RESUMEN
Cytotoxicity of nanoparticles and their sub-lethal effect on cell behavior and cell fate are a high topic of studies in the nanomaterial field. With an explosion of nanoparticle types (size, shape, polarity, stiffness, composition, etc.), Drosophila has become an attractive animal model for high throughput analysis of these nanocarriers in the drug delivery field with applications in cancer therapy, or simply to generate a fast and complete cytotoxic study of a peculiar nanoparticle. In respect to that, we have conducted an in cellulo study of poly(lactic acid) (PLA) nanoparticle cytotoxicity, and determined that near lethal nanoparticle doses, oxidative stress as well as P53 and ATP pathways may lead to cell cycle arrest at G1, and ultimately to cell death. Neither viability nor the development of Drosophila larvae are affected by the ingestion of PLA nanoparticles at sub-lethal concentrations. Drosophila will be a useful model to study PLA and PLA-modified nanoparticle toxicity, and nanoparticle fate after ingestion.
Asunto(s)
Materiales Biocompatibles/toxicidad , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Portadores de Fármacos/toxicidad , Nanopartículas/toxicidad , Poliésteres/toxicidad , Animales , Materiales Biocompatibles/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Drosophila melanogaster , Portadores de Fármacos/química , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Larva , Nanopartículas/química , Tamaño de la Partícula , Poliésteres/química , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Propiedades de Superficie , Pruebas de ToxicidadRESUMEN
Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Ácido Láctico/metabolismo , Nanopartículas/metabolismo , Polímeros/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Drosophila melanogaster/fisiología , Cuerpos de Inclusión , Ácido Láctico/química , Nanopartículas/química , Poliésteres , Polímeros/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Propiedades de Superficie , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
The development of safe and effective vaccines for cancer and infectious diseases remains a major goal in public health. Over the last two decades, controlled release of vaccine antigens and immunostimulant molecules has been achieved using nanometer or micron-sized delivery vehicles synthesized using biodegradable polymers. In addition to achieving a depot effect, enhanced vaccine efficacy using such delivery vehicles has been attributed to efficient targeting of antigen presenting cells such as dendritic cells. Biodegradable and biocompatible poly(lactic acid) and poly(lactic-co-glycolic acid) polymers belong to one such family of polymers that have been a popular choice of material used in the design of these delivery vehicles. This review summarizes research findings from ourselves and others highlighting the promise of poly(lactic acid)- and poly(lactic-co-glycolic acid)-based vaccine carriers in enhancing immune responses.
