CN111110657A - 可双模成像及靶向治疗乳腺癌的纳米微球及其制备方法 - Google Patents
可双模成像及靶向治疗乳腺癌的纳米微球及其制备方法 Download PDFInfo
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Abstract
本发明一种可双模成像及靶向治疗乳腺癌的纳米微球及其制备方法，包括以下组分：PLGA、Gd‑DTPA、PFP、ICG和AS1411，纳米微球以PLGA作为球壳膜，将Gd‑DTPA、PFP与ICG加载在PLGA壳膜内，利用PLGA微球所携带的羧基链接适配体AS1411。本发明制备的纳米微球外观为淡绿色乳状液，透射电镜下表现为形状规则，大小均一的微球，有明显的核壳结构。粒径均一，分散性好，包封率载药高，‑30 mV左右的电位保证了纳米微球在体内更稳定的存在。同时可以通过靶向作用和EPR效应使纳米微球在肿瘤部位聚集从而对肿瘤部位进行更好的US/MR成像和更精确的杀伤。制备方法中的各步骤反应所需的条件温和，易于操作。
Description
技术领域
本发明涉及一种可双模成像及靶向治疗乳腺癌的纳米微球及其制备方法，属于医学技术领域。
背景技术
乳腺癌是目前最常见的肿瘤之一。目前乳腺癌的诊断主要依靠影像学检查，如MR影像、US影像、CT等。然而，传统的单一成像模式已不能满足日益增长的乳腺癌早期诊断的医学需求。近年来，以多功能纳米颗粒为基础的纳米药物可以同时诊断和治疗癌症，可以提高临床疗效，减少副作用的治疗，为癌症的精确治疗提供创新的诊疗系统。
MRI是一种具有精细软组织对比和多平面成像能力的无创成像工具，但通常需要较长的采集时间，不能提供实时图像。相比之下，US可以提供实时图像，但在组织识别方面能力与磁共振相比略差。在许多临床应用中，US和MRI是互补的，以区别可能的病理改变组织。因此，两种模式的对比剂是必要的，而且比单模成像要方便得多。
目前，乳腺癌的治疗包括手术切除、化疗、放疗等。然而，这些方法也会对健康组织造成严重损害。因此，一种新型的肿瘤治疗方法-光热疗法(PTT)被提出。PTT的优势在于肿瘤组织特异性。在PTT治疗过程中，光敏剂在受到特定波长的光激发时将光转化为热。由于正常组织中血液循环正常，产生的热量可随血液循环及时扩散，而肿瘤组织中血管畸形、缺血缺氧，肿瘤组织产生的热量不利于增殖。因此，光敏剂可以将乳腺癌与正常组织区分开，提高其敏感性、特异性和准确性。同时，一些光敏剂可以吸收光，将能量传递给O2产生单态氧(1O2)，破坏细胞内各种生物大分子，最终导致肿瘤细胞坏死，实现光动力治疗(PDT)。为了达到更好的靶向性，除了利用EPR效应外，还可以通过在微球表面连接适配体达到更好的靶向效果。
发明内容
针对上述现有技术存在的问题，本发明提供一种可双模成像及靶向治疗乳腺癌的纳米微球及其制备方法。
为实现上述目的，本发明提供如下技术方案：一种可双模成像及靶向治疗乳腺癌的纳米微球，包括以下组分：PLGA、Gd-DTPA、PFP、ICG和AS1411，纳米微球以PLGA作为球壳膜，将Gd-DTPA、PFP与ICG加载在PLGA壳膜内，利用PLGA微球所携带的羧基链接适配体AS1411。
进一步的，所述组分用量为：
PLGA 10-100 mg；
Gd-DTPA 50-200 μL；
PFP 50-200 μL；
ICG 0.5-1.5 mg；
AS1411 1-10 OD。
组分优选用量为：
PLGA 20 mg；
Gd-DTPA 100 μL；
PFP 100 μL；
ICG 1 mg；
AS1411 2 OD。
一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，包括以下步骤：
