RESUMO
Overlapping synthetic long peptides (SLPs) hold great promise for immunotherapy of cancer. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are being developed as delivery systems to improve the potency of peptide-based therapeutic cancer vaccines. Our aim was to optimize PLGA NP for SLP delivery with respect to encapsulation and release, using OVA24, a 24-residue long synthetic antigenic peptide covering a CTL epitope of ovalbumin (SIINFEKL), as a model antigen. Peptide-loaded PLGA NPs were prepared by a double emulsion/solvent evaporation technique. Using standard conditions (acidic inner aqueous phase), we observed that either encapsulation was very low (1-30%), or burst release extremely high (>70%) upon resuspension of NP in physiological buffers. By adjusting formulation and process parameters, we uncovered that the pH of the first emulsion was critical to efficient encapsulation and controlled release. In particular, an alkaline inner aqueous phase resulted in circa 330 nm sized NP with approximately 40% encapsulation efficiency and low (<10%) burst release. These NP showed enhanced MHC class I restricted T cell activation in vitro when compared to high-burst releasing NP and soluble OVA24, proving that efficient entrapment of the antigen is crucial to induce a potent cellular immune response.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ácido Láctico/química , Ativação Linfocitária , Nanopartículas , Peptídeos/química , Ácido Poliglicólico/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoAssuntos
Lactatos/química , Polietilenoglicóis/química , Emulsões , Interações Hidrofóbicas e Hidrofílicas , Lactatos/síntese química , Luz , Espectroscopia de Ressonância Magnética , Peso Molecular , Nanopartículas/química , Polietilenoglicóis/síntese química , Espalhamento de Radiação , Água/químicaRESUMO
Previously, we showed that long-circulating polyethylene glycol (PEG)-liposomes are cleared rapidly from the circulation when injected repeatedly in the same animal. In this article, we describe the effects of PEG-coating, the circulation time, the lipid dose, and the presence of encapsulated doxorubicin on the pharmacokinetics upon repeated injection in rats. Furthermore, the role of liver and splenic macrophages was investigated. Liposomes without PEG-coating also showed the so-called "enhanced clearance effect": blood levels at 4 h post injection decreased from 62.8 +/- 13.7% of injected dose (%ID) after the first injection to 0.54 +/- 0.21%ID after the second injection. This decrease was independent of the circulation time of the first dose. Decreasing the first lipid dose of PEG-liposomes to 0.05 micromol/kg still led to enhanced clearance of a second dose of 5 micromol/kg. No changes in pharmacokinetics were observed when the second dose was 50 micromol/kg. When hepatosplenic macrophages were depleted, no enhanced clearance of repeated liposome injections was observed. A dose of doxorubicin containing PEG-liposomes (Doxil), injected 1 week after injection of empty PEG-liposomes, was cleared rapidly from the circulation in rats. Our results indicate that hepatosplenic macrophages play an essential role in the enhanced clearance effect and that the change in pharmacokinetic behavior upon repeated injection is a general characteristic of liposomes, unrelated to the presence of PEG. Therefore, these findings may have a considerable impact on the clinical application of liposomal formulations that are administered repeatedly.
Assuntos
Excipientes/farmacocinética , Polietilenoglicóis/farmacocinética , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Portadores de Fármacos , Excipientes/administração & dosagem , Radioisótopos de Índio , Lipossomos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Polietilenoglicóis/administração & dosagem , Ratos , Ratos Wistar , Tecnécio , Distribuição TecidualRESUMO
Intravenous injection of an endotoxin-free solution of poloxamine-908 to rats can enhance the phagocytic clearance capacity of tissue macrophages, particularly those of the liver and the spleen. Such stimulated cells were able to clear a significant portion of intravenously injected methoxypoly(ethyleneglycol)2000 liposomes (mean size of 87 nm), labelled with technetium-99m via the N-hydroxysuccinimidyl hydrazine nicotinate hydrochloride derivative of distearoyl phosphatidylethanolamine, within 4 h post administration. These liposomes, otherwise, exhibit long circulatory behaviour in control animals, with poor localization to the liver and spleen. We suggest that such technetium-99m-labelled engineered vesicles may be of aid for detection of the liver and spleen macrophages with enhanced phagocytic clearance capacity by gamma scintigraphy. Alterations in the phagocytic activity of liver and spleen macrophages is known to occur during cancer. Therefore, such diagnostic procedures may prove useful for patient selection or for monitoring the progress of treatment with long circulating nanoparticles carrying anti-cancer agents, thus minimizing damage to this important line of body's defence cells, and are discussed.