CN115025249B - 靶向探针及其制备方法和应用 - Google Patents
靶向探针及其制备方法和应用 Download PDFInfo
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- A61P35/00—Antineoplastic agents
Abstract
本发明公开了一种靶向探针及其制备方法和应用,靶向探针,包括以下质量份数的组分:1份~5份的靶向分子、0.1份~1份的药物分子、0.1份~1份的近红外染料、40份~60份的蛋白质载体和8份~12份的磁共振造影剂;所述靶向分子用于靶向脑胶质瘤,所述药物分子用于渗透血脑屏障,所述近红外染料用于近红外荧光成像,所述靶向分子、所述药物分子、所述近红外染料、所述蛋白质载体和所述磁共振造影剂复合形成所述靶向探针。本发明的靶向探针能够渗透血脑屏障靶向胶质瘤,且具有磁共振成像和近红外荧光成像的双模态成像,便于术前检测和术中精准定位,对胶质瘤的手术治疗具有重大意义。
Description
技术领域
本发明涉及生物医学技术领域,更具体地,涉及一种靶向探针及其制备方法和应用。
背景技术
手术治疗是脑胶质瘤的主要手段,但该肿瘤呈浸润性生长,与周围正常脑组织分界不明显,难以完全切除,因此,判断肿瘤的边界一直是神经外科领域亟待解决的难题,如果低估肿瘤边界,手术切缘就可能会残留肿瘤或者复发,反之,则会损伤正常脑组织,损害相应的脑功能。
目前,磁共振扫描配合钆造影剂(DOTA-Gd),再加上手术导航系统是识别胶质瘤边界“标配”方法,然而,它并不完美,原因在两个方面:①血脑屏障(BBB):因为胶质瘤在脑组织浸润生长,相应区域的血脑屏障常常保持完整,加上瘤区新生血管壁孔径细小,造影剂难以渗透,所以钆造影剂对胶质瘤的增强作用较身体其他部位的肿瘤弱很多,即EPR(Enhanced Permeability and Retention,高渗透长滞留)效应减弱,其结果就是肿瘤边界难以识别。②术中的“Brain shift”问题:在手术过程中,脑组织和肿瘤难免变形和移位,因此,即使术前导航的磁共振图像上能准确识别肿瘤边界,在手术中实际脑瘤区的位置可能与导航图像差距甚远。
发明内容
本发明的目的在于克服现有技术存在的上述缺陷,提供一种靶向探针及其制备方法和应用,能够渗透血脑屏障靶向胶质瘤,且具有磁共振成像和近红外荧光成像的双模态成像,便于术前检测和术中精准定位,对胶质瘤的手术治疗具有重大意义。
为实现上述目的,本发明的技术方案如下:
一种靶向探针,其特征在于,包括以下质量份数的组分:
1份~5份的靶向分子、0.1份~1份的药物分子、0.1份~1份的近红外染料、40份~60份的蛋白质载体和8份~12份的磁共振造影剂;
所述靶向分子用于靶向脑胶质瘤,所述药物分子用于渗透血脑屏障,所述近红外染料用于近红外荧光成像,所述靶向分子、所述药物分子、所述近红外染料、所述蛋白质载体和所述磁共振造影剂复合形成所述靶向探针。
本发明还提供了一种上述靶向探针的制备方法,包括以下过程:
提供以下质量份数的组分:
1份~5份的靶向分子、0.1份~1份的药物分子、0.1份~1份的近红外染料、40份~60份的蛋白质载体和8份~12份的磁共振造影剂;
将所述蛋白质载体、所述磁共振造影剂、所述近红外染料和所述药物分子复合形成纳米粒子;
将所述靶向分子结合在所述纳米粒子表面,得到所述靶向探针。
实施本发明实施例,将具有如下有益效果:
本发明的靶向探针在药物分子的作用下易渗透血脑屏障标记和治疗脑胶质瘤,且可以通过磁共振(MRI)成像和近红外荧光(FL)成像的双模态成像,不仅有效进行术前和术中定位,而且实现诊断和治疗的一体化。
