CN114558133B - 一种同时递送声敏剂和靶向抗体的超声靶向微泡及其制备方法和应用 - Google Patents
一种同时递送声敏剂和靶向抗体的超声靶向微泡及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种同时递送声敏剂和靶向抗体的超声靶向微泡及其制备方法和应用,属于生物医药技术领域。本发明用二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺‑聚乙二醇2000、二硬脂酰磷脂酰乙醇胺‑聚乙二醇‑活性酯、胆固醇、脂质化声敏剂制备出声敏剂纳米颗粒,然后偶联靶向抗体制成同时递送声敏剂和抗体的超声靶向微泡,通过微泡的超声靶向爆破提高肿瘤细胞对声敏剂和抗体的摄取。同时在超声作用下产生活性氧,实现靶向声动力治疗肿瘤的效果。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种同时递送声敏剂和靶向抗体的超声靶向微泡及其制备方法和应用。
背景技术
在胃癌中,有一种特殊类型的胃癌,人表皮生长因子2(HER2)过表达的胃癌,称为HER2阳性胃癌。HER2在所有胃癌中的阳性率为12%~35%。HER2通过异源二聚体和酪氨酸激酶的磷酸化介导信号转导,激活下游通路,调节细胞增殖和分化。因此,HER2阳性这一独特的疾病亚型,癌细胞生长更快,侵袭性更强,患者病情也相对更容易恶化。现有的主要治疗方法为化疗联合曲妥珠单抗(Mol.Cancer 2020,19,62),但是化疗以及曲妥珠单抗毒副作用明显,且易产生耐药,治疗效果不显著。
声动力疗法(SDT)是一种新兴的肿瘤治疗方法,其主要机制是超声激活声敏剂产生毒性活性氧(ROS)杀死癌细胞。现有技术(Biomater.Sci.2021,9,1945-1960)表明SDT产生的活性氧对于肿瘤细胞具有杀伤力,能够用于肿瘤的治疗。因此,使用SDT来治疗HER2阳性胃癌具有很好的运用前景。目前研究中,化疗以及声动力治疗面临的共同难点是药物全身分布到达肿瘤处的药量不足,导致药物生物利用度低疗效有限,为了提高声敏剂在肿瘤处的富集程度,现有技术(Theranostics 2021,11,9470-9491)采用了纳米载体来递送声敏剂,但是纳米颗粒携载的游离药物容易提前泄漏,因此到达肿瘤处的声敏剂仍然有限。
微泡(MBS)通常被用作超声成像的造影剂,近年来也被广泛用作不同化疗药物、光敏剂、声敏剂和其他小分子药物的载体。进行超声辐照时,微泡作为人工空化核进行扩张、压缩或爆破(即空化效应),能够增强细胞膜和血管系统的通透性。此外,空化效应产生的射流在细胞内释放能量,细胞膜将会瞬间开放“声孔”,这些“声孔”是促进药物进入细胞的良好通道。因此,超声结合载药微泡可以显著提高药物在肿瘤组织中的富集。然而,缺乏主动靶向能力,阻碍了微泡在药物递送方面的应用。因此,开发一种能够同时递送声敏剂和靶向抗体的超声微泡,实现声动力与靶向治疗联合治疗将是一种不错的策略。
发明内容
本发明的目的在于提供一种同时递送声敏剂和靶向抗体(例如曲妥珠单抗)的超声靶向微泡及其制备方法和应用。本发明提供的同时递送声敏剂和靶向抗体的超声靶向微泡具有提高声敏剂和抗体在肿瘤富集,并在超声作用下产生活性氧治疗肿瘤的效果。
为了实现上述技术目的,本发明提供以下技术方案:
一种同时递送声敏剂和靶向抗体的超声靶向微泡,其中,所述微泡由二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯、胆固醇和脂质化声敏剂构成,在微泡表面通过共价键偶联靶向抗体,所述微泡和靶向抗体的质量比为1:0.25~2.0。
上述超声靶向微泡中,可选择的声敏剂例如焦脱镁叶绿酸a、二氢卟吩e6、血卟啉单甲醚等。将声敏剂脂质化的方法通常是利用声敏剂上的羧基与1-棕榈酰-sn-甘油-3-磷酸胆碱上的羟基进行化学偶联,得到脂质化声敏剂。
在本发明的实施例中使用了如下结构式所示的焦脱镁叶绿酸a脂质:
将1-棕榈酰-sn-甘油-3-磷酸胆碱、焦脱镁叶绿酸a、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、4-(二甲氨基)吡啶和N,N'-二异丙基乙胺溶于无水二氯甲烷中,在氩气保护下常温磁力搅拌48小时,然后旋转蒸发溶液,进行薄层色谱纯化即得到上述焦脱镁叶绿酸a脂质。
上述超声靶向微泡中,所述靶向抗体可以是曲妥珠单抗、帕妥珠单抗等。
