The Effect Of Paclitaxel-chitosan Film On Inhibition Of Biliary Scar

[Background]Biliary scar is biliary fibrosis and scarring due to abdominal surgery, bile duct bacterium, virus infection, biliary calculi, inflammation, tumor and so on. Biliary scar may cause biliary stricture. How to effectively prevent pathological scar formation has been a major problem faced by biliary surgeon, which made patients often go on a secondary biliary surgery. The pathogenesis of biliary scar is unclear. Transforming growth factor betal (TGF-betal) is well-known as an important fibrosis cell factor. On the one hand, it promotes epithelial mesenchymal transformation (EMT); on the other hand, it promotes biliary fibroblast activation, proliferation, secretion of extracellular matrix, and prompted FB to MFB; In addition, TGF-betal also regulates the other cells in scar healing process. Some studies have reported that paclitaxel could effectively inhibit EMT and proliferation of fibroblast induced by TGF-beta 1, and the mechanism is mainly involved in the TGF-β1/Smads signal pathways. At present, in the study of bile duct scar, whether TGF-β1 can lead epithelial cells to EMT and regulate bile duct fibroblast to proliferate are less, more rare on the mechanism of its occurrence. This experiment is to observe the effect on duct epithelial cells and fibroblasts induced by TGF-β1 and confirm the biliary epithelial cells EMT and fibroblast proliferation.Paclitaxel (PTX), isolated from the bark of Pacific Yew (Taxus brevifolia), It is one of the most effective chemotherapeutic drugs and is mainly used to treat lung, ovarian, and breast cancer, etc. A few studies showed Paclitaxel has a good curative effect for nocancer clinical disease. The major limitation of PTX is its low water solubility (～0.4 μg/mL); thus, it is formulated in organic solvents of polyoxyethylated castor oil (Cremophor EL) under the trademark "Taxol". However, Cremophor EL is known to cause serious side effects, such as hypersensitivity reactions. As a result, prolonged infusion time and pretreatments are required. Moreover, the presence of Cremophor EL alters the pharmacokinetic profile of PTX in vivo which was described as unpredictable non-linear plasma pharmacokinetics when PTX was formulated in Cremophor EL. In addition, PTX is a substrate of P-glycoprotein (P-gp), which actively pumps PX out of the cells and induces drug resistance. Research reports, paclitax-el can inhibit EMT and the proliferation of fibroblasts induced by TGF-β1 and its mechanism was mainly TGF-β1/Smads signal pathway.At present, According to local tumor, local chemotherapy was wanted to be taken. It had a high local concentration, to avoid systemic multiple organ injury. Due to bile duct in the deep abdominal cavity and the scouring effect of bile, the chemotherapy drug is difficult to reach the lesion and the drug concentration was very low. How to make a slow releasing materials placed in the duct to release medicine, and reached the effective therapeutic dose every day?To overcome this problem, different sustained release dosage forms of paclitaxel were prepared as liposomes, microemulsions, microspheres, nanoparticles and so on. They could not only avoid the toxicity of Cremophor EL, inhibit toxicity of Taxol, also extend the period of drug action in vivo. We choose chitosan sustained-release system on our experiments, because the chitosan is a natural biodegradable material, non-toxic, no antigenicity, nonteratogenic and sensitization, good tissue compatibility, both antibacterial and anti-inflammatory and antifibrosis effect. Base on published reference, chitosan drug delivery system, we design to prepare two paclitaxel of chitosan membrane, to evaluate the effectiveness and safety of the drug delivery system, to contrast physical and chemical properties of two kinds of membranes, so it has good physical and chemical properties, good biological effect to meet the test requirements. We select one of the membranes as experimental membranes in the following experiment.At present, among the study of many slow-release forms of PTX, comparation of sustained-release form contrast to PTX alone has not been reported, therefore, this study proposed the taxolrelease membrane in the treatment effect were studied in vivo and in vitro experiments, to Confirm that paclitaxel can inhibit bile duct epithelial cells EMT and fibroblast proliferation;to evaluate paclitaxel release membrane in clinical efficacy, feasibility and safety of treatment of biliary tract scar.