【作者】 丁楠；

【导师】 项光亚；

【作者基本信息】 华中科技大学 ， 药物化学， 2010， 硕士

【摘要】 叶酸受体是一种与糖基化磷脂酰肌醇(glycosylp-hosphatidylinositol, GPI)连接的膜糖蛋白,对叶酸有高度亲和性。在多数肿瘤细胞中,叶酸受体高度表达,而在多数正常细胞中,叶酸受体不表达。因此,可将叶酸受体作为肿瘤标志物,设计叶酸偶联物,靶向叶酸受体。脂质体是一种生物相容性好的载药系统,便于携带药物或者诊断试剂。因此可以将叶酸配体连接到脂质体上,形成肿瘤靶向的脂质体。磁共振成像技术具有空间定位能力好的特点,但灵敏度不够高。荧光成像的灵敏度很高,但难以在组织中深层定位,因而将这两种方法结合起来,不仅可以更灵敏、精确的定位肿瘤,而且可以实现手术前用磁共振成像定位肿瘤,术中在荧光指导下行切除术的构想。本文利用脂质体作为生物相容的给药系统,将叶酸配合物作为靶向基团,同时包封MRI造影剂Gd-DTPA-bis(SA)和荧光染料Calcein,制备叶酸受体靶向的磁共振成像、荧光成像多功能脂质体。1. Gd-DTPA-bis(SA)的合成以DTPA为起始原料,制备出DTPA环酸酐cDTPAA。再用十八胺氨解酸酐制备酰胺DTPA-bis(SA) ,最后再在DTPA-bis(SA)上螯合Gd3+得到Gd-DTPA-bis(SA)。本文也制备了Gd-DTPA作为Gd-DTPA-bis(SA)的对照。两种产物经过红外图谱、紫外图谱的确证,并通过ICP-AES测得化合物中Gd的含量,其中Gd-DTPA-bis(SA)含Gd量为13.3%,与理论值14.9%较接近,Gd-DTPA含Gd量为26.1%,与理论值27.4%较接近。Gd-DTPA-bis(SA)的DSC图谱显示,其相变温度为65.32℃,相变和磷脂类似,可以与脂质材料一起成膜制备脂质体。2.叶酸靶向磁共振/荧光多功能脂质体的制备及表征通过薄膜分散法制备脂质体,用Gd-DTPA-bis(SA)、叶酸受体配体Folate-PEG3350-CHEMS,及HSPC, mPEG2000-DSPE, Cholesterol成膜,荧光染料Calcein溶液作为水化液。文中考察了Folate-PEG3350-CHEMS的投料量,通过细胞摄取实验,确定其含量占总脂质的摩尔量1%时,Hela细胞的特异性吸附及游离叶酸阻断实验结果明显。文中也考察了Gd-DTPA-bis(SA)的投料量,认为其含量占总脂质的摩尔量25%时,脂质体稳定而且磁共振成像清晰。同时也考察了乙醇注入法制备脂质体、脂质材料的总投料量、水化液的水化体积、Calcein水化液的配制方法等因素对脂质体稳定性的影响。所制备出的脂质体通过激光动态光散射技术考察其粒径、电位,测得粒径为125nm~150nm,而且4℃放置三个月后,脂质不沉降,粒径基本没有变化。采用荧光分光光度计考察脂质体的荧光光谱学特性,发现脂质体的包裹没有引起Calcein的发射波长的改变。Calcein在一定浓度范围内,紫外吸光度和浓度呈线性关系,从而测得脂质体破膜后Calcein的含量,计算Calcein的包封率。得出Calcein包封率低,1.2~4.7%,这是由于Calcein水溶性好、分子量很小,易于从脂质体中渗漏。采用ICP-AES测脂质体含Gd的量,计算得出Gd的包封率较高。对磁共振/荧光脂质进行T1加权成像,可以观察到脂质体明亮的磁共振信号,而且随着脂质体浓度的降低,磁共振信号逐渐减弱。3.荧光法和磁共振法考察体外细胞对脂质体的摄取荧光法考察体外细胞对脂质体的摄取3.1.1流式细胞术考察KB细胞对不含Gd、含Calcein的荧光脂质体的摄取将KB细胞与Calcein终浓度为3.3μM的Calcein-L、F-Calcein-L及F-Calcein-L+FA共培养1h后洗去药液,于流式细胞仪上检测。F-Calcein-L组的平均荧光强度是Calcein-L组的163倍,是加游离叶酸干预组的6.8倍。3.1.2荧光显微镜定性观察KB细胞对含Gd、含Calcein的磁共振/荧光脂质体的摄取将KB细胞与Calcein终浓度为15.2μM的Gd-Calcein-L、F-Gd-Calcein-L及F-Gd-Calcein-L+FA共培养1h后洗去药液,细胞在PBS中,使用荧光倒置显微镜观察。KB细胞与含叶酸配体的脂质体共培养后可以观察到明亮的绿色荧光,而没有偶联叶酸配体的脂质体几乎没有荧光。当在F-Gd-Calcein-L中加入游离叶酸后,可以看到细胞内的绿色荧光明显减弱。而且,从荧光照片上可以看到细胞的荧光主要集中在细胞膜上,由于叶酸受体是一种膜糖蛋白,而脂质体进入细胞是经过叶酸受体介导的内吞作用,所以细胞的荧光集中在膜上。3.1.3流式细胞术考察KB细胞、Hela细胞、A549细胞对含Gd、含Calcein磁共振/荧光脂质体的摄取将叶酸受体表达阳性的KB细胞与Calcein终浓度为12.5μM或5.8μM的Gd-Calcein-L、F-Gd-Calcein-L及F-Gd-Calcein-L+FA共培养1h后洗去药液(用了两种方法:六孔板法和Ep管法),于流式细胞仪上检测。F-Gd-Calcein-L组的平均荧光强度是Gd-Calcein-L组的几十甚至上百倍,是加游离叶酸干预组的10-31倍。将叶酸受体表达阳性的Hela细胞与Calcein终浓度为12.5μM的Gd-Calcein-L、F-Gd-Calcein-L及F-Gd-Calcein-L+FA共培养1h后洗去药液,于流式细胞仪上检测。F-Gd-Calcein-L组的平均荧光强度是Gd-Calcein-L组的33倍,是加游离叶酸干预组的17倍。将叶酸受体表达阴性的A549细胞与Calcein终浓度为5.8μM的Gd-Calcein-L、F-Gd-Calcein-L及F-Gd-Calcein-L+FA共培养1h后洗去药液,于流式细胞仪上检测。