Kyriakides Themis R, Cheung Charles Y, Murthy Niren, Bornstein Paul, Stayton Patrick S, Hoffman Allan S
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
J Control Release. 2002 Jan 17;78(1-3):295-303. doi: 10.1016/s0168-3659(01)00504-1.
Cytosolic delivery from endosomes is critical for those drugs that are susceptible to attack by lysosomal enzymes, such as DNA, RNA, oligonucleotides, proteins and peptides. Therefore, we have designed pH-sensitive, membrane-disruptive polymers to enhance the release of drugs from the acidic endosomal compartment to the cytoplasm. We have found that one polymer in particular, poly(propylacrylic acid) (PPAA), is very effective at membrane disruption at pHs below 6.5, based on hemolysis studies. PPAA also significantly enhances in vitro transfections of lipoplex formulations in cell culture, and does so in the presence of as much as 50% serum. In this study, we have extended our in vitro hemolysis and cell culture studies to an in vivo murine excisional wound healing model. A pilot study with a green fluorescent protein (GFP)-encoding plasmid indicated that injection of formulations containing PPAA into healing wounds resulted in increased GFP expression. Subsequently, by administering sense and antisense DNA for the angiogenesis inhibitor thrombospondin-2 (TSP2), we were able to alter the wound healing response in TSP2-null and wild type mice, respectively. Our findings showed that when PPAA was added to lipoplex formulations, expression of TSP2 was enhanced in TSP2-null mice compared to control formulations. These results show that PPAA can enhance in vivo transfections and that inhibition of TSP2 expression may lead to improved wound healing. These results suggest that PPAA can provide significant improvements in the in vivo efficacy of drugs such as DNA.
对于那些易受溶酶体酶攻击的药物，如DNA、RNA、寡核苷酸、蛋白质和肽而言，从内体进行胞质递送至关重要。因此，我们设计了对pH敏感的、可破坏膜的聚合物，以增强药物从酸性内体区室释放到细胞质中的能力。基于溶血研究，我们发现一种特别的聚合物，即聚（丙烯酸丙酯）（PPAA），在pH低于6.5时对膜破坏非常有效。PPAA还能显著增强细胞培养中脂质体配方的体外转染效果，并且在存在高达50%血清的情况下也是如此。在本研究中，我们将体外溶血和细胞培养研究扩展到了体内小鼠切除伤口愈合模型。一项使用编码绿色荧光蛋白（GFP）的质粒的初步研究表明，将含有PPAA的配方注射到愈合伤口中会导致GFP表达增加。随后，通过给予血管生成抑制剂血小板反应蛋白-2（TSP2）的正义和反义DNA，我们能够分别改变TSP2基因敲除小鼠和野生型小鼠的伤口愈合反应。我们的研究结果表明，当将PPAA添加到脂质体配方中时，与对照配方相比，TSP2基因敲除小鼠中TSP2的表达增强。这些结果表明，PPAA可以增强体内转染，并且抑制TSP2表达可能会导致伤口愈合改善。这些结果表明，PPAA可以显著提高诸如DNA等药物的体内疗效。
