Controlled Delivery of Single or Multiple Antigens in Tolerogenic Nanoparticles Using Peptide-Polymer Bioconjugates

Abstract

Polymeric nanoparticles (NPs) have demonstrated their potential to induce antigen (Ag)-specific immunological tolerance in multiple immune models and are at various stages of commercial development. Association of Ag with NPs is typically achieved through surface coupling or encapsulation methods. However, these methods have limitations that include high polydispersity, uncontrollable Ag loading and release, and possible immunogenicity. Here, using antigenic peptides conjugated to poly(lactide-co-glycolide), we developed Ag-polymer conjugate NPs (acNPs) with modular loading of single or multiple Ags, negligible burst release, and minimally exposed surface Ag. Tolerogenic responses of acNPs were studied in vitro to decouple the role of NP size, concentration, and Ag loading on regulatory T cell (Treg) induction. CD4⁺CD25⁺Foxp3⁺ Treg induction was dependent on NP size, but CD25 expression of CD4⁺ T cells was not. NP concentration and Ag loading could be modulated to achieve maximal levels of Treg induction. In relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a murine model of multiple sclerosis, acNPs were effective in inhibiting disease induced by a single peptide or multiple peptides. The acNPs provide a simple, modular, and well-defined platform, and the NP physicochemical properties offer potential to design and answer complex mechanistic questions surrounding NP-induced tolerance.

Keywords: autoimmune disease; bioconjugation; drug delivery; immune tolerance; multiple sclerosis; nanoparticle; regulatory T cells.

Graphical abstract

Figure 1

Synthesis and Characterization of PLG-Antigen Conjugates and Nanoparticle Formation (A) ¹H-NMR spectrum of PLG, OVA_323–339, and PLG-OVA_323–339 measured in DMSO-d6 (calibrated at 2.5 ppm). (B) The coupling efficiency of OVA_323–339 to PLG was calculated by comparing the integration values of the overlapping methyl proton peaks of leucine and isoleucine present at 0.8 ppm in OVA_323–339 (d, d’) to the methylene proton peak present at 5.3 ppm in PLG (b). (C) Schematic representation of nanoparticles used in this study. (D) PLG, NP(OVA_323–339), NP-OVA_323–339, and acNP-OVA_323–339 were incubated with FITC-labeled anti-OVA_323–339 IgG to identify the presence of peptide on the surface of the particles. Results are geometric mean fluorescence intensity. (E) Release profile of NP(OVA_323–339), NP-OVA_323–339, and acNP-OVA_323–339. Statistical differences between groups were determined by performing a one-way ANOVA and Tukey’s post hoc test (p < 0.05). Groups with similar letters indicate no statistical significance. Error bars represent SEM.

Figure 2

Regulatory T Cell Induction In Vitro Is Dependent on Nanoparticle Size and Ag Loading (A) Representative gating strategy for regulatory T cell induction. All cells, singlets, live, and CD4+ cells were examined for CD25 and Foxp3 expression. (B–E) BMDCs were treated for 3 hr with 300 μg/mL of 400 nm (B and D) and 80 nm (C and E) acNP-OVA_323–339 or 100 ng/mL of soluble OVA_323–339. Excess acNP-OVA_323–339 was subsequently washed from the wells prior to addition of naive CD4⁺ OT-II T cells and 2 ng/mL of TGF-β1. Cells were co-cultured for 4 days prior to flow cytometric analysis. The effect of antigen loading on CD25 (B and C) and Foxp3 expression of T cells (D and E) was measured. Although both particles formulations increased CD25 expression similarly, only 400 nm acNP-OVA_323–339 particles could efficiently induce CD4⁺CD25⁺Foxp3⁺ regulatory T cells. Statistical differences between groups were determined by performing a one-way ANOVA and Tukey’s post hoc test (p < 0.05). Groups with similar letters indicate no statistical significance. Error bars represent SEM.

Figure 3

Regulatory T Cell Induction Is Dependent on Nanoparticle Concentration BMDCs were treated for 3 hr with various concentrations of 400 nm of acNP-OVA_323–339 (2, 8, 25, and 150 μg/mg loading) formulation. Excess acNP-OVA_323–339 particles were subsequently washed from the cell surface prior to addition of OT-II T cells and 2 ng/mL of TGF-β1. (A and B) The cells were co-cultured for 4 days prior to using flow cytometry to measure CD25 (A) and Foxp3 expression (B) of T cells. OT-II splenocytes were treated with soluble OVA_323–339 (100 ng/mL) or acNP-OVA_323–339 (25-μg/mg loading; 75 μg/mL) in the presence or absence of 2 ng/mL of exogenous TGF-β1. (C) The cells were co-cultured for 4 days prior to using flow cytometry to measure CD25 and Foxp3 expression. Statistical differences between groups were determined by performing a one-way ANOVA and Tukey’s post hoc test (p < 0.05). Groups with similar letters indicate no statistical significance. Error bars represent SEM.

Figure 4

acNP-Ags Prophylactically Induce Tolerance in R-EAE Clinical scores for SJL/J mice treated with two doses of 2.5 mg of NP(OVA_323–339), 2.5 mg of NP(PLP_139–151) (2.6 μg/mg PLP_139–151), or 2.5 mg of acNP-PLP_139–151 (2.6 μg/mg PLP_139–151) and immunized with PLP_139–151/CFA to induce R-EAE. (A) Treatment of mice with nanoparticle formulations resulted in significantly abrogated clinical disease scores compared to the control. (B) Corresponding cumulative clinical score for mice treated with tolerogenic particles (n = 5). Differences between disease courses of different treatment groups were analyzed for statistical significance using the Kruskal-Wallis test (one-way ANOVA non-parametric test) with Dunn’s multiple comparisons test (p < 0.05). Error bars represent SEM.

Figure 5

acNP-Ag Particles Induce Protective Tolerance against R-EAE Induced with Cocktail of Autoantigens Clinical scores of SJL/J mice treated with 1.25 mg of acNP-OVA_323–339 (8 μg/mg OVA_323–339), acNP-PLP_139–151 (8 μg/mg PLP_139–151), acNP-PLP_178–191 (8 μg/mg PLP_178–191), or acNP-PLP_{139–151,178–191} (8 μg/mg PLP_139–151 and 8 μg/mg PLP_178–191) and immunized with PLP_139–151 and PLP_178–191 in CFA to induce R-EAE 7 days later. (A) Schematic representation of antigen-polymer conjugate nanoparticles delivering multiple Ags. (B) Treatment of mice with acNP-Ags formulated with both pathogenic epitopes resulted in significantly abrogated mean clinical disease scores compared to mice treated with only a single or irrelevant epitope. (C) Corresponding cumulative clinical score for mice treated with particles (n = 5). Differences between disease courses of different treatment groups were analyzed for statistical significance using the Kruskal-Wallis test (one-way ANOVA non-parametric test) with Dunn’s multiple comparisons test (p < 0.05). Error bars represent SEM.