F-Gd-Calcein-L组的平均荧光强度和Gd-Calcein-L组,加游离叶酸干预组的平均荧光强度基本一致,没有特异性的摄取作用。以上荧光实验可以看出:叶酸靶向的脂质体可以与叶酸受体表达阳性的KB细胞、Hela细胞特异性作用,并且这种作用可以被游离叶酸竞争性拮抗。对叶酸受体低表达的A549细胞,叶酸靶向的脂质体没有特异的作用。3.2磁共振成像法考察细胞对脂质体的摄取3.2.1磁共振成像定性考察Hela细胞对含Gd、不含Calcein的磁共振脂质体的摄取将叶酸受体表达阳性的Hela细胞与Gd-L、F-Gd-L及F-Gd-L+FA共培养2.5h后洗去药液,于3.0T磁共振成像仪上检测。F-Gd-L组的磁共振成像信号比Gd-L组强,且加游离叶酸干预后,信号减弱。3.2.2磁共振成像定性考察Hela细胞、KB细胞对含Gd、含Calcein的磁共振/荧光脂质体的摄取将叶酸受体表达阳性的Hela细胞与空白脂质体、Gd-Calcein-L、F-Gd-Calcein-L、F-Gd-Calcein-L+FA、Gd-DTPA共培养1h后洗去药液,于3.0T磁共振成像仪上检测。F-Gd-Calcein-L组的磁共振成像信号比Gd-Calcein-L组强,且加游离叶酸干预后,信号减弱。而且细胞孵育后,F-Gd-Calcein-L比Gd-DTPA磁共振信号强。说明脂质体包裹的Gd造影剂释放缓慢、滞留时间延长。将叶酸受体表达阳性的KB细胞与Gd-Calcein-L、F-Gd-Calcein-L及F-Gd-Calcein-L+FA共培养1h后洗去药液,于3.0T磁共振成像仪上检测。F-Gd-Calcein-L组的磁共振成像信号比Gd-Calcein-L组强,且加游离叶酸干预后,信号减弱。磁共振成像实验同样可以说明:叶酸靶向的脂质体可以与叶酸受体表达阳性的KB细胞、Hela细胞特异性作用,并且这种作用可以被游离叶酸竞争性拮抗。叶酸靶向的脂质体粒径较小,稳定性好。包裹水溶性荧光染料Calcein,不影响其最大发射波长;包裹磁共振造影剂Gd-DTPA-bis(SA),脂质体磁共振成像清晰。插入叶酸配体后,脂质体能靶向到叶酸受体表达阳性的细胞,并且这种作用可以被游离叶酸阻断,但是对于叶酸受体表达阴性的细胞,这种特异性作用并不存在。因此该脂质体可以作为模型药物,为进一步探索肿瘤靶向的磁共振/荧光多功能探针作铺垫。更多还原

【Abstract】 FR (folate receptor), which is a well-known tumor associated antigen, exhibits limited expression in normal tissues, but is greatly over-expressed in a variety of carcinomas. Due to its high binding affinity for cell surface FR, folate conjugation allows delivery of non-specific drugs selectively into pathologic cells without causing harm to normal tissues. Liposomes are versatile tools used in drugs and gene delivery. Therefore, folate-targeted liposomes were also prepared for tumor diagnosis and treatment.MRI is capable of producing three-dimensional images of tissues containing water with a high degree of spatial resolution. However, it suffers from a relatively low sensitivity. Optical imaging has higher sensitivity, but it has problems associated with tissue auto-fluorescence and tissue absorption/scattering of light. Combined the two technique together can complement each other. Tumor can be visualized by MRI and verified by fluorescence microscopy of histology samples. Moreover, the idea of delineate tumors both by preoperative MRI and by intraoperative optical imaging is attractive. Therefore, we design a multifunctional folate receptor targeted, paramagnetic and fluorescent liposomes for targeting and imaging cancer cells.1. Synthesis of Gd-DTPA-bis(SA)DTPA-bis(SA) was prepared from DTPA and SA, and then chelated by Gd3+ to form Gd-DTPA-bis(SA). Gd-DTPA was also prepared. The products were verified by