Diabetes and Obesity Drug Discovery & Therapy (Track)

Cationic Nanoparticles/Nanomicelles for delivery of pDNA encoding IL-10 and pDNA encoding IL-4 and IL-10 to prevent autoimmune diabetes

Jagdish Singh
Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA.

Abstract:

The purpose of this study was to deliver plasmid DNA encoding interleukin-10 (IL-10) and pDNA encoding IL-4 and IL-10 in mice using polymeric nanoparticles/nanomicelles for the prevention of type 1 diabetes. Cationic nanoparticles were prepared using PLGA (inherent visc. 0.17dL/g) in combination with cationic methacrylate copolymer Eudragit® E100 (50%w/w total polymer) using cetyl trimethylammonium bromide (CTAB) as external stabilizer. Cationic nanomicelles were prepared from the graft copolymers of soluble chitosan and unsaturated fatty acids. Nanoparticles/nanomicelles were characterized for size, polydispersity, surface charge, surface morphology, toxicity and transfection efficiency. Cellular uptake and endosomal buffering ability of the nanoparticles/nanomicelles were evaluated by confocal microscopy and titrimetric assay. pDNA encoding IL-10 or pDNA encoding IL-4 and IL-10 was loaded on the surface of nanoparticles/nanomicelles. Confocal microscopy and titrimetric assay indicated that greater proton buffering ability of nanoparticles/nanomicelles was responsible for faster endosomal escape and consequently greater transfection efficiency.  Multiple low-dose streptozotocin (MLD-STZ) was administered to Balb/c mice to induce autoimmune response against pancreatic beta cells typically observed in type 1 diabetes. Following MLD-STZ treatment, the animals were treated with pDNA encoding IL-10 or pDNA encoding IL-4 and IL-10 either alone (passive delivery) or loaded on nanoparticles/nanomicelles by injection into skeletal muscle. Blood samples were collected at weekly intervals for 6 weeks post-treatment. Blood glucose levels were determined by glucose-oxidase method. Serum levels of IL-4, IL-10 and interferon-gamma (IFN-γ) were measured by ELISA. At the end of 6-weeks, animals were euthanized and pancreas and skeletal muscle were excised. Sections of pancreas and muscle were prepared and observed by light microscopy. Nanoparticles/nanomicelles displayed positive zeta potential (62 ± 2 mV) and high plasmid loading efficiency (>90%). Blood glucose levels of animals treated with pDNA encoding IL-10 and pDNA encoding IL-4 and IL-10 loaded on nanoparticles/nanomicelles  were not significantly different (p>0.05) from healthy control animals throughout the study. Nanoparticles/nanomicelles-treated animals showed significantly higher (p<0.05) levels of serum IL-4 and IL-10 as compared to passive delivery group for all the time points. High expression of IL-4 and IL-10 in nanoparticles/nanomicelles-treated animals led to suppression of serum levels of IFN-γ. Histological sections of pancreas of animals treated with nanoparticles/nanomicelles did not show any signs of immune infiltration. Meanwhile, pancreas of animals receiving pDNA encoding IL-10 or pDNA encoding IL-4 and IL-10 alone showed mild inflammation. Histology of skeletal muscle showed that the injection of nanoparticles/nanomicelles did not lead to toxicity or chronic immune response. In conclusion, intramuscular delivery of gene encoding IL-10 or combination of IL-4 and IL-10 using nanoparticles/nanomicelles led to enhanced expression of IL-4 and IL-10 and efficient prevention of type 1 diabetes as compared to passive plasmid delivery.