Effects of Paclitaxel-conjugated N-Succinyl-Hydroxyethyl Chitosan Film for Proliferative Cholangitis in Rabbit Biliary Stricture Model Translated title: 紫杉醇偶联N-琥珀酰-羟乙基壳聚糖膜对兔胆道狭窄模型中增殖性胆管炎的影响

Abstract

Background:

Paclitaxel (PTX) could inhibit the growth of fibroblasts, which occurs in proliferative cholangitis and leads to biliary stricture. However, its use has been limited due to poor bioavailability and local administration for short time. This study designed and synthesized a new PTX-conjugated chitosan film (N-succinyl-hydroxyethyl chitosan containing PTX [PTX-SHEC]) and evaluated its safety and efficiency using in vivo and in vitro experiments.

Methods:

The SHEC conjugated with PTX was confirmed by nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FT-IR) measurements. Drug releases in vitro and in vivo were determined using high-performance liquid chromatography. Cell viability in vitro was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Rabbit biliary stricture model was constructed. All rabbits randomly divided into five groups ( n = 8 in each group): the sham-operated rabbits were used as control (Group A), Groups B received laparotomies and suture, Group C received laparotomies and covered SHEC suture without the PTX coating, Group D received laparotomies and covered PTX-SHEC suture, and Group E received laparotomies and 1000 μmol/L PTX administration. Liver function tests and residual dosage of PTX from each group were measured by enzyme-linked immunosorbent assay. Histological data and α-smooth muscle actin (SMA) immunohistochemical staining of common bile duct were examined.

Results:

NMR and FT-IR indicated that PTX was successfully introduced, based on the appearance of signals at 7.41–7.99 ppm, 1.50 ppm, and 1.03 ppm, due to the presence of aromatic protons, methylene protons, and methyl protons of PTX, respectively. No bile leak was observed. The PTX-conjugated film could slowly release PTX for 4 weeks (8.89 ± 0.03 μg at day 30). The in vitro cell viability test revealed significantly different levels of toxicity between films with and without PTX (111.7 ± 4.0% vs. 68.1 ± 6.0%, P < 0.001), whereas no statistically significant difference was observed among the three sets of PTX-contained films (67.7 ± 5.4%, 67.2 ± 3.4%, and 59.1 ± 6.0%, P > 0.05). Histological examinations revealed that after 28 days of implantment, Groups D and E (but not Group C) had less granulation tissue and glandular hyperplasia in the site of biliary duct injury than Group B. The pattern was more obvious in Group D than Group E. Less α-SMA-positive cells were found in tissue from Groups D and E. Comparing with Group E, the liver function was improved significantly in Group D, including total bilirubin (2.69 ± 1.03 μmol/L vs. 0.81 ± 0.54 μmol/L, P = 0.014), alanine aminotransferase (87.13 ± 17.51 U/L vs. 42.12 ± 15.76 U/L, P = 0.012), and alkaline phosphatase (60.61 ± 12.31 U/L vs. 40.59 ± 8.78 U/L, P < 0.001).

Conclusions:

PTX-SHEC film effectively inhibites the myofibroblast proliferation and extracellular matrix over-deposition during the healing process of biliary reconstruction. This original film might offer a new way for reducing the occurrence of the benign biliary stricture.

摘要

背景：

紫杉醇能抑制增殖性胆管炎中成纤维细胞的生长，其是导致胆管狭窄的重要原因。然而，由于生物利用度较差，局部用药时间过短，紫杉醇临床应用受到限制。本研究设计开发了一种新型紫杉醇缓释膜（紫杉醇偶联的N-琥珀酰-羟乙基壳聚糖膜，PTX-SHEC），并通过体内和体外实验评价其安全性和有效性。

方法：

通过核磁共振和傅立叶变换红外光谱测量验证紫杉醇与N-琥珀酰-羟乙基壳聚糖（SHEC）的偶联。使用高效液相色谱法测定体内和体外的药物释放。使用MTT实验测量体外细胞活力。构建胆道狭窄兔模型，并将SHEC或PTX-SHEC缓释膜分别植入模型中（分别定义为B，C，D组；每组8只）。同时将假手术组定义为A组，1000 μmol/L 紫杉醇作为E组（每组8只）。酶联免疫吸附试验检测各组肝功能和残留剂量。苏木素伊红染色和免疫组织化学染色检查胆总管的组织病理学和a-平滑肌肌动蛋白表达情况。

结果：

核磁共振和红外光谱表明紫杉醇成功偶联至缓释膜中，其于7.41–7.99 ppm、1.50 ppm和1.03 ppm检测到紫杉醇结构中的质子。动物模型中没有观察到胆汁泄漏。紫杉醇缓释膜可缓慢释放紫杉醇4周（第30天为8.89±0.03 μg）。体外细胞活力测试显示偶联或不偶联紫杉醇膜之间的毒性水平存在显著差异（111.7±4.0％ vs. 68.1±6.0％， P<0.001），而在三组含紫杉醇的膜中未观察到毒性水平存在显著差异（67.7±5.4％，67.2±3.4％和59.1±6.0％， P> 0.05）。组织病理学显示植入28天后，D和E组（非C组），胆道损伤部位的肉芽组织和腺体增生较B组明显减少，其中D组较E组变化更为明显。D组和E组中α-SMA阳性细胞率较少。与E组比较，D组肝脏功能明显改善，比较如下（E vs. D）：总胆红素 (2.69±1.03 μmol/L vs. 0.81±0.54 μmol/L, P=0.014)、谷草转氨酶(87.13±17.51 U/L vs. 42.12±15.76 U/L, P=0.012)、谷丙转氨酶(60.61±12.31 U/L vs. 40.59±8.78 U/L, P<0.001)。

结论：

紫杉醇缓释膜可有效抑制胆道重建愈合过程中肌成纤维细胞增殖和细胞外基质过度沉积。这种新型缓释膜为降低良性胆道狭窄的发生提供了新的途径。