A．将PLGA加入第一有机溶剂，得到第一混合物；
B．将Gd-DTPA与ICG混合，冰浴下加入PFP，得到第二混合物；
C．冰浴避光下，将第一混合物加入第二混合物超声乳化，得到第三混合物；
D．冰浴避光下，在高速（1500-2000r/min）磁力搅拌下，使用注射器将第三混合物于第二有机溶剂液面下缓慢注入,继续搅拌五分钟后，进行第二次超声乳化，得到第四混合物；
E．冰浴避光下，将第四混合物加入第三有机溶剂,低速（300-600r/min）磁力搅拌，使第一有机溶剂挥发；
F．超高速离心机4℃预冷，低速（1000-4000r/min，时间为20min）离心，收集上清；
G.上清高速（10000-15000r/min,时间为20min；离心洗涤的次数为3-5次）离心洗涤，收集沉淀；
H．沉淀真空冷冻干燥后收集备用；
I．冰浴避光下，将上述非靶向纳米微球溶解于缓冲液中，加入耦联活化剂，振荡孵育；
J. 离心（转速为10000-15000r/min, 时间为10min；离心洗涤的次数为3-5次）洗涤后，加入AS1411适配体孵育；
K．离心（10000-15000r/min,时间为20min；离心洗涤的次数为3-5次）洗涤次，得到靶向纳米微球。
进一步的，所述第一有机溶剂为1mL二氯甲烷。
进一步的，所述第二有机溶剂为质量百分比浓度是1%~10%的聚乙烯醇水溶液。
进一步的，所述第三有机溶剂为质量百分比浓度是0.1%~5%的聚乙烯醇水溶液。
进一步的，所述第一次超声乳化的方式为，脉冲式3s/3s,震荡时间为1-5min,能量输出为40%w。
进一步的，所述第二次超声乳化的方式为，脉冲式3s/3s,震荡时间为1-5min,能量输出为20%w。
进一步的，步骤I中缓冲液为MES缓冲液；使EDC/NHS的反应时具有更好的活性。
进一步的，所述步骤I中活化剂为EDC与NHS活化微球羧基端，为后续连接适配体做准备。
进一步的，所述步骤J中AS1411适配体3端修饰FAM为绿色荧光，便于后续直观检查适配体的连接与否于靶向效果靶向，5端修饰氨基用来和纳米微球表面羧基反应连接适配体。
与现有技术相比，本发明制备的纳米微球外观为淡绿色乳状液，透射电镜下表现为形状规则，大小均一的微球，有明显的核壳结构。粒径均一，分散性好，包封率载药高，-30mV左右的电位保证了纳米微球在体内更稳定的存在。同时可以通过靶向作用和EPR效应使纳米微球在肿瘤部位聚集从而对肿瘤部位进行更好的US/MR成像和更精确的杀伤。制备方法中的各步骤反应所需的条件温和，易于操作。
附图说明
图1为靶向纳米微球透射电镜图；
图2为靶向纳米微球扫描电镜图；
图3为靶向纳米微球粒径分布图；
图4 为ICG标准曲线图；
图5为在808 nm激光照射下(1.5W/cm2,3 min)，纯水和不同ICG浓度的靶向纳米微球的升温曲线图；
图6为在808 nm激光照射下(1.5W/cm2)，通过单态氧传感器(SOSG)分析1O2的产生曲线图；
图7 为在808 nm激光照射下(1.5W/cm2)，活死细胞染色示意图；A：Control, B：靶向纳米微球, C: Laser, D: 靶向纳米微球+Laser；
图8 为不同浓度MDA-MB-231细胞在808 nm NIR激光照射下(1.5 W/cm2)的细胞存活率以平均值±SD表示图；* p < 0.05，(n = 6)；
图9为显微镜下激光照射(808 nm, 1.5W/cm2)前后靶向纳米微球的变化图；
图10为胶管内激光照射(808 nm, 1.5W/cm2)前后靶向纳米微球的US成像图；
图11为使用3.0 T MR扫描仪进行不同Gd浓度下的T1W1和伪彩色图像；
