CN113398286B - 一种靶向载铁氧体多功能纳米粒及其制备方法和应用 - Google Patents
一种靶向载铁氧体多功能纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及纳米动脉粥样硬化斑块治疗药物以及显像剂技术领域，具体涉及一种靶向载铁氧体多功能纳米粒及其制备方法和应用。一种靶向载铁氧体多功能纳米粒，包括由聚乳酸‑羟基乙酸共聚物形成的外壳，外壳中包裹有尖晶石铁氧体和血卟啉单甲醚。本方案解决了现有的成像剂的安全性和灵敏度不能满足动脉粥样硬化易损斑块的检测需求的技术问题。该纳米粒是一种安全性和灵敏性均较为理想的MRI、光声和超声成像剂，可用于检测和识别动脉粥样硬化易损斑块；纳米粒还可以通过声动力疗法对动脉斑块新生血管进行有效干预，可用于动脉粥样硬化斑块新生血管的早期识别和治疗的医疗实践中。
Description
技术领域
本发明涉及纳米动脉粥样硬化斑块治疗药物以及显像剂技术领域，具体涉及一种靶向载铁氧体多功能纳米粒及其制备方法和应用。
背景技术
动脉粥样硬化(Atherosclerosis，AS)易损斑块破裂导致的脑卒中(脑梗死和脑出血)、急性心肌梗死及下肢动脉闭塞病变等疾病是造成人类致残、致死的主要原因。动脉粥样硬化易损斑块的形成与异常增殖的新生血管密切相关。因此，早期识别和有效干预动脉粥样硬化斑块新生血管对于预防易损斑块破裂、血栓形成以及急性心脑血管事件发生具有至关重要的意义。
动脉粥样硬化斑块新生血管成像技术包括体表多普勒血管超声造影，动态对比增强核磁成像以及光学相干断层成像。这些传统的影像技术都无法对斑块新生血管进行细胞和分子水平的成像，而纳米分子探针的构建很好的解决了这一问题。2003年，Winter等人构建了包载Gd-DTPA-BOA的靶向αvβ3整合素的顺磁性纳米粒，实现了对兔主动脉粥样硬化斑块新生血管的MRI分子成像。但是，顺磁性钆对比剂具有较大的体内毒性，导致肾功能不全病人发生肾源性系统性纤维化的风险明显增加。因此，Su等人构建了生物相容性好，低毒的Fe3O4纳米粒(68Ga-NGD-MNPs)，对兔主动脉的动脉粥样硬化斑块新生血管进行了MRI和PET双模态分子成像。但是，与钆对比剂的T1阳性成像相比，Fe3O4的T2阴性成像的空间分辨率和灵敏性存在明显的不足，而且对比信号容易与出血，钙化以及伪影相混淆。由此可见，寻找一种安全、灵敏性高的T1阳性对比剂用于MRI分子成像，对于早期特异性的检测新生血管进而识别动脉粥样硬化易损斑块具有重要意义。
发明内容
本发明意在提供一种靶向载铁氧体多功能纳米粒，以解决现有的成像剂安全性和灵敏度不能满足动脉粥样硬化易损斑块的检测需求的技术问题。
为达到上述目的，本发明采用如下技术方案：
一种靶向载铁氧体多功能纳米粒，其包括由聚乳酸-羟基乙酸共聚物形成的外壳，所述外壳上镶嵌有尖晶石铁氧体。
本方案的原理及优点是：本方案使用聚乳酸-羟基乙酸共聚物(PLGA)包裹尖晶石铁氧体，获得的多功能纳米粒具有核磁共振成像和光声成像的效果。相对于现有技术常规的Fe3O4纳米粒，在核磁共振成像(MRI)中，T1阳性成像的空间分辨率和灵敏性得到了较大的提升。相对于现有技术的顺磁性钆对比剂，对细胞和小鼠没有明显毒性，具有良好的生物相容性。因此，本方案的纳米粒能够安全有效的进行核磁共振T1阳性成像。以尖晶石铁氧体为核心的纳米粒可以代替现有技术的Fe3O4纳米粒和顺磁性钆纳米粒作为检测斑块新生血管的理想的核磁共振分子探针。尽管核磁共振具有很好的软组织分辨率，并且能够对斑块成分进行定量分析，但是，核磁共振较差的时间分辨率限制了它在快速运动血管中的使用，而使用尖晶石铁氧体可以克服上述问题。光声成像技术(Photoacoustic imaging,PAI)是一种具有高分辨率的无创成像技术。尖晶石铁氧体具有光声成像的性质，使用尖晶石铁氧体制备的纳米粒具有多模态成像的性质，可将光声成像和核磁共振成像结合起来，充分利用各个成像模式的优势，使得对斑块新生血管的分子成像更加精准。其中，尖晶石铁氧体(spinel ferrites)是晶体结构与天然矿物尖晶石MgAl2O4相同的铁氧体，又称磁性尖晶石。分子式为MFe2O4，式中M为二价金属离子，常见的M包括Mn、Mg、Co、Cu、Zn、Ni、Fe等。
进一步，所述外壳上还镶嵌有血卟啉单甲醚。
