纳米级液态氟碳靶向超声造影剂的制备及 体外寻靶的实验研究

目的制备一种新型的具备特异性肝细胞靶向性能的超声造影剂,即携半乳糖化多聚赖氨酸(Gal-PLL)的脂质包裹的纳米级液态氟碳微球超声造影剂,并研究其在体外肝细胞的寻靶能力。方法应用还原胺化法优化制备肝脏去唾液酸糖蛋白受体的配体Gal-PLL,将其磷脂膜洗脱下来并逐滴加入液态氟碳,通过超声波细胞粉碎机进行振荡获得目标造影剂,并观察其形态,检测其粒径和电位。将靶向造影剂和非靶向造影剂分别滴入贴壁生长的正常大鼠肝细胞及正常人肝细胞中,比较观察两组造影剂与肝细胞的靶向效果。结果成功制备Gal-PLL,并合成了靶向纳米液态氟碳脂质微球超声造影剂,电镜下其形态圆整、大小均一,性质稳定,平均粒径200 nm,平均电位35 m V。制备的靶向纳米液态氟碳脂质微球超声造影剂能靶向聚集于体外正常大鼠肝细胞和正常人肝细胞周围。结论自制携Gal-PLL的脂质包裹的纳米级液态氟碳微球超声造影剂具有性质稳定、靶向性等优点,为今后实时在体监测并定量评估肝脏损伤程度的超声显影研究提供了前期基础。     

LHRHa靶向微泡造影剂的制备及体外寻靶实验

目的 制备人卵巢癌靶向超声造影剂,观察其体外寻靶能力。方法 采用生物素-链霉亲和素法制备促黄体生成素释放激素类似物(LHRHa)靶向脂质微泡,以免疫荧光染色验证LHRHa与脂质微泡的结合情况,并以普通脂质微泡作为对照,在倒置显微镜下观察LHRHa靶向脂质微泡对人卵巢癌A2780/DDP的体外寻靶能力。结果 LHRHa靶向脂质微泡免疫荧光阳性;体外寻靶实验显示LHRHa靶向脂质微泡能够与表面存在LHRH受体的A2780/DDP细胞特异性结合,而普通脂质微泡则不能结合。结论 利用生物素-链霉亲和素方法可成功制备LHRHa靶向脂质微泡造影剂,该靶向造影剂具有体外寻靶能力。     

以HER2为靶点的靶向纳米级脂质微泡超声造影剂的制备及实验研究

目的 制备以HER2受体为靶点的靶向纳米级脂质微泡超声造影剂,并观察其体外寻靶能力及体外超声显像效果.方法 制备生物素化Herceptin单抗,检测其生物素化程度及生物学活性;薄膜水化-声振法制备生物素化纳米微泡,以生物素-亲和素为桥梁制备HER2为靶点的靶向纳米级脂质微泡超声造影剂,观察其对SKOV3卵巢癌细胞的体外寻靶能力及靶向结合的体外超声显像.结果 平均每分子Herceptin单抗可与16个生物素分子结合;与游离单抗相比,生物素化抗体的活性未见明显降低(P＞0.05).体外寻靶实验观察:靶向纳米微泡组SKOV3细胞表面有明显的红染纳米微泡与其牢固结合,沿细胞表面排列较规则;非靶向组SKOV3细胞表面未结合红染的纳米微泡.靶向纳米微泡体外超声显像:细胞爬片与靶向纳米微泡孵育后可明显增强超声显像效果,对照组均未见明显超声显像.结论 应用生物素-亲和素系统成功构建了以HER2为靶点的纳米级超声造影剂,与SKOV3细胞结合后有明显超声显像效果.     

靶向纳米脂质超声造影剂制备及其体外寻靶能力实验研究

目的 制备一种靶向乳腺癌的纳米脂质超声造影剂,并观察其体外寻靶能力.方法 制备生物素化抗人乳腺癌细胞单克隆抗体Neu(F-11),并检测其生物素化程度和活性;通过生物素-亲和素系统制备靶向纳米脂质超声造影剂,用免疫荧光染色定性检测造影剂与抗体的连接情况,通过靶向造影剂与不同细胞的结合实验检测其寻靶能力.结果 每分子抗体可结合的生物素数目平均为7个,生物素化抗体的活性与游离单抗相比未见明显降低;免疫荧光染色显示靶向纳米脂质超声造影剂表面可见明亮的红色环状荧光,细胞结合实验显示其可与乳腺癌细胞特异性牢固结合.结论 生物素-亲和素系统可使造影剂与抗体牢固结合,所制得的靶向纳米脂质超声造影剂在体外具有较强的寻靶能力.     