[Objective]Paclitaxel sustained-release membrane were prepared in different crosslinking ratio.The research content mainly includes paclitaxel chitosan membrane of prescription and preparation technology, drug release method research and comparison in vitro, the drug release evaluation in vitro; To study the possible mechanism of paclitaxel on bile duct scar; Tocompare paclitaxel chitosan membrane with paclitaxel on the inhibitory effect on fibroblast proliferation in vitro;Tocompare inhibition effect of paclitaxel chitosan membrane with paclitaxel on bile duct epithelial cells EMT in vitro;To research paclitaxel ch -itosan membrane on resistance to bile duct scar in vivo.[Methods] 1.The modified chitosan solvent evaporation method and chitosan embedding method were prepared to make two kinds of paclitaxel chitosan membrane. Prepare and purify hydroxyethyl chitosan (HECTS). N-Succinyl-hydroxyethyl chitosan (Suc-HECTS) is prepared by HECTS and succinic anhydride via ring-opening reaction. Then a drug loaded membrane is made of Suc-HECTS and paclitaxel (PTX). The conjugate prepared by the direct coupling of PTX with Suc-HECTS using EDC-HCL is obtained due to cross-linking among the polymer supports. Underdarklight condition,drug-loadedmembranes with three differentcross-linking degrees are synthesized by using BDDGE as apolymer cross-linker. The characteristics of the membranes, such as FTIR equilibriumwatercontent,swelling ratio, permeability, anti-Fibrosis in vitro, subcutaneous irritant reaction, biodegradability and biocompatibilityare determined. We successfully made paclitaxel-N-hydroxyethyl chitosan Succinylation drug-releasefilm (PTX-Suc-HECTS); To evaluate the infrared spectrum, sustained release membrane swelling ratio, drug loading and drug release kinetics in vitro as its physicochemical properties. Results show that we successfully make the PTX-Suc-HECTS; To Prepare the P-PTX-SRM, the 200mg chitosan dissolved in acetic acid solution, and added BDDGE at different crosslinking ratio to prepare chitosan solution. Then paclitaxel and 10mg poloxamer407 were dissolved in 1ml ethanol, and then mixed together, finally joined the chitosan solution prepared before and then mixed evenly. The suspension casted on Φ90mm (area63.6cm2) the glass plate, and vacuum dryed at 60 ℃ 12h. To evaluate the sustained release membrane swelling ratio, drug loading and drug release kinetics in vitro as its physicochemical properties;To compare the drug loading and drug release rate of two kinds of film in vitro.2.We successfully developed biliary fibroblasts (FB) in vitro. In addition to the comparative study inhibition of bile duct fibroblasts naked PTX and PTX-SRM, we also evaluated effect of PTX on proliferation of biliary fibroblast (BF) by MTT, cell migration, apoptosis and cell cycle experiment.3.We successfully developed biliary epithelial cells (BEC) in vitro, and use the TGF-beta 1 induced epithelial mesenchymal phenotype transformation (EMT) of BEC, then using pure paclitaxel injection and the paclitaxel chitosan membrane for processing. Effect of PTX and PTX-SRM on the EMT in BEC were observed.4.To investigate whether PTX-SRM in vivo could effectively slow the formation of bile duct scar, we chose to use big-ear rabbits to establish bile duct injury model, then in the corresponding time point, SRM-PTX was made into bile duct of rabbits. Mesenchymal marker expression of Vimentin and alpha SMA or epithelial marker expression of E-cadherin were detected by QPCR and immunohistochemical stain. Masson staining was used to observe the degree of biliary fibrosis.