上述超声靶向微泡中,所述微泡的平均粒径约为1~3微米。构成微泡的各脂类中,按总量为100摩尔计,所述二硬脂酰基磷脂酰胆碱的含量为60~70,所述二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000的含量为3~7,所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯的含量为3~10,所述胆固醇的含量为3~10,所述脂质化声敏剂的含量为10~30。在本发明的一个实施例中,二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇、二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯、胆固醇和脂质化声敏剂之间的摩尔比优选为65:5:5:5:20。
上述超声靶向微泡在超声作用下,微泡可转化为粒径均匀的纳米粒子,同时提高声敏剂和抗体在肿瘤处的富集,从而进行肿瘤治疗。其中,超声作用的超声频率范围为1-3MHz,占空比为10%-50%。
本发明还提供了上述同时递送声敏剂和靶向抗体的超声靶向微泡的制备方法,包括如下步骤:
步骤1、将二硬脂酰基磷脂酰胆碱溶解在无水乙醇或者三氯甲烷中得到溶液A;
步骤2、将二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶于无水乙醇或者三氯甲烷中得到溶液B;
步骤3、将二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯溶解到无水乙醇或者三氯甲烷中得到溶液C;
步骤4、将胆固醇溶解到无水乙醇或三氯甲烷中得到溶液D;
步骤5、将脂质化声敏剂溶解到四氢呋喃中得到溶液E;
步骤6、将溶液A、B、C、D、E混合,在水浴超声条件下注入到PBS溶液或者生理盐水中,使其形成纳米粒子,然后在PBS溶液中透析,除去有机溶剂;
步骤7、将步骤6所得的纳米粒子与靶向抗体在PBS溶液或者生理盐水中混合,避光条件下搅拌数小时,然后在PBS溶液或者生理盐水中透析除去未键合的靶向抗体,得到靶向声敏剂粒子分散液;
步骤8、将步骤7得到的靶向声敏剂粒子分散液与丙二醇和丙三醇混合均匀,然后充入SF6气体,机械震荡,得到同时递送声敏剂和靶向抗体的超声靶向微泡。
以所述脂质化声敏剂为焦脱镁叶绿酸a脂质、所述靶向抗体为曲妥珠单抗为例,上述步骤6的混合溶液中,二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯、胆固醇、焦脱镁叶绿酸a脂质之间的摩尔比为65:5:5:5:20,上述步骤7中,脂质纳米粒子与曲妥珠单抗的质量比优选2:1。
上述步骤6中,所述PBS溶液的pH值为7.2~7.4,水浴超声的超声波频率优选为25KHz~100KHz,温度为25~50℃。
上述步骤8中,优选的,所述靶向声敏剂粒子分散液的浓度优选为2~5mg/mL,其与丙二醇、丙三醇按体积比8:1:1混合。
本发明还提供了按上述技术方案制备的超声靶向微泡在癌症的诊断试剂或治疗试剂中的应用,例如曲妥珠单抗偶联的超声微泡用于治疗HER2阳性肿瘤。
在本发明中,用二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯、胆固醇、脂质化声敏剂制备出声敏剂纳米颗粒,然后偶联靶向抗体后,制备成同时递送声敏剂和抗体的靶向微泡。微泡的靶向爆破可以提高肿瘤细胞对声敏剂和抗体的摄取。同时,在超声作用下,产生活性氧,实现靶向声动力治疗肿瘤的效果。
本发明的有益效果是:
本发明通过使用脂质化声敏剂制备出载有声敏剂的微泡,有效避免了递送游离声敏剂提前泄漏的问题,降低毒副作用的同时提高了声敏剂的生物利用度。本发明通过共价键将抗体偶联到纳米颗粒后制备出靶向微泡,在不影响抗体活性的基础上,提高了抗体的递送效率,然后通过超声靶向微泡爆破,微气泡原位转化为纳米粒子,能够提高肿瘤组织对抗体和声敏剂的摄取,抗体本身的靶向作用也会进一步增强细胞对声敏剂的摄取,提高声动力和抗体治疗的效果。
附图说明
图1为本发明制备同时递送声敏剂和曲妥珠单抗的超声靶向微泡,并实现靶向声动力治疗肿瘤的示意图。
图2为实施例1制备的同时递送声敏剂和曲妥珠单抗的超声靶向微泡的显微镜图,其中a为明场图片,b为荧光图。
图3为实施例1制备的同时递送声敏剂和曲妥珠单抗的超声靶向微泡经超声靶向爆破后形成的纳米粒子的透射电镜图(a)和粒径分布图(b)。