本发明通过透射电子显微镜(TEM)、动态光散射、荧光光谱仪和HPLC分别测量了制备的靶向探针的形态、流体力学尺寸和表面Zeta电位、荧光强度和药物包封效率。在MRI/FL的双模态成像和细胞存活率测定的体外实验中,我们验证了本发明的靶向探针对胶质瘤细胞的靶向能力和细胞杀伤力都明显优于对照组。在MRI/FL的双模态成像的动物实验中,我们验证了本发明的靶向探针较对照组有更高肿瘤的聚集。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
其中:
图1A是本发明实施例1制得的靶向探针ANG-BSA/BCNU/ICG MNPs的TEM图像。
图1B是本发明实施例1制得的靶向探针ANG-BSA/BCNU/ICG MNPs的DLS分析。
图1C是本发明实施例1制得的靶向探针ANG-BSA/BCNU/ICG MNPs的水动力尺寸和表面Zeta电位图。
图1D是本发明实施例1制得的靶向探针ANG-BSA/BCNU/ICG MNPs的磁滞曲线。
图2是本发明实施例1制得的靶向探针ANG-BSA/BCNU/ICG MNPs在pH7.4(血浆)和pH5.5(肿瘤微环境)下BCNU的释放情况。
图3A是293T细胞分别在BCNU、BSA/BCNU/ICG MNPs和ANG-BSA/BCNU/ICG MNPs中的细胞存活率与剂量的关系。
图3B是U87MG细胞分别在BCNU、BSA/BCNU/ICG MNPs和ANG-BSA/BCNU/ICG MNPs中的细胞存活率与剂量的关系。
图4是U87MG细胞和293T细胞在BSA/BCNU/ICG MNPs和ANG-BSA/BCNU/ICG MNPs中的荧光图像。
图5是U87MG细胞和293T细胞在不同浓度ANG-BSA/BCNU/ICG MNPs中孵化的MRI图像。
图6A是分别注射ANG-BSA/BCNU/ICG MNPs和BSA/BCNU/ICG MNPs12h后动物体内的荧光信号。
图6B是分别注射ANG-BSA/BCNU/ICG MNPs和BSA/BCNU/ICG MNPs后不同时间的动物体内的荧光信号。
图7是分别注射PBS、BSA/BCNU/ICG、ANG-BSA/BCNU/ICG MNPs后的肿瘤部位的MRI图像。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明公开了一种靶向探针,包括以下质量份数的组分:
1份~5份的靶向分子、0.1份~1份的药物分子、0.1份~1份的近红外染料、40份~60份的蛋白质载体和8份~12份的磁共振造影剂;靶向分子用于靶向脑胶质瘤,药物分子用于渗透血脑屏障,近红外染料用于近红外荧光成像,靶向分子、药物分子、近红外染料、蛋白质载体和磁共振造影剂复合形成靶向探针。
本发明的靶向探针在药物分子的作用下易渗透血脑屏障标记和治疗脑胶质瘤,且可以通过磁共振成像和近红外荧光成像的双模态成像,不仅有效为胶质瘤进行术前和术中定位,而且实现诊断和治疗的一体化。
在一较优实施例中,药物分子可以选自亚硝脲类烷化剂,亚硝脲类烷化剂能够渗透BBB,且对胶质瘤细胞具有杀伤力,亚硝脲类烷化剂的分子中含有N+的缺电子原子,能够与蛋白质载体中的富电子基团(例如羟基、氨基、羧基、巯基等)相结合。
具体的,亚硝脲类烷化剂可以选自卡莫司汀、洛莫司汀、司莫司汀、尼莫司汀、链佐星和氯脲酶素等中的一种或两种以上。
当然,药物分子还可以选贝伐珠单抗等其它类治疗胶质瘤且能渗透BBB的药物。
蛋白质载体提供纳米探针的骨架,同时可以改善靶向分子和药物分子的药代动力学特性,可以改善多肽或蛋白质药物的药代动力学特性,在一较优实施例中,蛋白质载体可以选自白蛋白,白蛋白分子中含有氨基、羟基、羧基、巯基等多个活化反应位点,有利于药物分子、靶向分子、近红外染料和磁共振造影剂的嵌入和装载。应用白蛋白运输药物还可以提高药物的生物降解性和稳定性,从而使肿瘤和炎症组织缓慢释放和吸收。
在一较优实施例中,磁共振造影剂可以选自超顺磁性氧化铁(super-paramagnetism iron oxide,SPIO),SPIO是一种应用广泛且相对安全的磁共振T2造影剂,它在体内的半衰期短,容易被网状内皮细胞吸收和被巨噬细胞清除。当然,磁共振造影剂还可以选择现有技术中的其它磁共振造影剂,例如,GD-DTPA等。
