含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究

Study on the Delivery of RGD Modified Virus-Like Particles to ICG Targeted Tumors

目的:获取含RGD靶向肽的乙肝核心病毒样颗粒,为药物靶向纳米递送系统提供一种新型载体。方法:将实验室前期构建测序正确的含RGD修饰的乙肝核心病毒重组质粒转化入大肠杆菌BL21(DE3)中,单因素分析及正交试验探究重组蛋白最适表达条件。在最适表达条件下扩培,收集菌体超声破碎后离心,采用凝胶过滤层析、离子交换和蔗糖密度梯度离心进行纯化,利用透射电镜对形成的RGD-HBc VLPs的形态及稳定性进行鉴定。纯化的RGD-HBc VLPs利用其体外自组装的特性,将光敏剂ICG装载到颗粒的内部,通过静脉注射到4T1乳腺癌荷瘤小鼠,探究重组RGD-HBc VLPs作为纳米递送系统的靶向性。结果:RGD-HBc VLPs在温度32℃、IPTG0.5mmol/L、诱导4h时以可溶性蛋白的形式得到高效表达。经蔗糖密度梯度离心纯化后纯度到达95%以上。透射电镜下观察纯化的RGD-HBc VLPs形态、大小均一,直径约为32nm,通过近红外荧光活体成像证实了RGD-HBc作为纳米载体的靶向性。结论:经表达和纯化后,RGD-HBc VLPs具有较高的表达量和大小均一的形态外貌,近红外荧光活体成像证实具有较好的靶向性,这不仅为肿瘤的可视化诊断提供一种快速、精准、方便的方法,而且为今后靶向免疫治疗提供一种新型载体。

Objective:To obtain hepatitis B core virus-like particles containing RGD targeting peptide and provide a new carrier for drug targeting nanometer delivery system.Methods:RGD-modified recombinant plasmid of hepatitis B virus was transformed into E.coli BL21(DE3),and the optimal expression conditions of recombinant protein were investigated by single factor analysis and orthogonal test.Under the optimum expression conditions,the bacteria were cultured,collected after ultrasonic crushing,centrifuged,purified by Gel filtration chromatography,ion exchange and sucrose density gradient centrifugation,and the morphology and stability of RGD-HBc VLPs were identified by transmission electron microscopy.The purified RGD-HBc VLPs loaded the photosensitizer ICG into the inner part of the particle and injected it intravenously into 4T1 tumor-bearing mice of breast cancer to explore the targeting of recombinant RGD-HBc VLPs as a nano-delivery system.Results:RGDHBc VLPs was efficiently expressed in the form of soluble protein at 32℃and IPTG at 0.5 mmol/L for 4 h.After centrifugation with sucrose density gradient,the purity reached above 95%.The purified RGD-HBc VLPs were observed under transmission electron microscopy in uniform shape and size,with a diameter of about 32 nm.Near-infrared fluorescence in vivo imaging confirmed the targeting of RGD-HBc VLPs as a nano-carrier.Conclusion:After expression and purification,RGD-HBc VLPs has a high expression level and uniform appearance,and near-infrared fluorescence in vivo imaging has a good targeting property,which not only provides a fast,accurate and convenient method for the visual diagnosis of tumor,but also provides a new carrier for future targeted immunotherapy.