摘要
研究背景
肝纤维化(Hepatic fibrosis, HF)及肝硬化代表着各种慢性肝病的最终共同转归,目前尚缺乏有效治疗。氧化苦参碱(Oxymatrine, OM)是从特色中药材苦豆子中提取的生物碱之一,具有明确的抗HF作用,但半衰期短、肝脏分布少、剂量大、副作用明显。受体介导肝星状细胞(hepatic stellate cells, HSC)的纳米靶向给药为OM抗HF治疗提供了新的思路。聚合物泡囊(polymersomes, PM)是近20年发展的一种新型的药物载体,由具有两亲性质的聚合物分子自组装而成的类似于脂质体的双层分子结构。它不但具有脂质体和纳米粒等微粒载药系统的主要优点,而且因具有较大的中空亲水区,对水溶性药物的包封能力更强,可优于纳米粒；另外,通过聚合物分子的选择(分子量、嵌段比例、共聚物结构等)更易调控PM的理化特性(粒径、Zeta电位、载药量等)乃至体内行为,且具有表面修饰等优点。文献已报道亲水性二嵌段共聚物(PEG-b-PCL)可制备生物相容性且可生物降解的PM,且PM表面的PEG能连接多种靶向基团,可实现药物靶向传递。如将具有靶向功能配基的环状多肽RGD修饰于包载有OM的PM膜表面,就能将OM最大限度地运送到肝纤维化发生时肝脏活化的HSCs,使OM在靶区浓度超出传统制剂的数倍,治疗效果会明显提高,且可减少给药剂量及药物在其他脏器组织中的分布,降低毒副作用。
方法及结果
本文第一部分以MPEG、MalPEG及PCL为原料,辛酸亚锡为催化剂,通过改变mPEG种类与其ε-CL投料比,利用开环聚合法合成了系列不同分子量、嵌段比例的PEG-b-PCL两亲性嵌段共聚物MPEG2k-b-PCL3.1k MPEG2k-b-PCL4.0k、MPEG3k-b-PCL4.0k及MPEG5k-b-PCL9.1k, MalPEG3.4K-PCL9.1K。利用氢核磁共振('H-NMR)对产物进行结构鉴定与分析,用GPC测定了聚合物分散系数(PDI),用DSC测定了玻璃化转变温度(Tgs)。结果发现PEG占PEG-PCL链段比重(PEG)在0.33到0.42之间,Tgs在49.13到65.33之间,PDI为1.10到1.23之间。以粒径为主要考察指标,利用薄膜水化法制备了空白PM,探讨了形成PM的条件及特性,随着MPEG种类及PCL链段的不同,当水化温度高于Tgs时,形成的MPEG2k-b-PCL3.1k聚合物泡囊粒径为67nm、MPEG2k-b-PCL4.0k聚合物泡囊粒径为76nm、MPEG3k-b-PCL4.0k聚合物泡囊粒径为95nm及MPEG5k-b-PCL9.1k粒径为160nm,各种聚合物泡囊粒径分布较窄,Zeta电位测定接近中性,透射电镜(TEM)观察其形态为球形或近球形。
本文第二部分以载药量(DLC)、包封率(DLE)及粒径为主要考察指标,通过单因素分析、筛选处方及制备工艺,采用薄膜水化法被动载药制备氧化苦参碱聚合物泡囊(PM-OM),结果表明被动载药时,PM-OM聚合物泡囊DLC及DLE都比较低。以PEG3K-b-PCL4k为载体,采用薄膜水化法制备空白PM,通过pH梯度法主动载药法将OM包载于PM中,在内水相柠檬酸浓度为150mM,药脂比为0.4时,DLC可达6.6%,DLE为39.72%。以malPEG-PCL与mPEG-PCL按一定比例混合,以相同方法制备空白PM及PM-OM,将PM-OM与巯基化的RGD通过Maleiminde相连得RGD修饰的氧化苦参碱聚合物泡囊(RGD-PM-OM)。采用动态透析法考察不同制剂的体外释放行为,结果表明强亲水性OM在2h释达放94.46%,释药基本完全；PM-OM及RGD-PM-OM在2h内释有突释现象,随后OM释放相对缓慢,与OM相比PM-OM及RGD-PM-OM有一定的缓释效果。通过动态光散射(DLS)和TEM考察了PM、PM-OM及RGD-PM-OM各种不同聚合物泡囊的外观、粒径、Zeta电位及形态,结果表明各种聚合物泡囊均为带有蓝色乳光的澄清透明液体,粒径在100nm左右,且粒径分布较窄,Zeta电位电位测定接近中性,TEM观察形态为圆球形或近似球形。同时结果表明相同分子量、嵌段比例的mPEG-b-PCL聚合物制备所得的空白PM、PM-OM及RGD-PM-OM的粒径大小、Zeta电位、形态及体外释放趋势等理化特性并无明显差异。