在一较优实施例中,近红外染料可以选自吲哚菁绿(Indocyanine green,ICG,分子式为C43H49N2NaO6S2,化学结构式为吲哚菁绿的分子结构中也含有N+的缺电子原子,能够与蛋白质载体中的富电子基团(例如羟基、氨基、羧基、巯基等)相结合。ICG是FDA批准用于临床的近红外染料,不仅可用于近红外荧光成像(FL),还可将吸收的光能转化为活性氧和热能,分别实现光热疗法和光动力疗法,近红外荧光成像的高组织穿透深度和磁共振成像(MRI)的高分辨率,对术前肿瘤的准确定位、术中肿瘤边缘的客观界定和肿瘤切除都具有重要意义。ICG的激发和发射波长分别在785nm、810nm左右,比Cy系列(花菁类)染料(630-670nm、650-700nm)更长,可穿透更深的活体组织。
当然,近红外染料也可以选自Cy系列染料等。
在一较优实施例中,靶向分子选自Angiopep-2多肽(英文缩写为ANG,序列号为:TFFYGGSRGKRNNFKTEEY),其可以特异性地与低密度脂蛋白受体相关蛋白(low densitylipoprotein receptor-related protein,LRP)结合,后者在BBB和胶质瘤细胞中过度表达。
本发明还提供一种上述靶向探针的制备方法,包括以下步骤:
步骤1:提供以下质量份数的组分:1份~5份的靶向分子、0.1份~1份的药物分子、0.1份~1份的近红外染料、40份~60份的蛋白质载体和8份~12份的磁共振造影剂,上述各组分在前文已详述,参见前文。
步骤2:将蛋白质载体、磁共振造影剂、近红外染料和药物分子复合形成纳米粒子。
在本步骤中,在一具体实施例中,采用去溶剂化-化学交联法形成纳米粒子,具体的,包括以下步骤:
步骤21:将蛋白质载体、磁共振造影剂和近红外染料分散于溶剂中,得到第一混合液,具体的,可以采用搅拌机搅拌或超声分散等手段使蛋白质载体、磁共振造影剂和近红外染料均匀分散于溶剂中。
在一具体实施例中,将牛血清白蛋白(BSA)、SPIO、ICG溶解于去离子水中,采用超声进行分散。
步骤22:将药物分子加入第一混合液中,搅拌直至出现沉淀,得到第二混合液。
在一具体实施例中,将含有卡莫司汀(英文名称BCNU,分子式C5H9Cl2N3O2,化学结构式为)的无水酒精滴加到第一混合液中,在室温下搅拌直至出现沉淀。
优选的,将药物分子加入第一混合液之前,调节第一混合液的pH值为8~10,促进步骤22中沉淀的生成,以及促进步骤23中醛基与氨基之间的交联反应。
步骤23:将含有醛基的有机化合物加入第二混合液中,有机化合物中的醛基与蛋白质载体的氨基发生交联,得到纳米粒子。
含有醛基的有机化合物可以选自戊二醛。
具体的,在一具体实施例中,加入戊二醛以交联BSA的氨基,从而形成纳米粒子。
步骤3:将靶向分子结合在纳米粒子表面,得到靶向探针。
在一具体实施例中,采用碳二亚胺类化合物将靶向分子结合在纳米粒子表面,具体,包括以下步骤:
步骤31:将纳米粒子分散于缓冲溶液中,得到第三混合液。具体的,采用MES缓冲溶液(0.02M,pH6.5)。
步骤32:将碳二亚胺类化合物或者碳二亚胺类化合物与NHS和/或磺基-NHS的混合物加入第三混合液中,活化蛋白质载体中的羧基,得到第四混合液。
NHS和/或磺基-NHS与碳二亚胺类化合物联用,可以加快步骤33的偶联效率。
碳二亚胺类化合物可以选自碳二亚胺(EDC)等。
步骤33:将靶向分子加入第四混合液中,使靶向分子偶联至纳米粒子,得到靶向探针。
具体的,在一具体实施例中,将EDC水溶液和NHS水溶液加入第三混合液中,以活化BSA中的羧基,充分活化后,加入ANG水溶液,在室温下继续孵化2小时,然后转移到4℃下孵化过夜,最后用超滤管离心去除未耦合的ANG,最终获得ANG-BSA/BCNU/ICG靶向探针。
上述靶向探针可以应用于制备脑胶质瘤成像的检测试剂盒中。
