Wang Dong-an, Narang Ajit S, Kotb Malak, Gaber A Osama, Miller Duane D, Kim Sung Wan, Mahato Ram I
Department of Pharmaceutical Sciences, University of Tennessee, Memphis, Tennessee 38163, USA.
Biomacromolecules. 2002 Nov-Dec;3(6):1197-207. doi: 10.1021/bm025563c.
A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.
通过将胆固醇氯甲酸酯连接到1800和10000 Da的支链聚乙二胺（PEI）的仲氨基上，合成了一种新型水溶性脂聚合物。通过PEI仲胺进行共轭，使这种新合成的脂聚合物（简称为PEI-Chol）比我们之前合成的脂聚合物具有特殊优势，之前的脂聚合物利用伯氨基进行共轭，因为伯氨基在DNA缩合中起重要作用。同样重要的是，每个PEI分子上仅接枝了一个胆固醇分子（通过¹H NMR和基质辅助激光解吸电离飞行时间质谱法确认），为PEI伯胺与DNA的空间相互作用留出了足够空间。对PEI-Chol脂聚合物的临界胶束浓度（cmc）、缓冲容量、DNA缩合（通过凝胶阻滞和圆二色性）、体外转染效率和细胞活力进行了表征。PEI-Chol 1800和PEI-Chol 10000的cmc分别为496.6和1330.5 μg/mL。酸碱滴定表明聚合物在pH 5-7范围内具有高缓冲容量，这表明它们在内体酸性pH环境中具有缓冲潜力。凝胶阻滞研究表明，使用这些脂聚合物可以实现质粒DNA的有效缩合。圆二色光谱表明，当配制的N/P比为2.5/1或更高时，与这些脂聚合物缩合后，DNA构象发生变化并采用较低能量状态。这些复合物的平均粒径在110-205 nm范围内，但使用1800 Da的PEI制备的复合物除外，其平均粒径为384±300 nm。在N/P比为15/1时，使用PEI-Chol 1800、PEI-Chol 10000以及1800、10000和25000 Da的PEI制备的DNA复合物的ζ电位在23-30 mV范围内，并且取决于N/P比。通过流式细胞术测定，PEI-Chol/pCMS-EGFP复合物在Jurkat细胞中的体外转染显示出高水平表达的绿色荧光蛋白（GFP）且毒性很小。这些新型水溶性脂聚合物具有良好的转染效率以及其他理想特性，如水溶性、用于有效DNA缩合的游离伯氨基和高缓冲容量，这表明其具有有效内体释放的可能性。