图12 为MDA-MB-231细胞MR成像(T1W1)和不同浓度靶向纳米微球的伪彩色图像；
图13为靶向纳米微球摄入荧光示意图；
图14为靶向纳米微球分散在不同溶液中粒径变换图；
图15 为靶向纳米微球对细胞的毒性图。
具体实施方式
下面结合附图对本发明作进一步说明。
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做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。
本发明提供一种纳米微球，主要采用“水包油包水”的双乳化法，以PLGA作为球壳膜，将Gd-DTPA、PFP与ICG加载在PLGA壳膜内，利用PLGA微球所携带的羧基链接适配体AS1411；本发明选降最终解为二氧化碳和水的聚乳酸(PLGA)作为微球的载体。PLGA作为一个平台，可以很容易地将两个或多个组件合并到一个粒子系统中。Gadolinium-DTPA (Gd-DTPA)作为MRI T1加权造影剂包裹在PLGA内。Gd-DTPA是一种水溶性分子，进入体内后很快被清除。然而，将Gd-DTPA包埋在PLGA中可以增加其在体内的循环时间，为MRI成像提供足够的时间。
目前在US成像中研究最多的造影剂是微气泡，微气泡在血液中不稳定，无法长期成像发展，重复性差。大气泡难以通过肺毛细血管网，限制了其临床应用。为了克服这些缺点，我们使用了第三代对比剂全氟戊烷(PFP)。当相变温度达到29℃时，PFP由液态转变为气态，增强US成像，气相PFP具有较高的生物安全性和良好的稳定性。由于PFP包裹在PLGA中，相变温度升高。为了达到相变温度，我们加入了光敏剂吲哚菁绿(ICG)。近红外激光可以激发具有高光热转换效率的ICG。近红外波长范围位于人体组织的光学窗口(700-1000)nm处，可以保证足够的穿透深度，最大限度地减少对正常组织结构的损伤。在近红外激光照射下，ICG可以迅速提高温度，引起PTT/PDT，促进PFP的液气相变。
AS1411适配体（AS1411 aptamer）是含有26个碱基的G4序列核酸适配体，能够特异性识别核仁素。核仁素在持续增殖的细胞高度表达，如肿瘤细胞（肾癌、宫颈癌、乳腺癌等），已有研究结果表明，AS1411可以选择性靶向乳腺癌细胞。因此，以AS1411为靶向分子的磁共振纳米探针具有特异性识别肿瘤细胞的特性。
优选的，PLGA、Gd-DTPA、PFP、ICG与AS1411的用量为：
PLGA 10-100 mg；
Gd-DTPA 50-200 μL；
PFP 50-200 μL；
ICG 0.5-1.5 mg；
AS1411 1-10 OD。
优选的，PLGA选择LG:LA：50:50, MW: 10000，此种PLGA制备而成纳米微球不易产生粘连且微球形态好，包封率高，且PLGA是羧基修饰的便于后续的靶向修饰。
纳米微球的制备方法为：
A．将PLGA加入二氯甲烷，得到第一混合物；
B．将Gd-DTPA与ICG混合，冰浴下加入PFP，得到第二混合物；
C．冰浴避光下，将第一混合物加入第二混合物超声乳化，超声乳化条件为：脉冲式3s/3s,震荡时间为1-5min,能量输出为40%w，得到第三混合物；
D．冰浴避光下，在高速（1500-2000r/min）磁力搅拌下，使用注射器将第三混合物于聚乙烯醇水溶液液面下缓慢注入,继续搅拌五分钟后，进行第二次超声乳化，超声乳化条件为：脉冲式3s/3s,震荡时间为1-5min,能量输出为20%w，得到第四混合物；
E．冰浴避光下，将第四混合物加入聚乙烯醇水溶液,低速（300-600r/min）磁力搅拌，使二氯甲烷挥发；
F．超高速离心机4℃预冷，低速（1000-4000r/min）离心20min，离心3-5次，收集上清；
