Remote loading of an autoantigen in PLGA nanoparticles for the treatment of multiple sclerosis

Abstract

Autoimmune diseases like multiple sclerosis (MS) affect millions of people worldwide and have been growing in prevalence. Current therapeutic strategies either entirely suppress immune function or only offer modest efficacy. Research efforts have shifted focus more recently to antigen-specific therapies to promote immune tolerance and avoid compromising general immune function. Here, we show the application of a novel aqueous remote loading method using poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to load myelin oligodendrocyte glycoprotein (MOG) peptide at high loading and encapsulation efficiency. MOG is a target of autoreactive T cells in MS. These NPs (750 ± 200 nm and - 16.7 ± 0.4 mV zeta potential) slowly and continuously released MOG peptide and decreased costimulatory molecule expression on dendritic cells in vitro. A single dose of MOG-PLGA NPs administered either SC or IV exerted strong efficacy in a murine experimental autoimmune encephalomyelitis (EAE) model of MS. Prophylactic treatment with MOG-PLGA NPs prevented disease progression, while therapeutic treatment with MOG-PLGA NPs effectively reversed the EAE symptoms. MOG-PLGA NPs also induced long term tolerance against EAE re-challenge. Mechanistically, a single injection of MOG-PLGA NPs induced a 2-fold increase in the frequency of MOG-specific CD4+ T regulatory cells (Tregs) and anergic T cells, compared with PBS or free MOG peptide control groups. Additionally, histopathological analysis demonstrated a positive correlation between % demyelination and EAE score. Hence, autoantigens, such as MOG peptide, can be remote loaded into PLGA NPs from an aqueous solution at high loading and encapsulation efficiency for long-term controlled release.

Keywords: Controlled release; Experimental autoimmune encephalomyelitis; Long acting; Multiple sclerosis; Myelin oligodendrocyte glycoprotein; PLGA nanoparticles; Remote loading.

Fig. 1.

Characterization of MOG-PLGA NPs. A) Representative SEM images of MOG-PLGA NPs. These nanoparticles exhibit a dominant population centered in the 700–800 nm range as measured by DLS. B) Slow and continuous release of MOG peptide from MOG-PLGA NPs. Release studies were carried out in PBS + 0.02 % Tween 80 at pH 7.4, followed by analysis for MOG peptide by UPLC. Data includes mean ± SEM of % peptide released for each day, representing 3 separate batches of MOG-PLGA NPs.

Fig. 2.

Immune co-stimulatory markers on BMDCs. BMDCs were treated with 100 pg/mL of LPS and cultured with either 1.5 μg/mL of free MOG peptide or MOG-PLGA NPs for 48 h, followed by flow cytometric analysis. Blank PLGA NPs were added as a control group. Statistical significance was determined using Kruskal Wallis, followed by Dunn’s multiple comparison test for the last day of observations. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 with n = 9 for each group.

Fig. 3.

MOG-PLGA NPs prevent and reverse severe EAE disease progression. A) Naïve C57BL/6 mice were prophylactically treated once by SC administration of MOG-PLGA NPs (100 μg MOG per mouse), followed by EAE induction after 1 or 2 weeks. The experimental schematic is shown in Supplementary Fig. S1. B) When mice induced with EAE exhibited full paralysis of hind legs (EAE score of 3), they were treated once with free MOG peptide or MOG-PLGA NPs (100 μg MOG per mouse) administered via SC or IV route. The experimental schematic is shown in Supplementary Fig. S2. Shown are the mean EAE scores ± SEM over time. Statistical significance was determined using Kruskal Wallis, followed by Dunn’s multiple comparison test for the last day of observations. *p < 0.05 and **p < 0.01 with n = 4–6 replicates for each group. “ns” denotes no statistical significance.

Fig. 4.

