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CN116725965B - 一种双靶向血管vegf-vegfr的磁性纳米药物及其制备方法和应用 - Google Patents

一种双靶向血管vegf-vegfr的磁性纳米药物及其制备方法和应用 Download PDF

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CN116725965B
CN116725965B CN202310853646.3A CN202310853646A CN116725965B CN 116725965 B CN116725965 B CN 116725965B CN 202310853646 A CN202310853646 A CN 202310853646A CN 116725965 B CN116725965 B CN 116725965B
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CN116725965A (zh
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刘鑫
康然
谢林
陈博
王楠
王嫘
邓蓉蓉
王资涵
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Nanjing University of Chinese Medicine
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Abstract

本发明提供了一种双靶向血管VEGF‑VEGFR的磁性纳米药物及其制备方法和应用，属于生物医药技术领域。本发明的磁性纳米药物包括氨基化的ZnFe2O4中空多孔磁性纳米颗粒、LDH‑SF、EDC、NHS；所述氨基化ZnFe2O4中空多孔磁性纳米颗粒、EDC、NHS、LDH‑SF的质量比为(5～10)：(1～5)：(1～6):(1～8)。本发明的磁性纳米药物可通过中空铁酸锌颗粒的磁靶向定位VEGF配体，调控VEGF因子下调，抑制血管生成；同时，通过川芎嗪靶向血管受体VEGFR，结合磁热控制释放，发挥抑制作用；因此，该磁性纳米药物的双重靶向可有效抑制血管化，阻断神经营养供应，实现对盘源性疼痛的治疗。

Description

一种双靶向血管VEGF-VEGFR的磁性纳米药物及其制备方法和 应用
技术领域
本发明涉及生物医药技术领域，特别涉及一种双靶向血管VEGF-VEGFR的磁性纳米药物及其制备方法和应用。
背景技术
椎间盘退变(IDD)是一种严重影响患者身心健康的骨伤科常见疾病，主要临床症状包括腰骶部疼痛、下肢放射痛甚至导致患者严重的残疾。其发病机制涉及：纤维环破裂导致椎间盘内髓核的突出，刺激或/和压迫邻近的脊神经根/马尾，以及神经血管向椎间盘长入而引发的盘源性疼痛、麻木等系列症状。其中，退变症状之一的盘源性疼痛影响最为严重而广泛。目前，临床上用于缓解该疼痛的方法主要是使用镇痛药、手术、生物疗法以及一些传统的中医手法治疗。这些方法虽在缓解疼痛方面有一些作用，但仍然无法有效完全逆转/消除引起疼痛的关键原因——血管化，并且存在严重的不良反应。如镇痛药的长期使用会产生严重的毒副作用和耐受性；椎间盘融合手术后邻近节段损伤、狭窄等并发症；生物疗法的高成本以及中医手法治疗无法缓解严重疼痛。因此，针对盘源性疼痛的根本原因——血管化采取一定的抑制策略，是逆转/消除疼痛的重要方法之一。血管内皮生长因子(VEGF)及其受体信号(VEGFR)在椎间盘血管化过程中起着至关重要的作用，但目前临床尚缺乏针对VEGF和VEGFR双特异性免疫疗法。
