Development of hyaluronic acid-based nanogel platform for improved stability and polyvalent ligand display of poly(I:C) as an effective immunostimulatory adjuvant in cancer immunotherapy

Abstract

The aim of this research was to develop a nanogel formulation-based on modified natural polymer, hyaluronic acid (HA), as a biodegradable material for adjuvant delivery. Polyinosinic:polycytidylic acid (poly(I:C)) have been approved by FDA as a promising adjuvant candidate for the TLR3 (Toll-Like Receptor 3) activation. However, it suffers from being poor stability and is subjected to rapid enzymatic hydrolysis in serum, so that it requires high administered dose leading to adverse effects. To augment the adjuvant stability and protection from the degradation, the nano-particulate carriers were herein designed with self-assembly of HA scaffold grafted with poly (N-isopropylacrylamide), or pNIPAM. The grafting was processed through amide formation using the coupling agent (EDC/NHS). 1H NMR was carried out to confirm the modified products (HA-g-pNI). The physical incorporation of the nucleic acid into the grafted HA nanogel was achieved by incubation method with the poly(I:C) concentrations of 0.2, 1, and 10 (μg/mL) in formulations by using 0.1, 0.25, and 0.5 (% w/v) of HA-g-pNI to form the nanogel particles. The mean size, size distribution and surface charge of the nanogel particles were determined by dynamic light scattering (DLS). The particle morphology was investigated by transmission electron microscopy (TEM). Results demonstrated that HA-g-pNI with a 4 % degree of substitution were formed into nearly spherical nanogel particles with the size of approximately submicron range. The particles presented a negative value in zeta-potential. Moreover, we founded that the average size and polydispersity index (PDI) of the loaded particles were decreased significantly upon continuous incubation showing that poly(I:C) was entrapped. The stability against fetal bovine serum (FBS) degradation of the poly(I:C)-loaded nanogels was demonstrated by gel electrophoresis. Furthermore, cell viability of all HA-g-pNI formulations showed more than 80% of cell viability via RAW 264.7 macrophages. The poy(I:C)-loaded nanogel particles were excellent internalized by RAW 264.7 macrophages after 24 hrs of incubation. Therefore, the development of these poly(I:C)-loaded grafted HA nanogels will lead to a new generation of smart materials that can be functionalized and optimized for different medical purposes.