上述靶向探针由于结合有药物分子,也可以用于靶向失踪治疗胶质瘤,实现诊断和治疗的一体化,应用于制备脑胶质瘤靶向药物中。
以下为具体实施例。
实施例1
1)用无水苯甲醇制备5%(w/v)的乙酰丙酮铁溶液,然后加热到110℃,1小时后,之后在氮气环境下加热到退火温度,40小时后,将上述溶液冷却,用丙酮沉淀SPIO NPs(SPIO纳米粒子),彻底清洗并干燥。
2)将BSA(50毫克)、SPIO NPs(10毫克)和ICG(0.5毫克)溶解在10毫升去离子水中。经过超声分散后,将pH值调整到9.0。将含有BCNU(0.5毫克)的无水酒精(50毫升)滴加到溶液中,然后在室温下搅拌直到出现沉淀。加入25%(w/v)戊二醛(25μL)溶液以交联BSA的氨基,从而形成纳米粒子。
3)将步骤2)制得的的混合液以20,000rpm/min离心30分钟以去除多余的SPIONPs、ICG、BCNU和有机溶剂,得到纳米粒子(BSA/BCNU/ICG MNPs),再将纳米粒子重新分散在5mL的MES缓冲液(0.02M,pH6.5)中。
4)在1毫升BSA/BCNU/ICG MNPs溶液(10毫克/毫升)中加入8微升EDC溶液(10毫克/毫升,去离子水)和10微升NHS溶液(10毫克/毫升,去离子水)以激活羧基。孵化25分钟后,将ANG的溶液(0.1毫升,10毫克/毫升)加入到上述溶液中,混合物在室温下孵化2小时,然后转移到4℃下孵化过夜。
5)最后用超滤管(MWCO 30kD)以10,000rpm/min的离心速度去除未耦合的ANG,最终获得本发明的靶向探针(ANG-BSA/BCNU/ICG MNPs),以便进一步使用。
测试例1
表征实施例1制得的靶向探针ANG-BSA/BCNU/ICG MNPs。
采用透射电子显微镜(TEM)评估ANG-BSA/BCNU/ICG MNPs的形态,如图1A所示,ANG-BSA/BCNU/ICG MNPs表现出明确的球形形状,尺寸为85nm±10nm。
采用动态光散射(Dynamic light scattering,DLS)分析实施例1制得的ANG-BSA/BCNU/ICG MNPs的流体力学尺寸和表面Zeta电位,水动力尺寸和表面Zeta电位是评估ANG是否与BSA/BCNU/ICG MNPs共轭的辅助验证方法,具体的,将ANG-BSA/BCNU/ICG MNPs置于PBS缓冲液(0.01M,pH7.4)中24小时,结果分别如图1B和图1C所示。从图1B可见,ANG-BSA/BCNU/ICG MNPs平均流体力学直径为121nm±4.6nm。从图1C可见,ANG-BSA/BCNU/ICG MNPs在1周内的水动力尺寸和表面Zeta电位变化不大,且在生物培养基中分散良好,这表明ANG-BSA/BCNU/ICG MNPs具有良好的长期胶体稳定性。此外,ANG-BSA/BCNU/ICG MNPs在4℃的PBS缓冲液中储存3周后,其流体力学尺寸变化较小,表明其在水介质中的稳定性极佳。
参考图1D,通过振动样品磁强计的测量显示:实施例1制得的ANG-BSA/BCNU/ICGMNPs具有良好的磁性,饱和磁化达到16.3emu/g,磁滞曲线表明NPs具有超顺磁性。
测试例2
实施例1制得的靶向探针ANG-BSA/BCNU/ICG MNPs的药物包封效率和体外释放分析。
药物包封率由高效液相色谱仪(High Performance Liquid Chromatography,HPLC)(Waters,Alli-ance 2695,USA)测量,首先,将0.5mL ANG-BSA/BCNU/ICG MNPs分散到4.5mL 0.5%胃蛋白酶水溶液中,在37℃下消化5小时。其次,使用超滤管(MWCO 30kD)在8,000rpm下离心10分钟后收集渗透物,用HPLC在230nm处检测。
