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

Galectin-Anchored indoleamine 2,3-dioxygenase tissue-targeted therapeutic enzyme suppresses local inflammation in multiple animal models

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Chimeric protein and nano-construct for tissue-retained enzyme to locally suppress inflammation

There is considerable need for new retention strategies of immunomodulatory biologics for localized suppression of inflammation. We developed a chimeric protein as a well as a self-assembled nano-construct incorporating novel approaches for both retention and suppression to induce potent, confined metabolic programming. Immunosuppressive indoleamine 2,3 dioxygenase (IDO), which depletes tryptophan through the kynurenine pathway, was fused to Galectin 3 (Gal3), which binds extracellular glycans and provides tissue anchoring. Using a luciferase-Gal3 fusion reporter, tissue retention was prolonged to ~6 d whereas native luciferase is not retained and undetectable by 24 h. IDO-Gal3 injected subcutaneously controlled local LPS-challenged tissue inflammation. Furthermore, subgingival injection suppressed periodontal disease (PD) in a polymicrobial challenged mouse model. Multiplex analysis of gingival tissue revealed decreased inflammatory (IL-1β, IL-12p70, KC, IP10, MCP1, MIP2) and increased anti-inflammatory (IL-10, TGFβ3) proteins, indicating a shift toward homeostasis. Animals treated with IDO-Gal3 also showed significant decrease in bone loss commonly associated with PD, as determined by µCT analysis

Engineering surfaces to direct integrin binding and signaling to promote osteoblast differentiation

Cell adhesion to proteins adsorbed onto implanted surfaces is particularly important to host responses in biomedical and tissue engineering applications. Biomaterial surface properties influence the type, quantity and functional presentation (activity) of proteins adsorbed upon contact with physiological fluids, and modulate subsequent cell response. Cell adhesion to extracellular matrix proteins (e.g. fibronectin) is primarily mediated by the integrin family of cell-surface receptors. Integrins not only anchor cells, supporting cell spreading and migration, but also trigger signals that regulate survival, proliferation and differentiation. A fundamental understanding of the adhesive interactions at the biomaterial interface is critical to the rational design of biomaterial surfaces. Using model surfaces of self-assembled monolayers of alkanethiols on gold presenting well-defined surface chemistries (CH3, OH, COOH, NH2), we investigated the effects of surface chemistry on osteoblastic differentiation. We report that surface chemistry effectively modulates fibronectin adsorption, integrin binding, focal adhesion assembly and signaling to direct the osteoblast cellular functions of adhesion strength, gene expression and matrix mineralization. Specifically, surfaces presenting OH and NH2 functionalities provide enhanced functional presentation of adsorbed fibronectin, promoting specificity of integrin binding as well as elevating focal adhesion assembly and signaling. Furthermore, the OH and NH2 surfaces supported elevated levels of osteoblast differentiation as evidenced by osteoblast-specific gene expression and matrix mineralization. These results contribute to the development of design principles for the engineering of surfaces that direct cell adhesion for biomedical and tissue engineering applications. In particular, the understanding provided by this analysis may be useful in the engineering of surface properties for bone tissue repair and regeneration.Ph.D.Committee Chair: Andres J. Garcia ; Committee Members: Cheng Zhu, David Collard, Elliot Chaikoff, Harish Radhakrishna, and Robert Guldber

Dendritic cells in the host response to implanted materials

The role of dendritic cells (DCs) and their targeted manipulation in the body's response to implanted materials is an important and developing area of investigation, and a large component of the emerging field of biomaterials-based immune engineering. The key position of DCs in the immune system, serving to bridge innate and adaptive immunity, is facilitated by rich diversity in type and function and places DCs as a critical mediator to biomaterials of both synthetic and natural origins. This review presents current views regarding DC biology and summarizes recent findings in DC responses to implanted biomaterials. Based on these findings, there is promise that the directed programming of application-specific DC responses to biomaterials can become a reality, enabling and enhancing applications almost as diverse as the larger field of biomaterials itself