采用上述技术方案，血卟啉单甲醚(HMME)是一种稳定、安全的卟啉声敏剂，使用该声敏剂进行声动力治疗，相对于现有技术的华卟啉钠介导的声动力疗法(DVDMS-SDT)具有显著优势。在低强度聚焦超声(low-intensity focused ultrasound,LIFU)的激发下，超声激活纳米粒中的声敏剂HMME，产生大量活性氧(ROS)，诱导斑块新生血管内皮细胞凋亡，进而抑制斑块血管新生，最终稳定斑块。发明人在研究声动力治疗的过程中，在纳米粒中加入卟啉声敏剂以期获得理想的声动力治疗效果，意外发现，HMME和尖晶石铁氧体联合使用具有协同加强光声成像的效果，提高了纳米粒的显像效果，使得纳米粒可以作为一种更为优化的医学显像剂。
进一步，所述尖晶石铁氧体为MnFe2O4。
采用上述技术方案，相对于其他尖晶石铁氧体FeFe2O4，CoFe2O4，NiFe2O4的体内外核磁共振成像效果，MnFe2O4纳米粒的核磁共振T2成像灵敏度最好，而且对细胞和小鼠没有明显毒性。另外，HMME和MnFe2O4虽然分别具有光声成像作用，当HMME和MnFe2O4同时包载在纳米粒中时(详见实验例5)，所得到的光声成像信号要明显比HMME和MnFe2O4的光声成像信号值之和更高。这个现象提示HMME和MnFe2O4同时包载在PLGA上时产生了协同作用，明显提高了它们的光声成像能力，这种明显的光声成像增强作用更有利于斑块新生血管的早期诊断。
进一步，所述外壳上共价连接有雷莫芦单抗。
采用上述技术方案，雷莫芦单抗(Ramucirumab,Ram)是FDA批准的用于阻断VEGFR2的单克隆抗体(VEGF/VEGFR2是参与和促进斑块血管新生的重要通路)，被用于抑制肿瘤新生血管。含雷莫芦单抗的纳米粒能够主动靶向聚集在斑块新生血管内皮细胞的线粒体中，增加纳米粒在斑块新生血管中的聚集。
进一步，其还包括液态氟碳形成的内核。
采用上述技术方案，在超声激发下，声致相变作用使液态氟碳(PFP)气化为微泡，并作为超声成像对比剂。与光声成像相比，超声成像不仅无创，并且穿透能力更强。因此，使用多模态成像(MRI，光声成像，超声成像)的方式可以弥补MRI成像的不足，并且充分利用各个成像模式的优势，使得对斑块新生血管的分子成像更加精准。
综上所述，动脉粥样硬化斑块新生血管治疗手段的探索主要包括抗血管新生药物和VEGF-A(Vascular endothelial growth factor-A)阻断剂。抗血管新生药物Endostatin或TNP-470虽然可以抑制ApoE-/-小鼠斑块新生血管，但毒性较大无法临床使用。VEGF-A阻断剂临床用于抑制肿瘤病人的新生血管，但由于干扰了正常内皮细胞的稳态，导致肿瘤病人发生高血压，蛋白尿以及血栓栓塞等严重并发症，难以在动脉粥样硬化斑块病人中长期使用。由此可见，针对动脉粥样硬化斑块新生血管，目前还缺乏一种安全有效，无创靶向的治疗手段。另外，现有技术的华卟啉钠介导的声动力疗法(DVDMS-SDT)通过巨噬细胞凋亡诱导的内皮细胞凋亡途径有效的抑制了斑块新生血管。但是，我们无法实时监测活体内声敏剂的分布，不仅如此，DVDMS-SDT还不能直接作用到斑块新生血管内皮细胞，这些问题严重限制了声动力疗法的临床使用和治疗效率。
为了优化和监测声动力治疗斑块新生血管的效果，我们利用PLGA包裹液态氟碳(PFP)、MnFe2O4和HMME，并在PLGA表面键连抗VEGFR2抗体雷莫芦单抗，合成了本方案的(PFP-HMME@PLGA/MnFe2O4-ramucirumab纳米粒)。该纳米粒能够主动靶向聚集在斑块新生血管内皮细胞的线粒体中，并对斑块新生血管进行MRI/PA/US(核磁、光声和超声)多模态分子成像。在LIFU的激发下，超声激活纳米粒中的声敏剂，产生大量活性氧(ROS)，诱导斑块新生血管内皮细胞凋亡，进而抑制斑块血管新生，最终稳定斑块。
本发明还采用如下技术方案：
一种靶向载铁氧体多功能纳米粒的制备方法，包括如下依次进行的步骤：
S1：合成PLGA-PEG5000-ramucirumab；
S2：将PLGA-PEG5000-ramucirumab溶于三氯甲烷中，然后加入血卟啉单甲醚、MnFe2O4和液态氟碳，获得混合体系；然后对混合体系进行声震乳化，获得初乳液；
S3：在初乳液中滴加聚乙烯醇，然后再次进行声震乳化，获得复乳液；
S4：在复乳液中加入异丙醇溶液，冰浴后离心获得靶向载铁氧体多功能纳米粒。
进一步，在S1中，PLGA-PEG5000-ramucirumab由如下方法合成：将聚乳酸-羟基乙酸共聚物溶于二氯甲烷中，再加入N-羟基琥珀酰亚胺和催化剂，经反应获得PLGA-NHS；将PLGA-NHS溶解于二氯甲烷中，加入NH2-PEG-COOH和催化剂，经反应获得PLGA-PEG5000-COOH；将PLGA-PEG5000-COOH溶于二氯甲烷中，并加入雷莫芦单抗和催化剂，经反应获得PLGA-PEG-NHS-ramucirumab。
