based immunotherapies.

Biomaterials-based nanoparticles conjugated to regulatory T cells provide a modular system for localized delivery of pharmacotherapeutic agents.

Type 1 diabetes (T1D) presents with two therapeutic challenges: the need to correct underlying autoimmunity and restore ß-cell mass. We harnessed the unique capacity of regulatory T cells (Tregs) and the T cell receptor (TCR) to direct tolerance induction along with tissue-localized delivery of therapeutic agents to restore endogenous ß-cell function. Specifically, we designed a combinatorial therapy involving biomaterials-based poly(lactic-co-glycolic acid) nanoparticles co-loaded with the Treg growth factor, IL-2, and the ß-cell regenerative agent, harmine (a tyrosine-regulated kinase 1A [DYRK1A] inhibitor), conjugated to the surface of Tregs. We observed continuous elution of IL-2 and harmine from nanoparticles for at least 7 days in vitro. When conjugated to primary human Tregs, IL-2 nanoparticles provided sufficient IL-2 receptor signaling to support STAT5 phosphorylation for sustained phenotypic stability and viability in culture. Inclusion of poly-L-lysine (PLL) during nanoparticle-cell coupling dramatically increased conjugation efficiency, providing sufficient IL-2 to support in vitro proliferation of IL-2-dependent CTLL-2 cells and primary murine Tregs. In 12-week-old female non-obese diabetic mice, adoptive transfer of IL-2/harmine nanoparticle-conjugated NOD.BDC2.5 Tregs, which express an islet antigen-specific TCR, significantly prevented diabetes demonstrating preserved in vivo viability. These data provide the preclinical basis to develop a biomaterials-optimized cellular therapy to restore immune tolerance and promote ß-cell proliferation in T1D through receptor-targeted drug delivery within pancreatic islets.

Treatment with an antigen-specific dual microparticle system reverses advanced multiple sclerosis in mice.

Antigen-specific therapies hold promise for treating autoimmune diseases such as multiple sclerosis while avoiding the deleterious side effects of systemic immune suppression due to delivering the disease-specific antigen as part of the treatment. In this study, an antigen-specific dual-sized microparticle (dMP) treatment reversed hind limb paralysis when administered in mice with advanced experimental autoimmune encephalomyelitis (EAE). Treatment reduced central nervous system (CNS) immune cell infiltration, demyelination, and inflammatory cytokine levels. Mechanistic insights using single-cell RNA sequencing showed that treatment impacted the MHC II antigen presentation pathway in dendritic cells, macrophages, B cells, and microglia, not only in the draining lymph nodes but also strikingly in the spinal cord. CD74 and cathepsin S were among the common genes down-regulated in most antigen presenting cell (APC) clusters, with B cells also having numerous MHC II genes reduced. Efficacy of the treatment diminished when B cells were absent, suggesting their impact in this therapy, in concert with other immune populations. Activation and inflammation were reduced in both APCs and T cells. This promising antigen-specific therapeutic approach advantageously engaged essential components of both innate and adaptive autoimmune responses and capably reversed paralysis in advanced EAE without the use of a broad immunosuppressant.

Perspectives on immunometabolism at the biomaterials interface.

Productive engagement of the immune system is a persistent challenge for biomaterials scientists. Immune engineering offers a new perspective on biomaterial design, with immune cell interaction to modulate effector functions at the center. The effector functions of these cells are intimately linked to their metabolic needs and programming. Immune cell metabolism has received renewed attention in recent years, and with each new discovery there is opportunity for biomaterials scientists. This prospectus aims to provide an overview of the most recent advances in biomaterial engagement of immune cells alongside interrogation of immunometabolism, while looking to future avenues of coalescence. Four cell types are highlighted here: neutrophils, macrophages, dendritic cells, and T cells. Consideration of these two fields, and the tools within each, with a forward-looking mindset is the key to a new era of biomaterials.

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.

Immunosuppressive PLGA TGF-ß1 Microparticles Induce Polyclonal and Antigen-Specific Regulatory T Cells for Local Immunomodulation of Allogeneic Islet Transplants.