发明内容
有鉴于此，本发明目的在于提供一种双靶向血管VEGF-VEGFR的磁性纳米药物及其制备方法和应用，本发明提供的磁性纳米药物通过磁场作用双重靶向到达病区，抑制配体和受体作用进而抑制血管化，阻断神经营养供应，实现对疼痛的根治。
为了实现上述目的，本发明提供以下技术方案：
一种双靶向血管VEGF-VEGFR的磁性纳米药物，所述磁性纳米药物包括氨基化的ZnFe2O4中空多孔磁性纳米颗粒、盐酸川芎嗪纳米微球LDH-SF、EDC、NHS；所述LDH-SF包括丝素蛋白SF和盐酸川芎嗪LTH；所述氨基化ZnFe2O4中空多孔磁性纳米颗粒、EDC、NHS、LDH-SF的质量比为(5～10)：(1～5)：(1～6):(1～8)。
在某些实施方案中，所述氨基化的ZnFe2O4中空多孔磁性纳米颗粒的氨基化修饰为PEG-NH2。
在某些实施方案中，所述丝素蛋白和盐酸川芎嗪的质量比为10：(1～2.5)。
在某些实施方案中，所述氨基化的ZnFe2O4中空多孔磁性纳米颗粒通过EDC/NHS活化的酰胺键与LDH-SF纳米微球化学结合。
在某些实施方案中，所述氨基化的ZnFe2O4中空多孔磁性纳米颗粒的粒径为20nm～100nm；所述LDH-SF的粒径小于氨基化的ZnFe2O4中空多孔磁性纳米颗粒的粒径，LDH-SF可进入中空多孔磁性纳米颗粒中。
本发明还提供一种上述技术方案中所述的磁性纳米药物的制备方法，包括以下步骤：
S1、制备氨基化的ZnFe2O4中空多孔磁性纳米颗粒：将ZnCl2和FeCl3·6H2O溶解于有机溶剂中，加入NaAc和PEG 20000，磁力搅拌，反应后，加入PEG-NH2，再次搅拌，清洗，干燥，即得氨基化的ZnFe2O4中空多孔磁性纳米颗粒；
S2、制备LDH-SF：将SF溶液与LTH水溶液混合搅拌，加入无水乙醇，二次搅拌，孵育，乳化，透析，冻干，即得LTH-SF；
S3、将氨基化ZnFe2O4中空多孔磁性纳米颗粒和LTH-SF分别溶于MES缓冲液，并加入EDC/NHS活化，避光室温下搅拌混合反应，离心，冷冻干燥，即可获得磁性纳米药物。
在某些实施方案中，所述S1中的有机溶剂为乙二醇、DMF中的一种或两种；磁力搅拌的搅拌时间为30min～60min；反应为150℃～250℃下反应8h～12h；再次搅拌为50℃～60℃水浴下搅拌2h～4h；清洗为使用乙醇清洗3～6次；干燥为50℃～70℃干燥6h～12h；所述乙醇的体积浓度为75％～100％。
在某些实施方案中，所述S1中ZnCl2、FeCl3·6H2O、有机溶剂、NaAc、PEG 20000的mmol：mmol：mL：g：g比为2.5：5：(40～50)：(3.5～4)：(1～1.2)；磁力搅拌后需密封在容器中；反应后需冷却至室温。本发明利用改良的水热合成法制备中空多孔磁性纳米颗粒，经过原料比例、试剂种类、热处理时间等条件控制，合成出单分散性好、粒径均匀且呈中空多孔结构的ZnFe2O4纳米颗粒。随后，利用PEG-NH2对纳米颗粒进行氨基化修饰，最终获得氨基化ZnFe2O4中空多孔磁性纳米颗粒，并作为靶向配体VEGF物质。
在某些实施方案中，所述S2中的混合搅拌为20℃～25℃搅拌20min～30min；无水乙醇与SF的体积比为(2～3)：5；二次搅拌20℃～25℃下搅拌3min～10min；孵育为-20℃～-30℃孵育20h～24h；透析为使用12000Da～14000Da透析袋在去离子水中透析4h～6h。本发明通过将盐酸川芎嗪与丝素蛋白通过乳化法制备成纳米微球，可以增加川芎嗪的羧基化修饰，增加川芎嗪的代谢稳定性。