进一步，靶向载铁氧体多功能纳米粒的血卟啉单甲醚的包封率为83.56±2.47％，载药量为3.18±0.1％；靶向载铁氧体多功能纳米粒的MnFe2O4的包封率为80.51±2.21％，载药量为1.93±0.2％。
本方案的原理及优点是：本方法可制备获得本方案的纳米粒，该纳米粒具有多模态成像性质、可靶向斑块新生血管内皮细胞的线粒体、且能够诱导斑块新生血管内皮细胞凋亡，进而抑制斑块血管新生，最终稳定斑块。在合成的PFP-HMME@PLGA/MnFe2O4-ramucirumab纳米粒中，MnFe2O4可作为MRI和PA成像对比剂。HMME作为稳定、安全的卟啉声敏剂。在超声激发下，声致相变作用使PFP气化为微泡，并作为超声成像对比剂。雷莫芦单抗用于靶向斑块新生血管内皮细胞的线粒体。按照上述的合成方法，HMME的包封率可达83.56±2.47％，载药量可达3.18±0.1％；MnFe2O4的包封率可达80.51±2.21％，载药量可达1.93±0.2％。
本发明还采用如下技术方案：
一种靶向载铁氧体多功能纳米粒在制备显像剂中的应用。
本方案的原理及优点是：在LIFU的作用下，本方案的纳米粒具有MRI/PA/US多模态成像的效果，可以用于监测纳米粒在斑块中的分布并指导治疗，详见实验例4、实验例5和实验例6记载的内容。并且，HMME和MnFe2O4同时包载在PLGA上时产生了协同作用，明显提高了它们的光声成像能力，这种明显的光声成像增强作用更有利于斑块新生血管的早期诊断。
本发明还采用如下技术方案：
一种靶向载铁氧体多功能纳米粒在制备动脉粥样硬化易损斑块抑制药物中的应用。
本方案的原理及优点是：多模态成像指导下的声动力治疗最终能够有效的抑制斑块新生血管。本项研究的实施为临床早期诊断、实时监测以及有效治疗斑块新生血管提供了一种新的策略，详见实施例7中记载的内容。本方案合成的纳米粒介导的声动力疗法使兔股动脉斑块新生血管密度减少了59.1％，而DVDMS-SDT仅仅使兔股动脉斑块新生血管密度减少了52.9％，这个结果提示在抑制新生血管密度方面，本方案的纳米粒介导的声动力疗法要优于DVDMS-SDT。
附图说明
图1为本发明实验例1的PFP-HMME@PLGA/MnFe2O4-Ram纳米粒的粒径分布图。
图2为本发明实验例1的PFP-HMME@PLGA/MnFe2O4-Ram纳米粒的透射电镜图。
图3为本发明实验例1的不同纳米粒和处理条件下DPBF消耗量检测结果。
图4为本发明实验例1的纳米粒的高分辨透射电镜元素扫描结果。
图5为本发明实验例1的纳米粒的透射电镜线性扫描结果。
图6为本发明实验例1的不同纳米粒的紫外-可见吸收光谱图。
图7为本发明实验例1的纳米粒的磁滞回线图。
图8为本发明实验例2的纳米粒靶向兔主动脉内皮细胞的检测实验结果(DiI-标记的纳米粒和PLGA-PEG5000-ramucirumab共聚焦图像)。
图9为本发明实验例2的纳米粒靶向兔主动脉内皮细胞的检测实验结果(DiI-标记的靶向纳米粒和RAEC共孵育的共聚焦图像)。
图10为本发明实验例3的荧光共聚焦显微图像(Dil标记的PFP-HMME@PLGA/MnFe2O4-Ram纳米粒和线粒体探针)。
图11为本发明实验例3的荧光共聚焦显微图像(Dil标记的PFP-HMME@PLGA/MnFe2O4-Ram纳米粒和溶酶体探针)。
图12为本发明实验例3的荧光共聚焦显微图像(Dil标记的PFP@PLGA/MnFe2O4-Ram纳米粒和线粒体探针)。
图13为本发明实验例3的Mander共定位分析结果。
图14为本发明实验例3的procaspase 3和活化caspase 3的WB实验结果。
图15为本发明实验例3的活化的caspase 3蛋白相对于actin表达量的半定量分析结果。
图16为本发明实验例4的不同浓度的纳米粒子T1加权成像图和数值。
图17为本发明实验例4的兔股动脉斑块进行T1加权成像图像。
图18为本发明实验例4的不同组中和不同时间点斑块处的T1信号值的统计图。
图19为本发明实验例5的不同物质的光声成像图。
图20为本发明实验例5的不同物质的光声成像的定量分析结果。
图21为本发明实验例6的B型和造影(CEUS)图像。
图22为本发明实验例6的量化的回声强度统计图。
图23为本发明实验例6的激发前后兔股动脉斑块的B型和造影图像。
图24为本发明实验例6的LIFU激发前后各组的回声强度对比图。
图25为本发明实验例7的各组代表性的组织病理学染色股动脉斑块切片的显微图像。
图26为本发明实验例7的阳性区域定量分析统计图。
图27为本发明实验例8的各处理组血液检查结果统计图。
图28为本发明实验例8的各处理组实验兔不同时间的体重变化的统计图。