载ICAM-1抗体的靶向脂质纳米超声造影剂体外寻靶实验研究

目的 制备一种载细胞间黏附因子-1 (ICAM-1)单克隆抗体的脂质纳米超声造影剂,研究其体外寻靶能力.方法 采用机械振荡法制得普通脂质纳米超声造影剂和生物素化的脂质纳米超声造影剂,利用生物素-亲和素桥接法将ICAM-1抗体连接于脂质纳米超声造影剂上,制成靶向造影剂,检测两种造影剂的物理性质.将上述两种造影剂分别作用于普通HepG2细胞和经TNF-α处理的HepG2细胞,实时荧光定量PCR检测HepG2细胞上ICAM-1荧光表达强度,荧光显微镜观察造影剂与HepG2细胞靶向结合的情况.结果 普通造影剂粒径为(623.2±91.37) nm;靶向造影剂粒径为(760.6±145.0) nm.经TNF α刺激后HepG2细胞ICAM-1基因的表达量明显增高.普通造影剂组均未见造影剂与HepG2结合,而靶向造影剂组可见大量的造影剂聚集在经TNF-α刺激后的HepG2细胞周围.结论 本研究自制的载ICAM-1抗体的靶向脂质纳米超声造影剂,其粒径小,性质稳定,在体外能与高表达ICAM-1的HepG2细胞特异结合,可望成为一种良好的肝癌靶向造影剂.     

载紫杉醇的LHRH靶向相变型超声造影剂的制备及其一般特性

目的制备一种载紫杉醇的靶向相变型纳米级超声造影剂,并评价其一般特性。方法通过薄膜水化法、乳化法制备载紫杉醇的非靶向脂质纳米粒(PTX-LNP),通过生物素-亲和素法将生物素化的促黄体生成素释放激素(LHRH)连接于非靶向脂质微球制备载紫杉醇的LHRH靶向相变型脂质纳米粒(PTX-LNP-LHRH),检测PTX-LNP-LHRH的粒径以及表面电位,载药量及包封率,分别观察其声致相变及热致相变情况,检测纳米粒的LHRH靶连接率及其致人卵巢癌OVCAR3细胞的凋亡率。结果成功制备靶向相变型脂质纳米粒,LHRH的靶连接率为(98.85±0.75)%,共聚焦显微镜下可见PTX-LNP-LHRH聚集在OVCAR3细胞周围,PTX-LNP-LHRH组致OVCAR3细胞的凋亡率明显高于PTX-LNP组。结论成功制备靶连接率高且致靶细胞凋亡率较高的PTX-LNP-LHRH,其体外声致相变后可增强超声显影的效果。     

载10-羟基喜树碱靶向脂质微泡的制备及体外寻靶能力实验研究

目的 制备携人肝癌单抗Hab18载10-羟基喜树碱的脂质微泡,并观察其体外寻靶能力.方法 制备生物素化抗人肝癌Hab18单克隆抗体,检测其生物素化程度;用机械振荡法制备载药脂质微泡,以生物素-亲和素桥连方式构建携人肝癌单抗Hab18载10-羟基喜树碱的脂质超声微泡,检测其一般特性、包封率和载药量,以免疫荧光法检测微泡与抗体的连接情况,在光镜下观察靶向载药微泡的寻靶能力,并与载药非靶向微泡进行比较.结果 每个单抗分子平均可与13个生物素分子结合.载药靶向脂质超声微泡分布均匀,平均粒径为1.52 μm,包封率为76.32%,载药量为21.81%.免疫荧光法显示微泡表面可见明亮的红色环状荧光,体外寻靶实验显示该载药靶向微泡可与人肝癌7721细胞牢固结合.结论 携人肝癌单抗Hab18载10-羟基喜树碱的脂质超声载药靶向微泡可成功制备,其包封率和载药量较高,具有较强的体外寻靶能力.     

