以叶酸-BSA为载体的靶向性钆造影剂的制备及其体外弛豫和细胞摄取研究
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摘要
随着肿瘤病例的极速增加,肿瘤的早期诊断就变得尤为重要。磁共振成像技术就是一种临床上常用的肿瘤诊断技术。目前,临床上应用的大部分磁共振造影剂(包括常用的钆类造影剂)对肿瘤组织并无特异性的靶向作用,因而降低了造影效果。因此,磁共振成像技术应用于肿瘤诊断的关键在于研发靶向性的磁共振造影剂以提高肿瘤定位能力,并增强检测的灵敏性和准确性。叶酸-叶酸受体靶向就是近来备受关注的新型抗肿瘤机制,它是利用叶酸受体在某些肿瘤部位过度表达、而在正常组织中低水平表达的特征而实现叶酸偶联药物的靶向输送。基于叶酸受体的表达特征和叶酸配体的独特优势,叶酸受体已成为肿瘤诊断和治疗的理想靶点。
本文结合叶酸介导靶向输送的理念和磁共振造影成像的安全性与灵敏性,设计合成了叶酸介导的靶向性钆造影剂—叶酸-BSA- (Gd-DTPA)n。首先运用正交设计实验优化了牛血清白蛋白(BSA)与二乙三胺五乙酸环酸酐(cDTPAA)的偶联条件。接着在最佳条件的反应介质中,叶酸活化酯与BSA反应生成叶酸-白蛋白偶联物,该偶联物再与DTPA的酸酐反应,最后与氯化钆进行螯合并经Sephadex G-25分离纯化制得叶酸-BSA-(Gd-DTPA)n。通过紫外光谱法对造影剂的结构进行了鉴定,并定量测定了造影剂中叶酸、Gd-DTPA对BSA的偶联率。通过测定造影剂的体外弛豫时间T1,进一步分析其弛豫性能R1。
用异硫氰酸荧光素(FITC)对造影剂进行荧光标记,以叶酸受体阳性的KB、HeLa细胞和叶酸受体阴性的A549细胞为模型细胞,通过倒置荧光显微镜定性观察,研究了细胞对造影剂的特异性摄取作用;同时也通过钆含量的测定来分析细胞的摄取情况。
结果表明,叶酸-BSA-(Gd-DTPA)n造影剂中,叶酸对BSA的偶联比约为5,Gd-DTPA的偶联比随着酸酐投量的增加而增大;体外弛豫性能R1约为6×10-3 L·mmol-1ms-1,与未偶联叶酸的BSA-(Gd-DTPA)n的弛豫性能无显著性差异,但比小分子Gd-DTPA的弛豫性能提高了2倍左右。在细胞摄取实验中,荧光标记的叶酸-BSA-(Gd-DTPA)n在叶酸受体阳性细胞KB和HeLa的胞浆内有明亮的荧光,荧光强度随着加入的游离叶酸浓度的增大而减弱,而在叶酸受体阴性的A549细胞中荧光很弱;未偶联叶酸的BSA-(Gd-DTPA)n在三种细胞中荧光都很弱。在细胞摄取的影响因素实验中发现,随着培养时间的延长、给药浓度的增大,KB细胞中的荧光强度增强,但有饱和性;当蛋白上偶联的Gd-DTPA数目过大时,会因结合位阻增大而荧光减弱。钆含量的测定结果也初步显示了KB细胞与偶联叶酸的造影剂之间的特异性作用。
总之,本文制备的大分子造影剂—叶酸-BSA-(Gd-DTPA)n具有高弛豫性能和靶向叶酸受体阳性细胞的特异性。
The early diagnosis of cancer is becoming more and more important with the rapid increase of cancer cases. Magnetic resonance imaging (MRI) is one of the effective tumor diagnostic techniques in clinical use. But most of MR contrast agents (including Gd3+ contrast agents) could not specifically localize in tumor tissues, so the critical importance of the field is the development of targeted contrast agents which can localize to cancer cells through a passive or active mechanism. Among all those researches, targeted drug via the folate receptor (FR), based on the fact that folate receptor can be over-expressed on the surface of some kinds of tumor cells, but scarcely expressed on normal cells, has drawn great attentions by researchers. Based on specificity of FR expression and distinctive advantages of folate, folate receptor has become an ideal target of tumor diagnosis and therapy.
Based on the theory of folate-mediated delivery and the reliability and sensitivity of MRI, we have designed and prepared a new folate-mediated targeting Gd3+ contrast agent—“Folate-BSA-(Gd-DTPA)n”. First, we optimized the reacting conditions of conjugation of BSA and DTPA (Diethylene triamine pentaacetic dianhydride) anhydride. Then under the optimal condition, BSA was reacted with the activated folate; after that, the conjugates were coupled with DTPA anhydride to form Folate-BSA-DTPA; finally, these coupled compounds were chelated with GdCl3. All complexes were characterized by UV, the ratios of folate and Gd-DTPA to BSA were determined by UV and ICP-AES methods respectively.
