CN113797358A - 一种双靶向双模态显影纳米粒及其制备方法 - Google Patents
一种双靶向双模态显影纳米粒及其制备方法 Download PDFInfo
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- A61K49/225—Microparticles, microcapsules
Abstract
本发明涉及一种双靶向双模态显影纳米粒及其制备方法。具体提供了一种靶向双模态显影纳米粒,它是以聚乳酸‑羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽为原料制得的,所述靶向多肽选自cRGD和PP1中的一种或两种。该靶向双模态显影纳米粒形态规则、大小均匀、分散性好,具有超声、磁共振双模态显影功能,能靶向至易损斑块内的巨噬细胞,并在超声介导下诱发巨噬细胞凋亡,还能在超声介导下溶解易损斑块表面血栓,在制备诊断易损斑块(特别是易破裂斑块和蚀损斑块)的显像试剂,稳定易损斑块的药物,预防和/或治疗与易损斑块相关的心血管疾病的药物中具有广阔的应用前景。
Description
技术领域
本发明属于靶向药物领域,具体涉及一种双靶向双模态显影纳米粒及其制备方法。
背景技术
2018年《中国心血管病报告》统计数据推算,严重心血管疾病已成为我国最主要的死亡原因,现患人数至少2.9亿,其中脑卒中1300万、缺血性心肌病(心肌梗死)1100万;每年的住院总费用分别高达601.05亿元、190.85亿元,次均住院费用依次高达2.6万元、0.9万元。大大降低了患者生活质量和生存寿命,同时昂贵的治疗费用也给患者家庭和社会带来巨大经济负担。在致死、致残性的心脑血管疾病中,75%以上为动脉粥样硬化性疾病。动脉粥样硬化(Atherosclerosis,AS)以血管内斑块形成为典型病理特征,其致死、致残风险的高低,与斑块的易损性(即是指斑块迅速进展破裂或血栓形成导致严重心血管事件的风险)密切相关。故而,在临床症状出现前的早期病理阶段,筛查易损斑块(AtherosclerosisVulnerable Plaque,ASVP)、延缓或逆转斑块易损性是提高心血管疾病防治的关键环节,即为心血管疾病的一级预防。如何有效地筛选、干预并提高斑块的稳定性,建立有临床价值的心血管疾病防治模式,临床及社会经济学意义重大。
易损斑块是指那些不稳定和有血栓形成倾向的斑块,依据肇事斑块的病理可将易损斑块分为易破裂斑块(Plaque Rupture)、蚀损斑块(Plaque Erosion)和部分钙化结节性病变。大量急性冠脉心肌梗死患者尸检结果显示,肇事斑块中约60%为斑块破裂,30%-35%为蚀损斑块,2%~7%为钙化结节。其中,易破裂斑块以脂质核心、薄纤维帽、炎症细胞浸润为主要特征。大量浸润的炎症细胞中,巨噬细胞(Macrophage,)占80%以上,对斑块的形成及易损性起重要作用。而蚀损斑块又称糜烂斑块,1994年Van Der Wal等首次发现纤维帽未破裂的斑块,也可伴血栓形成;并将此类表面富含血小板和纤维蛋白、无脂质核心、炎症反应轻的斑块定义为蚀损斑块。蚀损斑块表面血小板聚集形成血栓,血栓堵塞管腔或脱落形成远端脉管栓塞,是致病的关键环节。
目前,易损斑块防治的关键是预防血栓形成,对具有明显症状的患者,可采用药物(如:抗血小板治疗、抗凝和溶栓治疗等)和介入手术治疗。但是,是否使用阿司匹林等药物进行抗血小板治疗一直存在很大争议,多项大规模研究结果显示,阿司匹林可使主要心血管病事件减少15%,疗效肯定;但其缺乏靶向性,局部血药浓度较低,消化道出血、颅外出血等全身并发症的相对危险增加69%;同样,口服药物进行抗炎治疗也因缺乏靶向性,治疗效果甚微,同时存在全身副作用大等缺点,无法用于常规治疗。