叶酸受体靶向液态氟碳纳米粒造影剂的制备及体外寻靶

目的 制备叶酸受体靶向的被脂质包裹的液态氟碳纳米粒造影剂,鉴定其基本性质,观察其体外靶向性能.方法 以氯仿将偶联叶酸的磷脂[DSPE-PEG(2000)]按照一定比例溶解在成膜材料中,用旋转蒸发仪去除有机溶剂成膜,加入磷酸盐缓冲液重新水合后,在悬液中逐滴加入适量液态氟碳,均质乳化后得到叶酸受体靶向的纳米粒造影剂.在人卵巢癌SKOV3细胞中验证该造影剂的靶向性能(靶向造影剂组),并与不带叶酸的普通造影剂组和游离叶酸干预组相比较.结果 叶酸受体靶向液态氟碳纳米粒造影剂外观与普通造影剂无明显差别.体外靶向实验发现此纳米粒造影剂聚集且牢固地黏附于SKOV3细胞,而普通造影剂组和游离叶酸干预组则未见明显特异性结合.结论 成功制备了偶联叶酸的被脂质包裹的液态氟碳纳米粒造影剂,该造影剂对高表达叶酸受体的SKOV3细胞具有较强的亲和力,有望成为卵巢癌靶向显影与治疗的理想分子探针.     

携RGD肽的靶向相变光声/超声双模态高分子造影剂的制备及体外实验研究

目的制备一种携RGD肽的靶向相变型光声/超声双模态造影剂(RNP),并观察其体外光致相变以及细胞靶向效果。方法制备包裹ICG及液态氟碳(PFH)的RGD肽靶向高分子微球,在荧光显微镜下观察RGD肽与微球的结合情况,流式细胞术量化其结合率,脉冲激光辐照观察微球相变情况,用乳腺癌MD-MB231细胞验证其体外细胞靶向能力。结果制备的RNP平均粒径为(627±28.5)nm,微球与RGD肽结合率为98.9%,脉冲激光辐照后微球发生液气相变。体外靶向实验结果显示,与对照组相比,更多的靶向微球聚集在MD-MB231细胞的周围。结论成功制备出携RGD肽的靶向相变型光声/超声双模态造影剂,该造影剂对高表达整合素的乳腺癌MD-MB231细胞具有较高的靶向结合能力。     

平行板流动腔法评价生物素化脂质微泡制备效果

目的 制备生物素化脂质微泡,并应用平行板流动腔检测流体切应力对生物素化脂质微泡与链亲和素结合稳定性的影响,为进一步开展超声分子成像研究奠定基础.方法 采用声振法制备出表面携有生物素分子的脂质微泡,与普通脂质微泡对照,应用平行板流动腔模型,设置不同流体剪切应力,观察与链亲和素靶向结合的生物素化微泡的黏附效果.结果 在不同浓度链亲和素包被的平行板流动腔中均见生物素化微泡结合;随着链亲和素包被浓度的提高,靶向结合的生物素化脂质微泡抗流体剪切应力能力明显提高.结论 应用平行板流动腔模型能成功检测生物素化微泡的靶向黏附效果,该模型可推广应用于其他靶向微泡制备成功后靶向黏附能力的体外检测.     

去唾液酸糖蛋白受体介导的肝靶向纳米脂质超声造影剂的制备及体外实验

目的 以肝细胞膜特异性受体去唾液酸糖蛋白受体为靶向受体,利用共价结合的原理,制备出肝靶向性液态氟碳纳米超声造影剂,观察该造影剂的一般特性、与人肝细胞L02的靶向结合及体外聚集显像效果.方法 利用还原胺法制备去唾液酸糖蛋白特异性配体半乳糖化多聚赖氨酸(Gal-PLL);利用旋转蒸发及声振法制备液态氟碳纳米脂质超声造影剂;培养人肝细胞L02,于不同时间点观察与L02细胞的靶向结合;Philips iU 22超声诊断仪L12-5探头对比观察靶向液态氟碳纳米脂质超声造影剂的体外显像效果.结果 靶向液态氟碳纳米脂质超声造影剂粒径极小,分布均匀,形态呈圆球形,且能与L02有效结合;体外乳胶囊显示:脱气水侧呈现无回声,靶向液态氟碳纳米脂质超声造影剂侧呈现高回声.结论 以去唾液酸糖蛋白受体为靶向受体,自制的携半乳糖化多聚赖氨酸的靶向液态氟碳纳米脂质超声造影剂能有效与人肝细胞LO2靶向结合,该靶向超声造影剂能在体外聚集显影.该靶向超声造影剂粒径极小,是细胞水平上肝靶向性超声分子显像的一种理想的超声分子探针.

