The nanomaterial-dependent modulation of dendritic cells and its potential influence on therapeutic immunosuppression in lupus.

Targeting dendritic cells with nanoparticles is an attractive modality for instigating immunity or inducing immunosuppression. An important aspect of successful delivery of antigen and immune modulators to these cells is the efficacy of nanoparticle internalization, which can dictate the strength and robustness of immune responses; optimizing particulate uptake is thus key. We compared the internalization of two nanoparticulate platforms: a vesicular "nanogel" platform with a lipid exterior, and the widely-used solid biodegradable poly(lactic-co-glycolic acid) (PLGA) system. We found that nanogels were more effectively internalized by dendritic cells in vitro, as demonstrated by fluorescent tracer measurements. Additionally, the magnitude of dendritic cell immunosuppression achieved by nanogels loaded with mycophenolic acid, an immunosuppressant, was greater than similarly drug-loaded PLGA. Although both types of particles could mitigate the production of inflammatory cytokines and the up-regulation of stimulatory surface markers, nanogels yielded greater reductions. These in vitro measurements correlated with in vivo efficacy, where immunosuppressive therapy with nanogels extended the survival of lupus-prone NZB/W F1 mice whereas PLGA particles did not. Our results highlight the importance of material on nanoparticle uptake by dendritic cells, which impacts the quality of therapeutic immunosuppression.

Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice.

The ability to selectively inactivate immune cells with immunosuppressants is a much sought-after modality for the treatment of systemic lupus erythematosus and autoimmunity in general. Here, we designed and tested a novel nanogel drug delivery vehicle for the immunosuppressant mycophenolic acid (MPA). Treatment with MPA-loaded nanogels increased the median survival time (MST) of lupus-prone NZB/W F1 mice by 3 months with prophylactic use (MST was 50 weeks versus 38 weeks without treatment), and by 2 months when administered after the development of severe renal damage (MST after proteinuria onset was 12.5 weeks versus 4 weeks without treatment). Equivalent and greater doses of MPA administered in buffer were not efficacious. Nanogels had enhanced biodistribution to organs and association with immune cells. CD4-targeted nanogels yielded similar therapeutic results compared with nontargeted formulations, with protection from glomerulonephritis and decreases in IFN-γ-positive CD4 T cells. DCs that internalized nanogels helped mediate immunosuppression, as they had reduced production of inflammatory cytokines such as IFN-γ and IL-12. Our results demonstrate efficacy of nanogel-based lupus therapy and implicate a mechanism by which immunosuppression is enhanced, in part, by the targeting of antigen-presenting cells.

Adsorption of multimeric T cell antigens on carbon nanotubes: effect on protein structure and antigen-specific T cell stimulation.

Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin.

Combination delivery of TGF-ß inhibitor and IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy.

The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-ß (TGF-ß), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-ß inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8(+) T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.

Development of a nanoparticulate formulation of retinoic acid that suppresses Th17 cells and upregulates regulatory T cells.

Retinoic acid (RA) is a small molecule capable of shunting developing T cells away from the Th17 lineage and towards the Treg phenotype, making it a potentially useful therapeutic for autoimmune and inflammatory diseases. However, therapy can be complicated by systemic toxicity and unpredictable bioavailability, making a targeted drug delivery vehicle for local therapy desirable. A promising approach is the use of nanoparticles, which have been demonstrated to increase potency and decrease toxicity of therapies in a variety of disease models including Th17 mediated diseases. Nanoparticles can also be targeted to specific cell types via surface modification, further increasing the potential specificity of this approach. We therefore constructed a nanoparticulate drug delivery platform from poly(lactic-co-glycolic acid) (PLGA) capable of encapsulating and releasing RA. Here we report the fabrication, characterization, and in vitro bioactivity of this platform. We demonstrate that RA containing PLGA nanoparticles suppress IL-17 production and ROR-γ(t) expression in T cells polarized towards the Th17 phenotype in vitro with similar potency to that of free drug. Furthermore, we show that these particles enhance TGF-ß dependent Foxp3 expression and IL-10 production of T cells in vitro with similar potency to free RA. Finally, we demonstrate that T cells polarized towards the Th17 phenotype in the presence of free and nanoparticulate RA have similarly suppressed ability to induce IL-6 production by fibroblasts. Our findings demonstrate the feasibility of RA delivery via biodegradable nanoparticles and represent an exciting technology for the treatment of autoimmune and inflammatory diseases.

Application of nanotechnologies for improved immune response against infectious diseases in the developing world.