IR and UV, and the concentrations of Gd were detected by ICP-AES. Gd-DTPA-bis(SA) contains Gd of 13.3%, which was similar to the theoretical value (14.9%). Gd-DTPA contains Gd of 26.1%, which was also similar to the theoretical value (27.4%). The phase-transition temperature of Gd-DTPA-bis(SA) was 65.32℃, which can be shown from the DSC spectrum. Phase-transition temperature was an important character of lipids, that is why Gd-DTPA-bis(SA) could form liposomes with other lipids. 2. Preparation of multifunctional liposomeLiposome was composed of HSPC, mPEG2000-DSPE, Cholesterol, Gd-DTPA-bis(SA), Folate-PEG3350-CHEMS. For MRI better imaging, 25% lipid amount of Gd-DTPA-bis(SA) that incorporated in the liposomal bilayer was chosen. To show the efficiency of targeting, the amount of Folate-PEG3350-CHEMS was found by cell uptake experiment, and confirmed that 1% total lipids of the amount of Folate-PEG3350-CHEMS was the best choice. Other factors influencing the stability of liposomes, like the total amount of lipids and the volume of Calcein solution were all analyzed.The liposomes were approximately 125-150nm with low polydispersity index, and could be stable at least for three months. The fluorescent character of Calcein did not change when encapsulating in liposome. The incorpotation rate of Calcein was pretty low, ranging from 1.2~4.7%, detected by UV. While the incorpotation rate of Gd was much higher, after being analyzed by ICP-AES. Liposomes were also imaged by MRI.3. Cell uptake of liposomes in vitro3.1 optical imaging of cells3.1.1 KB cells were incubated with fluorescent liposomes (containing Calcein but without Gd), analyzed by FCMThe average fluorescence intensity of KB cells after uptake of F-Calcein-L was 163 times more than the intensity of cells after uptake of Calcein-L. And it was 6.8 times more than the intensity of cells after uptake the mixture of F-Calcein-L and free FA.3.1.2 KB cells were incubated with biomodal liposomes (containing both Gd and Calcein), observed by fluorescence microscopeAfter 1h, cells incubated with F-Gd-Calcein-L showed greater green fluorescence than Gd-Calcein-L. In contrast, the fluorescence of cell reduced after adding free FA to F-Gd-Calcein-L. Additionally, it can be noticed that liposomes were primarily located on the surface of cell membrane, due to their preferential binding to FR on the membrane. This phenomenon demonstrated that liposomes were taken up by cells through an