在某些实施方案中，所述S3中的MES缓冲液的pH值为4～6；搅拌混合反应为20℃～25℃混合反应48h～60h；室温为20℃～25℃；离心为1000rpm～2000rpm离心1min。
本发明还提供一种上述技术方案中所述磁性纳米药物在制备抗血管生成和/或治疗盘源性疼痛药物中的应用。
有益技术效果：本发明提供了一种双靶向血管VEGF-VEGFR的磁性纳米药物及其制备方法和应用，所述磁性纳米药物包括氨基化的ZnFe2O4中空多孔磁性纳米颗粒、盐酸川芎嗪纳米微球LDH-SF、EDC、NHS；所述LDH-SF包括丝素蛋白SF和盐酸川芎嗪LTH；所述氨基化ZnFe2O4中空多孔磁性纳米颗粒、EDC、NHS、LDH-SF的质量比为(5～10)：(5～1)：(6～1):(8～1)。本发明提供的磁性纳米药物可通过中空铁酸锌颗粒的磁靶向定位VEGF配体，利用磁性颗粒调控椎间盘内巨噬细胞重编程来间接调控VEGF因子下调，抑制血管生成；同时，通过川芎嗪靶向血管受体VEGFR，结合磁热控制释放，发挥抑制作用；因此，该磁性纳米药物的双重靶向可有效抑制血管化，阻断神经营养供应，实现对疼痛的根治。
附图说明
图1为磁性纳米药物TEM表征图；
图2为不同浓度ZnFe2O4磁性纳米粒子与巨噬细胞共培养FACS检测图；其中，A～E分别对应20ng/mL IL-4(阴性对照)、100ng/mL LPS(阳性对照)、10μg/mL ZnFe2O4、50μg/mLZnFe2O4、100μg/mL ZnFe2O4、500μg/mL ZnFe2O4；
图3为不同浓度ZnFe2O4磁性纳米粒子与巨噬细胞共培养观察对VEGF表达影响的ELISA结果图；
图4为不同浓度ZnFe2O4磁性纳米粒子与巨噬细胞共培养CCK-8细胞活力检测图；
图5A为磁性纳米药物与巨噬细胞共培养观察对VEGF表达影响的ELISA结果图；图5B为磁性纳米药物与血管内皮细胞共培养观察对VEGFR2表达影响的免疫荧光图。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。本发明实施例及试验例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得；本发明实施例及试验例中所用的方法，如无特殊说明，均为常规方法。
实施例1
(1)氨基化ZnFe2O4中空多孔磁性纳米颗粒制备：将0.34g ZnCl2(2.5mmol)和1.35gFeCl3·6H2O(5mmol)溶解在40mL乙二醇中，加入3.6g NaAc和1.0g PEG20000，磁力搅拌30min，然后密封在容量为50mL的聚四氟乙烯高压反应釜中；将高压反应釜加热至200℃并保持8h，冷却至室温；加入PEG-NH2，50℃水浴下搅拌2h；将获得的黑色产品用乙醇清洗3次，在60℃下干燥6h，即可获得氨基化ZnFe2O4中空多孔磁性纳米颗粒ZnFe2O4@PEG-NH2。
(2)丝素蛋白修饰盐酸川芎嗪LTH-SF纳米微球的制备：分别取5mL浓度分别为10mg/mL、20mg/mL的丝素蛋白SF溶液，分别加入10mL浓度分别为0.625mg/mL、1.25mg/mL的盐酸川芎嗪LTH水溶液，轻轻搅拌20min，滴加2mL的无水乙醇；将混合物轻轻搅拌3min，在-20℃的冰箱中孵育20h；在室温下解冻后，形成乳白色的乳液；量取5mL制备好的乳化液，并用2000mL去离子水透析4h，去除大部分未被SF纳米微球包裹或仅吸附在表面的LTH。然后将乳液冻干，即可得到盐酸川芎嗪纳米微球LTH-SF。
