Mumper R J, Duguid J G, Anwer K, Barron M K, Nitta H, Rolland A P
Department of Gene Delivery, GENEMEDICINE, INC., The Woodlands, Texas 7738-4248, USA.
Pharm Res. 1996 May;13(5):701-9. doi: 10.1023/a:1016039330870.
DNA plasmids (pDNA) can be taken up by and expressed in striated muscle after direct intramuscular injection. We have developed interactive polymeric gene delivery systems that increase pDNA bioavailability to muscle cells by both protecting pDNA from nucleases and controlling the dispersion and retention of pDNA in muscle tissue.
A DNA plasmid, containing a CMV promoter and a galactosidase reporter gene (CMV-beta-gal), was injected either in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) solutions. Interactions between PVP and pDNA were assessed by dynamic dialysis, Isothermal Titration Calorimetry (ITC), and Fourier Transformed Infra Red (FT-IR) spectroscopy. Formulations (50 mu l) were injected into rat tibialis muscles after surgical exposure. Immunohistochemistry for beta-gal was used to visualize the sites of expression in muscle.
Beta-gal expression using pDNA in saline reached a plateau while beta-gal expression using PVP formulations increased linearly in the dose range studied (12.5-150 mu g pDNA injected) and resulted in an increase in the number and distribution of cells expressing beta-gal. The interaction between PVP and pDNA was found to be an endothermic process governed largely by hydrogen-bonding and results in protection of pDNA from extracellular nucleases.
Significant enhancement of gene expression using interactive polyvinyl-based delivery systems has been observed. The improved tissue dispersion and cellular uptake of pDNA using polyvinyl-based systems after direct injection into muscle is possibly due to osmotic effects.
直接肌内注射后，DNA质粒（pDNA）可被横纹肌摄取并表达。我们开发了交互式聚合物基因递送系统，该系统通过保护pDNA免受核酸酶的作用以及控制pDNA在肌肉组织中的分散和保留，提高了pDNA对肌肉细胞的生物利用度。
将含有巨细胞病毒（CMV）启动子和半乳糖苷酶报告基因（CMV-β-半乳糖苷酶）的DNA质粒，以生理盐水形式或用聚乙烯吡咯烷酮（PVP）和聚乙烯醇（PVA）溶液配制后进行注射。通过动态透析、等温滴定量热法（ITC）和傅里叶变换红外（FT-IR）光谱法评估PVP与pDNA之间的相互作用。手术暴露后，将制剂（50μl）注射到大鼠胫骨肌肉中。使用β-半乳糖苷酶的免疫组织化学来观察肌肉中的表达位点。
使用生理盐水形式的pDNA时，β-半乳糖苷酶的表达达到平台期，而使用PVP制剂时，β-半乳糖苷酶的表达在所研究的剂量范围内（注射12.5 - 150μg pDNA）呈线性增加，并导致表达β-半乳糖苷酶的细胞数量和分布增加。发现PVP与pDNA之间的相互作用是一个主要由氢键控制的吸热过程，可保护pDNA免受细胞外核酸酶的作用。
已观察到使用交互式聚乙烯基递送系统可显著增强基因表达。直接注射到肌肉后，使用聚乙烯基系统改善了pDNA在组织中的分散和细胞摄取，这可能是由于渗透作用。