此外,因为不同类型的易损斑块的病理基础存在较大差异,在治疗时也需要有针对性的对症治疗。目前尚未见采用无创的筛查手段来区分易损斑块的报道,也没有针对不同斑块病理类型的精准治疗方法。
因此,亟需研究出一种能够对易损斑块进行靶向精准的诊断和治疗的药物。
发明内容
本发明的目的在于提供一种能够对易损斑块进行靶向精准的诊断和治疗的药物。
本发明提供了一种靶向双模态显影纳米粒,它是以聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽为原料制得的,所述靶向多肽选自cRGD和PP1中的一种或两种。
进一步地,所述磁共振造影剂为Fe基磁共振造影剂,优选为四氧化三铁。
进一步地,所述超声造影剂选自全氟戊烷、全氟己烷、全氟庚烷或全氟辛溴烷,优选为全氟戊烷。
进一步地,所述聚乳酸-羟基乙酸共聚物分子量为6000Da~10000Da,优选为8000Da。
进一步地,所述靶向多肽为cRGD和PP1,其中cRGD和PP1的质量比为1:(0.8~1.2),优选为1:1。
进一步地,所述聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽的质量体积比为50mg:(0.8~1.2)mg:(180~220)μL:(2~6)mg,优选为50mg:1mg:200μL:4mg。
本发明还提供了上述靶向双模态显影纳米粒的制备方法,所述方法包括以下步骤:
(1)将聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂在有机溶剂中混合,超声震荡;
(2)在步骤(1)所得体系中加入乳化剂,超声震荡,得到非靶向纳米粒;
(3)将步骤(2)所得非靶向纳米粒与靶向多肽在pH=7~9的缓冲液中反应,即得。
进一步地,步骤(1)中,所述有机溶剂为二氯甲烷;所述聚乳酸-羟基乙酸共聚物与有机溶剂的质量体积比为20~30mg/mL,优选为25mg/mL;所述超声震荡的功率为50~70W,优选为60W,时间为3~5min,优选为3min,脉冲式为5秒on/5秒off;所述超声震荡的温度为0℃~4℃;
和/或,步骤(2)中,所述乳化剂为聚乙烯醇,优选为质量分数4%的聚乙烯醇水溶液;超声震荡功率为50~70W,优选为60W,时间为3~5min,优选为5min,脉冲式为5秒on/5秒off;所述超声震荡的温度为0℃~4℃;
和/或,步骤(2)中,所述超声震荡后,还包括在体系中加入醇类溶剂,搅拌,挥发有机溶剂,然后离心,弃上清;其中,所述醇类溶剂优选为异丙醇,更优选为体积分数2%的异丙醇水溶液;所述离心条件优选为1000rpm离心10min;
和/或,步骤(3)中,所述反应前,还包括将非靶向纳米粒与酰胺化反应活化剂混合,摇床孵育,离心,弃上清;优选的,所述酰胺化反应活化剂为EDC和NHS,所述摇床孵育条件为:在pH=5的MES缓冲溶液中于0℃~4℃孵育1小时,所述离心条件为1000rpm离心10min;
和/或,步骤(3)中,所述pH=7~9的缓冲液为pH=8的MES缓冲溶液,所述反应温度为0℃~4℃,反应时间为过夜。
本发明还提供了上述靶向双模态显影纳米粒在制备诊断易损斑块的显像试剂和/或稳定易损斑块的药物中的用途;优选的,所述易损斑块为易破裂斑块和/或蚀损斑块;更优选的,所述显像试剂和/或药物是在以下超声照射条件下使用的:功率3W,频率3HZ,时间3~4min,占空比30%。
本发明还提供了上述靶向双模态显影纳米粒在制备预防和/或治疗与易损斑块有关的心血管疾病的药物和/或诊断与易损斑块有关的心血管疾病的显像试剂中的用途;优选的,所述心血管疾病为动脉粥样硬化;更优选的,所述药物是在以下超声照射条件下使用的:功率3W,频率3HZ,时间3~4min,占空比30%。
cRGD,一种环状多肽,序列为cyclic(Arg-Gly-Asp-D-Phe-Lys)。