(3)磁性纳米药物的制备：将20mg(1)中获得的ZnFe2O4@PEG-NH2和3mg(2)中获得的LTH-SF溶于50mL的MES缓冲液(PH＝5.0)，加入过量的EDC/NHS(4mg/2mg)活化，室温下搅拌混合反应48h，2000rpm离心分离后，冷冻干燥，即可获得所需的磁性纳米药物。
取实施例1得到的磁性纳米药物进行透射电子显微镜扫描，扫描结果如图1所示。由图1可以看出，盐酸川芎嗪纳米微球很好的结合在ZnFe2O4中空多孔磁性纳米颗粒上，并呈现了多孔结构的磁性纳米药物。
实施例2不同浓度ZnFe2O4磁性纳米粒子与巨噬细胞共培养观察表型极化的影响
将实施例1制备的ZnFe2O4磁性纳米粒子分别配置成浓度10μg/mL、50μg/mL、100μg/mL、500μg/mL，进行高压蒸汽灭菌处理后，4℃保存、待用。准备3个6孔培养板放置于生物安全柜中紫外灭菌1h，将大鼠巨噬细胞(NR8383)制备成细胞悬液，接种到灭菌的6孔细胞培养板中，接种密度为1×105细胞/孔，加入2mL完全培养基(90％ DMEM+10％ FBS+1％ PS)；将细胞培养板转移至37℃，5％ CO2培养箱中，孵育4h，待细胞完全贴壁；将细胞分为6组，每组3个平行样，六组分别为：20ng/mL IL-4(阴性对照)，100ng/mL LPS(阳性对照)，10μg/mLZnFe2O4，50μg/mL ZnFe2O4，100μg/mL ZnFe2O4，500μg/mL ZnFe2O4，继续孵育72h后，除去培养基，并收集细胞，进行流式细胞仪检测。
流式细胞仪检测的具体步骤：1)在细胞中加入15mL细胞染色缓冲液(PBS(PH＝7.4)+1％ FBS+0.09％ NaN3)重悬，以350xg离心5min，弃去上清液；2)加入偶联的荧光一抗CD80(12-0800-82，鼠源，1:1000,Thermo Fisher,USA)，避光在冰上孵育15min～20min；3)用2mL的细胞染色缓冲液洗涤2次并离心(350xg，每次5min)；4)避光室温加入0.5mL固定液固定细胞20min；5)350xg离心5min，弃上清液；6)将固定的细胞重悬于细胞通透洗涤缓冲液中，并以350xg离心5min洗涤2次；7)加入偶联的荧光一抗CD206(PA5-114370，兔源，1:1000，Thermo Fisher,USA)，避光在冰上孵育20min；8)用2mL的细胞染色缓冲液350xg离心5min洗涤2次；9)采用流式细胞仪进行数据采集和分析。
流式细胞仪的检测结果如图2所示，由图2可以看出，较低浓度ZnFe2O4调控巨噬细胞M2极化，而高浓度调控巨噬细胞M1极化，可能下调VEGF表达。
实施例3不同浓度ZnFe2O4磁性纳米粒子与巨噬细胞共培养对VEGF表达影响
将实施例1制备的ZnFe2O4磁性纳米粒子分别配置成浓度0μg/mL、10μg/mL、100μg/mL、500μg/mL的溶液，高压蒸汽灭菌处理后4℃保存、待用；将12孔培养板放置于生物安全柜中紫外灭菌1h，将大鼠巨噬细胞制备成细胞悬液，接种到灭菌后的培养板中，接种密度为1×105细胞/孔，加入1mL完全培养基(90％ DMEM+10％ FBS+1％ PS)；
将细胞培养板转移至37℃，5％ CO2培养箱中，孵育4h，待细胞完全贴壁；将细胞分为4组，每组3个平行样，四组分别为：0μg/mL ZnFe2O4、10μg/mL ZnFe2O4，100μg/mL ZnFe2O4，500μg/mL ZnFe2O4，继续孵育24h后，收集上清液；将收集的上清液转移至高速离心机，12000rpm离心5min，再次收集上清液；采用大鼠血管内皮生长因子ELISA试剂盒(MM-0179R1，酶免生物，中国)对VEGF因子进行定量检测，每种样品测定设置3个平行样。