图29为本发明实验例8的对主要器官(心，肝，脾，肺，肾)进行切片HE染色的显微观察图。
具体实施方式
下面结合实施例对本发明做进一步详细的说明，但本发明的实施方式不限于此。若未特别指明，下述实施例以及实验例所用的技术手段为本领域技术人员所熟知的常规手段，且所用的材料、试剂等，均可从商业途径得到。
实施例1：靶向载铁氧体多功能纳米粒的制备
(1)PLGA-PEG5000-ramucirumab的合成
PLGA-PEG5000-ramucirumab的合成委托重庆浦诺维生物科技有限公司进行，PLGA(聚乳酸-羟基乙酸共聚物)通过PEG5000(聚乙二醇5000)共价连接上ramucirumab(雷莫芦单抗)，PLGA-PEG5000-ramucirumab的大致合成流程如下：
(1.1)PLGA-NHS制备
取PLGA干粉完全溶于DCM(二氯甲烷)中。加入适量的NHS(N-羟基琥珀酰亚胺)和相应催化剂，室温(20-25℃)条件下搅拌过夜。反向层析纯化，溶液低压抽干，得到PLGA-NHS样品。
(1.2)PLGA-PEG5000-COOH制备
将PLGA-NHS样品用DCM溶成原体积，加入适量NH2-PEG-COOH，补入适量催化剂反应，室温条件下搅拌过夜。然后，DDW(双蒸水)透析过夜。将透析后的样品取出，反向层析纯化，冻干。
(1.3)PLGA-PEG5000-NHS接ramucirumab
将PLGA-PEG5000-NHS溶于DCM，将过量的PLGA-PEG5000-NHS加入等体积的ramucirumab溶液(雷莫芦单抗)，补入相应催化剂，在冰水浴条件下搅拌过夜。将反应后的溶液低压抽干，复溶后离子层析和反向层析纯化。冻干得到PLGA-PEG5000-ramucirumab样品，-20℃保存。
(2)PFP-HMME@PLGA/MnFe2O4-ramucirumab纳米粒的合成
采用双乳化法，以水包油包水的原理制备PFP-HMME@PLGA/MnFe2O4-ramucirumab(简写为PFP-HMME@PLGA/MnFe2O4-Ram)纳米制剂。具体步骤如下：首先将PLGA-PEG5000-ramucirumab(50mg)溶于2mL的CHCl3溶液中；待PLGA-PEG5000-ramucirumab充分溶解后，先后向其中加入2mg HMME(血卟啉单甲醚)，3nm的MnFe2O4(160μl，8mg/ml)和PFP(液态氟碳，200μL)；使用超声波破碎仪(功率133W,乳化时长30秒)对上述混合溶液进行乳化，得到浅棕色初乳液。随后将初乳液缓慢滴加至8mL的PVA(聚乙烯醇，4％w/v)中；使用超声波破碎仪(功率133W,乳化时长30秒)再次乳化上述溶液，得到浅棕色复乳液；再向复乳液中加入10mL的异丙醇溶液(2％v/v)，4℃条件下冰浴6小时；待CHCl3充分挥发后，使用低温高速离心机，以11000rpm的转速离心上述乳剂7分钟，超纯水洗涤后储存于4℃环境下。
对比例1：PFP@PLGA/MnFe2O4-Ram纳米粒的制备
采用双乳化法，以水包油包水的原理制备PFP@PLGA/MnFe2O4-ramucirumab(简写为PFP@PLGA/MnFe2O4-Ram)纳米制剂。具体步骤如下：首先将PLGA-PEG5000-ramucirumab(50mg)溶于2mL的CHCl3溶液中；待PLGA-PEG5000-ramucirumab充分溶解后，先后向其中加入3nmMnFe2O4(160μl，8mg/ml)和PFP(液态氟碳，200μL)；使用超声波破碎仪(功率133W,乳化时长30秒)对上述混合溶液进行乳化，得到浅棕色初乳液。随后将初乳液缓慢滴加至8mL的PVA(聚乙烯醇，4％w/v)中；使用超声波破碎仪(功率133W,乳化时长30秒)再次乳化上述溶液，得到浅棕色复乳液；再向复乳液中加入10mL的异丙醇溶液(2％v/v)，4℃条件下冰浴6小时；待CHCl3充分挥发后，使用低温高速离心机，以11000rpm的转速离心上述乳剂7分钟，超纯水洗涤后储存于4℃环境下。
对比例2：PFP-HMME@PLGA–Ram纳米粒的制备
采用双乳化法，以水包油包水的原理制备PFP-HMME@PLGA-ramucirumab(简写为PFP-HMME@PLGA-Ram)纳米制剂。具体步骤如下：首先将PLGA-PEG5000-ramucirumab(50mg)溶于2mL的CHCl3溶液中；待PLGA-PEG5000-ramucirumab充分溶解后，先后向其中加入2mg HMME(血卟啉单甲醚)和PFP(液态氟碳，200μL)；使用超声波破碎仪(功率133W,乳化时长30秒)对上述混合溶液进行乳化，得到浅棕色初乳液。随后将初乳液缓慢滴加至8mL的PVA(聚乙烯醇，4％w/v)中；使用超声波破碎仪(功率133W,乳化时长30秒)再次乳化上述溶液，得到浅棕色复乳液；再向复乳液中加入10mL的异丙醇溶液(2％v/v)，4℃条件下冰浴6小时；待CHCl3充分挥发后，使用低温高速离心机，以11000rpm的转速离心上述乳剂7分钟，超纯水洗涤后储存于4℃环境下。