PP1,一种16氨基多肽,序列为LSLERFLRCWSDAPAK。
与现有技术相比,本发明具有以下有益效果:
(1)通过分别靶向巨噬细胞SR-A受体和血小板GPIIb/IIIa受体,实现了定点输送,对易破裂斑块和蚀损斑块具有双靶向特异性。
(2)通过超声触发相变,实现了US、MR双模态显像,可用于预防性筛查易损斑块、鉴别斑块类型,以及实时监测治疗效果。
(3)利用超声介导产生的生物学效应,实现易损斑块的防治一体化:
a.有利于增加血管内皮的通透性,协助双靶向双模态显影纳米粒高通量抵达斑块内部;
b.有利于诱导巨噬细胞凋亡,减轻炎症反应,稳定易破裂斑块,实现一级预防的作用;
c.有利于溶解斑块表面血栓,实现对蚀损斑块的精准治疗,从而避免抗炎或抗血小板药物的全身毒副反应。
总之,本发明以聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽为原料制得了一种形态规则、大小均匀、分散性好的双靶向双模态显影纳米粒,该双靶向双模态显影纳米粒具有超声、磁共振双模态显影功能,能靶向至易损斑块内的巨噬细胞,并在超声介导下诱发巨噬细胞凋亡,还能在超声介导下溶解易损斑块表面血栓,在制备诊断易损斑块(特别是易破裂斑块和蚀损斑块)的显像试剂,稳定易损斑块的药物,预防和/或治疗与易损斑块相关的心血管疾病的药物中具有广阔的应用前景。
本发明的制备方法简单,条件温和,适合工业化生产。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1的透射电镜图片(a)、平均粒径(b)、粒径及分布(c)、透射电镜元素分析伪彩图(d)。
图2为各纳米粒的透射电镜图片(a)、表面电荷(b)、激光共聚焦荧光显微镜图片(c)、红外谱图(d)。
图3为各样品的体外US成像结果:(a)B-mode和CEUS模式下的造影图片,(b)B-mode和CEUS模式下的回声强度,(c)Fe-PFP-PLGA@cRGD-PP1在不同超声条件下的造影图片,(d)Fe-PFP-PLGA@cRGD-PP1在不同超声条件下的回声强度。
图4为双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1的体外MR成像结果:(a)不同浓度下的T2信号;(b)浓度与R2的线性关系图。
图5为各荧光标记的样品与激活巨噬细胞或非激活巨噬细胞共培养后的激光共聚焦图片。
图6为不同处理条件下对巨噬细胞凋亡的影响,其中绿色表示活细胞,红色表示死细胞;“control”为空白对照组,“US”为仅超声辐照组,“non-PFP+US”为non-PFP+超声辐照组,“PFP+US”为PFP+超声辐照组。
图7为不同处理条件下对血栓的影响,其中,“US”为仅超声辐照组,“Fe-PLGA@cRGD-PP1”为Fe-PLGA@cRGD-PP1+超声辐照组,“Fe-PFP-PLGA@PP1”为Fe-PFP-PLGA@PP1+超声辐照组,“Fe-PFP-PLGA@cRGD-PP1”为Fe-PFP-PLGA@cRGD-PP1+超声辐照组。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
其中,聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid,PLGA)购买于济南岱罡生物科技有限公司,分子量8000Da;全氟戊烷(Perfluoropentane,PFP)购买于相对密度:1.63g/cm3;多肽cRGD购买于吉尔生化(上海)有限公司;多肽PP1购买于吉尔生化(上海)有限公司;聚乙烯醇(PVA)购买于平均摩尔质量30000~70000。
2-(N-吗啉)乙磺酸(MES),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC),N-羟基琥珀酰亚胺(NHS)均为市售产品。
