Convertine Anthony J, Benoit Danielle S W, Duvall Craig L, Hoffman Allan S, Stayton Patrick S
Department of Bioengineering, University of Washington, Seattle, WA 98125, USA.
J Control Release. 2009 Feb 10;133(3):221-9. doi: 10.1016/j.jconrel.2008.10.004. Epub 2008 Oct 17.
The gene knockdown activity of small interfering RNA (siRNA) has led to their use as target validation tools and as potential therapeutics for a variety of diseases. The delivery of these double-stranded RNA macromolecules has proven to be challenging, however, and in many cases, is a barrier to their deployment. Here we report the development of a new diblock copolymer family that was designed to enhance the systemic and intracellular delivery of siRNA. These diblock copolymers were synthesized using the controlled reversible addition fragmentation chain transfer polymerization (RAFT) method and are composed of a positively-charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA condensation, and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios, together with butyl methacylate (BMA). A related series of diblock compositions were characterized, with the cationic block kept constant, and with the ratio of DMAEMA and PAA to BMA varied. These carriers became sharply hemolytic at endosomal pH regimes, with increasing hemolytic activity seen as the percentage of BMA in the second block was systematically increased. The diblock copolymers condensed siRNA into 80-250 nm particles with slightly positive Zeta potentials. SiRNA-mediated knockdown of a model protein, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in HeLa cells generally followed the hemolytic activity trends, with the most hydrophobic second block (highest BMA content) exhibiting the best knockdown. This pH-responsive carrier designed to mediate endosomal release shows significant promise for the intracellular delivery of siRNA.
小干扰RNA（siRNA）的基因敲低活性使其被用作靶点验证工具以及多种疾病的潜在治疗药物。然而，这些双链RNA大分子的递送已被证明具有挑战性，并且在许多情况下，是其应用的障碍。在此，我们报告了一种新型二嵌段共聚物家族的开发，该家族旨在增强siRNA的全身递送和细胞内递送。这些二嵌段共聚物采用可控可逆加成-断裂链转移聚合（RAFT）方法合成，由带正电荷的甲基丙烯酸二甲氨基乙酯（DMAEMA）嵌段介导siRNA凝聚，以及第二个由大致等摩尔比的DMAEMA和丙基丙烯酸（PAA）与甲基丙烯酸丁酯（BMA）组成的内体释放嵌段。对一系列相关的二嵌段组合物进行了表征，其中阳离子嵌段保持不变，DMAEMA和PAA与BMA的比例有所变化。这些载体在内体pH范围内具有明显的溶血活性，随着第二个嵌段中BMA百分比的系统性增加，溶血活性也随之增加。二嵌段共聚物将siRNA凝聚成80 - 250 nm的颗粒，具有略微正的zeta电位。在HeLa细胞中，siRNA介导的模型蛋白甘油醛-3-磷酸脱氢酶（GAPDH）的敲低通常遵循溶血活性趋势，疏水性最强的第二个嵌段（BMA含量最高）表现出最佳的敲低效果。这种设计用于介导内体释放的pH响应载体在siRNA的细胞内递送方面显示出巨大的潜力。