本文第三部分采用改良的Friedman方法分离、培养原代肝星状细胞(HSC),0.4%的台盼兰染色检测,HSC成活率为90%以上。相差显微镜下观察刚分离出来的静息态HSC,呈小圆形,胞浆内因含有大量脂肪滴可为高折光性。10-14d后,细胞质中脂肪滴逐渐减少,细胞分化、增殖速度加快,逐渐成为成纤维样形态,为活化HSC,此时免疫组化染色鉴定显示大鼠HSC表达desmin阳性。采用体外细胞抑制实验对所制备的空白PM、OM溶液、PM-OM和RGD-PM-OM聚合物泡囊载药系统对活化HSC抑制作用进行评价。选择异硫氰酸荧光素(FITC)作为荧光标记物,采用激光共聚焦显微镜定性考察了FITC、 PM-FITC及RGD-PM-FITC的细胞摄取,以及通过Ⅵ型胶原受体的竞争抑制定性定量实验评价了RDG和Ⅵ型胶原受体靶向作用特性。此外,考察了OM溶液、PM-OM及RGD-PM-OM对活化活化HSC的α-平滑肌肌动蛋白(α-SMA)和collagen1α1表达的影响,为OM抗肝纤维化提供依据。结果表明,空白PM对活化HSC无抑制作用,OM、PM-OM及RGD-PM-OM对活化活化HSC有不同程度的抑制作用,且存在浓度依赖性,与OM溶液组及PM-OM相比,RGD-PM-OM对活化HSC的抑制作用更为显著(P<0.001)。细胞摄取实验表明PM-FITC及RGD-PM-FITC显著提高了FITC在HSC摄取能力,RGD-PM-FITC摄取能力最显著,进入细胞内主要分布在细胞核周围。Ⅵ型胶原受体竞争性抑制实验结果表明,相比于FITC溶液剂PM-FITC, RGD-PM-FITC通过与活化的HSC表面的Ⅵ型胶原受体相互作用,通过受体介导的内吞作用,增强聚合物泡囊被HSC的摄取能力。RT-PCR基因表达结果显示,与对照组相比,OM、PM-OM及RGD-PM-OM的a-SMA和collagen lal的mRNA表达都有不同程度的降低(P<0.001)。与OM相比,RGD-PM-OM组的a-SMA下降显著(P=0.03),与PM-OM相比下降不显著。RGD-PM-OM的a-SMA表达与OM组相比极其显著降低(P=0.006),与PM-OM相比下降显著(P=0.01),这有利于减轻肝纤维化程度。
本文第四部分建立了乙腈沉淀蛋白结合超高效液相色谱-串联质谱法(UPLC-MS/MS)分析方法,测定了大鼠血浆中OM浓度,研究OM溶液、PM-OM及RGD-PM-OM聚合物泡囊在大鼠体内的药动学行为。同时考察了OM的各种制剂在小鼠体内的组织分布,考察了PM-OM及RGD-PM-OM给药系统在改变OM组织分布方面的作用。结果发现在测定色谱及质谱条件下,内源性物质与OM及内标均能得到较好分离,不干扰OM的测定。同时该方法具有测定时间短,灵敏度高以及重现性好的优点,可以用于测定OM溶液、PM-OM及RGD-PM-OM给药后的大鼠体内血浆药物浓度以及小鼠组织中药物浓度水平,评价其体内药物代谢动力学过程和组织中药物分布情况。给大鼠尾静脉注射OM溶液、PM-OM及RGD-PM-OM后的药代动力学结果显示,OM溶液组的t1/2为1.41h,PM-OM和RGD-PM-OM t1/2分别是OM溶液组2.12和2.31倍,可见PM-OM和RGD-PM-OM均可延长OM在血液中循环时间。另外,PM-OM和RGD-PM-OM组的AUMC0-t分别是OM溶液组的2.51和3.15倍；与OM组相比,PM-OM和RGD-PM-OM的CL显著降低(P<0.05)。同时结果表明,PM-OM和RGD-PM-OM的主要药动学参数无显著性差别(P>0.05)。小鼠静脉注射OM溶液、PM-OM及RGD-PM-OM三种制剂后的药物组织分布研究结果表明,PM-OM及RGD-PM-OM组肝、脾及肺脏中的浓度显著高于OM溶液组,肾脏中显著低于OM溶液组；心脏中与OM溶液无显著性差异。
本研究第五部分建立胆总管结扎肝纤维化大鼠模型,从血清生化指标、肝脏病理检查、肝脏免疫组化等指标评价OM溶液、PM-OM和RGD-PM-OM对肝纤维化的治疗作用,并探讨其相关作用机制。
ALT及AST结果发现,与假手术组比较,模型组大鼠血清AST及ALT显著升高(P<0.001和P=0.049)：经不同组OM制剂治疗后,各组大鼠血清AST及ALT活性有不同程度下降,与模型组比较,RGD-PM-OM及PM-OM组ALT显著性降低(P<0.001和P=0.002)；与模型组相比,各组的AST有所降低,但差异并不显著(P>0.05)。
血清肝纤维化指标PCHI,Ⅳ-C,HA及LN各实验组之间有显著性差异(P<0.001)。与假手术组比较,大鼠血清中PCⅢ,Ⅳ-C,HA及LN极显著升高(P<0.001)。经各组治疗后有不同程度的降低,其中PCⅢ：与模型组比较,RGD-PM-OM组极显著降低(P=0.001),PM-OM组显著降低(P=0.022),OM组降低但差异不显著(P>0.05)；Ⅳ-C：与模型组比较,RGD-PM-OM组极显著降低(P=0.005),PM-OM和OM组显著降低(P=0.034和P=0.04)；HA：与模型组比较,RGD-PM-OM组显著降低(P=0.023),PM-OM组及OM组有所降低,但差异不显著(P>0.05)；LN：与模型组比较,RGD-PM-OM组、PM-OM组及OM组显著降低(P=0.019,P=0.025和P=0.044)。