对比例3：PFP-HMME@PLGA/MnFe2O4纳米粒的制备
采用双乳化法，以水包油包水的原理制备PFP-HMME@PLGA/MnFe2O4(简写为PFP-HMME@PLGA/MnFe2O4)纳米制剂。具体步骤如下：首先将PLGA-PEG5000(50mg)溶于2mL的CHCl3溶液中；待PLGA-PEG5000充分溶解后，先后向其中加入2mg HMME(血卟啉单甲醚)，3nmMnFe2O4(160μl，8mg/ml)和PFP(液态氟碳，200μL)；使用超声波破碎仪(功率133W,乳化时长30秒)对上述混合溶液进行乳化，得到浅棕色初乳液。随后将初乳液缓慢滴加至8mL的PVA(聚乙烯醇，4％w/v)中；使用超声波破碎仪(功率133W,乳化时长30秒)再次乳化上述溶液，得到浅棕色复乳液；再向复乳液中加入10mL的异丙醇溶液(2％v/v)，4℃条件下冰浴6小时；待CHCl3充分挥发后，使用低温高速离心机，以11000rpm的转速离心上述乳剂7分钟，超纯水洗涤后储存于4℃环境下。
实验例1：PFP-HMME@PLGA/MnFe2O4-Ram纳米粒的表征
使用动态光散射方法检测实施例1制备的纳米粒的粒径和zeta电位，实验结果如图1所示，粒径为347.4±5.93nm，zeta电位为-11.9±0.50nm；实施例1制备的纳米粒的透射电镜图如图2所示。
使用低强度聚焦超声(LIFU,1.5W cm2,1MHz))辐照1,3-二苯基异苯并呋喃DPBF(1,3-二苯基异苯并呋喃，50μM)和NPs(即纳米粒，125μg/mL)的混合溶液150s，对各组的DPBF的消耗量进行定量。NPs包括两种：PFP-HMME@PLGA/MnFe2O4-Ram(制备方法参见实施例1)和PFP@PLGA/MnFe2O4-Ram(制备方法参见对比例1)。实验结果如图3所示(mean±SD，n＝4，***表示与control相比，p<0.001；其中，control组中没有加入NPs且未进行LIFU处理；LIFU组中未加入NPs，但进行LIFU处理；PFP@PLGA/MnFe2O4-Ram+LIFU组中加入了PFP@PLGA/MnFe2O4-Ram，并进行LIFU处理；PFP-HMME@PLGA/MnFe2O4-Ram组中加入了PFP-HMME@PLGA/MnFe2O4-Ram，并未进行LIFU处理；PFP-HMME@PLGA/MnFe2O4-Ram+LIFU组中加入了PFP-HMME@PLGA/MnFe2O4-Ram，并进行LIFU处理)，实验结果表明LIFU能有效激活PFP-HMME@PLGA/MnFe2O4-Ram纳米粒中的声敏剂，产生大量活性氧。
实施例1制备的纳米粒中Mn,Fe和F的高分辨透射电镜元素扫描结果如图4所示；纳米粒透射电镜线性扫描结果如图5所示。不同纳米粒的紫外-可见吸收光谱如图6所示，三种纳米粒分别为：PFP-HMME@PLGA/MnFe2O4-Ram(制备方法参见实施例1)、PFP@PLGA/MnFe2O4-Ram(制备方法参见对比例1)和PFP-HMME@PLGA–Ram(制备方法参见对比例2)。在300K条件下，在-30kOe到+30kOe之间，纳米粒的磁滞回线如图7所示。
经检测和计算，PFP-HMME@PLGA/MnFe2O4-Ram纳米粒中HMME的包封率是83.56±2.47％，载药量是3.18±0.1％；MnFe2O4的包封率是80.51±2.21％，载药量是1.93±0.2％。包封率和载药量的具体测量以及计算方法如下：
对于MnFe2O4：使用2％的硝酸溶液充分溶解PFP-HMME@PLGA/MnFe2O4-Ram靶向纳米制剂(25mg)，采用电感耦合等离子体发射光谱仪(ICP-OES)检测纳米制剂中MnFe2O4的含量,计算MnFe2O4的包封率和装载量，计算公式如下:
包封率(％w/w)＝(MnFe2O4质量/MnFe2O4加入量)×100％
载药量(μg/mL)＝(MnFe2O4质量/纳米制剂总质量)×100％
对于HMME：使用DMSO充分溶解PFP-HMME@PLGA/MnFe2O4-Ram靶向纳米制剂(25mg)，采用紫外-可见-近红外分光光度计检测纳米制剂中HMME的含量,计算HMME的包封率和装载量，计算公式如下:
