Sys Rev Pharm 2020;11(11):247-253 A multifaceted review journal in the field of pharmacy 247 Systematic Reviews in Pharmacy Vol 11, Issue 11, Nov-Dec 2020 Grafted Hyaluronic Acid Nanogel for the Incorporation of Poly(I:C) as an Immunostimulatory Adjuvant Nararat Kotcharat 1 , Penpimol Charoenkanburkang 2 , Jittima Amie Luckanagul* 1,3 1 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand, 2 Nabsolute Co., Ltd., Bangkok 10700, Thailand, Penpimon 3 Biomaterial Engineering for Medical and Health Research Unit, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand *Corresponding author: Jittima.L@pharm.chula.ac.th ABSTRACT The aim of this research was to develop a nanogel formulation-based on modified natural polymer, hyaluronic acid (HA), as biodegradable material for adjuvant delivery. Polyinosinic:polycytidylic acid (poly(I:C)) have been approved by FDA as promising adjuvant candidate for the TLR3 (Toll-Like Receptor 3) activation to induce of effective immune system. 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 the 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-1) 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 4% degree of substitution were formed into nearly spherical nanogel particles with the size of approximately submicron range. The particles presented negative value in zeta-potential showing that poly(I:C) was entrapped. Moreover, we founded that the size and PDI of particles were decreased upon continuous incubation. The development of this poly(I:C)-loaded grafted HA nanogel will lead to the new generation of smart materials that can be functionalized and optimized for different medical purposes. Keywords: Adjuvant delivery, cancer Immunotherapy, Hyaluronic Acid, nanogels, poly(I:C). Correspondence: Jittima Amie Luckanagul 3Biomaterial Engineering for Medical and Health Research Unit, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand *Corresponding author: Jittima Amie Luckanagul email-address: Jittima.L@pharm.chula.ac.th INTRODUCTION In recent years, the novel innovation of nanotechnology has been progressing in the field of drug delivery. Among many types of sub-micron particulate drug carriers, the outstanding hydrophilic nanogel-based, non-viral vectors are the systems of interest [1] . Firstly, nanogels can provide special features from their physicochemical properties, compared to other traditional drug delivery systems [2] . Secondly, the nanoscale-sized hydrogels with three-dimensional network structures provide a high water-content property and high biocompatibility that leads to the stability of the colloidal system avoiding particle aggregation in the bloodstream [3] . Thirdly, the tunable polymeric network can be used to incorporate different types of therapeutic molecules including drugs, nucleic acids and proteins [4] . As a result, the payload can be protected from both enzymatic/chemical degradation with the extended the circulation time of the incorporated components [5] . In contrast to the conventional hydrogel or macrogel, the nano-sized hydrogels can be administered through intravenous injection giving the benefit of improved biodistribution. More importantly, hydrophilic nanogels are used to control release of the delivered therapeutic agents by incorporating within polymer networks. Hyaluronic acid (HA) is a naturally occurring polysaccharide that is mostly present in biological fluids and tissues, especially as a main component of the extracellular matrix and an important molecule for maintenance of cartilage structure [6] . Several research reports indicated that hyaluronic acid-based nanogels have been attractive as a biomaterial used for the development of drug delivery systems. The beneficial properties of these materials over other delivery platforms that are being studied for human clinical trials included 1) the extremely hydrophilic structure of the backbone with high water absorption making them safe for bioorganisms; 2) biodegradability; 3) simplicity for bioconjugation; 4) non-immunogenicity; and 5) high drug loading capacity from the porous polymer networks. One of the FDA approved potential immunostimulatory adjuvant candidate for novel vaccines is the synthetic double-stranded RNA (dsRNA) that mimics viral RNA, polyinosinic:polycytidylic acid (poly(I:C)). The adjuvant is being used in clinical studies for infectious diseases and cancers. It is recognized by Toll-like receptor 3 (TLR3), which is a transmembrane protein sited on endosomal of antigen-presenting cells (APCs) and many types of tumor cells. Poly(I:C) can encourage the production of the strongest type I interferons and inflammatory cytokines associated with innate and also adaptive immune system [7, 8] . Therefore, poly(I:C) has been promising adjuvants for the development of cancer immunotherapy. The soluble poly(I:C) possessed some concerns with low efficiency regarding the use in clinic. The molecule is inherently susceptible to rapid enzymatic degradation by RNase in the serum, resulting in a short half-life in human plasma. There are evidences for dose-dependent toxicity after administration of the adjuvant [9, 10] . Currently, besides an immunostimulant poly(I:C) that is widely used