Cationic and biocompatible polymeric nanoparticles: preparation, characterization, and immunoadjuvant activity

• Autor(a) principal: Pérez-Betancourt, Yunys Pérez
• Data de Publicação: 2022
• Tipo de documento: Tese
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: https://www.teses.usp.br/teses/disponiveis/46/46131/tde-23052022-152413/
• topic: Adjuvantes para vacinas; Artigen carriers; Carreadores de antígenos; Cationic lipids; Cationic polymers; Interação nanopartícula/proteína; Lipídios catiônicos; Nanoparticles; Nanoparticles/protein; Nanopartículas; Polímero/proteína; Polímeros catiônicos; Polymer/protein interactions; Vaccine adjuvants; Vaccines; Vacinas

Adjuvants activate innate immunity by diverse mechanisms simulating the activity of danger signals present in entire pathogens but absent in purified antigens used in vaccines. Cationic nanoparticles (NPs) are promising adjuvants as they may show high colloidal stability, they adsorb oppositely charged antigens, and they increase antigens uptake by antigen-presenting cells (APC). The high toxicity displayed by isolated cationic lipids or polymers has hampered their use in vaccines. In this work, the toxicity of cationic materials is overcome by combining them with biocompatible polymers. Herein is described the preparation, characterization, toxicity, and immunoadjuvant activity of three cationic NPs, poly(diallyldimethylammchloride)/ovalbumin (PDDA/OVA), poly(methyl methacrylate)/dimethyldioctadecylammonium bromide/PDDA (PMMA/DODAB/PDDA), and PMMA/DODAB. NPs of PDDA/OVA were prepared by mixing PDDA and OVA; NPs of PMMA/DODAB/PDDA and PMMA/DODAB NPs were synthesized by emulsion polymerization of methyl methacrylate (MMA) in the presence of cationic components. Dispersion of NPs had their physical characteristics (mean hydrodynamic diameter (Dz), polydispersity (P), mean zeta-potential (ζ), and conductance (G)) determined by dynamic light scattering (DSL), turbidimetry, and scanning electron microscopy (SEM). The content ofPMMA, PDDA, and DODAB in NPs also was characterized. PDDA, OVA, PDDA/OVA, PMMA/DODAB/PDDA, PMMA/DODAB, DODAB large vesicles (DODAB-LV), and DODAB bilayer fragment (DODAB-BF) toxicity was evaluated measuring cell viability against fibroblast and macrophages. Immunoadjuvant activity was assessed by determining antibody production (IgG1, IgG2a), delayed-type hypersensitivity reaction (DTH), and cytokines profile. PDDA/OVA (0.01/0.1 mg/mL) NPs displayed Dz=170±4 nm, ζ =+30±2 mV, and P=0.11±0.01. Free PDDA exhibited dose-dependent toxicity exerted by disrupting fibroblast membranes. PDDA/OVA NPs elicited a T helper 2 (Th2) immune response represented by high IgG1 production and DTH similar to Al(OH)3/OVA. On the other hand, PMMA/DODAB/PDDA NPs exhibited Dz=217±1 nm, ζ=+73±1 mV, and P=0.12±0.01. After 24h incubation, PMMA/DODAB/PDDA NPs at 0.05 mg/mL (used in mice immunization) were no toxic against fibroblast or macrophages. PMMA/DODAB/PDDA NPs elicited a dual Th1/Th2 immune response, IgG1 titers were four times higher than the titers quantified using Al(OH)3 as the adjuvant. DTH for PMMA/DODAB/PDDA NPs was significantly higher than the those registered for OVA or Al(OH)3/OVA. Th1/Th2 dual response for PMMA/DODAB/PDDA was confirmed by cytokines profile showing higher production of IFN-γ, IL-4, IL-2, and IL-10 than controls. NPs of PMMA/DODAB displayed Dz=80±1 nm, ζ=+52±3 mV, and P=0.07±0.01. PMMA/DODAB NPs were less toxic than DODAB-BF and DODAB-LV, both against fibroblast and macrophages, at the concentration used for immunizations. The immune response generated by PMMA/DODAB NPs was dual Th1/Th2, IgG1 titers were comparable to those elicited by Al(OH)3, IgG2a titers, as well as DTH, were significantly higher than the ones produced by OVA or Al(OH)3/OVA. Th1/Th2 dual response confirmation was attained by determining cytokines production, PMMA/DODABNPs generated both Th1 (IL-2, IFN-γ) and Th2 (IL-4) cytokines. In conslusion, obtained NPs were biocompatible, nanosized, showed high ζ and narrow particle size distribution. Cationic materials immobilization decreases their toxicity in vitro. NPs enhanced the immune response as compared with OVA and Al(OH)3/OVA. Bearing-PDDA NPs had a bias toward a Th2 type response with high titers of IgG1, while PMMA/DODAB NPs exhibited a dual Th1/Th2 response.