Allogeneic islet transplantation is a promising cell-based therapy for Type 1 Diabetes (T1D). The long-term efficacy of this approach, however, is impaired by allorejection. Current clinical practice relies on long-term systemic immunosuppression, leading to severe adverse events. To avoid these detrimental effects, poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were engineered for the localized and controlled release of immunomodulatory TGF-ß1. The in vitro co-incubation of TGF-ß1 releasing PLGA MPs with naïve CD4+ T cells resulted in the efficient generation of both polyclonal and antigen-specific induced regulatory T cells (iTregs) with robust immunosuppressive function. The co-transplantation of TGF-ß1 releasing PLGA MPs and Balb/c mouse islets within the extrahepatic epididymal fat pad (EFP) of diabetic C57BL/6J mice resulted in the prompt engraftment of the allogenic implants, supporting the compatibility of PLGA MPs and local TGF-ß1 release. The presence of the TGF-ß1-PLGA MPs, however, did not confer significant graft protection when compared to untreated controls, despite measurement of preserved insulin expression, reduced intra-islet CD3+ cells invasion, and elevated CD3+Foxp3+ T cells at the peri-transplantation site in long-term functioning grafts. Examination of the broader impacts of TGF-ß1/PLGA MPs on the host immune system implicated a localized nature of the immunomodulation with no observed systemic impacts. In summary, this approach establishes the feasibility of a local and modular microparticle delivery system for the immunomodulation of an extrahepatic implant site. This approach can be easily adapted to deliver larger doses or other agents, as well as multi-drug approaches, within the local graft microenvironment to prevent transplant rejection.

BCL11B is positioned upstream of PLZF and RORγt to control thymic development of mucosal-associated invariant T cells and MAIT17 program.

Mucosal-associated invariant T (MAIT) cells recognize microbial riboflavin metabolites presented by MR1 and play role in immune responses to microbial infections and tumors. We report here that absence of the transcription factor (TF) Bcl11b in mice alters predominantly MAIT17 cells in the thymus and further in the lung, both at steady state and following Salmonella infection. Transcriptomics and ChIP-seq analyses show direct control of TCR signaling program and position BCL11B upstream of essential TFs of MAIT17 program, including RORγt, ZBTB16 (PLZF), and MAF. BCL11B binding at key MAIT17 and at TCR signaling program genes in human MAIT cells occurred mostly in regions enriched for H3K27Ac. Unexpectedly, in human MAIT cells, BCL11B also bound at MAIT1 program genes, at putative active enhancers, although this program was not affected in mouse MAIT cells in the absence of Bcl11b. These studies endorse BCL11B as an essential TF for MAIT cells both in mice and humans.

Immunomodulatory Dual-Sized Microparticle System Conditions Human Antigen Presenting Cells Into a Tolerogenic Phenotype In Vitro and Inhibits Type 1 Diabetes-Specific Autoreactive T Cell Responses.

Current monotherapeutic agents fail to restore tolerance to self-antigens in autoimmune individuals without systemic immunosuppression. We hypothesized that a combinatorial drug formulation delivered by a poly-lactic-co-glycolic acid (PLGA) dual-sized microparticle (dMP) system would facilitate tunable drug delivery to elicit immune tolerance. Specifically, we utilized 30 µm MPs to provide local sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor ß1 (TGF-ß1) along with 1 µm MPs to facilitate phagocytic uptake of encapsulated antigen and 1α,25(OH)2 Vitamin D3 (VD3) followed by tolerogenic antigen presentation. We previously demonstrated the dMP system ameliorated type 1 diabetes (T1D) and experimental autoimmune encephalomyelitis (EAE) in murine models. Here, we investigated the system's capacity to impact human cell activity in vitro to advance clinical translation. dMP treatment directly reduced T cell proliferation and inflammatory cytokine production. dMP delivery to monocytes and monocyte-derived dendritic cells (DCs) increased their expression of surface and intracellular anti-inflammatory mediators. In co-culture, dMP-treated DCs (dMP-DCs) reduced allogeneic T cell receptor (TCR) signaling and proliferation, while increasing PD-1 expression, IL-10 production, and regulatory T cell (Treg) frequency. To model antigen-specific activation and downstream function, we co-cultured TCR-engineered autoreactive T cell "avatars," with dMP-DCs or control DCs followed by ß-cell line (ßlox5) target cells. For G6PC2-specific CD8+ avatars (clone 32), dMP-DC exposure reduced Granzyme B and dampened cytotoxicity. GAD65-reactive CD4+ avatars (clone 4.13) exhibited an anergic/exhausted phenotype with dMP-DC presence. Collectively, these data suggest this dMP formulation conditions human antigen presenting cells toward a tolerogenic phenotype, inducing regulatory and suppressive T cell responses.

Nano and Microparticle Emerging Strategies for Treatment of Autoimmune Diseases: Multiple Sclerosis and Type 1 Diabetes.

