Studies on Drug delivery system for gene therapy

我们研究了含血清的阳离子脂质体介导的DNA转染法，随着血清浓度的增加，转染率急剧下降，在含5%小牛血清的培养液中，转染率几乎是无血清时的一半。白蛋白的加入也降低了转染率，当白蛋白浓度为50 mg/ml时，转染率降至35%。进一步研究了DNA的稳定性和孵育时间的影响，以阐明血清对转染率的影响。DNA降解琼脂糖凝胶电泳法分析表明，阳离子脂质体包裹的DNA对血清中的核酸酶具有较强的抵抗力，而且在有血清存在的情况下，转染率随着时间的推移而增加。30h的转染率是3h的3倍，与无血清培养的正常转染率相当。通过降低dna-脂质体复合体在c-…中的细胞毒性，获得了更长的孵育时间。更有吸引力的血清。研究了合成多肽的转染力和细胞毒性，以及多肽的相对分子质量和浓度对转染率的影响。结果表明，L-鸟氨酸、L-精氨酸和L-鸟氨酸、L-亮氨酸均能诱导HeLa S3细胞中外源基因的高表达。而聚(L-赖氨酸)和聚(L-赖氨酸、L-丙氨酸)无效。在高浓度的多肽作用下，细胞毒性表现为：聚(L-鸟氨酸)&gt；聚(L-赖氨酸)&gt；聚(L-精氨酸)&lt；大于或等于&gt；聚(L-鸟氨酸，L-亮氨酸)&gt；聚(L-赖氨酸，L-丙氨酸)。多肽的相对分子质量对转染率也有很大影响。相对分子质量较高的L-鸟氨酸(MW203,400)最有效地介导了基因转移，其转染活性约为相对分子质量53,600的L-鸟氨酸的5倍，而相对分子质量较低的L-鸟氨酸(MW11,700)的活性较低。多肽的最佳浓度取决于应用溶液中的DNA水平。DNA/多肽的最佳质量比为0.4~0.8。多肽的转染活性取决于其氨基酸组成、相对分子质量和DNA/多肽的比例。高分子聚(L-鸟氨酸)和聚(L-精氨酸)是比商业上可获得的更少的脂联素更有效的DNA转染剂

We investigated cationic liposome-mediated DNA transfection in the presence of serum.Transfection efficiency decreased dramatically with an increasing of serum concentration and the efficiency in medium containing 5% calf serum was nearly half of that in serum-free medium. An addition of albumin to medium also lowered the efficiency and to 35% at a concentration of 50 mg/ml. DNA stability and the effect of incubation times were further studied to elucidate the effect of serum on transfection. Analysis of DNA degradation with agarose electrophoresis showed that DNA complexed with cationic liposomes were more resistant to nuclease in serum than free plasmid DNA.Furthermore, in the presence of serum, transfection efficiency increased over time. The efficiency at 30 hour was 3-fold higher than that at 3 hour, and it was almost comparable to that in normal transfection in serum-free medium. The longer incubation was achieved by reducing the cytotoxicity of DNA-liposome complexes in medium c … More ontaining serum. These results suggested that lowered transfection efficiency in the presence of serum is mainly due to a delay in the uptake process of DNA-liposome complexes in transfection, rather than an irreversible change in DNA.The transfection ability and cytotoxicity of the synthetic polypeptides and the effect of molecular weight and concentration of the polypeptide on DNA transfection were examined.Poly (L-ornithine) , poly (L-arginine) and poly (L-ornithine, L-leucine) induced very high expression levels of a foreign gene in HeLa S3 cells. However, poly (L-lysine) and poly (L-lysine, L-alanine) were not effective. Cytotoxicity was observed at high concentrations of polypeptides and increased in the order poly (L-ornithine) >poly (L-lysine) >poly (L-arginine) <greater than or equal>poly (L-ornithine, L-leucine) >poly (L-lysine, L-alanine) . The molecular weight of polypeptide also greatly influenced on the transfection activity. The higher molecular weight of poly (L-ornithine) (MW203,400) mediated genc transfer most effectively, and its transfection activity were approximately 5-fold higher than that of poly (L-ornithine) with a molecular weight of 53,600, whereas the poly (L-ornithine) of a lower molecular weight (MW11,700) exhibited little activity. The optimal concentrations of the polypeptides depended on the DNA level in an applied solution. The optimal mass ratio of DNA/polypeptide for transfection was 0.4-0.8. The transfection activity of a polypeptide depends on its amino acid composition , molecular weight and DNA/peptide ratio. High molecular poly (L-ornithine) and poly (L-arginine) are more efficient mediators of DNA transfection than Lipofectin, a commercially available Less