【作者】 陈华兵；

【导师】 杨祥良； 徐辉碧；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2008， 博士

【摘要】 经皮给药系统是药剂学中快速发展一个领域。在降低不良反应、提高治疗指数和用药顺应性方面具有明显的优势。经皮给药系统的研究主要包括药物的透皮吸收、局部组织和局部皮肤吸收等方面。近年来,以胰岛素为代表的多肽蛋白类药物的透皮给药和皮肤疾病治疗药物的皮肤靶向给药是经皮给药系统研究中的两个热点问题。随着纳米技术的快速发展,纳米载药系统已开始应用于解决上述两个问题。在本论文中,制备、表征了不同粒径和电位的胰岛素纳米囊泡,评价了微针、离子导入及两者联合应用对胰岛素纳米囊泡透皮的协同促渗作用,并考察了不同促渗条件下胰岛素纳米囊泡对糖尿病大鼠的降糖作用及其透皮机理。在皮肤靶向研究方面,构建并表征了鬼臼毒素微乳凝胶(HTM)和固体脂质纳米粒(SLN),考察其皮肤靶向性,并探讨了其皮肤靶向机制。在此基础上,在微乳凝胶中引入固体脂质材料,构建了一种新型的微乳凝胶,用于皮肤靶向给药。主要完成了以下研究工作:(1)采用高剪切法、超声波法、高压匀质法等制备了纳米囊泡。通过改变制备工艺条件,制备了粒径分别为91.0 nm、143.0 nm、175.9 nm的胰岛素纳米囊泡。进一步对纳米囊泡表面电荷进行修饰,制得了zeta电位分别为+27.8 mV、-25.3 mV、-50.5 mV的胰岛素纳米囊泡。采用量子点示踪技术和高分辨透射电镜观测了纳米囊泡的微区结构,纳米囊泡脂质双层膜厚度在3～5 nm之间,并观测到胰岛素聚集体在囊泡内外均有分布。Sephadex G25凝胶柱法测得胰岛素纳米囊泡的包封率为(89.05±0.91)%。纳米囊泡能促进胰岛素以被动扩散方式透过离体皮肤,但其透皮速率较低,仅为0.19±0.01μg/(cm2·h)。(2)微针预处理皮肤后,形成了皮肤微通道,有效克服了角质层的屏障作用,胰岛素纳米囊泡的透皮速率增加了86～166倍,最高可达31.68±0.79μg/(cm2·h),透皮时滞在0.9～1.2 h之间,透皮释药行为符合Fick’s第一扩散定律。而纳米囊泡在离子导入促渗时,其透皮速率仅为被动扩散的3.9～4.3倍,最高透皮速率为0.97±0.05μg/(cm2·h),其促渗能力作用明显弱于微针。在微针促渗时,上述胰岛素纳米囊泡的透皮速率分别是胰岛素溶液的3.3～13.0倍,表明纳米囊泡也具有重要的促渗作用。微针主要通过提高胰岛素的扩散系数来提高透皮能力,纳米囊泡则可能对扩散系数和分配系数均有提高。表面的正电荷修饰增强了胰岛素纳米囊泡在微针或离子导入促渗时的透皮能力,而表面负电荷修饰对胰岛素透皮影响较小。(3)微针和离子导入联合促渗作用下,胰岛素纳米囊泡的透皮释药行为符合Fick’s第一扩散定律,透皮时滞范围为1.0～1.3 h,其离体透皮速率在60.23～106.99μg/(cm2·h)之间,为胰岛素PBS溶液对照组透皮速率的3.9～7.0倍,是微针单独促渗时的3.4～7.1倍,离子导入单独促渗时的92.5～134.9倍,被动扩散时的359.6～713.3倍。微针、离子导入和纳米囊泡在胰岛素透皮过程中具有明显的协同促渗作用。微针联合离子导入促进胰岛素纳米囊泡透皮时具有显著降糖作用(P<0.01),在第3～6 h时血糖水平为初始值的28.3～41.7%,与皮下注射组无显著性区别,明显高于微针、离子导入以及纳米囊泡单独促渗时的降糖作用,与离体透皮实验结果一致。荧光成像实验表明,在微针和离子导入联合促渗时,纳米囊泡导致了荧光标记的胰岛素在皮肤中的富集,皮肤微通道和角质层途径均有较好的通透作用。联合促渗的主要透皮机制包括以下几个方面:微针预处理皮肤所诱导的微通道成为胰岛素纳米囊泡透皮的主要透皮途径之一;纳米囊泡在电场推动下沿微通道向皮肤深部渗透,可引起胰岛素纳米囊泡在皮肤层中的富集,进而降低角质层的屏障作用;纳米囊泡较高的包封率可显著提高胰岛素在皮肤中的分配系数,同时还可避免胰岛素分子在离子导入过程中存在的电荷反转问题。(4)制备、表征了鬼臼毒素微乳凝胶,微乳凝胶中的凝胶对微乳结构和透皮释药行为无明显影响。通过调节微乳凝胶中Tween 80的比例,鬼臼毒素的透皮速率和皮肤滞留量可同时下降或升高,其皮肤靶向性有待于进一步提高。此外,分别制备了泊洛沙姆188稳定的鬼臼毒素SLN(P-SLN)和Tween 80稳定的鬼臼毒素SLN(T-SLN),两者的平均粒径分别为73.4 nm、123.1 nm。原子力显微镜和扫描电镜研究表明两种SLN均为球形纳米粒, P-SLN具有更较好的物理稳定性。离体透皮实验结果显示,两种SLN均未透过皮肤,避免了鬼臼毒素的透皮吸收,而酊剂则有明显的透皮吸收。P-SLN和T-SLN的皮肤滞留量分别为23.38±0.55μg、6.82±0.34μg,比微乳凝胶有更好的皮肤靶向性。荧光成像研究发现SLN可促使药物分布于表皮层,表明P-SLN具有表皮靶向性。在SLN皮肤靶向机制方面,除现有的闭合膜和缓控释机理之外,推测SLN也可能进入角质层,改变药物在角质层中分配系数,从而实现皮肤靶向。(5)以熔融的脂质材料Compritol 888 ATO为微乳凝胶的油相,制成了热微乳凝胶,在-20℃和5℃时冷却可得到粒径分布分别为20～50 nm和20～200 nm的新型微乳凝胶(SHTM)。前者粒径分布窄、具有较好的单分散性,而后者在冷却过程中可能存在液滴融合的现象,导致粒径明显增加、分布较宽。离体小鼠透皮实验表明,雷公藤内酯SHTM具有较强的皮肤滞留能力,可明显降低药物的透皮速率,具有较好的靶向性。有关SHTM的纳米结构、释药行为及其皮肤靶向机制等还有待于深入研究。本论文的研究结果表明微针、离子导入协同促进胰岛素纳米囊泡透皮可有效克服皮肤的屏障作用,对胰岛素的透皮给药研究具有重要意义,对其它多肽蛋白药物的透皮给药也具有借鉴意义。有关皮肤疾病治疗药物的皮肤靶向载体研究为开发高效、低毒的局部给药新制剂提供了新的思路。更多还原

【Abstract】 Recently, the rapid development in transdermal drug delivery systems (TDDS) has been made in pharmaceutical science. TDDS are of advantage to reduce adverse side effects and to enhance therapy index and convenience for clinical uses. Much attention has been paid on transdermal delivery, topical delivery and dermal delivery in TDDS. The transdermal delivery of peptides and proteins with large molecular weights and skin targeting delivery of drugs for the therapy of skin diseases are attractive challenges in TDDS. Nano drug delivery systems (NDDS) are used to conquer these challenges due to the rapid development of nanotechnology.In this dissertation, the insulin-loaded nanovesicles were prepared, characterized and used to improve the permeation rates of insulin through skins with the enhancement of microneedle, iontophoresis or their combination. The change of blood glucose levels of diabetic rats were evaluated using in vivo animal experiments. The penetration mechanisms of insulin-loaded nanovesicles through skins were also explored