Autoimmune diseases affect 10% of the world's population, and 1 in 200 people worldwide suffer from either multiple sclerosis (MS) or type 1 diabetes (T1D). While the targeted organ systems are different, MS and T1D share similarities in terms of autoreactive immune cells playing a critical role in pathogenesis. Both diseases can be managed only symptomatically without curative remission, and treatment options are limited and non-specific. Most current therapies cause some degree of systemic immune suppression, leaving the patients susceptible to opportunistic infections and other complications. Thus, there is considerable interest in the development of immunotherapies not associated with generalized immune suppression for these diseases. This review presents current and preclinical strategies for MS and T1D treatment, emphasizing those aimed to modulate the immune response, including the most recent strategies for tolerance induction. A central focus is on the emerging approaches using nano- and microparticle platforms, their evolution as immunotherapeutic carriers, including those incorporating specific antigens to induce tolerance and reduce unwanted generalized immune suppression.

Dendritic Cells Treated with Exogenous Indoleamine 2,3-Dioxygenase Maintain an Immature Phenotype and Suppress Antigen-specific T cell Proliferation.

Indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme responsible for catalyzing the rate limiting step of tryptophan catabolism, plays a critical role in immune cell suppression and tolerance. Indoleamine 2,3-dioxygenase-mediated depletion of the essential amino acid tryptophan increases susceptibility of T cells to apoptosis, while kynurenine and its downstream metabolites, such as 3-hydroxyanthranilic acid and quinolinic acid, have a direct cytotoxic effect on conventional effector T cells. Additionally, IDO-expressing antigen presenting cells (APCs) induce proliferation of regulatory T cells. When expressed by an APC, the immunosuppressive effects of IDO may act directly on the APC as well as indirectly upon local T cells. One approach to elicit immune tolerance or reduce inflammation therefore is to promote expression of IDO. However, this approach is constrained by several factors including the potential for deleterious biologic effects of conventional IDO-inducing agents such as interferon gamma (IFNγ), and the potential limitations of constitutive gene transfection. Alternatively, direct action of recombinant IDO enzyme supplied exogenously as a potential therapeutic in the extracellular space has not been investigated previously, and is the focus of this work. Results indicate exogenous recombinant human IDO supplementation influences murine dendritic cell (DC) maturation and ability to suppress antigen specific T cell proliferation. Following treatment, DCs were refractory to maturation by LPS as defined by co-stimulatory molecule expression (CD80 and CD86) and major histocompatibility complex II (MHC-II) expression. Dendritic cells exhibited skewing toward an anti-inflammatory cytokine release profile, with reduced secretion of IL-12p70 and maintained basal level of secreted IL-10. Notably, IDO-treated DCs suppressed proliferation of ovalbumin (OVA) antigen-specific CD4+ and CD8+ T cells in the presence of cognate antigen presentation in a manner dependent on active enzyme, as introduction of IDO inhibitor 1-methyl-tryptophan, restored T cell proliferation. Defined media experiments indicate a cumulative role for both tryptophan depletion and kynurenine presence, in the suppressive programming of DCs. In sum, we report that exogenously supplied IDO maintains immunoregulatory function on DCs, suggesting that IDO may have potential as a therapeutic protein for suppressive programming with application toward inflammation 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.

Locally anchoring enzymes to tissues via extracellular glycan recognition.

Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames. Here we report a general strategy to anchor enzymes at injection sites via fusion to galectin-3 (G3), a carbohydrate-binding protein. Fusing G3 to luciferase extended bioluminescence in subcutaneous tissue to ~7 days, whereas unmodified luciferase was undetectable within hours. Engineering G3-luciferase fusions to self-assemble into a trimeric architecture extended bioluminescence in subcutaneous tissue to 14 days, and intramuscularly to 3 days. The longer local half-life of the trimeric assembly was likely due to its higher carbohydrate-binding affinity compared to the monomeric fusion. G3 fusions and trimeric assemblies lacked extracellular signaling activity of wild-type G3 and did not accumulate in blood after subcutaneous injection, suggesting low potential for deleterious off-site effects. G3-mediated anchoring to common tissue glycans is expected to be broadly applicable for improving local pharmacokinetics of various existing and emerging enzyme drugs.

Enzymes as Immunotherapeutics.

Enzymes are attractive as immunotherapeutics because they can catalyze shifts in the local availability of immunostimulatory and immunosuppressive signals. Clinical success of enzyme immunotherapeutics frequently hinges upon achieving sustained biocatalysis over relevant time scales. The time scale and location of biocatalysis are often dictated by the location of the substrate. For example, therapeutic enzymes that convert substrates distributed systemically are typically designed to have a long half-life in circulation, whereas enzymes that convert substrates localized to a specific tissue or cell population can be more effective when designed to accumulate at the target site. This Topical Review surveys approaches to improve enzyme immunotherapeutic efficacy via chemical modification, encapsulation, and immobilization that increases enzyme accumulation at target sites or extends enzyme half-life in circulation. Examples provided illustrate "replacement therapies" to restore deficient enzyme function, as well as "enhancement therapies" that augment native enzyme function via supraphysiologic doses. Existing FDA-approved enzyme immunotherapies are highlighted, followed by discussion of emerging experimental strategies such as those designed to enhance antitumor immunity or resolve inflammation.