Abstract：

Objective To prepare the liver targeting nano-liquid perfluorocarbon ultrasound contrast agent and observe its general characteristics;to observe the targeting combined effects of the human liver cells L02 and the targeted ultrasound contrast agent ;to evaluate the gathering imaging effects of the targeted ultrasound contrast agent. Methods Amine method was used to prepared asialoglycoprotein Gal specific ligand polylysine (Gal-PLL), rotary evaporator and sonicated liquid fluorocarbon were used to prepare nano lipid ultrasound contrast agent. Human liver cell L02 were cultured, the combined effects were observed according to the reacting time of the cells and the targeted nano-lipid ultrasound contrast agent. The nanolipid ultrasound contrast agent and the degassed_ water_ were loaded into cysts and their ultrasound imaging effects were observed by ultrasound diagnostic apparatus Philips iU22. Results The particle size of the liquid fluorocarbon nano-targeted lipid ultrasound contrast agent was extremely small, uniform, cylindrical and spherical. The cysts in vitro showed that the side of the targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent showed high echo. Conclusions The targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent can be effectively targeted to the human liver cells L02 due to carrying home-made Gal-PLL. The targeted ultrasound contrast agents can be imaging by ultrasound and be confirmed in vitro.The size of the contrast agent was small, therefore, it can be considered an ideal ultrasonic molecular probe and achieve the ultrasound molecular imaging in cell level.     

携载PSMA单抗靶向前列腺癌的纳米级脂质微泡的制备及体外寻靶能力

目的制备携载PSMA单抗靶向人前列腺癌的纳米级脂质微泡,并观察其体外寻靶能力。方法利用静电吸附法制备携载PSMA单抗靶向前列腺癌的脂质纳米级微泡,检测其一般特性;免疫荧光法检测抗体与纳米级微泡的结合情况;用细胞免疫荧光分析鉴定PSMA的表达及其在细胞膜上的定位;普通光镜下观察靶向微泡对前列腺癌细胞的寻靶能力,同时以胃癌细胞作对照。结果携载PSMA单抗的靶向纳米级微泡分布均匀,平均粒径为(623.70±66.05)nm,免疫荧光法显示微泡表面可见绿色荧光,细胞免疫荧光检测前列腺癌细胞膜高表达PSMA,体外寻靶实验显示该靶向微泡可与人前列腺癌LNCAP及C4-2细胞牢固结合,而与胃癌MKN45细胞不结合。结论本实验成功制备的携载PSMA单抗靶向人前列腺癌的纳米级脂质微泡具有较强的体外寻靶能力,能特异性地与前列腺癌细胞结合。     

抗VEGF抗体介导靶向超声造影剂的体外实验研究

目的以抗血管内皮因子（VEGF）抗体为配体研制能与血管内皮细胞特异性结合的靶向脂质体超声造影剂并检测其体外寻靶能力。 方法以静电吸附法将抗VEGF抗体连接到脂质体造影剂微泡的表面；体外培养ECV304人血管内皮细胞，用免疫荧光法检测靶向造影剂与其体外结合能力，以普通造影剂为对照组。 结果所制备的靶向超声造影剂与普通微泡无显著差异；免疫荧光实验结果显示靶向造影剂能在体外与血管内皮细胞特异性结合。 结论携抗VEGF抗体的脂质体靶向造影剂能通过静电吸附法成功制备，且在体外能与血管内皮细胞特异性结合。     

Metabolic biotinylation provides a unique platform for the purification and targeting of multiple AAV vector serotypes.

The development of rationally designed targeted gene delivery vectors is an important focus for gene therapy. While genetic modification of AAV can produce vectors with modified tropism, incorporation of targeting peptides into the structural context of the AAV virion often results in loss of function or loss of virion integrity. To address this issue, we have developed a targeting system using metabolically biotinylated AAV. We generated serotype 1, 2, 3, 4, and 5 AAV capsids with small peptide insertions that are metabolically biotinylated in packaging cells during vector production by coexpression of the Escherichia coli BirA, biotin ligase, gene. Biotin moieties are exposed on the surface of assembled AAV particles and can interact with avidin. Metabolically biotinylated AAV vectors produced in this manner maintained endogenous titer and tissue tropism, could be purified on monomeric avidin resin, and could be retargeted to cells engineered to express an artificial avidin-biotin receptor. This technology provides not only a single platform for the purification of multiple AAV vector serotypes, but also a means for the development of multiple targeted AAV vectors utilizing a single capsid modification via straightforward avidin-biotin ligand coupling.     