G.上清高速（10000-15000r/min）离心洗涤20min，离心3-5次，收集沉淀；
H．沉淀真空冷冻干燥后收集备用；
I．冰浴避光下，将上述非靶向纳米微球溶解于MES缓冲液中，加入EDC与NHS活化微球羧基端，振荡孵育30min；
J. 离心（转速为10000-15000r/min）洗涤10min，离心洗涤3-5次后，加入AS1411适配体孵育；
K．离心（10000-15000r/min）洗涤20min，离心洗涤3-5次后，得到靶向纳米微球。
下面结合实施列，进一步阐明本发明
一、制备材料：
聚乳酸-羟基乙酸共聚物（PLGA，分子量10000，聚合比 50：50， Evonik），氯化亚锰（MnCl2，北京，北陆药业），全氟戊烷(PFP，上海，阿拉丁)，ICG（上海，麦克林），聚乙烯醇（PVA; Sigma）；AS1411(上海，生工生物工程)；单线态氧试剂盒(SOSG)、2,7 -二氯二氢荧光素(DCFH-DA)、钆喷酸普安 (Magnevist, Gd-DTPA)（德国，Bayer Pharma AG)。MDA-MB-231,NIH-3T3细胞株（中科院上海细胞库），细胞计数试剂盒(CCK-8，大连，美仑)。
二、纳米微球的制备：
将20 mg PLGA加入1 mL 二氯甲烷，得到第一混合物；
将100 μL Gd-DTPA与100 μL ICG混合，冰浴下加入100 μL PFP，得到第二混合物；
冰浴避光下，将第一混合物加入第二混合物超声乳化，超声乳化条件为：脉冲式3s/3s,震荡时间为1min,能量输出为40%w，得到第三混合物；
冰浴避光下，在高速2000r/min磁力搅拌下，使用注射器将第三混合物于质量百分比浓度是4%的聚乙烯醇水溶液液面下缓慢注入，继续搅拌五分钟后，进行第二次超声乳化，超声乳化条件为：脉冲式3s/3s，震荡时间为4min，能量输出为20%w，得到第四混合物；
冰浴避光下，将第四混合物加入质量百分比浓度是1%的聚乙烯醇水溶液，低速400r/min磁力搅拌，使二氯甲烷挥发；
超高速离心机4℃预冷，低速1500r/min离心20min，离心3次后收集上清；
上清高速11000r/min离心洗涤20min，离心3次后收集沉淀即非靶向纳米微球；
沉淀真空冷冻干燥后收集备用；
冰浴避光下，将上述非靶向纳米微球溶解于0.1 mol/L，pH=5的MES缓冲液中，加入EDC∶NHS 摩尔比为 1∶1，PLGA∶EDC 摩尔比为 1∶5的EDC与NHS活化微球羧基端，振荡孵育30min；
在转速为11000r/min条件下离心洗涤10min，离心洗涤3次后，加入PLGA∶AS1411摩尔比为 1∶1的AS1411适配体孵育；
在转速为11000r/min条件下离心洗涤20min，离心洗涤3次后，得到靶向纳米微球。
三、纳米微球的表征：
透射电镜和扫描电镜表征靶向纳米微球的结构和形貌。DSL分析仪测量纳米微球尺寸分布和zeta电位。
结果：
如图1-4所示，采用改进的双乳液法合成了靶向纳米微球。所制备的纳米粒子具有明显的核/壳结构，粒径均匀，分散性强，靶向纳米微球的尺寸为217±4.1 nm左右，电位-31mV左右，ICG的包封率为76.38±0.32%，载药量为6.99±3.98%。
四、纳米微球光热光动力效果：
以超纯水为对照组，将靶向纳米微球根据ICG浓度，配制成5μg/mL，15μg/mL，25μg/mL，35μg/mL和45μg/mL的溶液，取100μL 置于96孔板中。给予808nm激光辐照（1.5w/cm2，5min），用红外热成像仪记录各组温度变化。
纳米微球用808nm近红外激光辐照(1.5 W/cm2)后使用单态氧传感器(SOSG)探针检测体外ROS生成。