肝组织Masson染色结果表明,假手术组染色可见肝小叶结构正常,肝索排列规则,无纤维间隔形成,汇管区及中央静脉周围偶见少许细索状的绿色胶原纤维：模型组肝组织胶原纤维增生,有片状假小叶形成,汇管区及中央静脉周围可见宽而疏松绿染胶原纤维带,局部肝窦周围可见网状胶原纤维。经不同制剂组治疗后,情况得到明显改善。按肝纤维化组织学半定量计分系统(semiquantitative scoring system, SSS)标准计分,模型组胶原显色指数明显高于其他各组(F=24.996,P<0.001)。经治疗后各组胶原显色指数有不同程度降低,与模型组比较,RGD-PM-OM极显著降低(P<0.001),PM-OM组胶原显色指数显著降低(P=0.011),OM组降低不显著(P>0.05)。
大鼠肝脏组织a-SMA免疫组化染色阳性表达显黄棕色,假手术组有轻微阳性表达；肝纤维化动物模型组a-SMA阳性表达明显增加,表达主要位于汇管区纤维细胞,肝窦管内皮细胞及少量肝细胞胞浆。对阳性表达进行半定量分析结果发现,与假手术组比较,模型组表达显著增加(P<0.001)；经治疗后表达有不同程度降低,与模型组比较,OM组(P=0.037)及PM-OM组(P=0.043)显著降低,RGD-PM-OM组(P=0.001)阳性表达极显著降低。TGF-β1阳性表达呈深棕黄色细颗粒状,模型组TGF-β1阳性细胞增多,在增生的成纤维细胞、汇管区纤维组织中血管内皮细胞呈表达,间质细胞也有表达；与假手术组比较模型组表达有显著性增高(P<0.01),经治疗后与模型组比较,OM组(P=0.02)和PM-OM组(P=0.011)明显降低,RGD-PM-OM显著性降低(P=0.003)。
结论
本文成功制备了RGD修饰的氧化苦参碱聚合物泡囊靶向给药系统,该系统粒径约96nm,Zeta电位接近中性,透射电镜观察其形态为球形或近球形；体外释药结果表明RGD-PM-OM前2h内有突释放现象,后期释放相对缓慢,与OM相比有明显缓释效果。体外细胞实验结果表明与OM及PM-OM相比,RGD-PM-OM对HSCs抑制作用明显,且能显著降低α-SMA及collagen1α1型胶原mRNA表达水平,有利于减轻肝纤维化程度；细胞摄取及受体竞争抑制实验证实RGD修饰的PM可与活化HSC表面的Ⅵ型胶原受体相互作用增强其在活化HSC的摄取水平。药动学及组织分布实验结果可知,RGD-PM-O可延长OM在血液中循环时间,且改变药物组织分布,在肝、脾及肺脏中的药物浓度显著高于OM组,肾脏中显著低于OM组。从肝纤维化大鼠动物模型结果可知RGD-PM-OM的抗肝纤维化效果优于PM-OM组及普通OM溶液组,表明受体介导的RGD-PM-OM靶向活化HSC有利于增强OM的治疗肝纤维化作用,这与RGD修饰的氧化苦参碱聚合物泡囊具有HSC靶向性有关。
Background
Hepatic fibrosis represents the final common pathway of most types of chronic liver diseases. The end-stage of liver fibrosis is characterized by the disposition of excess extracellular matrix (ECM) components and the appearance of regenerative nodules. The key factor in the pathogenesis of hepatic fibrosis is the activation and proliferation of hepatic stellate cells (HSC) and their transformation into myofibroblasts. Up to date, there are no effective anti-fibrotic drugs for liver fibrosis in patients.