包封率(％w/w)＝(HMME质量/HMME加入量)×100％
载药量(μg/mL)＝(HMME质量/纳米制剂总质量)×100％
实验例2：靶向实验
对纳米粒进行靶向兔主动脉内皮细胞(RAEC)的检测实验，实验结果如图8和图9所示。图8(实验对象为实施例1和对比例3的纳米粒)展示了DiI-标记的纳米粒(红色荧光)和FITC-标记的PLGA-PEG5000-ramucirumab(绿色荧光)的共聚焦图像。在图8的合成图像中，纳米粒和雷莫芦单抗重叠的地方显示黄色。图9中展示了，DiI-标记的靶向纳米粒和RAEC共孵育2小时后,共聚焦显示各组细胞对纳米粒的摄取情况。结果发现，PFP-HMME@PLGA/MnFe2O4-Ram靶向纳米粒能够大量被RAEC吞噬，而PFP-HMME@PLGA/MnFe2O4非靶向纳米粒被RAEC吞噬的比例很少，此外，预先用ramucirumab与RAEC孵育，PFP-HMME@PLGA/MnFe2O4-Ram靶向纳米粒则很难再被RAEC吞噬，此结果说明PFP-HMME@PLGA/MnFe2O4-Ram靶向纳米粒通过结合RAEC上的VEGFR2受体，进而被RAEC大量吞噬。
实验例3：体外声动力治疗效果研究
本实验例进行了PFP-HMME@PLGA/MnFe2O4-Ram纳米粒介导的声动力治疗(SDT)促进兔主动脉内皮细胞(RAEC)线粒体途径凋亡的实验，验证PFP-HMME@PLGA/MnFe2O4-Ram纳米粒的线粒体靶向性能。
将RAEC和DiI-标记的纳米粒孵育2，4和6小时后，进行实验观察，实验结果如图10-图15所示。图10-图12为荧光共聚焦显微镜下观察结果，图10使用的是Dil标记的PFP-HMME@PLGA/MnFe2O4-Ram纳米粒和线粒体探针，图11使用的是Dil标记的PFP-HMME@PLGA/MnFe2O4-Ram纳米粒和溶酶体探针，图12使用的是Dil标记的PFP@PLGA/MnFe2O4-Ram纳米粒和线粒体探针。对共定位情况进行了Mander共定位分析(Mander's overlap coefficient，MOC)，实验结果如图13所示(mean±SD，n＝5，每一个时间点的数据柱从左往右依次为PFP-HMME@PLGA/MnFe2O4-Ram和线粒体探针、PFP-HMME@PLGA/MnFe2O4-Ram和溶酶体探针、PFP@PLGA/MnFe2O4-Ram和线粒体探针)。MOC展示了纳米粒和线粒体探针或溶酶体探针的共定位程度，数值范围介于0和1之间，0代表完全没有共定位，1代表完全共定位。实验结果显示：本方案的纳米粒(PFP-HMME@PLGA/MnFe2O4-Ram)与线粒体探针的共定位程度高，而PFP@PLGA/MnFe2O4-Ram纳米粒显示出的共定位程度不理想。说明了PFP-HMME@PLGA/MnFe2O4-Ram能够更加特异地引导RAEC的线粒体途径凋亡。
本实验例研究了PFP-HMME@PLGA/MnFe2O4-Ram纳米粒介导的SDT诱导RAEC凋亡，RAECs先与活性氧清除剂NAC孵育1小时，然后进行PFP-HMME@PLGA/MnFe2O4-Ram纳米粒介导的SDT实验。实验结束后进行WB测试，RAEC中procaspase 3和活化caspase 3代表性的western条带图详见图14。对Cleaved caspase 3蛋白相对于actin表达量的半定量分析，实验结果参见图15(mean±SD，n＝3，数据柱从左往右依次为：control、纳米粒+LIFU、NAC、纳米粒+LIFU+NAC)。在图10-15中，*表示p<0.05,**表示p<0.01,***表示p<0.001。实验结果显示，纳米粒和超声联合处理细胞，可显著增加活化的caspase 3的蛋白量。从而促进了线粒体凋亡途径。
实验例4：体内和体外MRI成像研究
本实验例在兔股动脉斑块新生血管模型中，研究了PFP-HMME@PLGA/MnFe2O4-Ram纳米粒的体内和体外MRI成像。图16展示了在MnFe2O4不同投入量下(0,160,320,640,1280和2560μL，即纳米粒的投入量以MnFe2O4计)，PFP-HMME@PLGA/MnFe2O4-Ram纳米粒溶液的T1加权成像图和数值(mean±SD，n＝3)。分别注射PFP-HMME@PLGA/MnFe2O4和PFP-HMME@PLGA/MnFe2O4-Ram纳米粒溶液后不同时间点，对兔股动脉斑块进行T1加权成像，实验结果如图17所示，图片中的红色箭头代表股动脉斑块。图18展示了在不同组中，不同时间点斑块处的T1信号值(mean±SD，n＝3，*表示p<0.05，其中每个时间点，从左往右依次为PFP-HMME@PLGA/MnFe2O4、PFP-HMME@PLGA/MnFe2O4-Ram和钆喷酸葡胺MRI造影剂Gd-DTPA)。实验结果显示，本方案的纳米粒能够显著加强体内和体外MRI成像。
