基于Endoglin靶大鼠胶质瘤的磁共振分子影像学实验研究

【作者】 邱龙华；

【导师】 冯晓源；

【作者基本信息】 复旦大学 ， 影像医学与核医学， 2011， 博士

【摘要】 目的：合成制备基于Endoglin靶的空间稳定脂质体磁共振分子探针并评价其理化性质；各种对比剂大鼠胶质瘤磁共振分子影像学研究,验证探针特异性显示胶质瘤形态及边界的有效性。材料与方法：一)空间稳定脂质体的制备：(1)脂质体膜材料,二硬脂酰磷脂酰胆碱(DSPC).胆固醇(CHOL).甲氧基聚乙二醇-二硬脂酰乙醇胺(mPEG-DSPE).钆-二乙三胺五醋酸-二硬脂酰胺(Gd-DTPA-BSA)按36：36：3：25摩尔比例溶于氯仿甲醇混合溶剂(2：1容积)中,经40℃旋转蒸发,60℃水化,高压均质仪挤压过膜,制备包载顺磁性物质(Gd)的空间稳定脂质体(Gd-SLs);(2)评价脂质体的粒径、稳定性、钆含量、磁共振驰豫率及体外MR成像作用等性质。二)基于Endoglin靶的空间稳定免疫脂质体及预定位脂质体分子探针的制备：(1)Gd-SLs成膜过程中,加入膜材料吡啶二硫丙酰基-聚乙二醇(2000)-二硬脂酰乙醇胺(PDP-PEG(2000)-DSPE),制备表面修饰结合PDP基团脂质体(PDP-SLs), PDP-SLs脂质体经二硫苏糖醇(DTT)还原,得表面结合游离巯基的脂质体(HS-SLs):(2)CD105单克隆抗体(CD105～MAb)与丁二酰亚胺-4-(对-马来酰亚胺-苯基)-丁酸酯(SMPB)反应制备马来酰亚胺苯基丁酰基单克隆抗体(MPB-MAb);(3)将HS-SLs与MPB-MAb反应获得的表面连接抗体的空间稳定脂质体(MAb-SLs);(4)生物素氨基己糖酸-3-磺酸基-N-羟基琥珀酰亚胺酯(Sulfo-NHS-LC-biotin)与CD105-MAb反应可得生物素化单克隆抗体(Bio-MAb);(5)链霉亲和素结合空间稳定脂质体(SAv-SLs)的制备过程与MAb-SLs基本相同,将HS-SLs与马来酰亚胺苯基丁酰基链霉亲和素(MPB-SAv)作用,可得SAv-SLs;(6)测定免疫脂质体抗体蛋白密度及抗体结合率,各种脂质体探针透射电镜观察形态。三)大鼠胶质瘤新生血管内皮细胞Endoglin靶的MR分子成像：(1)大鼠C6胶质瘤细胞培养及胶质瘤动物模型的建立；(2)25只模型动物随机分为A-E共5组,每组5只,分别尾静脉注射钆喷酸葡胺(Gd-DTPA).空白脂质体(Gd-SLs).同种型对照IgG脂质体(IgG-SLs).结合Endoglin单克隆抗体的免疫脂质体探针(MAb-SLs)行MR成像,两步法预定位成像大鼠预先给予生物素化单克隆抗体(Bio-MAb)后再给予链霉亲和素脂质体探针(SAv- SLs)进行成像；(3)比较各组大鼠MR T1加权增强图像肿瘤强化农现的差异,分析各组动脉血管、正常脑组织及肌组织、肿瘤组织时间-信号增强特点；(4)比较各组间肿瘤强化程度及强化范围差异、比较组内肿瘤中央部及周边部强化差异；(5)比较各组间肿瘤强化形态的差异；(6)大鼠胶质瘤CD105免疫组织化学染色肿瘤中央部及周边部新生血管计数,比较两者差异。结果：一)所制备之钆结合空间稳定脂质体(Gd-SLs)经高压均质仪过膜后粒径控制于117.4±31.8nm；脂质体Gd载药率达87%～100%；常温下(26℃)Gd-SLs磁共振驰豫率为Gd-DTPA的1.16倍,37℃为1.25倍；体外MR成像显示Gd-SLs与Gd-DTPA两对比剂MR T1加权成像作用接近。二)空间稳定脂质体结合单克隆抗体后粒径由116.1nm±33.9nm增加至129.9nm±40.9nm,于4℃保存7、14、42天,平均粒径及分布变化较小；将Bio-MAb与SAv-SLs混合后,平均粒径显著增大(267nm±142.8nm),分布增宽；免疫脂质体抗体结合率为52～67%,相应抗体/磷脂比例约为47～60μg/μmol；透射电镜显示,脂质体为均匀大小囊泡样结构,将Bio-MAb与SAv-SLs混合后,SAv-SLs有相互聚集作用。三)荷瘤大鼠MR成像(1)动脉增强表现：注射Gd-DTPA后,动脉血液快速明显强化,即刻达峰值,2小时基本消退,时间-信号曲线呈快进-快出类型；B～E组大鼠注射相应分子探针后,血管强化表现接近,早期快速明显强化,强化峰值于20～60min出现,之后强化信号缓慢下降,于48小时恢复至增强前水平,其时间-信号曲线表现为快进-平台-缓出类型。(2)肿瘤强化表现：注射Gd-DTPA后,肿瘤迅速强化达峰值,20min强化即有减退,2小时强化信号约为峰值强度30%,24小时完全退出；注射Gd-SLs及IgG-SLs,肿瘤早期强化不明显,60～120min达峰值呈轻度强化,48小时强化信号为峰值强度42～58%；注射MAb-SLs及Bio-MAb/SAv-SLs两步法成像,肿瘤周边及中央信号迅速增高,于8小时达峰值,后缓慢下降,注射MAb-SLs后肿瘤中央周边强化程度相仿,而两步法成像周边强化明显高于中央部(P=0.032)。(3)肿瘤强化形态分析显示,注射Gd-DTPA整个肿瘤不均匀强化,Gd-SLs及IgG-SLs组以肿瘤边缘点状强化为主,MAb-SLs及Bio-MAb/SAv-SLs两步法成像肿瘤增强以边缘环形强化为主。免疫组织化学染色显示,CD105阳性微血管主要沿肿瘤周边分布。结论：(1)顺磁性空间稳定脂质体能有效提高磁共振分子探针体内成像信号,制备方法简单,实验操作可控性好,在一定温度范围内性质稳定,具有较高的磁共振弛豫作用,是理想的MR分子影像学对比剂载体。(2)免疫脂质体结合抗体方法简单,效果确切可靠,体外相关实验能够验证生物素-亲和素介导的两步法成像的有效性。(3)大鼠胶质瘤MR分子成像,临床用Gd-DTPA肿瘤强化周边及中央强度无差异,非特异性脂质体探针肿瘤强化程度低,肿瘤边界强化形态不完整,因此均不能用于肿瘤边界显示。MAb-SLs及Bio-MAb/SAv-SLs两步法成像显示肿瘤边界较明确,两步法成像强化程度更高,实验证明为特异性靶向成像作用,因此可以用来进行肿瘤边界分析。更多还原