Oxymatrine (OM) is an alkaloid extracted from traditional Chinese herb Sophora alopecuraides L.(Kudouzi) and the root of Sophora flavescesn Ait.(Kushen). Approved OM capsule and injection have been clinically applied for treating viral hepatitis B, hepatitis C and hepatoprotective in China. In recent years, OM has been proved to have anti-hepatic fibrosis effect in rat and human. However, OM injection, the short elimination half-life and poor distribution in liver result in low biological availability. To enhance the treatment effect, patients have to be administered too large a dose, yet followed increasing some side-effects on non-target tissues or cells. To improve drug effectiveness and reduce the side-effects on non-target tissues or cells, targeted drug delivery systems have been employed in recent years.
HSC are identified as key roles in the processes of liver fibrosis, which are an essential target for the development of therapeutic strategies. Selectively targeting HSCs have shown a powerful anti-fibrotic effect both in vitro and in vivo experiments. The cyclic Arg-Gly-Asp (RGD) peptides have been demonstrated to recognize collagen type VI receptors, which selectively interfere with collagen type Ⅵ-mediated cell adhesion, can be used as a homing device to target to HSC in liver fibrosis.
Over the last decade, nanocarriers (e.g., nanoparticles, micelles, dendrimers) have been developed as promising alternatives for the delivery of hydrophobic therapeutic agents. OM is a very hydrophilic drug. It is important to select a different approach which can provide high payloads of OM in drug delivery system (DDS). Polymersomes (PM), self-assembled polymeric vesicles, currently attract growing interest for potential applications as DDS as a novel class of nanocarriers. With a similar structure to the liposomes, polymersomes can carry high payloads of hydrophilic drug in their aqueous core. Moreover, compared with liposomes, the physical and chemical properties of polymersomes including particles size, drug loading, surface modification, and offering a stealth character in vivo may be broadly controlled and modulated by varying block lengths, block chemistry, and functionality. Therefore, polymersomes is good candidates for drug delivery carriers, which are currently being developed by many groups.