实施例1、本发明双靶向双模态显影纳米粒的制备
1.非靶向纳米粒的制备
50mg PLGA和1mg四氧化三铁(Fe3O4)充分溶于2mL二氯甲烷(CH2Cl2)中,加入200μLPFP;利用超声波震荡仪进行超声震荡,震荡功率为60W,震荡时间为3min(脉冲式5s on/5soff),超声震荡的温度为0℃。
然后将6mL的质量分数4%的聚乙烯醇水溶液,在0℃下再次超声震荡5min(震荡功率60W,脉冲式5s on/5s off)。最后,加入10mL的体积分数2%的异丙醇水溶液,冰浴下搅拌3小时直到有机溶剂完全挥发;1000rpm离心10min,弃上清,用双蒸水清洗固体三次,即得非靶向纳米粒Fe-PFP-PLGA。
2.双靶向双模态显影纳米粒的制备
将步骤1制得的Fe-PFP-PLGA全部分散在10mL 0.1M的MES缓冲溶液(pH=5)中,加入活化剂EDC 8mg和NHS 24mg,0℃下摇床孵育1小时;1000rpm离心10min,弃上清;将剩余体系分散于10mL l 0.1M的MES缓冲液(pH=8)中,加入2mg PP1和2mg cRGD,0℃下摇床反应过夜;1000rpm离心10min,弃上清,所得固体即双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1。
将上述双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1加入双蒸水重悬,即得Fe-PFP-PLGA@cRGD-PP1的纳米乳液。
将所得Fe-PFP-PLGA@cRGD-PP1纳米粒和纳米乳液收集并储存在4℃冰箱中。
以下为对照纳米粒的制备:
对照例1、Fe-PFP-PLGA纳米粒的制备
采用与实施例1步骤1相同的方法,制得Fe-PFP-PLGA纳米粒。对照例2、Fe-PLGA纳米粒的制备
按照实施例1步骤1的方法,将原料中的200μL PFP替换为200μL双蒸水,制得Fe-PLGA纳米粒。
对照例3、Fe-PFP-PLGA@PP1纳米粒的制备
按照实施例1的方法,将步骤2中的2mg PP1和2mg cRGD替换为2mg PP1,制得Fe-PFP-PLGA@PP1纳米粒。
对照例4、Fe-PFP-PLGA@cRGD纳米粒的制备
按照实施例1的方法,将步骤2中的2mg PP1和2mg cRGD替换为2mg cRGD,制得Fe-PFP-PLGA@cRGD纳米粒。
对照例5、Fe-PLGA@cRGD-PP1纳米粒的制备
按照实施例1的方法,将步骤1原料中的200μL PFP替换为200μL双蒸水,制得Fe-PLGA@cRGD纳米粒。
以下通过实验例证明本发明的有益效果。
实验例1:本发明双靶向双模态显影纳米粒的理化性质测试
1、测试对象:
实施例1制得的Fe-PFP-PLGA@cRGD-PP1,对照例1~5制得的Fe-PFP-PLGA、Fe-PLGA、Fe-PFP-PLGA@PP1、Fe-PFP-PLGA@cRGD、Fe-PLGA@cRGD-PP1。
2、测试方法:
动态光散射:利用动态光散射仪(马尔文)检测各纳米粒的粒径和电位,重复3次,取平均值;
纳米粒形态:利用透射电镜、扫描电镜检测各纳米粒的形态;并对透射电镜图进行伪彩色处理,进行元素分析。
红外光谱检测:利用红外光谱检测各纳米粒的结构;
激光共聚焦荧光显微镜:采用荧光标记的多肽(cRGD连接荧光罗丹明B、红色,PP1连接荧光FITC、绿色)制备荧光标记的材料,激光共聚焦显微镜下,分别使用PE、FITC通道观察纳米乳液形态,并将两个通道图像融合,分析两种荧光多肽与纳米粒的位置关系。
3、实验结果
表1各纳米粒的动态光散射测试结果