BCNU的包封率和载药率按下列公式计算:包封率(Ee,%)=(1-Wt/WP)x100%;载药率(LC,%)=Mp/Mt*100%,其中Wt和WP分别代表制作过程中所用BCNU的总重量和渗透过程中BCNU的重量,Mp和Mt分别代表封装在ANG-BSA/BCNU/ICG MNPs中的BCNU重量和ANG-BSA/BCNU/ICG MNPs的总重量。
使用动态透析法评估BCNU在37℃下分别在pH7.4(血浆)和pH5.5(肿瘤微环境)下的释放。将样品(1mL)置于透析袋(3-5KD)中,然后将其浸入含有50mL PBS的50mL离心管中,置于温度为37℃的水平摇动培养箱中,以120rpm/min的速度摇动。之后,从离心管中取出上述1mL样品,每隔一段时间用1mL新鲜缓冲液替换。每个样品中的BCNU浓度通过HPLC(Waters)在230nm处进行定量。
BCNU累积释放率(cumulative release rate,CR)(%)用以下公式计算:CR=[(50Cn+ΣCn-1)]/W0×100%,其中Cn和Cn-1分别为第n次和(n-1)次采样时释放溶液中相应的药物浓度,而W0为透析袋中ANG-BSA/BCNU/ICG MNPs封装的BCNU总重量。
通过HPLC检测BCNU的载药率,载药率结果为30微克BCNU/毫克ANG-BSA/BCNU/ICGMNPs,效率约为15%,这表明ANG-BSA/BCNU/ICG MNPs能容纳足够数量的BCNU。
图2显示了ANG-BSA/BCNU/ICG MNPs在pH7.4(血浆)和pH5.5(肿瘤微环境)下BCNU的释放情况,随着时间的推移,释放率逐渐增加;特别是在注射后118小时,pH7.4(血浆)和pH5.5(肿瘤微环境)的释放率分别高达44.84%和63.22%。
测试例3
测试实施例1制得的ANG-BSA/BCNU/ICG MNPs对U87MG和293T细胞存活率的影响。
人原代GBM细胞系U87MG和293T细胞在含有10%胎牛血清(FBS,Thermo FisherScien-tific)和100U/ml青霉素/链霉素(Invitrogen;Thermo Fisher Scien-tific)的Dulbecco改良Eagle培养基(DMEM,Thermo Fisher Scien-tific,Waltham,MA)中培养,加湿培养箱温度为37℃,CO2 5%。
至于细胞活力的检测,将细胞置于96孔板中(每孔5×103),然后与不同浓度(0、50、100、150和200纳克/毫升)的BCNU、BSA/BCNU/ICG MNPs(非靶向)和ANG-BSA/BCNU/ICGMNPs(靶向)培养48小时,通过MTT法检测评估每种药剂的生长抑制率。光密度(opticaldensity,OD)是用多功能酶标仪(PerkinElmer,MA,USA)在490nm波长下测量的。细胞存活率按以下公式计算:细胞存活率(%)=样品的OD/对照品的OD×100%。
参考图3A~图3B,分别评估了U87MG和293T细胞在BCNU、非靶向NPs(BSA/BCNU/ICGMNPs)和靶向NPs(ANG-BSA/BCNU/ICG MNPs)中的体外抗肿瘤能力。在U87MG细胞中,BCNU和BSA/BCNU/ICG MNPs之间的细胞存活率无明显差异,而与BSA/BCNU/ICG MNPs组或BCNU组相比,ANG-BSA/BCNU/ICG MNPs组的细胞存活率明显下降;此外,BCNU、BSA/BCNU/ICG MNPs和ANG-BSA/BCNU/ICG MNPs对细胞存活率呈剂量依赖改变(如图3A所示)。而在293T细胞中,BCNU、BSA/BCNU/ICG MNPs和ANG-BSA/BCNU/ICG MNPs组的细胞存活率没有明显差异;BCNU、BSA/BCNU/ICG MNPs和ANG-BSA/BCNU/ICG MNPs的细胞存活率也是剂量依赖的(图3B)。
