RESUMO
PURPOSE: This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates. MATERIALS AND METHODS: Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model. RESULTS: The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice. CONCLUSIONS: This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy.
Assuntos
Resinas Acrílicas/química , Portadores de Fármacos , Nanopartículas , Polimetil Metacrilato/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/metabolismo , Vacinas contra a AIDS/toxicidade , Resinas Acrílicas/toxicidade , Animais , Formação de Anticorpos , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Relação Dose-Resposta a Droga , Composição de Medicamentos , Estabilidade de Medicamentos , Feminino , Células HeLa , Humanos , Imunidade Celular , Camundongos , Camundongos Endogâmicos C57BL , Muramidase/química , Oxirredução , Tamanho da Partícula , Polimetil Metacrilato/toxicidade , Ligação Proteica , Desnaturação Proteica , Solubilidade , Fatores de Tempo , Tripsina/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidadeRESUMO
Peptide nucleic acids (PNA) are very promising antisense agents, but their in vivo application is often hampered by their low bioavailability, mainly due to their limited uptake through cellular and nuclear membranes. However, PNA chemical synthesis easily allows modification with functional structures able to improve the intrinsically low permeability and great interest is arising in finding specific and efficient delivery protocols. Polymeric core-shell microspheres with anionic functional groups on the surface were tested for their ability to reversibly bind lysine modified PNA sequences, whose antisense activity against COX-2 mRNA was already demonstrated in murine macrophages.