ELISA检测结果如图3所示，由图3可以看出，较低浓度ZnFe2O4可以促进VEGF表达，而高浓度ZnFe2O4显著抑制VEGF表达，表现血管生成抑制潜能。
实施例4不同浓度ZnFe2O4磁性纳米粒子与巨噬细胞共培养观察生物相容性检测
将实施例1制备的ZnFe2O4磁性纳米粒子分别配置成浓度0μg/mL、10μg/mL、100μg/mL、500μg/mL，高压蒸汽灭菌处理后，4℃保存、待用；将24孔培养板放置于生物安全柜中紫外灭菌1h，然后将大鼠巨噬细胞制备成细胞悬液，随后接种到培养板中，接种密度为1×104细胞/孔，加入1mL完全培养基(90％ DMEM+10％ FBS+1％ PS)；
将细胞培养板转移至37℃，5％ CO2培养箱中，孵育4h，待细胞完全贴壁；将细胞分为4组，每组3个平行样，四组分别为：0μg/mL ZnFe2O4，10μg/mL ZnFe2O4，100μg/mL ZnFe2O4，500μg/mL ZnFe2O4，四组分别孵育24h、48h后，除去培养基，用PBS(0.01M，PH＝7.4)洗涤非贴壁细胞三次；
将体积比为10：1的新鲜完全培养基和CCK-8试剂的混合物加入到孔板中，转移至细胞培养箱孵育2h，将悬浮液转移至96孔板中(200μL/孔)，用酶标仪在450nm测量吸光度。
不同浓度ZnFe2O4磁性纳米粒子与巨噬细胞共培养CCK-8细胞活力检测结果如图4所示，由图4可以看出，在24h时，10μg/mL、100μg/mL实验组相比于空白对照组细胞活力略有降低，但差异不显著(P＞0.05)，甚至500μg/mL实验组表现出更高的细胞活力；到48h，各实验组相比于空白对照组细胞活力均略有降低，但均没有表现出显著性差异(P＞0.05)，这表明ZnFe2O4磁性纳米粒子良好的生物相容性。
实施例5磁性纳米药物与巨噬细胞和血管内皮祖细胞共培养抑制VEGF-VEGFR表达影响
将实施例1制备的磁性纳米药物配置成浓度为400μg/mL，放置于生物安全柜中进行紫外线灭菌处理后，4℃保存、待用。将2个24孔培养板放置于生物安全柜中紫外灭菌1h，分别将大鼠巨噬细胞和血管内皮细胞制备成细胞悬液，分别接种到培养板中，接种密度为5×104细胞/孔，加入1.5mL完全培养基(大鼠巨噬细胞完全培养基：80％ F12+20％ FBS+1％PS；血管内皮细胞完全培养基：90％ DMEM+10％ FBS+1％ PS)；
将细胞培养板转移至37℃，5％ CO2培养箱中，孵育4h，待细胞稳定或贴壁；将细胞分为4组，每组6个平行样，四组分别为：Blank control、400μg/mL氨基化ZnFe2O4磁性纳米颗粒、400μg/mL LTH-SF纳米微球、400μg/mL磁性纳米药物，继续孵育48h后，巨噬细胞培养板收集上清液进行ELISA检测，内皮细胞培养板去除上清，固定细胞，进行免疫组化染色。
ELISA检测：将收集的上清液转移至高速离心机，12000rpm离心5min，再次收集上清液；采用ELISA试剂盒检测纳米药物对巨噬细胞分泌VEGF细胞因子的影响，每种样品测定6个平行样。ELISA检测结果如图5A所示，由图5A可以看出，磁性纳米药物显著降低VEGF表达，证明了通过调控巨噬细胞重编程向M1极化下调VEGF抑制血管生成的潜力。