【Abstract】 Objectives:The objectives of this study was (1) to develop paramagnetic sterical stabilized liposomes conjugated with monoclonal antibodies targeted to Endoglin on neovascular endothelial cells and evaluate their physical characteristics. (2) to quantify and compare the magnitude of signal intensity enhancement on a rat glioma model applying different contrast agent or molecular tracer using a clinical MRI (3.0 Tesla) scanner. (3) to demonstrate the use of targeted paramagnetic nanoparticles to delineate the glioma margins.Materials and Methods:1. Preparation of paramagnetic sterical stabilized liposomes:(1) A mixture of the appropriate amounts of lipids (DSPC/CHOL/mPEG-DSPE/Gd-DTPA-BSA at a molar ratio of 36/36/3/25) was dissolved in chloroform/methanol 2/1 (v/v) and evaporated to dryness by rotary evaporation at 40℃. The lipid film was subsequently hydrated in HEPES-buffered saline at 60℃. The resulting lipid dispersion was extruded sequentially 10 times through polycarbonate membrane filters with a pore diameter of 400,200, and 100 using a high-pressure homogenizer. The temperature during extrusion was 60℃. The paramagnetic sterical stabilized liposomes were obtained. (2) The size, stability, Gd containing, magnetic resonance relaxivity of the liposomes was evaluated in vitro.2. Coupling the antibodies to liposomes and synthesizing of pretargeting reagents. (1) For monoclonal antibodies conjugated liposomes, PDP-PEG-DSPE were added to the lipid during the preparation of liposomes (at a molar ratio of mPEG-DSPE/PDP-PEG-DSPE=4/1). The pyridyldithio groups on the PDP-PEG-DSPE incorporated liposomes were reduced by adding DTT and the thiolated liposomes (HS-SLs) were obtained. (2) CD105 monoclonal antibodies reacted with SMPB to prepare the MPB-MAb (maleimidophenylbutyrate-MAb). (3) Thiolated liposomes were incubated with MPB-MAb. The antibodies conjugated immunoliposomes (MAb-SLs) were obtained. (4) Biotinylated monoclonal antibodies were synthesized by added Sulfo-NHS-LC-biotin to the antibodies. (5) The preparation of Streptavidin coupling liposomes (SAv-SLs) was similar to that of MAb-SLs. HS-SLs were incubated with MPB-SAv. Gel filtration or centrifugation was used to purify the product in these above-mentioned reactions. (6) The antibodies protein to lipid ratio which denoted the density of antibodies conjunct to the liposomes and antibodies coupling efficiency were evaluated. The morphology of liposomes was studied by Transmission electron microscopy (TEM).3. MR imaging in vivo. (1) 27 male Sprague-Dawley rats were inoculated with 1.5×106 C6 glioma cells in the right caudate nucleus to develop animal model. (2)25 tumor-bearing rats were classified into five groups randomly (5 rates pre group). Gd-DTPA, Gd-SLs, IgG-SLs, MAb-SLs were injected through tail veins respectively in corresponding groups. For two steps pretargeting group, Bio-MAb were administrated 24 hours before SAv-SLs contrast enhancement. (3) MR T1-weighted images of each group were compared to evaluate the enhancement features, the signal enhancement time-intensity curves of arteries, contralateral normal brain tissues, muscular tissues and tumor tissues were analyzed. (4)The degree of contrast enhancement and notable enhancement regions were compared between each group. The degree of enhancement of the tumor central and periphery were also compared for each tumor. (5) The enhancement morphology of the tumors was analyzed. (6) CD 105 immunohistochemistry was performed after MR imaging to detect the difference of microvessel density of the tumor central and periphery.Results:1. Features of Gd-SLs. The size of Gd-SLs was 117.4±31.8nm after homogenized. The incorporation efficiency of Gd-DTPA-BSA was 87%-100%. At 26℃(room temperature), the T1 relaxivity of Gd-SLs was 1.16 times that of the solution with free Gd-DTPA. It was 1.25 times at 37℃. MR T1 imaging of Gd-SLs in vitro presented similar enhancement