结果如表1和图1、图2所示。可以看出,本发明制得的双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1为壳核结构,其形态规则、大小均匀、分散性好,表面带负电(-34.90mV),平均粒径为289.54nm,而且粒径分布较均匀。
从图2c的激光共聚焦荧光显微镜图片可以看出,多肽cRGD(红色)、多肽PP1(绿色)都成功连接到了Fe-PFP-PLGA@cRGD-PP1纳米粒上。进一步从图1d的透射电镜伪彩图可以看出,本发明成功将金属Fe3O4(图中的Fe)、PFP(图中的F)、多肽cRGD和PP1(图中的S)包载到了PLGA材料中,形成了以PLGA为外壳,液态PFP、大量致密的金属Fe3O4颗粒为内核、多肽cRGD和PP1连接于PLGA外壳上的壳核结构纳米粒。
实验例2:体外US成像
1、测试方法
(1)验证超声(Ultrasound,简称US)照射触发相变后,纳米材料在超声下的成像效果
a.测试样品:Fe-PFP-PLGA@cRGD-PP1、生理盐水、Fe-PLGA@cRGD-PP1、Fe-PFP-PLGA;
b.超声相变条件:(超声治疗仪)3W,3HZ,3min,占空比30%;
c.记录B-mode和CEUS模式下的造影图片+回声强度。
(2)验证显影强度与诱发相变时超声照射强度或时间的关系
a.测试样品:Fe-PFP-PLGA@PP1-cRGD
b.超声相变条件:超声治疗仪,频率3HZ,占空比30%,功率和时间组合为3W 2min、3W 3min、3W 4min、3W 5min、3W 6min;
c.记录B-mode和CEUS模式下的造影图片+回声强度。
2、实验结果
结果如图3所示。从图3a和3b可以看出,相同相变条件下,与Fe-PLGA@cRGD-PP1和Fe-PFP-PLGA相比,本发明制得的双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1在CEUS模式下的超声信号更强,能更清楚展示局部信息,在CEUS模式下的回声强度最高。说明本发明制得的Fe-PFP-PLGA@cRGD-PP1在超声照射触发相变后,具有优异的超声显影效果。
从图3c和3d可以看出,本发明的双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1随着超声时间的增加,局部信息先清晰后模糊,回声强度先增加后降低。在以下超声相变条件下,显影效果最佳:3W,3HZ,3~4min,占空比30%。
实验例3:体外磁共振(MR)成像
1、测试方法
a.测试样品:Fe-PFP-PLGA@cRGD-PP1,利用双蒸水配置成梯度浓度0,0.2,0.4,0.6,0.8,and 1.0mg/mL,根据Fe的实际包载率计算相应Fe含量(单位:mM)的理论值;
b.磁共振成像:1.5T MRI scanner,head coil;
c.参数设置:
T2加权成像的参数:使用梯度回波序列;回波时间(TE),10.7毫秒;重复时间(TR),520毫秒;成像野(FOV),180毫米;翻转角(FA),45°。
横向弛豫速率(R2)的参数:回波时间(TE)1.5毫秒~18.8毫秒;重复时间(TR):35毫秒;脉冲重复激发次数(NEX)1;矩阵,320×192;切片厚度,1.5毫米。
d.记录图像,计算不同浓度纳米材料的R2数值并进行线性拟合。
2、实验结果
结果如图4所示。可以看出,随着Fe-PFP-PLGA@cRGD-PP1溶液浓度的降低,T2信号逐渐降低(图4a),说明本发明制得的双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1具有良好的核磁共振造影功能。而且,Fe-PFP-PLGA@cRGD-PP1的横向弛豫速率(R2)与其浓度具有良好的线性关系,说明该材料顺磁性良好(图4b),可以作为磁共振造影剂使用。
实验例4:对易破裂斑块的靶向性