免疫荧光染色：去除4％多聚甲醛固定液，PBS洗涤三次，每次5min,滴加一抗VEGFR2，4℃过夜；倾去一抗，PBS洗涤三次，每次5min；滴加二抗试剂(SA00003-2,Fluorescein(FITC)–conjugated Affinipure Goat Anti-Rabbit IgG(H+L)，Proteintech,China)，室温下孵育2h；PBS洗第一次，DAPI复染10min，PBS洗涤两次，每次5min；最后，显微镜下观察并采集图片；免疫荧光染色结果如图5B所示，由图5B所示，400μg/mL浓度的LTH-SF和磁性纳米药物均表现出显著抑制VEGFR2表达，展现出阻断VEGF配体激活受体，从而抑制血管化疼痛传递而缓解疼痛的潜力。
综合所述，本发明所得的双靶向血管VEGF-VEGFR磁性纳米药物具有抑制退变椎间盘内血管化并降低神经支配的潜力，在治疗盘源性疼痛方面具有广泛的应用前景。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (9)

1.一种双靶向血管VEGF-VEGFR的磁性纳米药物，其特征在于，所述磁性纳米药物包括氨基化的ZnFe2O4中空多孔磁性纳米颗粒、盐酸川芎嗪纳米微球LDH-SF、EDC、NHS；所述LDH-SF包括丝素蛋白和盐酸川芎嗪；所述氨基化ZnFe2O4中空多孔磁性纳米颗粒、EDC、NHS、LDH-SF的质量比为(5～10)：(1～5)：(1～6):(1～8)。
2.根据权利要求1所述的磁性纳米药物，其特征在于，所述氨基化的ZnFe2O4中空多孔磁性纳米颗粒的氨基化修饰为PEG-NH2。
3.根据权利要求1所述的磁性纳米药物，其特征在于，所述丝素蛋白和盐酸川芎嗪的质量比为10：(1～2.5)。
4.根据权利要求1所述的磁性纳米药物，其特征在于，所述氨基化的ZnFe2O4中空多孔磁性纳米颗粒通过EDC/NHS活化的酰胺键与LDH-SF纳米微球化学结合。
5.根据权利要求1所述的磁性纳米药物，其特征在于，所述氨基化的ZnFe2O4中空多孔磁性纳米颗粒的粒径为20nm～100nm。
6.一种权利要求1～5任一所述的磁性纳米药物的制备方法，其特征在于，包括以下步骤：
S1、制备氨基化的ZnFe2O4中空多孔磁性纳米颗粒：将ZnCl2和FeCl3·6H2O溶解于有机溶剂中，加入NaAc和PEG 20000，磁力搅拌，反应后，加入PEG-NH2，再次搅拌，清洗，干燥，即得氨基化的ZnFe2O4中空多孔磁性纳米颗粒；
S2、制备LDH-SF：将丝素蛋白溶液与盐酸川芎嗪水溶液混合搅拌，加入无水乙醇，二次搅拌，孵育，乳化，透析，冻干，即得LDH-SF；
S3、将氨基化ZnFe2O4中空多孔磁性纳米颗粒和LDH-SF分别溶于MES缓冲液，并加入EDC/NHS活化，避光室温下搅拌混合反应，离心，冷冻干燥，即可获得磁性纳米药物。
7.根据权利要求6所述的制备方法，其特征在于，所述S1中的有机溶剂为乙二醇、DMF中的一种或两种；磁力搅拌的搅拌时间为30min～60min；反应为150℃～250℃下反应8h～12h；再次搅拌为50℃～60℃水浴下搅拌2h～4h；清洗为使用乙醇清洗3～6次；干燥为50℃～70℃干燥6h～12h。
8.根据权利要求6所述的制备方法，其特征在于，所述S2中的混合搅拌为20℃～25℃搅拌20min～30min；无水乙醇与丝素蛋白的体积比为(2～3)：5；二次搅拌20℃～25℃下搅拌3min～10min；孵育为-20℃～-30℃孵育20h～24h；透析为使用12000Da～14000Da透析袋在去离子水中透析4h～6h。
9.根据权利要求6所述的制备方法，其特征在于，所述S3中的MES缓冲液的pH值为4～6；搅拌混合反应为20℃～25℃混合反应48h～60h。
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