Formulation and characterization of poly(propylacrylic acid)/poly(lactic-co-glycolic acid) blend microparticles for pH-dependent membrane disruption and cytosolic delivery.

Poly(lactic-co-glycolic acid) (PLGA) is widely used as a vehicle for delivery of pharmaceutically relevant payloads. PLGA is readily fabricated as a nano- or microparticle (MP) matrix to load both hydrophobic and hydrophilic small molecular drugs as well as biomacromolecules such as nucleic acids and proteins. However, targeting such payloads to the cell cytosol is often limited by MP entrapment and degradation within acidic endolysosomes. Poly(propylacrylic acid) (PPAA) is a polyelectrolyte polymer with the membrane disruptive capability triggered at low pH. PPAA has been previously formulated in various carrier configurations to enable cytosolic payload delivery, but requires sophisticated carrier design. Taking advantage of PPAA functionality, we have incorporated PPAA into PLGA MPs as a simple polymer mixture to enhance cytosolic delivery of PLGA-encapsulated payloads. Rhodamine loaded PLGA and PPAA/PLGA blend MPs were prepared by a modified nanoprecipitation method. Incorporation of PPAA into PLGA MPs had little to no effect on the size, shape, or loading efficiency, and evidenced no toxicity in Chinese hamster ovary epithelial cells. Notably, incorporation of PPAA into PLGA MPs enabled pH-dependent membrane disruption in a hemolysis assay, and a three-fold increased endosomal escape and cytosolic delivery in dendritic cells after 2 h of MP uptake. These results demonstrate that a simple PLGA/PPAA polymer blend is readily fabricated into composite MPs, enabling cytosolic delivery of an encapsulated payload. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1022-1033, 2018.

An antigen-specific semi-therapeutic treatment with local delivery of tolerogenic factors through a dual-sized microparticle system blocks experimental autoimmune encephalomyelitis.

Antigen-specific treatments are highly desirable for autoimmune diseases in contrast to treatments which induce systemic immunosuppression. A novel antigen-specific therapy has been developed which, when administered semi-therapeutically, is highly efficacious in the treatment of the mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). The treatment uses dual-sized, polymeric microparticles (dMPs) loaded with specific antigen and tolerizing factors for intra- and extra-cellular delivery, designed to recruit and modulate dendritic cells toward a tolerogenic phenotype without systemic release. This approach demonstrated robust efficacy and provided complete protection against disease. Therapeutic efficacy required encapsulation of the factors in controlled-release microparticles and was antigen-specific. Disease blocking was associated with a reduction of infiltrating CD4+ T cells, inflammatory cytokine-producing pathogenic CD4+ T cells, and activated macrophages and microglia in the central nervous system. Furthermore, CD4+ T cells isolated from dMP-treated mice were anergic in response to disease-specific, antigen-loaded splenocytes. Additionally, the frequency of CD86hiMHCIIhi dendritic cells in draining lymph nodes of EAE mice treated with Ag-specific dMPs was reduced. Our findings highlight the efficacy of microparticle-based drug delivery platform to mediate antigen-specific tolerance, and suggest that such a multi-factor combinatorial approach can act to block autoimmunity.

Combinatorial drug delivery approaches for immunomodulation.

Immunotherapy has been widely explored for applications to both augment and suppress intrinsic host immunity. Clinical achievements have seen a number of immunotherapeutic drugs displace established strategies like chemotherapy in treating immune-associated diseases. However, single drug approaches modulating an individual arm of the immune system are often incompletely effective. Imperfect mechanistic understanding and heterogeneity within disease pathology have seen monotherapies inadequately equipped to mediate complete disease remission. Recent success in applications of combinatorial immunotherapy has suggested that targeting multiple biological pathways simultaneously may be critical in treating complex immune pathologies. Drug delivery approaches through engineered biomaterials offer the potential to augment desired immune responses while mitigating toxic side-effects by localizing immunotherapy. This review discusses recent advances in immunotherapy and highlights newly explored combinatorial drug delivery approaches. Furthermore, prospective future directions for immunomodulatory drug delivery to exploit are provided.