endocytic pathway.3.1.3 Cell lines of KB, Hela, A549 were incubated with biomodal liposomes (containg both Calcein and Gd), analyzed by FCMThe average fluorescence intensity of KB cells and Hela cells after uptake of F-Gd-Calcein-L was much higher than the intensity of cells after uptake of Gd-Calcein-L. And the uptake could be blocked by free FA.However, the average fluoresce intensity of A549 cells were nearly the same after incubation with different liposomes.Form the fluorescent experiments, it can be seen that folate-decorated liposomes could be targetd delived to folate receptor positive cells but not the negative ones. And this targeted effect can be blocked by free folic acid.3.2 MRI imaging of cells3.2.1 Hela cells were incubated with liposomes containing Gd without Calcein, imaged by MRIHela cells were incubated with Gd-L, F-Gd-L and F-Gd-L+FA for 2.5h. Then the drugs was washed and cells were imaged by MRI. F-Gd-L showed the highest signal intensity and the action could be blocked by free FA.3.2.2 Hela cells and KB cells were incubated with liposomes containing both Gd and Calcein.Hela cells were incubated with L, Gd-Calcein-L, F-Gd-Calcein-L, F-Gd-Calcein-L+FA and Gd-DTPA for 1h. Imaging from MRI showed that cells incubated with F-Gd-Calcein-L obtain highest signal intensity and the action could be blocked by free FA. Comparing with Gd-DTPA, which enter cells by diffusion and can be removed soon, liposomes prolong the circulation time and enhance the signal.KB cells were incubated with L, Gd-Calcein-L, F-Gd-Calcein-L, F-Gd-Calcein-L+FA and Gd-DTPA for 1h. Imaging from MRI showed that cells incubated with F-Gd-Calcein-L obtain highest signal intensity and the action could be blocked by free FA.Form the MRI imaging, it also can be demonstrated that folate-decorated liposomes could be targetd delived to folate receptor positive cells but not the negative ones. And this targeted effect can be blocked by free folic acid.In conclusion, a folate-decorated, MR- and fluorescence- detectable liposome was prepared with narrow size distribution and can be imaged by MRI imaging and fluorescent imaging. It could be targeted deliverd to folate receptor positive cells (KB cells and Hela cells). In contrast, for the folate receptor negative cells, it did not show any preferential uptake of folate-decorated liposomes. This multifunctional liposome may serve as a useful diagnostic tool to co-localize tumors both by MRI and fluorescence.更多还原