喜树碱衍生物通过透明质酸纳米微乳经皮给药作用于瘢痕修复的研究

发布时间：2018-09-04 11:19

【摘要】：透明质酸纳米微乳经皮药物释放体系，在皮肤渗透、定量给药、药物缓释、延长药效和浅表层病灶区定位方面体现了诸多优势。10,11-亚甲二氧基喜树碱（MD-CPT）是喜树碱衍生物的代表，具有很高的生物活性，能够有效地阻碍DNA复制，阻止有丝分裂，抑制细胞增殖，是良好的抗肿瘤制剂。但是，MD-CPT的疏水性、内酯环不稳定性等理化性质限制了其体内应用。透明质酸纳米微乳作为MD-CPT的载体，可以克服MD-CPT的水难溶性、促进机体吸收、提高生物利用度、增加药物靶向性、减少毒副作用、维持血药浓度、延长半衰期和提高药效。瘢痕疙瘩（KD）被认为是一种良性肿瘤，其特征是成纤维细胞过度增殖，且受刺激易复发和加重病情，因此本文将载MD-CPT透明质酸纳米微乳应用于瘢痕疙瘩的修复研究，建立了一种温和无创的经皮给药治疗途径。对透明质酸进行修饰，，成功制备了双亲性透明质酸单甘脂衍生物（HA-GMS）。通过红外、核磁检测证实了单甘酯成功地交联到HA多糖主链上，以FITC为荧光标记，以乙二胺为链接壁，利用硫脲键将FITC共价修饰在EDA-HA-GMS多糖主链上，得到FITC-HA-GMS，可以对HA-GMS载体材料进行定位示踪与定量分析。溶血实验表明，HA-GMS的溶血率均小于5%，符合生物医用材料对溶血率的要求，具有较好的血液相容性。通过MTT法检测了HA-GMS的细胞毒性。采用超声乳化法，成功制备了不同组分配比的载MD-CPT的HANs纳米微乳，平均粒径是50~200nm，zeta电位绝对值是10~20mV，带负电，均一性较好。通过透射电镜观察，纳米粒均为完整的球形，大小均一，分布均匀，几乎没有聚集的现象，稳定性良好。通过体外模拟实验，考察了HANs纳米微乳在皮肤表面的稳定性，HANs纳米微乳在pH4.0~7.4形态几乎没有变化，在pH7.4是纳米微乳表现出不规则的形状，而且部分颗粒出现崩解的现象，说明HANs纳米微乳在皮肤弱酸性环境下可以有效地递送药物。 MD-CPT/HANs纳米微乳用于透皮扩散研究，角质层透过试验：0~4h呈时间依赖性递增，达到平衡时累积渗透量Qt为660.7±20.5μg/cm2，明显高于对照组。全皮渗透性测定：Qt为51.7±9.5μg/cm2明显低于角质层透过量，随时间呈S型变化趋势。不同HA-GMS：MD-CPT质量配比制得的载药纳米微乳的透皮扩散效率不同。Rhodamine B标记HA-GMS，利用荧光显微镜观察到载药纳米乳透过角质层到达真皮层的拟动态过程，4h内，部分载药纳米微乳已经到达真皮层，实现透皮。血药浓度与尿样检测，证明了HANs纳米载体对MD-CPT具有缓释的作用，多余的MD-CPT进入了血液循环，参与体内的新陈代谢，最终被排出体外，不会成为机体负担。活体成像研究体内分布，进一步验证了MD-CPT在给药区皮下组织的滞留时间较长，药物浓度较高，形成了局部临时药物贮库，可以不断渗透到浅表层病灶区，实现持续给药、延长药效的目的。另外，药物含量：肾肝脾，进一步说明MD-CPT参与了新陈代谢，并避开了对喜树碱类药物十分敏感的消化道等组织器官。与HSF、HUVECS、MCF-7、N2a等不同细胞株作用比较，证明了MD-CPT对细胞生长抑制、细胞吞噬和细胞周期调节都是有选择的，效果明显倾向于分裂活跃的增生性细胞。 MD-CPT对瘢痕成纤维细胞（KF）的抑制呈浓度依赖性。KF对FITC-HANs-MD-CPT细胞吞噬是一个时间依赖性的动态的过程，由胞外扩散到胞内再到细胞核内。将MD-CPT/HANs作用于瘢痕皮肤，角质层变得膨胀、疏松，局部呈现筛孔的结构，促进渗透。荧光显微镜证明了Rhodamine-HANs-MD-CPT透过瘢痕疙瘩表皮直达真皮层的病变部位。 MD-CPT/HA-GMS作用KF细胞12h，处于S期的细胞增加，24h，48h，处于S与G2/M的细胞均急剧减少，超过90%的细胞处于G0/G1期，停止有丝分裂，出现了老化和凋亡的趋势。实时荧光定量RT-PCR的结果，PAI-1表达水平下调，但是TGF-β、Smad3、Smad7上调，推测TGF-β/SMAD—PAI-1被Smad7阻断。Western Blot显示Smad2/3磷酸化水平显著下降，印证了Smad7通过阻止Smad2/3磷酸化进而阻断TGF-β/SMAD信号通路，使下游基因PAI-1表达水平下降，从而抑制瘢痕疙瘩再生。综上所述，通过体外、体内实验评价了双亲性的透明质纳米微乳经皮给药体系的生物安全性，皮肤渗透性，细胞特异性及药代动力学特性，并应用于治疗瘢痕。研究表明，喜树碱衍生物通过透明质酸纳米微乳经皮给药有望成为瘢痕修复的治疗途径。
[Abstract]:Hyaluronic acid nano-microemulsion transdermal drug delivery system has many advantages in skin penetration, quantitative drug delivery, drug sustained release, prolonged efficacy and superficial lesion localization. 10,11-methylenedioxycamptothecin (MD-CPT) is a representative of camptothecin derivatives with high biological activity, which can effectively hinder DNA replication and prevent filaments. Hyaluronic acid nanoemulsion as the carrier of MD-CPT can overcome the water insolubility of MD-CPT, promote the body absorption, improve bioavailability, increase drug targeting, reduce toxicity and side effects. Keloids (KD) are considered to be benign neoplasms characterized by excessive proliferation of fibroblasts and prone to recurrence and aggravation by stimulation. In this paper, MD-CPT-loaded hyaluronic acid nanoemulsions were applied to keloid repair and a mild non-invasive skin graft was established. Route of administration.
Amphiphilic hyaluronic acid monoglyceride derivatives (HA-GMS) were successfully prepared by modification of hyaluronic acid. It was confirmed by IR and NMR that monoglyceride was successfully crosslinked onto the main chain of HA polysaccharide. FITC was labeled with fluorescence and linked with ethylenediamine. FITC was covalently modified on the main chain of EDA-HA-GMS by thiourea bond. Haemolysis test showed that the hemolysis rate of HA-GMS was less than 5%, which accorded with the requirement of biomedical materials for hemolysis rate and had good blood compatibility. The cytotoxicity of HA-GMS was detected by MTT method.
