RESUMO
Polyplexes consisting of a standard CMV promoter driven luciferase plasmid condensed with PAMAM starburst dendrimers (generation 4 and 5) efficiently transfected tumor cells in vitro. Tail vein injection of PAMAM polyplexes into immune competent mice bearing subcutaneous, well vascularized murine neuroblastoma tumors (Neuro2A) led to predominant luciferase reporter gene expression in the tumor, and negligible transgene expression levels in other organs. Repeated PAMAM polyplex applications were well tolerated and prolonged transgene expression in the tumor. In vivo imaging studies using polyplexes fluorescently labeled with near infrared emitting semiconductor quantum dots (quantoplexes) revealed lung accumulation for both PAMAM and linear PEI (LPEI) based polyplexes, but only LPEI polyplexes induced high luciferase expression in lung, demonstrating that biodistribution and transgene expression of polyplexes does not necessarily correlate. With a luciferase plasmid devoid of immune modulatory CpG sequences and a combination of human CMV enhancer and human elongation factor 1 alpha promoter elements, Neuro2A tumor transgene expression after a single intravenous injection of generation 5 PAMAM polyplexes was observed for up to 1week as measured by luciferase bioluminescence imaging. Utilizing a human xenograft model (HUH7) in immune compromised nude mice, a low level of luciferase activity in the tumor area was observed after systemic PAMAM polyplex application.
Assuntos
Ilhas de CpG/genética , Dendrímeros/química , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética/métodos , Neuroblastoma/terapia , Plasmídeos/genética , Transgenes , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Corantes Fluorescentes , Humanos , Luciferases/genética , Medições Luminescentes , Camundongos , Camundongos Nus , Neuroblastoma/genética , Neuroblastoma/patologia , Plasmídeos/administração & dosagem , Plasmídeos/química , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extracellular stability of electrostatically formed siRNA polyplexes is a significant concern in the delivery process. To overcome the risk of polyplex dissociation in the extracellular environment, siRNA was covalently incorporated into a pH- and redox-responsive polymer conjugate. The novel siRNA conjugate consists of polylysine (PLL) as RNA binding and protecting polycation, polyethylene glycol (PEG) as solubilizing and shielding polymer, the lytic peptide melittin masked by dimethylmaleic anhydride (DMMAn) removable at endosomal pH, and the siRNA attached at the 5'-end of the sense strand via a bioreducible disulfide bond. The purified siRNA conjugate was stable in the presence of the polyanion heparin at conditions where the analogous electrostatic siRNA polyplexes disassemble. Only the combination of heparin plus a reducing agent such as glutathione triggered the release of siRNA from the conjugate. High in vitro biocompatibility (absence of cytotoxicity or hemolytic activity at neutral pH) and efficient and sequence-specific gene silencing was found at > or =25 nM siRNA, comparable to the corresponding electrostatic polyplexes. In vivo toxicity studies of this formulation demonstrated that conjugates remain to be optimized for therapeutic application.