There is an urgent need for new strategies to combat infectious diseases in developing countries. Many pathogens have evolved to elude immunity and this has limited the utility of current therapies. Additionally, the emergence of co-infections and drug resistant pathogens has increased the need for advanced therapeutic and diagnostic strategies. These challenges can be addressed with therapies that boost the quality and magnitude of an immune response in a predictable, designable fashion that can be applied for wide-spread use. Here, we discuss how biomaterials and specifically nanoscale delivery vehicles can be used to modify and improve the immune system response against infectious diseases. Immunotherapy of infectious disease is the enhancement or modulation of the immune system response to more effectively prevent or clear pathogen infection. Nanoscale vehicles are particularly adept at facilitating immunotherapeutic approaches because they can be engineered to have different physical properties, encapsulated agents, and surface ligands. Additionally, nanoscaled point-of-care diagnostics offer new alternatives for portable and sensitive health monitoring that can guide the use of nanoscale immunotherapies. By exploiting the unique tunability of nanoscale biomaterials to activate, shape, and detect immune system effector function, it may be possible in the near future to generate practical strategies for the prevention and treatment of infectious diseases in the developing world.

Clustering of stimuli on single-walled carbon nanotube bundles enhances cellular activation.

Functionalized single-walled carbon nanotube bundles (f-bSWNT) adsorbed with T-cell-stimulating antibodies are shown to enhance both the kinetics and magnitude of T cell stimulation compared to the same concentration of free antibodies in solution. This enhancement is unique to f-bSWNT compared to other artificial substrates with high surface area and similar chemistry. We explored the origins of this enhanced activity with FRET microscopy and found the preferential formation of large antibody stimuli clusters (5 to 6 microm) on the surface of functionalized versus untreated nanotubes. This highlights the important aspect that antigen clusters can be formed on f-bSWNT, impacting the potency of the T cell stimulus. Clustering of T cell antigens on artificial substrates impacts the avidity of interaction with cells facilitating rapid stimulation dynamics and an overall greater magnitude of response. These findings support the use of chemically treated nanotube bundles as an efficient substrate for the presentation of antigens and point to their potential in clinical applications involving artificial antigen-presentation for ex vivo T cell expansion in adoptive immunotherapy.

Demonstration of multi-analyte patterning using piezoelectric inkjet printing of multiple layers.

Patterning substrates with biological reagents is a critical component of biosensor development. Many applications require multi-analyte patterning capabilities, with a need to deposit several species reproducibly with a high degree of precision. We demonstrate a piezoelectric inkjet printing system that is capable of creating sub-millimeter (down to 150microm) patterns of aqueous and nonaqueous reagents with precise placement for biosensor applications. The size, shape, and density of the patterns may be modified by simple adjustments of the patterning parameters. Using this system, two methods of multi-analyte protein patterning for use in biosensor assays are demonstrated. The first method involves the deposition of multiple proteins directly onto a gold substrate. Specific binding of an antibody to the deposited antigen is demonstrated, although nonspecific adsorption of the antibody may limit the utility of this simple method in quantitative biosensor applications. A second, more sophisticated multi-analyte patterning method involves two sequential patterning steps, consisting of an initial deposition onto gold of a mixed thiol layer to provide oriented binding capabilities in a nonfouling background and a second deposition of multiple biotinylated proteins. Highly specific antibody binding to this patterned multi-analyte surface was demonstrated, with minimal nonspecific adsorption to the surrounding regions. Thus, this method produces high-quality, localized, and customizable sub-millimeter patterns in a nonfouling background for multi-analyte bioassay development.

The use of deoxycholic acid to enhance the oral bioavailability of biodegradable nanoparticles.

Oral delivery of nanoparticles encapsulating drugs and proteins remains a challenging route for administration due to the many barriers in the gastrointestinal tract that limit bioavailability. We hypothesized that bile salts could be used to improve the bioavailability of poly(lactide-co-glycolide) (PLGA) nanoparticles by protecting them during their transport through the gastrointestinal tract and enhancing their absorption by the intestinal epithelia. A deoxycholic acid emulsion is shown to protect PLGA nanoparticles from degradation in acidic conditions and enhance their permeability across a Caco-2 cell monolayer, an in vitro model of human epithelium. Oral administration of loaded PLGA nanoparticles to mice, using a deoxycholic acid emulsion, produced sustained levels of the encapsulant in the blood over 24-48 h with a relative bioavailability of 1.81. Encapsulant concentration was highest in the liver, demonstrating a novel means for targeted delivery to the liver by the oral route.