c(RGDyk)环肽修饰的脂质体包载量子点荧光成像脑胶质瘤引导手术精准切除效果评价

目的：构建c(RGDyk)环肽修饰的脂质体包载量子点，用于脑胶质瘤的精准成像，实现术中荧光成像引导的肿瘤精准切除。方法：以氢化大豆磷脂，二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氨基和胆固醇为材料，经逆向蒸发法，制备包载ZnCdSe/ZnS量子点（QDs）的脂质体（QDs-Lip）；经EDC/NHS介导的缩合反应，将c(RGDyk)环肽与QDs-Lip表面氨基键合，制备c(RGDyk)环肽修饰的量子点脂质体QDs-c(RGDyk)-Lip]。动态光散射（DLS）以及透射电镜（TEM）技术对QDs-c(RGDyk)-Lip进行了粒径与形态表征。同时，荧光光谱和DLS考察QDs-c(RGDyk)-Lip在生理相关介质中的荧光稳定性与粒径稳定性。体外MTT和细胞摄取实验评价QDs-c(RGDyk)-Lip对PC12 和C6 的细胞毒性及其对脑胶质瘤细胞的靶向性。原位脑胶质大鼠经尾静脉注射QDs-c(RGDyk)-Lip后，对脑胶质瘤激光照射，进行荧光成像并实施手术切除，HE染色确证手术切除精准度。。结果：DLS和TEM表明QDs有效包裹在脂质体内。DLS显示游离QDs粒径大小约为35 nm，而QDs-c(RGDyk)-Lip粒径增大至175 nm，显示为较窄分布特征（PDI=0.12）；TEM可见QDs-c(RGDyk)-Lip呈囊泡状，其中心可见数个微小QDs聚集。QDs-c(RGDyk)-Lip在激光照射下发射紫色荧光，最大发射波长610 nm。而且，QDsc(RGDyk)-Lip在生理介质中展现出较好的荧光稳定性和粒径稳定性。体外细胞毒性实验表明QDs-c(RGDyk)-Lip仅在高浓度对PC12或C6细胞具有微弱毒性。C6细胞能特异性摄取QDs-c(RGDyk)-Lip，进而发射强荧光。而且预先用游离RGDyk孵育封闭靶位，C6细胞对QDs-c(RGDyk)-Lip摄取被明显抑制，表明QDs-c(RGDyk)-Lip对脑胶质瘤特异靶向性。动物实验表明QDs-c(RGDyk)-Lip静脉注射后能高效靶向原位荷瘤大鼠脑胶质瘤，使其发射强荧光引导手术精准切除。结论：RGDyk环肽修饰的脂质体结合量子点构建QDs-c(RGDyk)-Lip，可以作为一种安全、稳定的荧光探针，能够特异靶向脑胶质瘤组织，引导手术精准切除。

The value of fluorescent imaging by c(RGDyk) modified liposome encapsulating quantum dots in the accurate delineation of glioma margin for the precise surgical resection of tumor

Objective: To prepare c(RGDyk) cyclic peptide modified liposome encapsulating quantumdots (QDs) and evaluate its ability to delineate accurately glioma margin by fluorescent imaging for the accurate urgical resection of tumor. Methods: The mixed lipid materials including hydrogenated soybean phospholipid/distearoyl phosphoethanolamine-PEG2000/cholesterol were used to encapsulate ZnCdSe/ZnS quantum dots(QDs) to prepare QDs-liposome (QDs-Lip) through the reverse evaporation method. Afterwards, c(RGDyk) cyclic peptide as a targeting ligand was conjugated to the amino group on the surface of QDs-Lip by EDC/NHS condensation reaction to obtain QDs-c(RGDyk)-Lip. Dynamic light scatting (DLS) and Transmission electron microscope (TEM) were used to characterize particle size and morphology of QDs-c(RGDyk)-Lip in vitro.Meanwhile, the stability of QDs-c(RGDyk)-Lip in physiologic relevant medium was also evaluated in terms of both fluorescent emission spectrum and particle size. In vitro CCK-8 assay was used to evaluate the toxic effect of QDs-c(RGDyk)-Lip on PC12 cells and C6 cells. Besides, the cellular uptake of QDs-c(RGDyk)-Lip by C6 cells was evaluated by laser scanning confocal microscopy in presence of free c(RGDyk) or not. Upon intravenous injection of QDs-c(RGDyk)-Lip, fluorescent imaging of brain of glioma-bearing rats was collected to evaluate its ability to accurately delineate the margin between tumor tissue and normal tissue and the accurate surgical resection of tumor was confirmed by HE staining. Results: The successful encapsulation of QDs was confirmed by TEM and DLS. The particle size of free QDs was about 35nm, but the particle size of QDs-c(RGDyk)-Lip increased to 175 nm with narrow particle distribution (PDI=0.12). Moreover, QDs-c(RGDyk)-Lip displayed the vesicle-like morphology with small aggregate of QDs in center. QDs-c(RGDyk)-Lip exhibited the maximum peak at 610 nm by the fluorescent emission spectrum. The good stability of QDs-c(RGDyk)-Lip in in physiologic relevant medium was confirmed by both fluorescent emission spectrum and particle size. In vitro cytotoxicity study showed that QDs-c(RGDyk)-Lip had the weak toxicity toward PC12 or C6 cells only at high concentration. QDs-c(RGDyk)-Lip was effectively up-taken by C6 cells, resulting in the strong fluorescence in their cytoplasm.Moreover, the fluorescence of QDs-c(RGDyk)-Lip in C6 cells was obviously inhibited by pre-incubating with free c(RGDyk), indicating that its specific targeting toward C6 cells. Animal experiments show that QDsc( RGDyk)-Lip can effectively target the glioma-bearing rats after intravenous injection for the accurate surgical resection of tumor. Conclusion: c(RGDyk) cyclic peptide modified liposome in combination with quantum dots constructs QDs-c(RGDyk)-Lip as a safe and stable fluorescent probe, which can specifically target glioma tissue and accurately guide surgical resection.