HANS nanoemulsions loaded with MD-CPT with different composition ratios were successfully prepared by ultrasonic emulsification. The average particle size was 50-200 nm, the absolute value of zeta potential was 10-20 mV, and the nanoparticles were negatively charged with good homogeneity. The stability of HANs nanoemulsion on the skin surface was investigated in vitro. The morphology of HANs nanoemulsion was almost unchanged at pH 4.0-7.4. The shape of HANs nanoemulsion was irregular at pH 7.4, and some particles disintegrated. The results showed that HANs nanoemulsion could deliver drugs effectively in weak acidic environment.
MD-CPT/HANs nanoemulsion was used in the study of transdermal diffusion. The cuticle penetration test showed that the cumulative penetration of MD-CPT/HANs nanoemulsion increased in a time-dependent manner from 0 to 4 hours. The cumulative penetration of Qt at equilibrium was 660.7+20.5 ug/cm 2, which was significantly higher than that of the control group. Rhodamine B labeled HA-GMS was used to observe the pseudo-dynamic process of drug-loaded nanoemulsions through the cuticle to the dermis by fluorescence microscopy. Within 4 hours, part of the drug-loaded nanoemulsions had reached the dermis and achieved transdermal penetration.
Blood concentration and urine samples showed that HANs nanocarriers could slow the release of MD-CPT. Excess MD-CPT entered the blood circulation, participated in metabolism in vivo, and eventually was excluded from the body. In vivo imaging study of the distribution of MD-CPT in the subcutaneous tissue of the drug delivery area further confirmed the longer retention time of MD-CPT. In addition, the content of MD-CPT in kidney, liver and spleen further indicates that MD-CPT participates in metabolism and avoids organs such as digestive tract which are sensitive to camptothecin.
Compared with HSF, HUVECS, MCF-7, N2a and other cell lines, MD-CPT has selective effects on cell growth inhibition, cell phagocytosis and cell cycle regulation, and the effect is obviously inclined to divide active proliferative cells.
MD-CPT inhibits scar fibroblasts (KF) in a concentration-dependent manner. KF phagocytosis of FITC-HANs-MD-CPT cells is a time-dependent dynamic process, from extracellular diffusion to intracellular and then to the nucleus. Microscopic examination showed that Rhodamine-HANs-MD-CPT penetrated the keloid epidermis directly to the dermal lesion site. KF cells in S phase increased at 12h, and decreased sharply at 24h and 48h. More than 90% of the cells in S and G2/M were in G0/G1 phase, stopped mitosis, and appeared the trend of aging and apoptosis. Western Blot showed a significant decrease in Smad2/3 phosphorylation, confirming that Smad7 blocked the TGF-beta/SMAD signaling pathway by inhibiting Smad2/3 phosphorylation, thereby inhibiting the downstream gene PAI-1 expression and thereby inhibiting scar formation. Lump regeneration.
In conclusion, in vitro and in vivo experiments were carried out to evaluate the bio-safety, skin permeability, cell specificity and pharmacokinetics of the amphiphilic hyaluronic nano-microemulsion transdermal delivery system and its application in the treatment of scars. Therapeutic approach.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

【参考文献】