材料与细胞孵育6h后处理四个分组，激光照射后用calcein-AM和PI处理细胞，采用钙黄绿素染色法定性考察光热/光动力治疗效果。用CCK-8定量考察光热/光动力治疗效果。
结果：
如图5所示，经过808 nm激光辐照后，靶向纳米微球升温显著，与ICG的浓度呈正相关。如图6所示，可以看激光照射后ROS产生且与照射时间成正相关。已知calcein-AM染色后活细胞呈绿色，PI染色死细胞/凋亡细胞呈红色。如图7-8所示，仅激光照射，靶向纳米微球处理对细胞无明显杀伤作用。然而，仅在近红外激光照射和靶向纳米微球治疗联合使用时，有大量的死细胞，说明近红外激光照射联合使用靶向纳米微球具有良好的肿瘤消融能力。
五、纳米微球超声成像效果：
为了观察到靶向纳米微球液气相变过程，使用去离子水稀释靶向纳米微球乳剂，滴于载玻片上。将载玻片置于显微镜下，激光照射(1.5W/cm2)下观察靶向纳米微球的变化。
为了观察靶向纳米微球超声成像效果，将靶向纳米微球转入透明塑胶软管(d=2mm)中，将软管浸入水中，采用808激光照射(1.5W/cm2)，使用超声成像仪观察是否成像。
结果：
如图9所示，在显微镜下，可以观察到，在808nm近红外激光照射前，靶向纳米微球没有发生相变。激光照射后，小颗粒逐渐变成微气泡。随着辐照时间的延长，微泡膨胀到一定体积后开始破裂。
如图10所示，在808nm 近红外激光照射前，透明塑胶软管中靶向纳米微球的超声成像效果没有增强。当激光照射后，PFP气化，产生气泡，并发生明显的超声成像增强。
六、纳米微球磁共振成像：
为验证靶向纳米微球磁共振增强成像效果，将靶向纳米微球配制成不同浓度的水溶液。在3.0T Discovery 750 W MR系统上对靶向纳米微球进行t1加权成像研究。用自旋回波序列对稀释后的样品进行MRI扫描。
此外，利用MDA-MB-231细胞的体外MR成像试验评价其对乳腺癌细胞的磁共振成像能力。MDA-MB-231细胞以1×105细胞/孔在接种在6孔板37℃孵育12 h,然后加入不同浓度(0、20、40、80、160μg /mL)靶向纳米微球孵育6小时。将细胞消化离心收集并用PBS洗涤，使用前面描述的磁共振成像系统进行磁共振成像。
如图11所示，靶向纳米微球的MRI信号强度随着Gd浓度的增加而增强，证明靶向纳米微球具有良好的磁共振成像效果。
如图12所示，可以看出靶向纳米微球在细胞中具有很好的磁共振成像能力，并且是浓度依赖性的，得到的结果与之前的结果一致。
七、纳米微球靶向效果：
将靶向纳米微球于MDA-MB-231与NIH-3T3细胞共同孵育6h,制片后于倒置荧光显微镜观察适配体连接效果和靶向性。
如图13所示，由于适配体表面修饰有绿色荧光，在和细胞孵育后，于倒置荧光显微镜下可以观察到，乳腺癌细胞出现了明显的绿色荧光，而NIH-2T3细胞则具有比较少的荧光，证明了适配体的成功连接和纳米微球的靶向性。
八、稳定性和生物安全性：
将靶向纳米微球分散在DI水、PBS、FBS、DMEM等不同溶液中测量其七天内粒径变化。
选用MDA-MB-231和NIH-3T3细胞在与纳米微球共处理，采用细胞计数试剂盒(CCK-8)检测纳米微球的细胞毒性。
结果：
如图14所示，靶向纳米微球在DI水、PBS、FBS、DMEM等不同溶液中均表现出良好的分散性，保证了血液循环时间较长。此外，与PBS、DMEM和FBS混合后7天内未观察到明显的粒径变化。
如图15所示，CCK-8试验结果显示当浓度到达160μg/mL时，靶向纳米微球对MDA-MB-231和NIH-3T3细胞均无明显细胞毒性，经单因素方差分析比较，不同浓度吸光度值差异无统计学意义，且细胞活力都在90%以上。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其它的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