effect to that of Gd-DTPA. 2. Characterization of immunoliposomes. The mean size of immunoliposomes after antibodies coupling increased from 116.1nm±33.9nm (Gd-SLs) to 129.9nm±40.9nm (MAb-SLs) and the polydispersity index showed little increase. Hypothermia (4℃) showed little influence on the size of the immunoliposomes. The mean size of SAv-SLs increased dramatically to 267nm after incubated with Bio-MAb with a polydispersity index of 0.286. The antibodies coupling efficiency ranged from 52% to 67% and corresponding antibodies protein to lipid ratio was 47～60μg/μmol. Transmission electron microscopy revealed spherical structures of liposomes with homogeneous size. SAv-SLs aggregated after mixed with Bio-MAb.MR imaging and analysis. (1) Arterials enhancement:Cervical arterial showed instantly significantly enhancement after Gd-DTPA was given and declined dramatically after that. The signal intensity returned to precontrast level at 2-hr time point. Thus, time-intensity curve represented as quick washin-early washout pattern. The features of arterials enhancement after liposomes tracers (Gd-SLs. IgG-SLs. MAb-SLs, Bio-MAb/SAv-SLs) injection were similar. Notable contrast enhancement was detected soon after injection and continued to improve through 20 min. It decreased slowly and washout absolutely at 48-hr time point. The time-intensity curve was described as quick washin-platform-slow washout pattern. (2) Characteristics of tumor enhancement:The signal intensity of the tumor peaked soon after the Gd-DTPA injection and sharply decreased subsequently. The intensity was about 30% that of the peak at 2-hr time point and return to the precontrast level at 24-hr. After Gd-DTPA or IgG-DTPA was administrated, the tumor showed no enhancement at early phase. It reached to maximum at 60～120 min and appeared mildly enhancement. As for MAb-SLs and Bio-MAb/SAv-SLs groups, the central and periphery of tumor signal intensity increased post-injection and reached to peak at 8-hr time point. It decreased slowly after that. The signal enhancement of the central and periphery of tumor after MAb-SLs injection were not different. In contrast, the periphery enhancement was significantly higher than that of central in Bio-MAb/SAv-SLs group (P=0.032). (3) The morphology of enhancement:T1 weighted imaging revealed an intensive entire tumor heterogeneous enhancement after Gd-DTPA injection. As for Gd-SLs and IgG-SLs groups, dots or spots enhancement scattered in or around the tumor. Ring-like marked enhancement occurred predominately along the tumor periphery after MAb-SLs or Bio-MAb/SAv-SLs were given. Immunohistochemical assessments of the tumors corroborated that angiogenesis was predominately distributed along the tumor periphery.Conclusions:1. It is easy to prepare the paramagnetic sterical stabilized liposomes which present high relaxivity to improve the MR contrast signal. The liposomes are thermo-sensitive but stable at common temperature. It may potential serve as an ideally useful contrast agent for MR molecular imaging.2. The applied covalent coupling of targeting antibodies is efficient. The biotin-avidin mediate antibodies and liposomes crosslink are certified in vitro.3. MR imaging in a glioma rat model confirmed that:no difference was found in degree between the enhancement of tumor central and periphery after Gd-DTPA contrast, and the tumor showed low enhancement while using non-targeting liposomes. The results demonstrate the limitation of these contrast agents to define the tumor morphology. The tumor extent was well delineated after targeting liposomes (MAb-SLs and Bio-MAb/SAv-SLs) contrast enhancement and the specificity was supported by the competition study in vivo. Moreover, the two-step imaging using biotin-avidin interaction was certified to induce more intensive enhancement, which imply that this novel MR molecular tracer is more suitable for detecting the tumor margins.更多还原