Nonionic polymeric micelles for oral gene delivery in vivo
Nonionic polymeric micelles for oral gene delivery in vivo
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DOI：
10.1089/10430340460745801
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发表时间：
2004-05-01
影响因子：
4.2
通讯作者：
Liaw, JH
中科院分区：
文献类型：
--
作者：
Chang, SF;Chang, HY;Liaw, JH
本研究的主要目的是探讨聚环氧乙烷-聚环氧丙烷-聚环氧乙烷(PEO-PPO-PEO)非离子聚合物胶束作为口服DNA载体的可行性。用动态光散射法和原子力显微镜分别考察了DNA/PEO-PPO-PEO聚合物胶束的大小和形貌，并测量了它们的Zeta电位。用5-溴-4-氯-3-吲哚-β-D-半乳糖苷染色，以氯酚红-β-D-半乳糖苷为底物，通过组织提取液中酶活性的测定，定性地评价了LacZ基因在裸鼠不同组织中的表达。此外，通过组织学分析鉴定了表达LacZ基因的十二指肠细胞的类型。DNA/PEO-PPO-PEO聚合物胶束是一组圆形胶束，平均直径为170 nm，Zeta电位为-4.3 mV。通过计算表观渗透系数来评价DNA/PEO-PPO-PEO聚合物胶束对十二指肠的体外透皮性能。结果表明，在低浓度(0.026-0.26mug/mul)时，药物的穿透速率与剂量无关，为(5.75+/-0.37)×10(-5)cm/s，而在浓度为1.3mug/mul时，穿透速率下降到(2.89+/-0.37)×10(-5)cm/s。此外，在给予DNA/PEO-PPO-PEO聚合物胶束之前和同时给予10 mM RGD多肽或10 mM EDTA时，转运分别通过阻断内吞作用而被抑制([0.95+/-0.57]x 10(-5)cm/sec)或通过开放紧密连接而增强([29.8+/-5.7]x 10(-5)cm/sec)。每隔8小时口服6次后，在第一次给药后48小时，转导基因LacZ的表达最高，在十二指肠的绒毛、隐窝、杯状细胞和胃的隐窝细胞中检测到该基因的表达。报告基因在十二指肠、胃和肝脏中有活性。当小鼠在给予DNA/PEO-PPO-PEO聚合物胶束之前和同时给予10 mM EDTA时，小鼠的大脑和睾丸也有活动。逆转录-聚合酶链式反应检测到5个脏器和血液中LacZ基因的表达。综上所述，这些结果表明，通过口服PEO-PPO-PEO聚合物胶束可以在小鼠体内实现高效、稳定的基因转移。
The main aim of this study was to investigate the feasibility of using nonionic polymeric micelles of poly( ethylene oxide) -poly(propylene oxide) - poly(ethylene oxide) (PEO-PPO-PEO) as a carrier for oral DNA delivery in vivo. The size and appearance of DNA/PEO-PPO-PEO polymeric micelles were examined, respectively, by dynamic light scattering and atomic force microscopy, and their zeta potential was measured. Expression of the delivered lacZ gene in various tissues of nude mice was assessed qualitatively by 5-bromo-4-chloro-3-indolyl-beta-D- galactopyranoside staining of sections and quantitatively by measuring enzyme activity in tissue extracts, using the substrate of beta-galactosidase, chlorophenol red-beta-D-galactopyranoside. In addition, the types of cells expressing the lacZ gene in the duodenum were identified by histological analysis. DNA/PEO-PPO-PEO polymeric micelles are a single population of rounded micelles with a mean diameter of 170 nm and a zeta potential of -4.3 mV. Duodenal penetration of DNA/PEO-PPO- PEO polymeric micelles was evaluated in vitro by calculating the apparent permeability coefficient. The results showed a dose-independent penetration rate of (5.75 +/- 0.37) x 10(-5) cm/sec at low DNA concentrations (0.026 - 0.26 mug/mul), but a decrease to (2.89 +/- 0.37) x 10(-5) cm/sec at a concentration of 1.3 mug/mul. Furthermore, when 10 mM RGD peptide or 10 mM EDTA was administered before and concurrent with the administration of DNA/PEO-PPO-PEO polymeric micelles, transport was inhibited ([0.95 +/- 0.57] x 10(-5) cm/sec) by blocking endocytosis or enhanced ([29.8 +/- 5.7] x 10(-5) cm/sec) by opening tight junctions, respectively. After oral administration of six doses at 8-hr intervals, the highest expression of transferred gene lacZ was seen 48 hr after administration of the first dose, with gene expression detected in the villi, crypts, and goblet cells of the duodenum and in the crypt cells of the stomach. Reporter gene activity was seen in the duodenum, stomach, and liver. Activity was also seen in the brain and testis when mice were administered 10 mM EDTA before and concurrent with DNA/PEO-PPO- PEO polymeric micelle administration. lacZ mRNA was detected in these five organs and in the blood by reverse transcription-polymerase chain reaction. Taken together, these results show efficient, stable gene transfer can be achieved in mice by oral delivery of PEO-PPO-PEO polymeric micelles.