Clement Jule, Kiefer Karin, Kimpfler Andrea, Garidel Patrick, Peschka-Süss Regine
Department of Pharmaceutical Technology, Albert-Ludwigs-University, Freiburg, Germany.
Eur J Pharm Biopharm. 2005 Jan;59(1):35-43. doi: 10.1016/j.ejpb.2004.06.001.
The instability of lipoplex formulations is a major obstacle to overcome before their commercial application in gene therapy. In this study, a continuous mixing technique for the large-scale preparation of lipoplexes followed by lyophilisation for increased stability and shelf-life has been developed. Lipoplexes were analysed for transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMC) and a rat smooth muscle cell line (A-10 SMC). Homogeneity of lipid/DNA-products was investigated by photon correlation spectroscopy (PCS) and cryotransmission electron microscopy (cryo-TEM). Studies have been undertaken with DAC-30, a composition of 3beta-[N-(N,N'-dimethylaminoethane)-carbamoyl]-cholesterol (DAC-Chol) and dioleylphosphatidylethanolamine (DOPE) and a green fluorescent protein (GFP) expressing marker plasmid. A continuous mixing technique was compared to the small-scale preparation of lipoplexes by pipetting. Individual steps of the continuous mixing process were evaluated in order to optimise the manufacturing technique: lipid/plasmid ratio, composition of transfection medium, pre-treatment of the lipid, size of the mixing device, mixing procedure and the influence of the lyophilisation process. It could be shown that the method developed for production of lipoplexes on a large scale under sterile conditions led to lipoplexes with good transfection efficiencies combined with low cytotoxicity, improved characteristics and long shelf-life.
在脂质体配方用于基因治疗的商业应用之前，其不稳定性是需要克服的主要障碍。在本研究中，已开发出一种连续混合技术，用于大规模制备脂质体，随后进行冻干以提高稳定性和保质期。分析了脂质体在人主动脉平滑肌细胞（HASMC）和大鼠平滑肌细胞系（A-10 SMC）中的转染效率和细胞毒性。通过光子相关光谱法（PCS）和低温透射电子显微镜（cryo-TEM）研究了脂质/DNA产物的均匀性。使用DAC-30进行了研究，DAC-30是由3β-[N-（N，N'-二甲基氨基乙烷）-氨基甲酰基]-胆固醇（DAC-Chol）和二油酰磷脂酰乙醇胺（DOPE）以及表达绿色荧光蛋白（GFP）的标记质粒组成。将连续混合技术与通过移液管小规模制备脂质体进行了比较。评估了连续混合过程的各个步骤，以优化制造技术：脂质/质粒比例、转染培养基的组成、脂质的预处理、混合装置的尺寸、混合程序以及冻干过程的影响。结果表明，所开发的在无菌条件下大规模生产脂质体的方法可产生具有良好转染效率且细胞毒性低、特性改善且保质期长的脂质体。