靶向BST2微泡造影剂的制备及其与肿瘤细胞的体外结合能力

目的 制备骨髓基质抗原蛋白(BST2)靶向微泡造影剂,观察其与小鼠前列腺癌细胞(RM-1)和小鼠乳腺癌细胞(4T1)的体外结合能力,探讨BST2作为前列腺癌潜在靶点的可行性.方法 通过免疫荧光染色和蛋白质印迹法对BST2在两种细胞中的表达进行对比分析.采用生物素-亲和素桥连技术制备BST2靶向脂质微泡,普通光镜下观察BST2靶向微泡造影剂,并采用Accu Sizer 780A粒度仪进行表征,以非靶向微泡作为对照,比较其与RM-1和4T1两种肿瘤细胞系的结合特性及结合率.结果 BST2在RM-1细胞中的表达高于在4T1细胞中的表达;BST2靶向微泡与RM-1细胞的黏附率明显高于其与4T1细胞的黏附率,并远远高于非靶向微泡的黏附率.结论 BST2靶向微泡造影剂可与RM-1细胞特异性结合,有望作为前列腺癌的特异性超声分子探针用于前列腺癌的靶向分子成像.     

Sonic activation of molecularly-targeted nanoparticles accelerates transmembrane lipid delivery to cancer cells through contact-mediated mechanisms: implications for enhanced local drug delivery

Liquid perfluorocarbon nanoparticles serve as sensitive and specific targeted contrast and drug delivery vehicles by binding to specific cell surface markers. We hypothesized that application of acoustic energy at diagnostic power levels could promote nanoparticle-associated drug delivery by stimulating increased interaction between the nanoparticle's lipid layer and the targeted cell's plasma membrane. Ultrasound (mechanical index = 1.9) applied with a conventional ultrasound imaging system to nanoparticles targeted to alpha(v)beta3-integrins on C32 melanoma cancer cells in vitro produced no untoward effects. Within 5 min, lipid delivery from nanoparticles into cell cytoplasm was dramatically augmented. We also demonstrate the operation of a potential physical mechanism for this effect, the acoustic radiation force on the nanoparticles, which may contribute to the enhanced lipid delivery. Accordingly, we propose that local delivery of lipophilic substances (e.g., drugs) from targeted nanoparticles directly into cell cytoplasm can be augmented rapidly and safely with conventional ultrasound imaging devices through nondestructive mechanisms.     

Interaction between heat shock protein 72 and alpha-fetoprotein in human hepatocellular carcinomas

BACKGROUND: AFP in adult serum often signals pathological conditions, particularly the presence of hepatocellular carcinoma (HCC) and germ cell tumors containing yolk sac cell elements. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to overexpress in epithelial carcinoma cells. There may be a possible correlation between the expression of HSP72 and AFP during the growth and differentiation of hepatocellular carcinoma cells. We investigated the interaction between heat shock protein 72 (HSP72) and alpha-fetoprotein (AFP) in human hepatocellular carcinomas. METHODS: The expression and localization of HSP72 and AFP in human hepatocellular carcinomas were determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and AFP in hepatocellular carcinoma cells was analyzed by immunoprecipitation and Western immunoblots. RESULTS: Hepatocellular carcinoma synchronously co-expressed higher level of HSP72 and AFP than in adjacent normal liver tissues. HSP72 were stained in cell nuclei and cytoplasm respectively, while AFP stained in cell plasma. Based on Western blotting methods, AFP was detected in the immunoprecipitate of anti-HSP72 monoclonal antibody (mAb), while HSP72 existed in the immunoprecipitate of anti-AFP mAb. CONCLUSIONS: HSP72 and AFP expression are higher in hepatocellular carcinoma tissues. HSP72 was associated with alpha-fetoprotein in human hepatocellular carcinoma cells. The interaction between HSP72 and AFP in human hepatocellular carcinoma cells can be a new route for studying the pathogenesis and immunotherapy of hepatocellular carcinoma.     

结合链霉亲和素的高分子造影剂的制备及其初步应用

目的 制备一种结合链霉亲和素的高分子超声造影剂,并与生物素化抗体结合,考察其体外寻靶能力.方法 采用双乳化法制备高分子材料PLGA-COOH超声造影剂;并用碳二亚胺法将造影剂与亲和素耦联,制备出结合链霉亲和素的高分子超声造影剂.检测该造影剂一般特性;采用红外与免疫荧光检测亲和素与PLGA-COOH超声造影剂连接的情况.检测生物素-亲和素技术使该造影剂与生物素化抗体结合后其体外寻靶能力.结果 红外检测与免疫荧光检测显示亲和素被成功地连接在高分子造影剂上,并存在活性.体外寻靶实验显示,与生物素化单抗结合后的靶向高分子造影剂较多并牢固地聚集到肝癌细胞表面.结论 成功制备了结合链霉亲和素的高分子超声造影剂,该造影剂与生物素化抗体结合后于体外对肝癌细胞具有较强的亲和力.