RESUMO
Protein transduction domains (PTDs), such as HIV-derived Tat, have been successfully used as functional biomaterials for intracellular delivery of anti-cancer macromolecular drugs (protein, peptides, and oligonucleotides). Although there were therefore great expectations regarding the therapeutic potential of PTDs for the development of anti-cancer therapeutics, their clinical application so far has been extremely limited because of the relatively high concentrations required to mediate any effects on cancer cells in vitro or in vivo. In this context, improving the transduction efficiency of PTDs using phage display-based molecular evolution techniques may be useful for creating artificial PTDs with high efficiency and safety. Here, we report an evaluation of transduction efficiency and toxicity of such artificial PTDs (designated mT02 and mT03) compared with Tat. The internalization of mT02 was the most rapid and efficient by a mechanism different from the usual macropinocytosis. Furthermore, we found that artificial PTDs fused with survivin antagonistic peptide potentiate tumor cell-cytostatic activity. Thus, the results of this work provide new insights for designing new-generation peptide therapeutics for a wide variety of cancers as well as those expressing survivin.
Assuntos
Peptídeos/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Transdução Genética/métodos , Sequência de Aminoácidos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Linhagem Celular , Citocalasina D/metabolismo , Citostáticos/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/uso terapêutico , Pinocitose/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , beta-Ciclodextrinas/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
We reported previously that fusogenic liposome (FL) introduced antigen protein encapsulated in the liposome directly into the cytoplasm of the antigen presenting cells, and that it induced immune responses. In the present study, we encapsulated TAX38-46, an HTLV-I derived protein and an antigen peptide model, into FL. The ability to induce effective cytotoxic T lymphocytes (CTL) responses in immunized mice was evaluated. Results showed FL could induce CTL response effectively and suggested that FL is a potential peptide vaccine carrier.
Assuntos
Portadores de Fármacos/administração & dosagem , Linfócitos T Citotóxicos/efeitos dos fármacos , Vacinas de Subunidades Antigênicas/administração & dosagem , Animais , Linhagem Celular , Feminino , Lipossomos , Camundongos , Camundongos Endogâmicos C3H , Linfócitos T Citotóxicos/imunologia , Vacinação/métodos , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen.