RESUMO
The lack of efficient in vivo gene delivery is a well-known shortcoming of nonviral delivery vectors, in particular of chemical vectors. We developed a series of novel nonviral carriers for plasmid-based in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with synthetic derivatives of naturally occurring molecules-fatty acid-spermine conjugates (or lipospermines). We tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA) and found that they formed DNA nanocomplexes, which are protected from DNase I degradation. This protection was shown to directly correlate with the length of the aliphatic component. However, this increase in the length of the hydrocarbon chain resulted in increased toxicity. The cationic lipids used for transfection typically have a C(16) and C(18) hydrocarbon chain. Interestingly, toxicity studies, together with further characterization studies, suggested that the two most suitable candidates for in vivo delivery are those with the shortest hydrocarbon chain, butanoyl- and decanoylspermine. Morphological characterization of DNA nanocomplexes resulting from these lipospermines showed the formation of a homogenous population, with the diameter ranging approximately from 40 to 200 nm. Butanoylspermine was found to be the most promising carrier from this series, resulting in a significantly increased gene expression, in relation to naked plasmid, in both tissues herein targeted (dermis and M. tibialis anterior). Thus, we established a correlation between the in vitro properties of the ensuing DNA nanocarriers and their efficient in vivo gene expression.
Assuntos
Ácidos Graxos , Técnicas de Transferência de Genes , Vetores Genéticos , Espermina , Animais , DNA/química , Ácidos Graxos/farmacologia , Ácidos Graxos/toxicidade , Ácidos Graxos Insaturados/farmacologia , Ácidos Graxos Insaturados/toxicidade , Expressão Gênica , Vetores Genéticos/biossíntese , Vetores Genéticos/toxicidade , Células Hep G2 , Humanos , Camundongos , Plasmídeos , Espermina/análogos & derivados , Espermina/farmacologia , Espermina/toxicidadeRESUMO
The amyloid-beta peptide (Abeta) can generate cytotoxic oligomers, and their accumulation is thought to underlie the neuropathologic changes found in Alzheimer's disease. Known inhibitors of Abeta polymerization bind to undefined structures and can work as nonspecific aggregators, and inhibitors that target conformations that also occur in larger Abeta assemblies may even increase oligomer-derived toxicity. Here we report on an alternative approach whereby ligands are designed to bind and stabilize the 13-26 region of Abeta in an alpha-helical conformation, inspired by the postulated Abeta native structure. This is achieved with 2 different classes of compounds that also reduce Abeta toxicity to cells in culture and to hippocampal slice preparations, and that do not show any nonspecific aggregatory properties. In addition, when these inhibitors are administered to Drosophila melanogaster expressing human Abeta(1-42) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed. We conclude that stabilization of the central Abeta alpha-helix counteracts polymerization into toxic assemblies and provides a strategy for development of specific inhibitors of Abeta polymerization.