1、测试样品:FITC荧光素标记的Fe-PFP-PLGA@cRGD-PP1、Fe-PFP-PLGA@cRGD。
2、实验方法:
(1)巨噬细胞的培养
激活巨噬细胞:对数生长期巨噬细胞,共计2.0×105个,接种于mm的共聚焦小皿中培养24小时;加脂多糖(Lipopolysaccharide,LPS;100ng/mL)于培养基中(200ng加入2mL新鲜培养基)孵育24小时。
脂多糖可促进骨髓来源的巨噬细胞分化为更接近于斑块内炎性巨噬细胞的表型,更好的模拟易破裂斑块内的巨噬细胞。
(2)分组共培养:
(a)不加PP1溶液:将荧光标记的样品(1mg/mL,1mL)分别加入非激活巨噬细胞、激活巨噬细胞的培养皿中,共培养2h后,用PBS洗3次,洗去多余的荧光纳米材料;Hoechst33258染色细胞核后,用激光共聚焦显微镜观察,并定量计算细胞内样品的荧光强度值(重复测5次,取平均值)。
(b)加PP1溶液:加入荧光标记的样品前,先在激活巨噬细胞的培养皿中加入PP1(50μg/mL,1mL),然后将荧光标记的样品(1mg/mL,1mL)加入,共培养2h后,用PBS洗3次,洗去多余的荧光纳米材料和PP1;染色细胞核后,用激光共聚焦显微镜观察,并定量计算细胞内样品的荧光强度值。
3、实验结果
表2各组细胞内样品的荧光强度值
结果如图5和表2所示,可以看出,在不加PP1溶液时,与Fe-PFP-PLGA@cRGD相比,本发明制得的Fe-PFP-PLGA@cRGD-PP1在非激活和激活巨噬细胞中的荧光强度都更高。特别是在激活巨噬细胞中,Fe-PFP-PLGA@cRGD-PP1的荧光强度比Fe-PFP-PLGA@cRGD显著提高。另外,与在加PP1溶液相比,不加PP1溶液时Fe-PFP-PLGA@cRGD-PP1在激活巨噬细胞中的荧光强度更高。
说明与Fe-PFP-PLGA@cRGD相比,本发明制得的Fe-PFP-PLGA@cRGD-PP1对巨噬细胞,特别是激活巨噬细胞具有显著提高的靶向作用,有利于提高对易破裂斑块的靶向性。
实验例5:在超声触发下诱导巨噬细胞凋亡
1、测试样品:PE荧光标记的Fe-PFP-PLGA@cRGD-PP1、PE荧光标记的Fe-PLGA@cRGD-PP1。
2、实验方法:
(1)激活巨噬细胞的培养:培养方法如实验例4所述。
(2)分组处理:
空白对照组:使用完全培养基培养48小时;
仅超声辐照组:培养24小时后,超声辐照(3W,3HZ,3min,占空比30%),再培养12小时;
non-PFP+超声辐照组:培养基培养24小时后,加入PE荧光标记的Fe-PLGA@PP1-cRGD(1mg/mL,1mL),共同孵育4小时后,PBS洗三次,更换新鲜培养基,超声辐照(3W,3HZ,3min,占空比30%),辐照后再培养12小时;
PFP+超声辐照组:培养24小时后,加入PE荧光标记的Fe-PFP-PLGA@cRGD-PP1,共同孵育4小时后,PBS洗三次,更换新鲜培养基,超声辐照(3W,3HZ,3min,占空比30%),辐照后再培养12小时。
(3)制样:PBS洗三次,染色(Calcein-AM,10mM in PBS;PI,2mM in PBS)15min(37℃,5%CO2);
(4)观察:CLSM观察,Calcein-AM和PI的激发波长分别为490±10nm、545nm。
3、实验结果:
结果如图6所示,可以看出,空白对照组、仅超声辐照组、non-PFP+超声辐照组的巨噬细胞细胞在共培养后基本均为活细胞,不能诱导细胞凋亡;而PFP+超声辐照组的巨噬细胞细胞在共培养后基本都已凋亡。说明只有将本发明制得的Fe-PFP-PLGA@cRGD-PP1联合超声辐照,才能够诱导激活巨噬细胞的凋亡,减轻炎症反应。所以,本发明制得的Fe-PFP-PLGA@cRGD-PP1能够通过诱发斑块内巨噬细胞凋亡,实现靶向稳定易损斑块和治疗斑块的目的。
实验例6:在超声触发下诱导血栓溶解