using fluorescence imaging. In addition, podophyllotoxin-loaded hydrogel-thickened microemulsions (HTM) and solid lipid nanoparticles (SLN) were constructed and characterized. The dermal delivery of podophyllotoxin-loaded HTM and SLN was evaluated and their permeation mechanisms were also explored. Furthermore, Compritol 888 ATO was used to costruct novel HTM based on solid lipid for dermal delivery. The following are main results:(1) The nanovesicles were respectively prepared using high shear, ultrasound and high pressure homogenization methods. The nanovesicles with average diameters of 91.0 nm, 143.0 nm and 175.9 nm were constructed. The nanovesicles with zeta potentials of +27.8 mV, -25.3 mV and -50.5 mV were also prepared by modifying the surface charges of nanovesicles. The microstructures of nanovesicles were investigated by quantum dots (QDs)-based trace method and high resolution transmission electron microscopy (HR-TEM). The lipid membranes were found to have the thickness of 3～5 nm and insulin molecules distributed both inside and outside membranes. The entrapment efficiency of insulin-loaded nanoveiscles from Sephadex G25 column was (89.05±0.91)%. Insulin can passively penetrate through skins from nanovesicles at a low permeation rate of 0.19±0.01μg/(cm2·h).(2) The microneedles-pretreated microchannels could reduce the barrier of stratum corneum and increase the permeation rate of insulin from nanovesicles 86～166 times and the highest permeation rate was 31.68±0.79μg/(cm2·h). The permeation time lag ranged from 0.9 h to 1.2 h and the penetration accorded with Fick’s first law of diffusion. The iontophoresis could also enhance the permeation rates of insulin from nanovesicles 3.9～4.3 times and the highest permeation rate was 0.97±0.05μg/(cm2·h). The microneedles had more powerful ability to enhance the permeation rates of insulin from nanovesicles when compared with iontophoresis. The permeation rates of insulin from nanovesicles combined with the microneedles or iontophoresis were 3.3～13.0 times higher than those from control solution and the nanovesicles significantly contributed to the permeation enhancement. Microneedles could enhance the diffusion coefficient of insulin in skins and nanovesicles could improve both diffusion coefficient and partition coefficient of insulin. The nanovesicles with positive charges had more powerful permeation ability when compared with those with negative charges, when microneedles or iontophoresis were used.(3) The penetration of insulin from nanovesicles enhanced by the combination of microneedles and iontophoresis followed with Fick’s first law of diffusion and the permeation time lag ranged from 1.0 h to 1.3 h. The permeation rates of insulin were 60.23～106.99μg/(cm2·h), which were 3.9～7.0, 3.4～7.1, 92.5～134.9 and 359.6～713.3 times, respectively, when compared with those from control solution at passive diffusion, nanovesicles combined with microneedles alone, nanovesicles combined with iontophoresis alone and nanovesicles at passive diffusion. The combination of microneedles, iontophoresis and nanovesicles resulted in synergetic effects on the penetration of insulin (P<0.01). The synergetic effect could result in significant decrease of blood glucose levels (BGL) (P<0.01). The values of BGL at 3～6 h were 28.3～41.7% of initial values, which were comparable to those of rats administrated by subcutaneous injection and higher than that of the other groups. Fluorescence imaging showed that the synergetic effects of nanovesicles, microneedles and iontophoresis resulted in the enrichment of