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Dual-Responsive Glycopolymers for Intracellular Codelivery of Antigen and Lipophilic Adjuvants


De Mel JJudith , Hossain MMehjabeen , Shofolawe-Bakare OOluwaseyi , Mohammad SASk Arif , Rasmussen EEmily , Milloy KKhadeeja , Shields MMicaela , Roth EWEric W , Arora KKaran , Cueto RRafael , Tang SCShou-Ching , Wilson JTJohn T , Smith AEAdam E , Werfel TAThomas A . Molecular pharmaceutics. 2022 11 14; ().


Traditional approaches to vaccines use whole organisms to trigger an immune response, but they do not typically generate robust cellular-mediated immunity and have various safety risks. Subunit vaccines composed of proteins and/or peptides represent an attractive and safe alternative to whole organism vaccines, but they are poorly immunogenic. Though there are biological reasons for the poor immunogenicity of proteins and peptides, one other key to their relative lack of immunogenicity could be attributed to the poor pharmacokinetic properties of exogenously delivered proteins and peptides. For instance, peptides often aggregate at the site of injection and are not stable in biological fluids, proteins and peptides are rapidly cleared from circulation, and both have poor cellular internalization and endosomal escape. Herein, we developed a delivery system to address the lack of protein immunogenicity by overcoming delivery barriers as well as codelivering immune-stimulating adjuvants. The glycopolymeric nanoparticles (glycoNPs) are composed of a dual-stimuli-responsive block glycopolymer, poly[2-(diisopropylamino)ethyl methacrylate]–poly[(pyridyl disulfide ethyl methacrylate)–(methacrylamidoglucopyranose)] (p[DPA–(PDSMA–MAG)]). This polymer facilitates protein conjugation and cytosolic release, the pH-responsive release of lipophilic adjuvants, and pH-dependent membrane disruption to ensure cytosolic delivery of antigens. We synthesized p[DPA–(PDSMA–MAG)] by reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by the formation and physicochemical characterization of glycoNPs using the p[DPA–(PDSMA–MAG)] building blocks. These glycoNPs conjugated the model antigen ovalbumin (OVA) and released OVA in response to elevated glutathione levels. Moreover, the glycoNPs displayed pH-dependent drug release of the model hydrophobic drug Nile Red while also exhibiting pH-responsive endosomolytic behavior as indicated by a red blood cell hemolysis assay. GlycoNPs coloaded with OVA and the toll-like receptor 7/8 (TLR-7/8) agonist Resiquimod (R848) activated DC 2.4 dendritic cells (DCs) significantly more than free OVA and R848 and led to robust antigen presentation of the OVA epitope SIINFEKL on major histocompatibility complex I (MHC-I). In sum, the dual-stimuli-responsive glycopolymer introduced here overcomes major protein and peptide delivery barriers and could vastly improve the immunogenicity of protein-based vaccines.