Method and Result
In the present work, diblock copolymers of MPEG and PCL were synthesized with various compositions for OM delivery. We investigated the relationships between the copolymer composition and the OM-loading content (DLC) as well as the physicochemical properties of these OM-loaded polymersomes, including the polymersomes size and OM-release profiles. Furthermore, we coupled RGD to the surface of polymersomes (RGD-PM-OM) as a homing ligand to HSC. We evaluated the anti-fibrotic properties of this new drug carrier on cultured HSCs, by testing inhibition of HSCs proliferation, cellular uptake efficiency, and the expression of profibrotic genes. In addition, the influence of labeled RGD to OM-loaded polymersomes on the fibrotic process in the liver was also evaluated in a bile duct ligation (BDL) rat model of liver fibrosis. We demonstrate that OM, a hydrophilic antifibrotic drug was successfully encapsulated in polymersoems by a pH-gradient method and the potential of using RGD-PM-OM as a cell-specific drug that may effectively lead to reduced activation of HSC and may markedly attenuate hepatic fibrosis in rats.
The MPEG-PCL and Maleimide-PEG-PCL block copolymers were synthesized by ring-opening polymerization of ε-CL using MPEG or Maleimide-PEG as the initiator in the presence of stannous octoate as a catalyst, and method described in full detail elsewhere. The molecular weight and composition of MPEG-PCL and Maleimide-PEG-PCL were investigated by1H-NMR using CDCl3as the solvent, respectively. The Tgs or PDI of diblock polymer were detected by DSC or GPC, respectively. The hydrophilic fraction used in this study adjust from0.33to0.42, while mPEG Mw was fixed at2,000,3000and5,000. PDI was from1.10to1.23. Tgs was from49.13to65.33.
Polymersomes were formed using the film hydration method. The effect of varying hydrophilic weight fraction and mPEG chain length on polymersomes was studied under the same or different conditions of preparation. It is important to underscore that the polymer films are hydrated for0.5-16hours at above the melting point of the diblock polymer with vigorous stir. The size of polymersomes increased with increasing molecular weight of PCL part when fixing Mw of PEG part at2,000, the size increased from67±4.3nm to78±2.1nm, as the molecular weight of PCL segment was increased from3100to4000. Also, the size of polymersomes increases with increasing molecular weight of mPEG part. They are78±2.1,95±1.9,160±6.3(nm), for PEG2K, MEG3K and PEG5K and, respectively. Zeta potential of polymersomes were near neutral. vesicle morphology of polymersomes presented Spherical or spheroidic nanoparticle with uniform size.
Drug loading efficacy (DLE) and drug loading contents (DLC) capacities are two critical parameters for evaluating the capacity of polymersomes to entrap the drug. In the presence of a pH gradient, increasing the ratio of drug to polymer from0.1to0.6led to an increased DLC from2.98%to11.71%, while a slight DLC increased from1.44%to3.73%was observed in no a pH gradient, reflecting the ability of the pH gradient to enhance loading. In the MPEG3k-b-PCL4k vesicles, at a ratio of drug to polymer of0.4, the highest DLC (6.6%) achieved at200Mm in the presence of a pH gradient. The cyclic RGD peptide was coupled via a sulfhydryl group at the cysteine residue to OM-loaded polymersomes formulation. After labeled with RGD and OM-loading in polymersomes, the size distribution, Zeta potential and shape of polymersomes were relatively identical to that before drug loading. The release of doxorubicin micelles was investigated by dynamic dialysis. The results shown that OM solution dispersions show a fast release of OM. At the2hours, the release was already94.5%. PM-OM and RGD-PM-OM showed a fast release profile at the beginning of2hours, followed slow steady release profiles were observed for these polymersomes formulations after2hours.
In vitro HSC proliferation experiments suggested PM-OM and RGD-PM-OM exhibited significantly higher inhibitor of HSCs proliferation compared to OM solution. RGD-PM-OM was the most potent inhibitor of HSCs proliferation, whereas incubation of HSCs with the empty polymersomes alone did not have any effect on the inhibitory of HSCs. The cellular uptake tests were conducted by confocal laser scanning microscopy of FITC solution, PM-FITC and RGD-PM-FITC. The qualitative cellular uptake experiments showed that polymersomes systems possessed highed cellular uptake capacity compared with the free FITC solution. And the majority of visible fluorescence of RGD-PM-FITC was mainly in the nuclear compartmen. The collagen Ⅵ receptors competitive inhibition studies performed by fluorescence microscopy imaging suggested intracellular delivery of RGD-PM-FITC was efficiently taken up via collagen Ⅵ receptors-mediated endocytosis. The VI receptors receptor-mediated endocytosis further enhanced internalized amounts of RGD-PM-FITC in HSC, as compared with FITC and PM-FITC.
