共载溶瘤病毒HSV和阿霉素的多功能电纺纳米纤维植入膜对于肝癌手术治疗的研究

To overcome the problems met by resection operation of hepatocellular carcinoma, such as high recurrence rate, serious side-effect by normal chemotherapy, multi-drug resistance and peritoneal adhesion, a multifunctional electrospun membrane, loading with oncolytic virus, chemotherapeutic and anti-infective drugs, is established by us. The multifunctional membrane is consisted of two nanofibrous electrospun layers, which are called anti-tumor layer and anti-infective & anti-adhesive layer separately. Implantable membrane could increase the concentration of drugs in the tumor and decrease the side-effect of the body. Using electrospun membrane to load oncolytic virus provides a new platform for the application of oncolytic virus therapy. Through this method, deeply seated tumor and residual cancer cell after tumorectomy could be treated by oncolytic virus, which was impossible in the past. Due to the different anti-tumor mechanism of oncolytic virus and doxorubicin, both normal cancer cells and cancer stem cells are cured by the synergistic effect of oncolytic virus therapy and chemotherapy. The outer layer of the bilayered fibrous membranes, separating the wound and the abdominal wall, serves as a physical barrier to prevent adhesions, meanwhile avoiding the direct contact between doxorubicin and abdominal wall to reduce the toxic effect. This multifunctional implantable membrane could solve multiple problems met by resection operation of hepatocellular carcinoma. It has great medical research value and broad prospect of clinical application.

针对肝癌手术治疗面临的易复发、化疗药物全身给药毒副反应大、肿瘤细胞耐药性强和腹膜粘连等多重问题，本项目拟构建一种共载溶瘤病毒、化疗药物和抗菌药物的多功能电纺纳米纤维植入膜。该复合膜由分别负载抗肿瘤药物和抗菌药物的两层电纺纤维膜组成。植入缓释膜可以提高肿瘤部位抗肿瘤药物的浓度，降低全身毒副反应。研究电纺纳米纤维膜富集并释放溶瘤病毒的机制，突破溶瘤病毒高浓度治疗和瘤内注射的技术瓶颈，发展一种用于体内深部肿瘤和术后残留肿瘤的溶瘤病毒疗法的新型给药方式。研究化疗药物和溶瘤病毒不同的抗肿瘤协同作用，实现对普通肝癌细胞、耐药性肝癌细胞以及肿瘤干细胞的治疗，解决肝癌细胞耐药性问题。研究双层膜中抗菌防粘连层的组成结构，达到抗菌防粘连和控制药物释放方向的目的，减少化疗药物与腹膜的接触，降低毒副作用。本项目构建的复合膜解决了肝癌手术治疗面临的多重问题，具有重要的生物医学研究价值和广泛的临床应用前景。

本项目研究了一种肝癌手术治疗的多功能植入膜，该膜具有抗肿瘤层和抗菌防粘连层两层结构，分别研究了各层的制备工艺并对其进行表征和评价。获得的同时负载溶瘤性单纯疱疹病毒（HSV）和化疗药物阿霉素（DOX）的HSV&DOX@CS纳米纤维膜将溶瘤病毒治疗和化疗结合起来，可以维持25天左右的阿霉素缓释。实时定量PCR检测证明，在治疗浓度范围内阿霉素不会影响溶瘤病毒在肝癌细胞中的复制。细胞水平药效评价证明，该复合膜对人肝癌细胞Bel-7402，Bel-7402耐化疗药物5-氟尿嘧啶(5-fu)的耐药细胞株7402-5fu，用3D细胞培养技术培养的具有干细胞性质的Bel-7402细胞，人肝癌细胞HepG2以及HepG2耐化疗药物阿霉素的耐药细胞株HepG2-ADM都具有很强的杀伤作用，并且其杀伤作用强于单独负载溶瘤病毒的HSV@CS纳米纤维膜或者单独负载阿霉素的DOX@CS纳米纤维膜。体内评价证明，该复合膜可以有效控制肿瘤的生长，并且残余肿瘤中干细胞的数目明显低于其余各组。对重要器官的组织切片分析证明该复合膜具有良好的生物安全性。获得的负载阿司匹林（ASA）的聚乳酸乙醇酸共聚物(ASA@PLAG) 可以维持7天左右的阿司匹林缓释，能有效减少肝脏切除时的出血量，提高肝功能的恢复并减少腹腔粘连情况。本项目构建的复合膜解决了肝癌手术治疗面临的易复发、化疗药物治疗全身毒副反应大、肿瘤细胞耐药性强和腹膜粘连等多重问题，具有重要的生物医学研究价值和广泛的临床应用前景。