Studies on Drug delivery system for gene therapy

我们研究了在血清存在下阳离子脂质体介导的DNA转染。转染效率随着血清浓度的增加而急剧下降，在含有5%牛血清的培养基中转染效率接近无血清培养基的一半。在培养基中加入白蛋白也降低了效率，当浓度为50 mg/ml时，效率降至35%。进一步研究了DNA稳定性和孵育时间的影响，以阐明血清对转染的影响。琼脂糖电泳分析表明，与阳离子脂质体配合的DNA比游离质粒DNA对血清核酸酶的抗性更强。此外，在血清存在下，转染效率随着时间的推移而增加。30小时的转染效率是3小时的3倍，与无血清培养基中正常转染的效率几乎相当。通过降低dna -脂质体复合物在培养基c和更多含血清中的细胞毒性，实现了更长的孵育。这些结果表明，在血清存在的情况下，转染效率降低主要是由于转染过程中DNA-脂质体复合物的摄取过程延迟，而不是DNA的不可逆变化。研究了合成的多肽的转染能力和细胞毒性，以及多肽的分子量和浓度对DNA转染的影响。聚（l -鸟氨酸）、聚（l -精氨酸）和聚（l -鸟氨酸、l -亮氨酸）诱导HeLa S3细胞中外源基因的高表达水平。而聚赖氨酸和聚赖氨酸-丙氨酸均无效果。在高浓度多肽下观察到细胞毒性，并按poly （l -鸟氨酸）>poly （l -赖氨酸）>poly （l -精氨酸）的顺序增加，<大于或等于>poly （l -鸟氨酸，l -亮氨酸）>poly （l -赖氨酸，l -丙氨酸）。多肽的分子量对转染活性也有很大影响。分子量较高的聚l -鸟氨酸（MW203,400）介导基因转移效果最好，其转染活性比分子量为53,600的聚l -鸟氨酸高约5倍，而分子量较低的聚l -鸟氨酸（MW11,700）几乎没有转染活性。多肽的最佳浓度取决于应用溶液中的DNA水平。转染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、分子量和DNA/肽比。高分子聚（l -鸟氨酸）和聚（l -精氨酸）是比脂脂素更有效的DNA转染介质，脂脂素是一种市售的Less

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