实验例5：体外光声成像研究
对不同纳米粒溶液的体外光声成像(PAI)研究，包括PFP-HMME@PLGA-Ram纳米粒(HMME的载药量为3.69±0.20％，包封率为99.03±0.72％)、PFP@PLGA/MnFe2O4-Ram纳米粒(MnFe2O4的载药量为2.74±0.004％，包封率为88.29±0.13％)、PFP-HMME@PLGA/MnFe2O4-Ram纳米粒以及3nm MnFe2O4纳米粒(8mg/ml)。用双乳化法分别合成PFP-HMME@PLGA-Ram，PFP@PLGA/MnFe2O4-Ram以及PFP-HMME@PLGA/MnFe2O4-Ram纳米粒。根据不同纳米粒的载药量和包封率的不同，分别计算投入量，使PFP-HMME@PLGA/MnFe2O4-Ram纳米粒中HMME和MnFe2O4的含量与PFP-HMME@PLGA-Ram纳米粒中的HMME的含量，PFP@PLGA/MnFe2O4-Ram纳米粒中的MnFe2O4含量相同。图19展示了水,PFP-HMME@PLGA-Ram纳米粒(10mg/mL纳米粒溶液含有1.1272mg HMME),PFP@PLGA/MnFe2O4-Ram纳米粒(10mg/mL纳米粒溶液含有1.029mgMnFe2O4),MnFe2O4溶液(1.029mg)以及PFP-HMME@PLGA/MnFe2O4-Ram纳米粒溶液(10mg/mL纳米粒溶液含有1.1272mgHMME和1.029mgMnFe2O4)的光声成像图。对光声成像图像进行了定量分析，如图20所示(数据柱从左往右依次为water、PFP-HMME@PLGA-Ram、PFP@PLGA/MnFe2O4-Ram、MnFe2O4、PFP-HMME@PLGA/MnFe2O4-Ram)，结果显示，PFP-HMME@PLGA/MnFe2O4-Ram纳米粒的光声信号值(2.47±0.12)分别是PFP@PLGA/MnFe2O4-Ram纳米粒(1.43±0.19)和PFP-HMME@PLGA-Ram纳米粒(0.60±0.01)的1.73倍和4.1倍(mean±SD，n＝3，***表示p<0.001)。PFP-HMME@PLGA/MnFe2O4-Ram纳米粒的光声信号值比PFP@PLGA/MnFe2O4-Ram纳米粒和PFP-HMME@PLGA-Ram纳米粒的光声信号值之和更高，这说明MnFe2O4和HMME在同一个纳米粒中联合使用，可以获得协同增效的作用，而不是两种物质的作用的简单相加，获得了预料不到的技术效果。
实验例6：体外和体内超声成像
LIFU(1.5W/cm2,1MHz)辐照PFP-HMME@PLGA/MnFe2O4-Ram纳米粒后不同时间后，B型和造影(CEUS)图像如图21所示，量化的回声强度统计图如图22所示(mean±SD，n＝5；*表示p<0.05,**表示p<0.01,***表示p<0.001，均与0min相比较)。向实验兔静脉注射PFP-HMME@PLGA/MnFe2O4-Ram纳米粒、PFP-HMME@PLGA/MnFe2O4纳米粒和HMME@PLGA/MnFe2O4-Ram纳米粒，90min后使用LIFU(1.5Wcm2,1MHz,15min)激发，图23展示了激发前后兔股动脉斑块的B型和造影图像，红色箭头表示斑块内的微泡。图24展示了LIFU激发前后各组的回声强度(mean±SD，n＝5；每种处理的两个数据柱中左侧为辐射前，右侧为辐射后；***表示p<0.001)。
实验例7：体内声动力治疗实验研究
1)建立兔股动脉进展期斑块模型：雄性新西兰大白兔(2.5-3kg)在股动脉球囊损伤前的2周和损伤后的6周给予1.5％的高胆固醇饮食(1.5％胆固醇，7.5％蛋黄粉，10％猪油)，然后再给予4周普通饲料的喂养。在全身麻醉(3％戊巴比妥[30mg/kg]和氯胺酮[10mg/kg]静脉注射)的条件下，在兔右侧股动脉的一个侧枝上剪口，把2.0×15mm的球囊扩张导管插入股动脉。当球囊完全进入股动脉后，迅速使压力扩张到8个大气压(ATM)，维持20s后撤销压力，充气放气的过程重复3次，以达到使股动脉内皮损伤的目的。手术结束后，结扎侧枝，把兔子送回动物中心单笼喂养，并且自由饮食和饮水。2)静脉注射PFP-HMME@PLGA/MnFe2O4-Ram纳米粒(1mg/ml,50ml)后90min,麻醉成模的兔子后，将兔子平躺，将超声探头放置在兔股动脉搏动最明显处，治疗15min.治疗结束后，将兔子送回动物中心。治疗后28天，PFP-HMME@PLGA/MnFe2O4-Ram介导的SDT治疗能够抑制兔股动脉斑块新生血管，进而稳定斑块。