以上所述，仅为本发明的较佳实施例，并不用以限制本发明，凡是依据本发明的技术实质对以上实施例所作的任何细微修改、等同替换和改进，均应包含在本发明技术方案的保护范围之内。
Claims (10)
1.一种可双模成像及靶向治疗乳腺癌的纳米微球，其特征在于，包括以下组分：PLGA、Gd-DTPA、PFP、ICG和AS1411，纳米微球以PLGA作为球壳膜，将Gd-DTPA、PFP与ICG加载在PLGA壳膜内，利用PLGA微球所携带的羧基链接适配体AS1411。
2.根据权利要求1所述的一种可双模成像及靶向治疗乳腺癌的纳米微球，其特征在于，所述组分用量为：
PLGA 10-100 mg；
Gd-DTPA 50-200 μL；
PFP 50-200 μL；
ICG 0.5-1.5 mg；
AS1411 1-10 OD。
3.根据权利要求1或2所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于包括以下步骤：
A．将PLGA加入第一有机溶剂，得到第一混合物；
B．将Gd-DTPA与ICG混合，冰浴下加入PFP，得到第二混合物；
C．冰浴避光下，将第一混合物加入第二混合物超声乳化，得到第三混合物；
D．冰浴避光下，在高速磁力搅拌下，使用注射器将第三混合物于第二有机溶剂液面下缓慢注入,继续搅拌五分钟后，进行第二次超声乳化，得到第四混合物；
E．冰浴避光下，将第四混合物加入第三有机溶剂,低速磁力搅拌,使第一有机溶剂挥发；
F．超高速离心机4℃预冷，低速离心，收集上清；
G．上清高速离心洗涤，收集沉淀；
H．沉淀真空冷冻干燥后收集备用；
I．冰浴避光下，将上述非靶向纳米微球溶解于缓冲液中，加入耦联活化剂，振荡孵育；
J．离心洗涤后，加入AS1411适配体孵育；
K．离心洗涤次，得到靶向纳米微球。
4.根据权利要求3所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于：所述第一有机溶剂为1mL二氯甲烷。
5.根据权利要求3所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于：所述第二有机溶剂为质量百分比浓度是1%~10%的聚乙烯醇水溶液。
6.根据权利要求3所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于：所述第三有机溶剂为质量百分比浓度是0.1%~5%的聚乙烯醇水溶液。
7.根据权利要求3所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于：所述第一次超声乳化的方式为，脉冲式3s/3s,震荡时间为1-5min,能量输出为40%w。
8.根据权利要求3所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于：所述第二次超声乳化的方式为，脉冲式3s/3s,震荡时间为1-5min,能量输出为20%w。
9.根据权利要求3所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于：所述步骤D中高速磁力搅拌的转速为1500-2000r/min。
10.根据权利要求3所述的一种可双模成像及靶向治疗乳腺癌的纳米微球的制备方法，其特征在于：所述步骤E中低速磁力搅拌的转速为300-600r/min。
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