The spin-lattice relaxivity of the Gd complexes in vitro were analyzed by T1-weighted magnetic resonance images.
The contrast agents were labeled with fluorescein isothiocyanate (FITC) under weak base. Using KB (FR+), HeLa (FR+) and A549 (FR-) cells as model cells, the intracelluar uptake of contrast agents was observed under inverted fluorescent microscope. Besides, intracelluar uptake of contrast agents was also detected by assay of Gd3+.
As a result, the ratio of folate to BSA was about 5 in every molecular of Folate-BSA-(Gd-DTPA)n , and the relaxivity was about 6×10-3 L·mmol-1ms-1 enhanced twice compared to the small molecular Gd-DTPA, though there was no difference in relaxivity between Folate-BSA-(Gd-DTPA)n and BSA-(Gd-DTPA)n. The investigation of the specific cellular uptake showed that there was a greater fluorescence intensity when FITC-labelled Folate-BSA-(Gd-DTPA)n were incubated with KB and HeLa cells than FITC-labelled BSA-(Gd-DTPA)n, and the intracellular uptake of folate complexes in KB and HeLa cells could be blocked by free folate; but there was weak fluorescent in A549 cells incubated folate-conjugated or non-conjugated contrast agents. In the test of influent factors of KB cellular uptake incubated with FITC-labelled Folate-BSA-(Gd-DTPA)n, the result showed that the intensity of cell-associated fluorescence increased with time and concentration, and decreased with ratio of Gd-DTPA to BSA. And the result of ICP-AES assay of Gd3+ also indicated that the specific cellular uptake of FR(+) with folate-conjugated contrast agents.
In all, Folate-BSA-(Gd-DTPA)n thus obtained had high relaxivity and potential for targeting FR-positive tumor.
本文结合叶酸介导靶向输送的理念和磁共振造影成像的安全性与灵敏性,设计合成了叶酸介导的靶向性钆造影剂—叶酸-BSA- (Gd-DTPA)n。首先运用正交设计实验优化了牛血清白蛋白(BSA)与二乙三胺五乙酸环酸酐(cDTPAA)的偶联条件。接着在最佳条件的反应介质中,叶酸活化酯与BSA反应生成叶酸-白蛋白偶联物,该偶联物再与DTPA的酸酐反应,最后与氯化钆进行螯合并经Sephadex G-25分离纯化制得叶酸-BSA-(Gd-DTPA)n。通过紫外光谱法对造影剂的结构进行了鉴定,并定量测定了造影剂中叶酸、Gd-DTPA对BSA的偶联率。通过测定造影剂的体外弛豫时间T1,进一步分析其弛豫性能R1。
用异硫氰酸荧光素(FITC)对造影剂进行荧光标记,以叶酸受体阳性的KB、HeLa细胞和叶酸受体阴性的A549细胞为模型细胞,通过倒置荧光显微镜定性观察,研究了细胞对造影剂的特异性摄取作用;同时也通过钆含量的测定来分析细胞的摄取情况。
结果表明,叶酸-BSA-(Gd-DTPA)n造影剂中,叶酸对BSA的偶联比约为5,Gd-DTPA的偶联比随着酸酐投量的增加而增大;体外弛豫性能R1约为6×10-3 L·mmol-1ms-1,与未偶联叶酸的BSA-(Gd-DTPA)n的弛豫性能无显著性差异,但比小分子Gd-DTPA的弛豫性能提高了2倍左右。在细胞摄取实验中,荧光标记的叶酸-BSA-(Gd-DTPA)n在叶酸受体阳性细胞KB和HeLa的胞浆内有明亮的荧光,荧光强度随着加入的游离叶酸浓度的增大而减弱,而在叶酸受体阴性的A549细胞中荧光很弱;未偶联叶酸的BSA-(Gd-DTPA)n在三种细胞中荧光都很弱。在细胞摄取的影响因素实验中发现,随着培养时间的延长、给药浓度的增大,KB细胞中的荧光强度增强,但有饱和性;当蛋白上偶联的Gd-DTPA数目过大时,会因结合位阻增大而荧光减弱。钆含量的测定结果也初步显示了KB细胞与偶联叶酸的造影剂之间的特异性作用。
总之,本文制备的大分子造影剂—叶酸-BSA-(Gd-DTPA)n具有高弛豫性能和靶向叶酸受体阳性细胞的特异性。
The early diagnosis of cancer is becoming more and more important with the rapid increase of cancer cases. Magnetic resonance imaging (MRI) is one of the effective tumor diagnostic techniques in clinical use. But most of MR contrast agents (including Gd3+ contrast agents) could not specifically localize in tumor tissues, so the critical importance of the field is the development of targeted contrast agents which can localize to cancer cells through a passive or active mechanism. Among all those researches, targeted drug via the folate receptor (FR), based on the fact that folate receptor can be over-expressed on the surface of some kinds of tumor cells, but scarcely expressed on normal cells, has drawn great attentions by researchers. Based on specificity of FR expression and distinctive advantages of folate, folate receptor has become an ideal target of tumor diagnosis and therapy.