图25为各组代表性的组织病理学染色股动脉斑块切片的显微图像，红色箭头代表外膜异常增生的新生血管，白色箭头代表斑块内出血。
图26为阳性区域定量分析(mean±SD，n＝7；每个统计图的数据柱从左往右依次为：control、LIFU、PFP-HMME@PLGA/MnFe2O4-Ram、PFP@PLGA/MnFe2O4-Ram+LIFU、PFP-HMME@PLGA/MnFe2O4+LIFU和PFP-HMME@PLGA/MnFe2O4-Ram+LIFU；*表示p<0.05,**表示p<0.01,***表示p<0.001)。
本实验证明本方案的PFP-HMME@PLGA/MnFe2O4-Ram在LIFU作用下，对实验动物的体内声动力治疗效果显著优于其他处理组。
实验例8：体内生物安全性评价
实验兔处理方法参见实验例7，治疗前和治疗后28天，对各处理组血液检查(mean±SD，n＝6)，实验结果如图27所示。图27中每种处理包括两个数据柱，左侧为治疗前，右侧为治疗后28天。图28展示了各处理组实验兔不同时间的体重变化(mean±SD，n＝5)，图中每个时间点的数据柱从左往右依次为：
control、LIFU、PFP-HMME@PLGA/MnFe2O4-Ram、PFP@PLGA/MnFe2O4-Ram+LIFU、PFP-HMME@PLGA/MnFe2O4+LIFU和PFP-HMME@PLGA/MnFe2O4-Ram+LIFU。
各处理组治疗后28天，处死实验兔，对主要器官(心，肝，脾，肺，肾)进行切片HE染色，实验结果如图29所示。
体内生物安全性评价实验证明了本方案的纳米粒对实验动物的毒性小，对主要器官组织未造成明显损伤。
以上所述的仅是本发明的实施例，方案中公知的具体技术方案和/或特性等常识在此未作过多描述。应当指出，对于本领域的技术人员来说，在不脱离本发明技术方案的前提下，还可以作出若干变形和改进，这些也应该视为本发明的保护范围，这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准，说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (8)
1.一种靶向载铁氧体多功能纳米粒，其特征在于：其包括由聚乳酸-羟基乙酸共聚物形成的外壳，所述外壳上镶嵌有MnFe2O4和血卟啉单甲醚。
2.根据权利要求1所述的一种靶向载铁氧体多功能纳米粒，其特征在于：所述外壳上共价连接有雷莫芦单抗。
3.根据权利要求2所述的一种靶向载铁氧体多功能纳米粒，其特征在于：其还包括液态氟碳形成的内核。
4.根据权利要求3所述的一种靶向载铁氧体多功能纳米粒的制备方法，其特征在于：包括如下依次进行的步骤：
S1：合成PLGA-PEG5000-ramucirumab；
S2：将PLGA-PEG5000-ramucirumab溶于三氯甲烷中，然后加入血卟啉单甲醚、MnFe2O4和液态氟碳，获得混合体系；然后对混合体系进行声震乳化，获得初乳液；
S3：在初乳液中滴加聚乙烯醇，然后再次进行声震乳化，获得复乳液；
S4：在复乳液中加入异丙醇溶液，冰浴后离心获得靶向载铁氧体多功能纳米粒。
5.根据权利要求4所述的一种靶向载铁氧体多功能纳米粒的制备方法，其特征在于：在S1中，PLGA-PEG5000-ramucirumab由如下方法合成：将聚乳酸-羟基乙酸共聚物溶于二氯甲烷中，再加入N-羟基琥珀酰亚胺和催化剂，经反应获得PLGA-NHS；将PLGA-NHS溶解于二氯甲烷中，加入NH2-PEG-COOH和催化剂，经反应获得PLGA-PEG5000-COOH；将PLGA-PEG5000-COOH溶于二氯甲烷中，并加入雷莫芦单抗和催化剂，经反应获得PLGA-PEG-NHS-ramucirumab。
6.根据权利要求5所述的一种靶向载铁氧体多功能纳米粒的制备方法，其特征在于：靶向载铁氧体多功能纳米粒的血卟啉单甲醚的包封率为83.56±2.47％，载药量为3.18±0.1％；靶向载铁氧体多功能纳米粒的MnFe2O4的包封率为80.51±2.21％，载药量为1.93±0.2％。
7.根据权利要求1-3中任一项所述的一种靶向载铁氧体多功能纳米粒在制备显像剂中的应用。
8.根据权利要求1-3中任一项所述的一种靶向载铁氧体多功能纳米粒在制备动脉粥样硬化易损斑块抑制药物中的应用。
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