Effects Of The RGD Modified With Oxymatrine-loaded Polymersomes On Liver Fibrosis

BackgroundHepatic 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 ResultIn 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.ConclusionA 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.