Single MOG-PLGA NP IV therapy reduces EAE scores and induces long-term immune tolerance against EAE rechallenge. When full paralysis of hind legs was observed (EAE score of 3), mice were treated once with MOG-PLGA NPs (100 μg MOG per mouse) administered via the SC or IV route. On day 56, the mice were rechallenged with MOG EAE. Naïve mice challenged with EAE on day 56 were included as a control group. The experimental schematic is shown in Supplementary Fig. S3. Shown are the mean EAE scores ± SEM of mice over time. Statistical significance was determined using Kruskal Wallis, followed by Dunn’s multiple comparison test on each group on day 55 prior to rechallenge and again on day 88 as the last day of observation. **p < 0.01 and ****p < 0.0001 with n = 4–7 replicates for each group. Arrow indicates day 56 when EAE rechallenge was performed. “ns” denotes no statistical significance.

Fig. 5.

A low dose of MOG-PLGA NPs given IV potently reverses EAE. A) Comparison between the SC or IV route of administration for MOG-PLGA NPs. When full paralysis of hind legs was observed (EAE score of 3), mice were treated once with free MOG peptide or MOG-PLGA NPs (10 μg MOG per mouse) administered via the SC or IV route. The experimental schematic is shown in Supplementary Fig. S4. B) When mice induced with EAE exhibited full paralysis of hind legs (EAE score of 3), mice were treated once with MOG-PLGA NPs (100 μg MOG per mouse) or with blank PLGA NPs IV. The experimental schematic is shown in Supplementary Fig. S2. Shown are the mean EAE scores ± SEM of mice over time. Statistical significance was determined using Kruskal Wallis, followed by Dunn’s multiple comparison test for the last day of observations. *p < 0.05 and **p < 0.01 with n = 4–6 replicates for each group.

Fig. 6.

Treatment with MOG-PLGA NPs decreases % demyelination in EAE mice. EAE mice in A and B) were treated as in Fig. 4, and 30 days after the treatment, cross-sections of spinal cords were stained with luxol fast blue and analyzed for the extent of demyelination. Shown are A) luxol fast blue stained spinal cord sections and B) average % demyelination for each treatment group with n = 4 replicates for each group. C) Positive correlation between % demyelination and EAE scores was observed across the lumbar region and at the caudal end of the lumbar region (closest to the tail) in spinal cords of mice.

Fig. 7.

Biodistribution of MOG-PLGA NPs. After administration of either MOG(Cy5.5)-PLGA NPs or free MOG(Cy5.5) for 48 h, spleen, liver, and lungs of all mice were collected and imaged using IVIS. Fluorescent signal was quantified using living image software. Statistical significance was determined using Kruskal Wallis, followed by Dunn’s multiple comparison tests, where *p < 0.05 with n = 5 replicates for each group.

Fig. 8.

Treatment with MOG-PLGA NPs significantly increases MOG-specific Tregs and anergic T cells in spleens. The experimental schematic is shown in Supplementary Fig. S5. Two complementary gating strategies are shown: A) Frequency (%) of MOG-tetramer+ cells among live CD3 + CD4 + Foxp3+ (Tregs) and CD3 + CD4 + FR4 + CD73 + Foxp3- (Anergic T cells). B) Frequency (%) of Foxp3+ cells among live CD3 + CD4 + MOG-Tet + cells and FR4 + CD73+ cells among live CD3 + CD4 + MOGTet+ Foxp3- cells. Statistical significance was determined using Kruskal Wallis, followed by Dunn’s multiple comparison tests, where *p < 0.05 with n = 5 replicates for each group.

Fig. 9.

Treatment with MOG-PLGA NPs significantly increases MOG-specific anergic T cells in spinal cords. The experimental schematic is shown in Supplementary Fig. S5. Shown here is the frequency (%) of Foxp3+ cells among live CD3 + CD4 + MOG-Tet + cells and FR4 + CD73+ cells among live CD3 + CD4 + MOGTet+ Foxp3- cells. Statistical significance was determined using Kruskal Wallis, followed by Dunn’s multiple comparison tests, where *p < 0.05 with n = 5 replicates for each group.