In order to support pharmacokinetic study for OM-loaded polymersomes (OM-PM) based mPEG-PCL in rat, a rapid, highly selective ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-ESI-MS/MS) to quantify OM in rat plasma was developed and validated. Acetonitrile emulsion breaking method established as a blood extraction of oymatrine combined with UPLC-MS/MS was used to determine plasma concentrations of oxymatrine. Intravenous injection of OM solution and oxymatrine loaded polymersomes, the in vivo pharmacokinetic results showed that, t1/2of was1.41h. PM-OM and RGD-PM-OM was2.12and2.13times. Oxymatrine loaded polymersomes improved the blood circulation time of oxymatrine (P<0.01), compared with OM solution. Tissue biodistribution behavior was also investigated in normal mice. The results showed OM-loaded polymersomes and RGD-PM-OM were altered the distribution performances of OM in mice.OM in PM-OM and RGD-PM-OM more distributed into liver, spleen and lung, while decreased the distribution of OM in heart and than OM solution.
In order to examine whether RGD-PM-OM attenuate the fibrotic processes within the liver, we performed a study using bile duct-ligated rats as the model of liver fibrosis. Collagen fiber detection of the livers in those rats was assessed by analysis of Masson staining. Few collagen depositions or fibrous septa formations were observed in the sham-operated group. A marked collagen deposition was present in the periportal areas and areas of bridging fibrosis in the model group, which resulted in obvious changes in the hepatic architecture, including pseudo lobule formation, extensive portal-portal and portal-central fibrotic linkages. Treatment with OM, PM-OM or RGD-PM-OM groups had a tendency to modulate the production of collagen. It was notable that BDL rats receiving RGD-PM-OM displayed very low levels of collagen deposition detected in the liver compared with model BDL groups (P<0.05).
The effects of the carrier were determined in cultured HSCs and in bile duct-ligated rats (BLD). Targeted RGD-PM-OM and PM-OM displayed better suppression on HSC proliferation and significantly reduced the expression of the genes for α-SMA and collagen lal in cultured HSCs. Compared with free OM, RGD-PM-OM and PM-OM decreased serum ALT, indicating improved functionality of the hepatocyte. Targeted RGD-PM-OM exhibited distinctly superior anti-fibrosis activity by reducing the levels of PC-Ⅲ, Ⅳ-C, HA and LN in serum, decreasing gene expression of α-SMA and TGF-β1and lessening connective tissue deposition in the BLD compared PM-OM and free OM. These results indicated that targeted PM containing OM markedly attenuate hepatic fibrosis. This approach may provide a new means to treat chronic hepatic diseases.
Conclusion
A series of nano-sized polymersomes based on biodegradable PEG-PCL were prepared with a narrow size distribution. OM could be successfully loaded into the aqueous core of the polymersomes by a pH-gradient method. The peptide-labeled polymersomes are selectively taken up by activated HSCs via receptor-mediated endocytosis and that RGD modified with OM-loaded polymersomes displayed better suppression on HSC proliferation and reduced the expression of the genes for a-SMA and collagen1α1, important markers of fibrosis in vitro. Compared with PM-OM and free OM, RGD-PM-OM exhibited the most powerful anti-fibrosis activity by reducing the levels of bioactive markers in serum, decreasing gene expression of a-SMA and lessening connective tissue deposition in the BLD. These results demonstrate the potential of using the peptide RGD labeled with OM-loaded polymersomes as a cell-specific carrier that may effectively lead to attenuate in vitro and in vivo in rats with hepatic fibrosis. The in vivo pharmacokinetic results showed that Oxymatrine loaded polymersomes improved the blood circulation time of oxymatrine, compared with OM solution. Tissue biodistribution results showed OM-loaded polymersomes and RGD-PM-OM were altered the distribution performances of OM in mice.OM in PM-OM and RGD-PM-OM more distributed into liver, spleen and lung, while decreased the distribution of OM in heart and than OM solution.
引文
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