insulin in skins. Both the skin microchannels and stratum corneum showed high permeability for the penetration of insulin. The synergetic effect might attribute to several factors. The microneedle-pretreated skin microchannels acted as one of the main routes for insulin-loaded nanovesicles. The nanovesicles with charges propelled by iontophoresis could penetrate into deep skins along the microchannels and also lead to the enrichment of insulin in skins and reduce the barriers of stratum corneum. The high entrapment efficiency of nanovesicles could also improve the partition coefficient and avoid the charge reversal of insulin under the electric field of iontophoresis.(4) The podophyllotoxin-loaded hydrogel-thickened microemulsions (HTM) were constructed and characterized. The hydrogel have no significant influence on the microstructure of microemulsion and the permeation ability. The permeation rates of podophyllotoxin through skins increased with the increase of the accumulative amounts of drug in skin when the concentrations of Tween 80 were changed. The further studies should be performed for enhancing the skin targeting ability of HTM. In addition, podophyllotoxin-loaded solid lipid nanoparticles (SLN) were constructed using poloxamer 188 (P-SLN) and Tween 80 (T-SLN) as stabilizers. The average diameters of P-SLN and T-SLN were 73.4 nm and 123.1 nm, respectively. Atomic force microscopy and scanning electron microscopy showed that both SLN had spherical morphology and P-SLN had good physical stability. The in vitro permeation studies showed both P-SLN and T-SLN coult not penetrate through skins and avoid the systemic absorption of podophyllotoxin. But the tincture resulted in high permeation rates of podophyllotoxin. The accumulative amounts of podophyllotoxin from P-SLN and T-SLN in skins were 23.38±0.55μg and 6.82±0.34μg, respectively. Both SLN showed more powerful skin targeting than HTM. The fluorescence imaging found that P-SLN resulted in the local distribution of podophyllotoxin in epidermis and showed an epidermal targeting ability. The epidermal targeting might contribute to the penetration of SLN into stratum corneum and further change the partition coefficient besides their occlusive effects and sustained release.(5) The melt Compritol 888 ATO as an oily phase was used to construct a hot HTM. A novel HTM based on solid lipid (SHTM) were obtained by cooling the hot HTM at various temperature. The size distributions of SHTM obtained at the cooling temperature of -20℃and 5℃were 20～50 nm and 20～200 nm, respectively. SHTM obtained at -20℃had a narrow size distribution. But SHTM obtained at 5℃showed significant increases of diameters and broad size distribution because of possible coalescence between droplets during cooling. The in vitro permeation studies showed that SHTM could enhance the accumulative amounts of triptolide in skins, reduce the permeation rates and show a good skin targeting. The studies about microstructure, drug release and skin targeting of SHTM should be valuable in future.The results of this dissertation show that the penetration strategy of insulin-loaded nanovesicles enhanced by the combination of microneedles and iontophoresis would be significantly valuable for transdermal delivery of insulin and also be promising for transdermal delivery of the other peptides and proteins with large molecular weights. The research on the drug-loaded nanocarriers with skin targetings for the therapy of skin diseases affords a novel strategy in development of novel formulations with low toxicity and good curative effects.更多还原