Based on the theory of folate-mediated delivery and the reliability and sensitivity of MRI, we have designed and prepared a new folate-mediated targeting Gd3+ contrast agent—“Folate-BSA-(Gd-DTPA)n”. First, we optimized the reacting conditions of conjugation of BSA and DTPA (Diethylene triamine pentaacetic dianhydride) anhydride. Then under the optimal condition, BSA was reacted with the activated folate; after that, the conjugates were coupled with DTPA anhydride to form Folate-BSA-DTPA; finally, these coupled compounds were chelated with GdCl3. All complexes were characterized by UV, the ratios of folate and Gd-DTPA to BSA were determined by UV and ICP-AES methods respectively.
The spin-lattice relaxivity of the Gd complexes in vitro were analyzed by T1-weighted magnetic resonance images.
The contrast agents were labeled with fluorescein isothiocyanate (FITC) under weak base. Using KB (FR+), HeLa (FR+) and A549 (FR-) cells as model cells, the intracelluar uptake of contrast agents was observed under inverted fluorescent microscope. Besides, intracelluar uptake of contrast agents was also detected by assay of Gd3+.
As a result, the ratio of folate to BSA was about 5 in every molecular of Folate-BSA-(Gd-DTPA)n , and the relaxivity was about 6×10-3 L·mmol-1ms-1 enhanced twice compared to the small molecular Gd-DTPA, though there was no difference in relaxivity between Folate-BSA-(Gd-DTPA)n and BSA-(Gd-DTPA)n. The investigation of the specific cellular uptake showed that there was a greater fluorescence intensity when FITC-labelled Folate-BSA-(Gd-DTPA)n were incubated with KB and HeLa cells than FITC-labelled BSA-(Gd-DTPA)n, and the intracellular uptake of folate complexes in KB and HeLa cells could be blocked by free folate; but there was weak fluorescent in A549 cells incubated folate-conjugated or non-conjugated contrast agents. In the test of influent factors of KB cellular uptake incubated with FITC-labelled Folate-BSA-(Gd-DTPA)n, the result showed that the intensity of cell-associated fluorescence increased with time and concentration, and decreased with ratio of Gd-DTPA to BSA. And the result of ICP-AES assay of Gd3+ also indicated that the specific cellular uptake of FR(+) with folate-conjugated contrast agents.
In all, Folate-BSA-(Gd-DTPA)n thus obtained had high relaxivity and potential for targeting FR-positive tumor.
引文
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[32] Pan XQ, Wang H, Lee RJ. Boron delivery to a murine lung carcinoma using folate receptor-targeted liposomes[J]. Anticancer Res, 2002, 22: 1629-1633.
[33] Pan X, Wang H, Shukla S, et al. Boron-containing folate receptortargeted liposomes as potential delivery agents for neutron capture therapy[J]. Bioconjug Chem 2002,13:435-442.
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[43] Ward CM, Pechar M, Oupicky D, et al. Modification of PLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo[J]. J Gene Med, 2002, 4(5): 536-47.
[44] Zhao XB, Lee RJ. Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor[J]. Adv Drug Deliv Rev, 2004, 56(8): 1193-1204.
[45] Gosselin MA, Guo W, Lee RJ. Incorporation of reversibly cross-linked polyplexes into LPDII vectors for gene delivery[J]. Bioconjug Chem, 2002, 13(5):1044-53.
[46] Olivier Z, Lisa SU , Lee GB , et al . Effect of serum components on the physico chemical properties of cationic lipid/oligonucleotide complexes and on theirinteractions with cells [J] . Biochim Biophys Acta, 1998, 1390 (2): 119-133.
[47] Ki Hyun Bae, Yuhan Lee, Tae Gwan Park , et al. Oil-Encapsulating PEO-PPO-PEO/PEG Shell Cross-Linked Nanocapsules for Target-Specific Delivery of Paclitaxel[J]. Biomacromolecules, 2007, 8:650-656.
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[51]葛泉波,何淑兰,毛津淑,等.生物材料与细胞相互作用及表面修饰[J].化学通报,2005,1: 43-48.