如表1所示,BCNU和BSA/BCNU/ICG MNPs在U87MG细胞或293T细胞中的IC50没有明显差异,而与BSA/BCNU/ICG MNPs组或BCNU组相比,ANG-BSA/BCNU/ICG MNPs组在U87MG细胞中的IC50明显较低(48小时为三分之一),但293T细胞没有此改变。
总之,上述结果表明,与BSA/BCNU/ICG MNPs和BCNU相比,本发明的ANG-BSA/BCNU/ICG MNPs对胶质瘤的抗肿瘤性最好,同时具有靶向和杀伤作用。
表1:抗肿瘤活性指标
n.s:差异无统计学意义,BSA/BCNU/ICG MNPs与BCNU相比、ANG-BSA/BCNU/ICGMNPs与BSA/BCNU/ICG MNPs相比,差异无统计学意义;##:p<0.01,ANG-BSA/BCNU/ICG MNPs与BCNU相比,差异有统计学意义,**:p<0.01,ANG-BSA/BCNU/ICG MNPs与BSA/BCNU/ICGMNPs相比,差异有统计学意义。
测试例4
测试实施例1制得的ANG-BSA/BCNU/ICG MNPs的靶向能力。
当U87MG和293T细胞达到80%融合时,将ANG-BSA/ICG MNPs和BSA/ICG MNPs加入细胞中孵育4小时。PBS清洗后,用Hoechst试剂盒对细胞进行染色。使用共聚焦激光扫描显微镜系统(Leica,TCS SP8,德国)捕捉图像。
对于体外MRI成像,将部分细胞重新悬浮在1%琼脂糖中,扫描序列为T2WI(多回波脉冲序列),具体参数为:TR=3000毫秒,TE=22-352毫秒(16个TE),FOV=100×120mm2,图像矩阵=280×216,层厚=5mm,间距=1mm。
用荧光光谱仪评估纳米探针的荧光强度,以评价近红外荧光成像的性能。通过设置750nm波长的激发,可以获得800-900nm范围内的发射峰光谱。
参考图4,在U87MG细胞中,与BSA/BCNU/ICG MNPs组相比,ANG-BSA/BCNU/ICG MNPs组的红色荧光信号更强。然而,在用BSA/BCNU/ICG MNPs或ANG-BSA/BCNU/ICG MNPs处理的293T细胞中几乎观察不到红色荧光信号。
参考图5,我们分别将不同浓度的ANG-BSA/BCNU/ICG MNPs与293T和U87MG细胞孵化,然后收集处理后的细胞进行MRI检测,MRI图像显示了与荧光图像类似的结果(图5)。
测试例5
在动物体内测试实施例1制得的ANG-BSA/BCNU/ICG MNPs的靶向能力。
动物模型为雄性成年的异种原位GBM裸鼠,体重18-22g,购自云桥生物技术有限公司。所有的动物实验都获得了深圳市第二人民医院的机构动物护理委员会批准。
体内荧光成像
为了进行体内成像,将GBM裸鼠随机分两组,之后将ANG-BSA/BCNU/ICG MNPs和BSA/BCNU/ICG MNPs分别通过尾静脉注射到相应组的每只小鼠体内。在注射前和注射后0.5h、6h、12h、24h、48h分别采集图像。对于光学成像,使用IVIS成像光谱系统(PerkinElmer),其激发和发射波长分别为797nm和835nm。
结果参见图6A和图6B,可见:BSA/BCNU/ICG MNPs组的荧光信号在注射后6h首次检测到,24h达到峰值,然后逐渐减弱,在注射后48h几乎消失;而ANG-BSA/BCNU/ICG MNPs组的荧光信号在注射后30min首次检测到,此后,在12h达到峰值,在注射后48h保持稳定。此外,ANG-BSA/BCNU/ICG MNPs组的荧光信号在注射后12h和24h明显高于BSA/BCNU/ICG MNPs组。这些结果表明,ANG-BSA/BCNU/ICG MNPs可以明显穿过BBB,促进近红外成像能力。
体内MRI成像
作为补充,我们进行了体内MRI成像,结合荧光成像来证明ANG-BSA/BCNU/ICGMNPs的靶向性能。GBM裸鼠被随机分为三组。PBS;BSA/BCNU/ICG MNPs和ANG-BSA/BCNU/ICGMNPs。将上述各组小鼠经尾静脉注射,给药后12小时进行MRI检查。