1、测试样品:Fe-PFP-PLGA@cRGD-PP1、Fe-PFP-PLGA@PP1、Fe-PLGA@cRGD-PP1。
2、实验方法:
(1)血栓切片制备:
取新西兰大耳白兔(雌性2.5Kg)耳缘动脉采集动脉血10mL,37℃水浴4小时,得血栓模型;
将血栓模型切成1×0.3×0.3cm大小、约200mg重,使用PBS洗三次后,制作为冰冻切片;为了保证血栓结构,使用冰丙酮固定10min,并用双蒸水再洗两次。
(2)分组处理:
空白对照组:将25mL PBS溶液加入上述血栓切片,摇床共同孵育2小时;
以下各组各自超声辐照不同时间(超声辐照时间如表3所示):
仅超声辐照组:将25mLPBS溶液加入上述血栓切片,超声辐照(3W,3HZ,3min,占空比30%),摇床共同孵育;
Fe-PFP-PLGA@cRGD-PP1+超声辐照组:将25mL Fe-PFP-PLGA@cRGD-PP1溶液(2mg/mL)加入上述血栓切片,超声辐照(3W,3HZ,3min,占空比30%),摇床共同孵育;
Fe-PFP-PLGA@PP1+超声辐照组:将25mLFe-PFP-PLGA@PP1溶液(2mg/mL)加入上述血栓切片,超声辐照(3W,3HZ,3min,占空比30%),摇床共同孵育;
Fe-PLGA@cRGD-PP1+超声辐照组:将25mLFe-PLGA@cRGD-PP1溶液(2mg/mL)加入上述血栓切片,超声辐照(3W,3HZ,3min,占空比30%),摇床共同孵育。
(3)观察:共同孵育结束后,去除上清液,PBS洗涤3次后,滤纸去除表面水分,留取大体图像,称重。
上述实验为多组平行实验,n=6,重量取平均值。
3、实验结果:
表3超声辐照不同时间后各组血栓切片重量平均值
结果如图7和表3所示。可以看出,超声辐照3小时后,仅超声辐照组、Fe-PLGA@cRGD-PP1+超声辐照组和Fe-PFP-PLGA@PP1+超声辐照组的血栓重量降低程度相当,均只降低了9.77%~10.66%;但是,Fe-PFP-PLGA@cRGD-PP1+超声辐照组的血栓重量降低了31.41%,降低程度显著高于3个对照组。超声辐照30小时后,仅超声辐照组、Fe-PLGA@cRGD-PP1+超声辐照组和Fe-PFP-PLGA@PP1+超声辐照组的血栓重量也仅仅降低了28.36%~30.36%,而Fe-PFP-PLGA@cRGD-PP1+超声辐照组的血栓重量降低了73.98%,降低程度显著高于3个对照组。
所以,只有将本发明制得的双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1在超声诱导下与血栓切片共培养,才能够显著降低血栓重量,诱导血栓溶解;而且,与Fe-PLGA@cRGD-PP1+超声辐照组和Fe-PFP-PLGA@PP1+超声辐照组相比,本发明制得的双靶向双模态显影纳米粒Fe-PFP-PLGA@cRGD-PP1在超声诱导对血栓的诱导溶解起到了协同增效的作用,取得了意料不到的技术效果。说明本发明制得的Fe-PFP-PLGA@cRGD-PP1能够在超声触发下有效诱导血栓溶解,有利于靶向溶解蚀损斑块表面血栓,实现蚀损斑块局部的抗血小板治疗。
综上,本发明以聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽为原料制得了一种形态规则、大小均匀、分散性好的双靶向双模态显影纳米粒,该双靶向双模态显影纳米粒具有超声、磁共振双模态显影功能,能靶向至易损斑块内的巨噬细胞,并在超声介导下诱发巨噬细胞凋亡,还能在超声介导下溶解易损斑块表面血栓,在制备诊断易损斑块(特别是易破裂斑块和蚀损斑块)的显像试剂,稳定易损斑块的药物,预防和/或治疗与易损斑块相关的心血管疾病的药物中具有广阔的应用前景。
Claims (10)
