载10-羟基喜树碱靶向相变纳米粒联合高强度聚焦超声治疗裸鼠肝癌移植瘤

目的探讨叶酸受体靶向的载10-羟基喜树碱(10-HCPT)相变纳米粒(FR-HCPT-PNPCA)造影剂联合高强度聚焦超声(HIFU)对裸鼠肝癌移植瘤的治疗效果。方法采用皮下注射人肝癌7721细胞的方法制备荷瘤裸鼠模型120只,随机分为4组,每组30只,HIFU治疗前1 d分别通过尾静脉注射药物或生理盐水200μl,其中A组注射生理盐水+HIFU辐照,B组注射HCPT+HIFU辐照,C组注射靶向非载药相变纳米粒(FR-PNPCA)+HIFU辐照,D组注射FRHCPT-PNPCA+HIFU辐照。辐照后即刻应用超声观察各组裸鼠肿瘤内灰度变化。2 h后每组处死10只裸鼠,取肿瘤组织行TTC染色观察大体病理并进行组织切片,HE染色后于显微镜下观察肿瘤细胞坏死情况,然后行组织匀浆检查瘤内HCPT浓度。48 h后每组再处死10只裸鼠,取肿瘤组织切片行PCNA和TUNEL染色观察各组肿瘤细胞增殖和凋亡情况。2周后处死各组剩余裸鼠,测量肿瘤大小,并取主要脏器观察肿瘤转移情况。结果辐照后即刻超声显示A、B组辐照区域几乎无灰度变化,C、D组辐照前后灰度变化面积和变化值与A、B组比较,差异均有统计学意义(均P<0.05)。辐照后2h,B、D组瘤内均可检测到HCPT,且D组瘤内HCPT浓度高于B组,差异有统计学意义(P<0.05);A、B组均未见明显凝固性坏死,C、D组肿瘤内均可见明显凝固性坏死,坏死范围与A、B组比较差异均有统计学意义(均P<0.01)。辐照后48 h,D组细胞增殖率最低,细胞凋亡率最高,与A、B、C组比较差异均有统计学意义(均P<0.05)。辐照后2周,D组肿瘤几乎消失,转移灶最少,与A、B、C组比较差异均有统计学意义(均P<0.05)。结论FR-HCPT-PNPCA联合HIFU不仅能实时监控肿瘤HIFU消融并增强消融效果,且释放的药物能辅助治疗肿瘤,有望为肿瘤治疗提供一种精准、高效的方法。

Treatment of xenograft hepatoma in nude mice by high intensity focused ultrasound combined with targeted phase-change nanoparticles loading 10-HCPT

Objective To investigate the therapeutic effect of high intensity focused ultrasound(HIFU)combined with folic acid receptor-targeted 10-hydroxycamptothecin(10-HCPT)-loaded phase-change nanoparticle contrast agent(FRHCPT-PNPCA)on xenograft hepatoma in nude mice.Methods One hundred and twenty tumor-bearing nude mice injected subcutaneoushy by human hepatocellular cancer cell(SMCC-7721)were randomly divided into 4 groups,and 200μl of drug(or saline was injected into the tail vein one day before HIFU treatment respectively.Group A was injected by normal saline+HIFU irradiation,irradiation,group B was injected by HCPT+HIFU irradiation,group C was injected by targeted non-drug loaded phase change nanoparticles FR-PNPCA+HIFU irradiation,and group D was injected by targeted drug loaded phase change nanoparticles HCPT-PNPCA+HIFU irradiation.Immediately after irradiation,the grayscale changes in the tumor area were observed by ultrasound.2 h later,10 nude mice were killed in each group.TTC staining of tumors and HE staining of tumor sections were performed to observe coagulation necrosis of tumor cells respectively.The intratumor HCPT concentration was examined by tissue homogenate examination.48 h later,10 nude mice were killed in each group again.The tumor sections were stained with PCNA and TUNEL to observe the effect of treatment on tumor cell proliferation and apoptosis respectively.2 weeks later,the remaining nude mice were killed,the size of the tumor was measured,and the main organs were taken out to observe the tumor metastasis.Results There was no change in grayscale in the irradiated areas of groups A and B,and significant grayscale changes were found in groups C and D.The area and value of grayscale change before and after irradiation of groups C and D were statistically different from those in groups A and B(all P<0.05).After 2 h irradiation,HCPT can be detected in tumors of groups B and D,and the concentration of HCPT in tumors of group D was higher,which was statistically different from that of group B(P<0.05).Furthermore,no obvious coagulation necrosis was found in groups A and B,while obvious coagulative necrosis was found in the tumors of groups C and D,which were statistically different from that of groups A and B(all P<0.01).The tumor cell proliferation rate was the lowest and apoptosis rate was the highest in group D,the difference compared with A,B,C groups were statistically significant(all P<0.05).After irradiation 2 weeks,the tumor in group D almost disappeared,and the metastasis was the least,and the difference compared with A,B,C groups were statistically significant(all P<0.05).Conclusion Not only the targeted phase-change nanoparticles loading 10-HCPT combined with HIFU treatment can monitor the HIFU ablation of tumor in real time and enhance the HIFU ablation effect,but also can release the loading drug to assist the treatment of the tumor,which is expected to provide a precise and efficient tumor treatment method.