(T1530-12-67) Remote Loading of Auto-Antigens in PLGA Nanoparticles for the Treatment of Autoimmune Diseases

Purpose: FDA approved therapies for autoimmune diseases focus on widespread immunosuppression or general immunomodulatory functions which have shown some success. However, these therapeutic strategies either come with potentially serious side effects or modest efficacy. Recently, research efforts have shifted focus toward exploring more specific treatment strategies by inducing tolerance using self-peptide antigens. By delivering a self-peptide antigen without co-stimulatory signals, tolerance can theoretically be induced by priming antigen presenting cells with a tolerogenic profile. We describe the development of a polymer nanoparticle formulation using an aqueous absorption remote loading method to encapsulate two self-antigen peptides with the ultimate goal of inducing tolerance in patients with multiple sclerosis and type 1 diabetes. Poly(D,L-lactic-co-glycolic acid) or PLGA is a biodegradable, biocompatible polymer that has been used in numerous FDA approved long-acting release formulations. PLGA encapsulated self-peptide antigens are useful to induce tolerance via subcutaneous injection targeting lymph nodes. Variations of the nanoparticle formulation were characterized for size and surface morphology as well as in vitro for release profiles.

Methods: MOG 38-50 (GWYRSPFSRVVHL) and NRPA7 (KYNKANAFL) peptides were purchased from Genemed Synthesis (San Antonio, TX). Uncapped PLGA 75:25 with molecular weight 13 kDa was purchased from Wako Chemicals (Osaka, Japan). Poly vinyl alcohol (PVA – 88% hydrolyzed) was purchased from Sigma Aldrich (St. Louis, MO). PLGA nanoparticles were prepared by dissolving polymer in dichloromethane and adding aqueous PVA prior to single emulsification using a homogenizer. The resulting emulsion was then transferred into an aqueous PVA bath and mixed for 3 h to allow for solvent evaporation. After mixing, nanoparticles were centrifuged for collection and washed 3 times with water before freeze drying. The resulting peptide-free PLGA nanoparticles were then incubated with an aqueous peptide loading solution at 37 °C for 24 h. These loaded nanoparticles were centrifuged for collection, washed 3 times with water, and freeze dried. Nanoparticle size was determined by dynamic light scattering (DLS) using a Malvern Zetasizer. The loading percentage and encapsulation efficiency for the nanoparticle formulation was determined by a multiple extraction protocol and UPLC. Additionally, each formulation was incubated in PBS and 0.02% Tween 80 at pH 7.4 and placed in an incubator at 37 °C. At predetermined time points, samples were centrifuged, and 80% of the buffer was collected for analysis by UPLC for determination of release kinetics before replacing with fresh media.

Results: After several iterations of the blank PLGA nanoparticle formulation, the volume average size of the nanoparticles was measured at 702 ± 30 nm. The dispersibility of the nanoparticles was confirmed by measuring a similar size and polydispersity index before and after lyophilization. As reported previously by our group, remote loading of net cationic peptides using uncapped PLGA allows for simple peptide encapsulation at competitive loading and encapsulation efficiencies without the need for additional excipients like dextran sulfate or aluminum adjuvants used in remote self-healing encapsulation^1,2. Both MOG 38-50 and NRPA7 are cationic peptides, which make them good candidates for the novel absorption remote loading method. MOG 38-50 and NRPA7 were efficiently loaded in blank PLGA nanoparticles at 9.5 ± 0.3% and 10.4 ± 0.6% w/w, respectively. The peptide loaded nanoparticles also exhibited similar size and dispersibility as compared to the blank nanoparticles with a volume average size of 745 ± 199 nm. Additionally, both MOG 38-50 and NRPA7 loaded PLGA nanoparticles exhibited slow and continuous release over the course of 56 days. The MOG 38-50 formulation released 88% of the encapsulated peptide over the release study, and the NRPA7 formulation released 72% of the encapsulated peptide. Both formulations exhibited a desirable minimal burst release with less than 10% of the peptide released after one day.

Conclusion: Remotely loaded MOG 38-50 and NRPA7 PLGA nanoparticles show a promising controlled release formulation strategy for inducing tolerance. Utilizing a remote encapsulation process allows for flexibility in the manufacturing process to modify blank PLGA nanoparticles without the peptide of interest. The blank PLGA nanoparticles can be sterilized, and the polymer structure can be adjusted. Additionally, the peptide does not have to be exposed to organic solvents like dichloromethane or ethyl acetate which is required in a standard double emulsion process. This can improve the scalability, stability, and cost of goods of the final product.

References: [1] Giles, M. B., Hong, J. K. Y., Liu, Y., Tang, J., Li, T., Beig, A., Schwendeman, A. and Schwendeman, S. P., Efficient aqueous remote loading of peptides in poly(lactic-co-glycolic acid), Nat Commun 13, 3282 (2022).
[2] Reinhold, S. E., Desai, K.-G. H., Zhang, L., Olsen, K. F. and Schwendeman, S. P. Self-healing microencapsulation of biomacromolecules without organic solvents, Angew. Chem. Int. Ed., 51, 10800-10803 (2012).
[3] Prasad, S. et al. Tolerogenic Ag-PLG Nanoparticles Induce Tregs to Suppress Activated Diabetogenic CD4 and CD8 T Cells. J. Autoimmun. 89, 112–124 (2018).

Cationic peptides are remotely loaded by incubating the peptide-free PLGA nanoparticles with an aqueous peptide solution for 24 hours.



Slow and continuous release of peptides from remote loaded PLGA nanoparticles. Release studies were carried out in PBS + Tween 80 at pH 7.4 and analysis was done by UPLC.