1.一种靶向双模态显影纳米粒,其特征在于:它是以聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽为原料制得的,所述靶向多肽选自cRGD和PP1中的一种或两种。
2.根据权利要求1所述的靶向双模态显影纳米粒,其特征在于:所述磁共振造影剂为Fe基磁共振造影剂,优选为四氧化三铁。
3.根据权利要求1所述的靶向双模态显影纳米粒,其特征在于:所述超声造影剂选自全氟戊烷、全氟己烷、全氟庚烷或全氟辛溴烷,优选为全氟戊烷。
4.根据权利要求1所述的靶向双模态显影纳米粒,其特征在于:所述聚乳酸-羟基乙酸共聚物分子量为6000Da~10000Da,优选为8000Da。
5.根据权利要求1所述的靶向双模态显影纳米粒,其特征在于:所述靶向多肽为cRGD和PP1,其中cRGD和PP1的质量比为1:(0.8~1.2),优选为1:1。
6.根据权利要求1~5任一项所述的靶向双模态显影纳米粒,其特征在于:所述聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽的质量体积比为50mg:(0.8~1.2)mg:(180~220)μL:(2~6)mg,优选为50mg:1mg:200μL:4mg。
7.权利要求1~6任一项所述靶向双模态显影纳米粒的制备方法,其特征在于:所述方法包括以下步骤:
(1)将聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂在有机溶剂中混合,超声震荡;
(2)在步骤(1)所得体系中加入乳化剂,超声震荡,得到非靶向纳米粒;
(3)将步骤(2)所得非靶向纳米粒与靶向多肽在pH=7~9的缓冲液中反应,即得。
8.根据权利要求7所述得方法,其特征在于:步骤(1)中,所述有机溶剂为二氯甲烷;所述聚乳酸-羟基乙酸共聚物与有机溶剂的质量体积比为20~30mg/mL,优选为25mg/mL;所述超声震荡的功率为50~70W,优选为60W,时间为3~5min,优选为3min,脉冲式为5秒on/5秒off;所述超声震荡的温度为0℃~4℃;
和/或,步骤(2)中,所述乳化剂为聚乙烯醇,优选为质量分数4%的聚乙烯醇水溶液;超声震荡功率为50~70W,优选为60W,时间为3~5min,优选为5min,脉冲式为5秒on/5秒off;所述超声震荡的温度为0℃~4℃;
和/或,步骤(2)中,所述超声震荡后,还包括在体系中加入醇类溶剂,搅拌,挥发有机溶剂,然后离心,弃上清;其中,所述醇类溶剂优选为异丙醇,更优选为体积分数2%的异丙醇水溶液;所述离心条件优选为1000rpm离心10min;
和/或,步骤(3)中,所述反应前,还包括将非靶向纳米粒与酰胺化反应活化剂混合,摇床孵育,离心,弃上清;优选的,所述酰胺化反应活化剂为EDC和NHS,所述摇床孵育条件为:在pH=5的MES缓冲溶液中于0℃~4℃孵育1小时,所述离心条件为1000rpm离心10min;
和/或,步骤(3)中,所述pH=7~9的缓冲液为pH=8的MES缓冲溶液,所述反应温度为0℃~4℃,反应时间为过夜。
9.权利要求1~6任一项所述靶向双模态显影纳米粒在制备诊断易损斑块的显像试剂和/或稳定易损斑块的药物中的用途;优选的,所述易损斑块为易破裂斑块和/或蚀损斑块;更优选的,所述显像试剂和/或药物是在以下超声照射条件下使用的:功率3W,频率3HZ,时间3~4min,占空比30%。
10.权利要求1~6任一项所述靶向双模态显影纳米粒在制备预防和/或治疗与易损斑块有关的心血管疾病的药物和/或诊断与易损斑块有关的心血管疾病的显像试剂中的用途;优选的,所述心血管疾病为动脉粥样硬化;更优选的,所述药物是在以下超声照射条件下使用的:功率3W,频率3HZ,时间3~4min,占空比30%。
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