[Results]1. Design and technology research for prescription of paclitaxel-chitosan film Paclitaxel sustained-release film has some in vitro drug delivery capabilities with good hydrophilicity and biodegradability. As lower degree of crosslinking, the drug release rate is gradually increased. Two kinds of drug membrane can meet the experimental requirements.2. Paclitaxel chitosan membrane inhibits proliferation of biliary fibroblasts in vitroDrug paclitaxel and low, mid, high chitosan release membrane loading drug paclitaxel can increase the bile duct fibroblast apoptosis rate, and with increasing drug loading conce-ntration, cell apoptosis rate increased; chitosan membrane without drug can inhibit bile-duct fibroblastsproliferation.When using naked PTX or SRM-PTX to treat BF with TGF-betal stimulation, we found they both inhibit the proliferation of BF. The result of MTT show SRM-PTX has longer inhibiting time of proliferation of BF than naked PTX. The results of Flow cytometry show higher apoptosis rate of SRM-PTX in long time. The cell cycle results show mechanism of action of PTX is that high concentration of PTX-SRM caused G2/M cell cycle arrest; low and middle concentration of PTX-SRM caused G0/G1 cell cycle arrest.3. Paclitaxel Chitosan Film inhibits EMT of biliary epithelial cells in vitroLow, mid, high chitosan release membrane loading drug paclitaxel were able to inhibit the bile epithelial cells proliferation, and with increasing carrier concentration, the inhibition of cell proliferation rate also increased, but chitosan membranewithout drug paclitaxel can not inhibit the bile epithelial cells proliferationThe expression of TGF-beta 1 significantly increased after TGF-beta 1 stimulation. We used QPCR and Western blot to find that with TGF-betal stimulation, the expression of E-cadherin significantly decreased, the expression of Vimentin and alpha SMA increased obviously. It indicated EMT can be induced by TGF-beta in BEC.When using paclitaxel slow-release membranes or pure taxol to treat BEC with TGF-betal stimulation, we found the expression of mesenchymal markers Vimentin and alpha SMA decreased obviously, and the expression of E-cadherin obviously increased, especially in 48 h. It suggested that paclitaxel could effectively inhibit the TGF-beta of EMT. But after 72 h, in the BEC with naked PTX, the expression of Vimentin and alpha SMA increased slightly, and the expression of E-cadherin slightly decreased.It suggested that the reverse effects of EMT werereduced;And in the BEC with PTX-SRM, PTX-SRM had the continuous inhibitory effect. It suggested that the SRM-PTX had longer EMT inhibition effect.In addition, we tested TGF/Smads signaling pathways, the results showed that both naked PTX and PTX-SRM could inhibit the protein levels of TGF-beta 1 and phosphorylation Smad2/3. Interesting, no significant differences of inhibition of signal pathway between them, we suspected SRM-PTX may involve other mechanisms including TGF/Smads signaling pathways.4. PTX-SRM can inhibit bile duct scar in animalsAfter operation stimulation, the bile epithelial cellsduct mesenchymal markers (Vimentin and alpha SMA) abnormal expressed positive, and epithelial markers E-cadherin expressed weakly and fibrous tissue increased in bile duct. After encapsulated paclitaxel chito-san membrane, Vimentin and alpha SMA protein expression significantly decreased, and E-cadherin high positive expressionand fibrous tissue was significantly reduced in the bile duct. Paclitaxel chitosan membrane can inhibit the transformation and fibrosis without non-toxicity.[Conclusion]1. PTX-Suc-HECTS and P-PTX-SRM prepared in this experiment have good biological properties, consistentwith bio implant material requirements.2. Bile duct scar may be combined outcome of EMT and myofibroblast proliferation.3. PTX-SRM can inhibit the proliferation of BF. PTX-SRM can be more effectively than PTX in inhibiting the proliferation of BF. The mechanism of action of PTX is that low and middle concentration of PTX-SRM caused G0/G1 cell cycle arrest; high concentration of PTX-SRM causes G2-M cell cycle arrest.4. TGF-beta 1 induces EMT in biliary epithelial cells via the TGF-β1/Smad2/3 signal pathway. Paclitaxel can inhibit the EMT and efficacy and durationof PTX-SRM form are better than PTX. In this process, PTX-SRM may involve other signaling pathways include TGF-β1/Smad2/3 signaling pathway.5. In vivo, operation can induce EMT of BEC and biliary fibrosis, which could be inhibited by PTX-SRM.