参考图7,可见,与荧光靶向成像的结果一致,ANG-BSA/BCNU/ICG MNPs组在脑瘤区域出现了明显的强化,而BSA/BCNU/ICG MNPs则出现了相对较少的强化,在PBS组没有出现强化。结果显示,ANG-BSA/BCNU/ICGMNPs对胶质瘤有很强的靶向成像能力,能清晰显示肿瘤的早期大小和边界,而加载BCNU并不影响靶向MRI成像能力。也证实ANG-BSA/BCNU/ICGMNPs有助于胶质瘤的早期诊断和准确评估。
综上,本发明成功构建了针对胶质瘤的BBB通透性的双模态ANG-BSA/BCNU/ICGMNPs,既能在术前MRI中显影,又能在术中产生荧光,可以在诊疗的全过程中了解肿瘤的位置、轮廓和大小。
新合成的ANG-BSA/BCNU/ICG MNPs具有良好的生物相容性、较大的胶体稳定性、出色的BBB渗透能力和较强胶质瘤细胞的靶向性,且ANG-BSA/BCNU/ICG MNPs比BSA/BCNU/ICGMNPs或BCNU对胶质瘤细胞生长的抑制作用更强。
我们在细胞实验和动物实验中也证明了:ANG-BSA/BCNU/ICG MNPs可以在肿瘤部位聚集,提高药效,以及对胶质瘤的增殖有较好的抑制作用,有望成为治疗胶质瘤的重要候选药物。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对申请专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (2)
1.一种靶向探针,其特征在于,包括以下质量份数的组分:
1份~5份的靶向分子、0.1份~1份的药物分子、0.1份~1份的近红外染料、40份~60份的蛋白质载体和 8份~12份的磁共振造影剂;
所述靶向分子用于靶向脑胶质瘤,所述药物分子用于渗透血脑屏障,所述近红外染
料用于近红外荧光成像,所述靶向分子、所述药物分子、所述近红外染料、所述蛋白质载体和所述磁共振造影剂复合形成所述靶向探针;
所述药物分子选自亚硝脲类烷化剂;
所述靶向分子选自 Angiopep-2 多肽;
所述亚硝脲类烷化剂选自卡莫司汀;
所述蛋白质载体选自白蛋白;
所述磁共振造影剂选自超顺磁性氧化铁;
所述近红外染料选自吲哚菁绿;
所述靶向探针的制备方法,包括以下过程:
1)用无水苯甲醇制备 5%w/v的乙酰丙酮铁溶液,然后加热到 110℃,1 小时后,之后在氮气环境下加热到退火温度,40 小时后,将上述溶液冷却,用丙酮沉淀 SPIO NPs,彻底清洗并干燥;
2)将50mg BSA、10mg SPIO NPs和0.5mg ICG溶解在10毫升去离子水中;经过超声分散后,将pH值调整到9.0;将含有0.5mg BCNU的50mL无水酒精滴加到溶液中,然后在室温下搅拌直到出现沉淀;加入25%w/v 25μL的戊二醛溶液以交联BSA的氨基,从而形成纳米粒子;
3)将步骤 2)制得的的混合液以20000rpm/min离心30分钟以去除多余的 SPIONPs、ICG、BCNU和有机溶剂,得到纳米粒子 BSA/BCNU/ICG MNPs,再将纳米粒子重新分散在5mL的0.02M pH6.5的MES 缓冲液中;
4)在1mL 10mg/mL的BSA/BCNU/ICG MNPs 溶液中加入8微升 10mg/mL的EDC去离子水溶液和10微升 10mg/mL的NHS去离子水溶液以激活羧基;孵化25分钟后,将0.1mL 10mg/mL的ANG溶液加入到上述溶液中,混合物在室温下孵化2小时,然后转移到4℃下孵化过夜;
5)最后用超滤管MWCO 30kD以10000rpm/min的离心速度去除未耦合的ANG,最终获得靶向探针ANG-BSA/BCNU/ICG MNPs。
2.如权利要求1所述的靶向探针在制备脑胶质瘤成像的检测试剂盒或制备脑胶质瘤靶向药物中的应用。
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