RESUMO
Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.
Assuntos
Materiais Biocompatíveis/química , Nanopartículas/metabolismo , Polietilenoglicóis/química , Poliglactina 910/química , RNA Interferente Pequeno/metabolismo , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/toxicidade , Carbocianinas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Humanos , Lipídeos/química , Microscopia de Fluorescência , Nanopartículas/química , Nanopartículas/toxicidade , Tamanho da Partícula , Poliésteres , Polietilenoglicóis/metabolismo , Polietilenoglicóis/toxicidade , Poliglactina 910/metabolismo , Poliglactina 910/toxicidade , Interferência de RNA , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
BACKGROUND: A novel small interfering RNA (siRNA) delivery method based on the combined use of nanoparticles (NPs) with ultrasound (US) and/or microbubbles (MBs) was introduced in the present study. We investigated the efficacy and safety of US and/or MBs-enhanced delivery of monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly l-lysine (mPEG-PLGA-PLL) NPs loading platelet-derived growth factor BB (PDGF-BB) siRNA to rat retinal pigment epithelium (RPE)-J cells. METHODS: The effect of US and/or MBs on the delivery of NPs containing Cy3-labeled siRNA was evaluated by fluorescence microscopy and flow cytometry. Potential toxicity of NPs and cell viability under different conditions of US and/or MBs were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: The results obtained showed that low intensity US or 15-20% MBs could increase the delivery efficiency of a lower concentration of mPEG-PLGA-PLL NPs loading siRNA to RPE-J cells, whereas the combination of US with MBs under the optimal conditions for the enhancement of NPs delivery did not further increase the cellular uptake of NPs compared to either US or MBs alone (p = 0.072 and p = 0.488, respectively). Under the optimal condition for US-enhanced NPs delivery, the enhanced PDGF-BB gene silencing with a combination of US and NPs encapsulating siRNA resulted in a significant decrease of mRNA and protein expression levels compared to NPs alone. CONCLUSIONS: US and/or MBs could be used safely to enhance the delivery of NPs loading siRNA to rat RPE-J cells. A combination of the chemical (mPEG-PLGA-PLL NPs loading siRNA) and physical (US) approaches could more effectively downregulate the mRNA and protein expression of PDGF-BB.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Células Epiteliais/metabolismo , Microbolhas/uso terapêutico , Nanopartículas/administração & dosagem , Fator de Crescimento Derivado de Plaquetas/genética , RNA Interferente Pequeno/genética , Ultrassom , Animais , Becaplermina , Técnicas de Cultura de Células/métodos , Primers do DNA/genética , Lisina/administração & dosagem , Lisina/metabolismo , Poliésteres/administração & dosagem , Poliésteres/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Proteínas Proto-Oncogênicas c-sis , Interferência de RNA , Ratos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
Gene therapy is a potentially viable approach for treating hormone-refractory prostate cancer (HRPC), it requires efficient delivery systems and a target gene. Inducing carcinoma cell apoptosis by inhibition of heat shock protein 70 (HSP70) overexpression has been emerging as an attractive strategy for cancer therapy. In our study, the high tumor-specificity of human telomerase reverse transcriptase (HTERT) expression prompted the use of an HTERT/cytomegalovirus (CMV) chimeric promoter to drive HSP70-ShRNA expression to induce HRPC 22RV1 cell apoptosis. At the same time, sonoporation induced by ultrasound-targeted microbubble destruction (UTMD) was utilized for delivery of plasmid loaded with HTERT/CMV promoter. Our results indicated the combination of sonoporation, low-dose liposomes and HTERT/CMV chimeric promoter as a delivery system has the potential to promote efficient gene transfer with lower cytotoxicity.
Assuntos
Terapia Genética , Proteínas de Choque Térmico HSP70/genética , Neoplasias da Próstata/genética , Transfecção/métodos , Apoptose , Linhagem Celular Tumoral , Citomegalovirus/genética , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Lipossomos/administração & dosagem , Masculino , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , RNA Interferente Pequeno , Telomerase/genéticaRESUMO
A potentially viable approach for treating late-stage prostate cancer is gene therapy. Successful gene therapy requires safe and efficient delivery systems. In this study, we report the efficient delivery of small interfering RNA (siRNA) via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. On the basis of previous findings, cyclic Arg-Gly-Asp (cRGD) peptides were conjugated to NPs to recognize the target site, integrin αvß3, expressed in high levels in PC-3 prostate cancer cells. The suppression of angiogenesis by the downregulation of vascular endothelial growth factor (VEGF) expression has been widely used to inhibit the growth of malignant tumors. In our study, human VEGF (hVEGF)-siRNA was encapsulated in NPs to inhibit VEGF expression in PC-3 cells. Concurrently, sonoporation induced by ultrasound-targeted microbubble destruction (UTMD) was utilized for the delivery of siRNA-loaded NPs. Our results showed low cytotoxicity and high gene transfection efficiency, demonstrating that the targeted delivery of biodegradable NPs with UTMD may be potentially applied as new vector system for gene delivery.