图4为实施例2测得的递送声敏剂的微泡与同时递送声敏剂和曲妥珠单抗的超声靶向微泡经超声靶向爆破后形成的纳米粒子的电势结果图。
图5为实施例3测量同时递送声敏剂和曲妥珠单抗的超声靶向微泡在超声作用下产生活性氧的结果图。
图6为实施例4对同时递送声敏剂和曲妥珠单抗的超声靶向微泡进行超声增强造影的结果图。
图7为实施例5中同时递送声敏剂和曲妥珠单抗的超声靶向微泡在超声作用下提高HER2阳性NCI-N87细胞对声敏剂摄取的实验结果图。
图8为实施例6中同时递送声敏剂和曲妥珠单抗的超声靶向微泡在超声作用下通过靶向声动力杀伤肿瘤细胞的实验结果图。
图9为实施例7中同时递送声敏剂和曲妥珠单抗的超声靶向微泡处理HER2阳性NCI-N87细胞后细胞信号通路表达结果图。
图10为实施例8中同时递送声敏剂和曲妥珠单抗的超声靶向微泡在超声作用下通过声动力和抗体治疗抑制肿瘤生长曲线图,以单纯注射生理盐水、单纯超声、游离曲妥珠单抗、靶向声敏剂微泡、声敏剂微泡加超声处理、靶向纳米粒子加超声处理作为对照。
具体实施方式
下面结合附图,通过实施例对本发明提供的一种同时递送声敏剂和曲妥珠单抗的超声靶向微泡及其制备方法和应用进行详细的说明,但是不能将它们理解为对本发明保护范围的限定,本发明的保护范围由所附权利要求书界定。
HER2阳性胃癌发病率高,患者病情也相对更容易恶化,加之早期症状不明显,多数患者确诊时已属于中晚期。对于进展期HER2阳性胃癌目前主要的治疗方法为化疗联合曲妥珠单抗,但是此治疗方案易产生耐药且毒副作用明显,减少耐药提高治疗效果的同时降低毒副作用具有重要意义。
超声不仅可以用于疾病的诊断,也可以用于疾病的治疗及药物的递送。超声介导的声动力治疗是一种由光动力治疗发展而来的无创新兴的治疗手段,目前声动力治疗的主要机制为超声激活声敏剂产生有毒的活性氧来杀死癌细胞,因此声敏剂在声动力治疗中具有重要作用,但是卟啉等小分子声敏剂疏水性强在血液循环中会被快速清除,因此声动力治疗的难点在于声敏剂缺乏靶向性,到达肿瘤处的声敏剂少,治疗效果不显著。开发一种递送平台将治疗抗体和声敏剂高效的递送到肿瘤组织,能够有效的降低毒副作用提高肿瘤的治疗效果。
如图1所示,制备同时递送声敏剂和曲妥珠单抗的超声靶向微泡,通过静脉注射注入体内,利用超声造影观察微泡到达肿瘤处后利用超声靶向微泡爆破技术将微气泡原位转化为纳米颗粒,利用EPR效应(增强渗透滞留效应)及抗体靶向作用增加细胞对声敏剂的吸收和摄取实现靶向治疗和SDT的联合治疗,实现高效的肿瘤治疗。
实施例1、同时递送声敏剂和曲妥珠单抗的超声靶向微泡的制备
步骤1、将艾伟拓(上海)医药科技有限公司购买的二硬脂酰基磷脂酰胆碱溶解在乙醇中得到浓度为20mg/mL的溶液A;
步骤2、将艾伟拓(上海)医药科技有限公司购买的二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶于无水乙醇中得到浓度为20mg/mL的溶液B;
步骤3、将上海源叶生物科技有限公司购买的二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯溶解到无水乙醇中得到浓度为20mg/mL的溶液C;
步骤4、将胆固醇溶解到无水乙醇中得到浓度为20mg/mL的溶液D;
步骤5、将100nmol 1-棕榈酰-sn-甘油-3-磷酸胆碱,50nmol焦脱镁叶绿酸a,25nmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐,4-(二甲氨基)吡啶,25μL N,N'-二异丙基乙胺溶于10mL无水二氯甲烷中,在氩气保护下常温磁力搅拌48小时,然后旋转蒸发溶液后,进行薄层色谱纯化,得到焦脱镁叶绿酸a脂质;将焦脱镁叶绿酸a脂质(C58H84N5O10P)溶解到四氢呋喃中得到浓度为10mg/mL的溶液E;
步骤6、将A、B、C、D、E溶液按摩尔比65:5:5:5:20混合,然后在水浴超声条件(30℃,40KHz)下注入到pH为7.4的PBS溶液中,使其形成纳米粒子,然后在PBS溶液中透析半小时除去有机溶剂乙醇和四氢呋喃;
步骤7、将步骤6所得的纳米粒子与曲妥珠单抗混合,纳米粒子与抗体质量比为2:1,避光条件下搅拌6小时,然后在PBS缓冲液中透析除去未键合的曲妥珠单抗,得到靶向声敏剂粒子分散液。
步骤8、将步骤7得到的4mg/mL的靶向声敏剂粒子分散液与丙二醇和丙三醇以体积比8:1:1混合均匀,然后充入SF6气体,机械震荡45秒,得到同时递送声敏剂和曲妥珠单抗的超声靶向微泡。
图2为本实施例所得同时递送声敏剂和曲妥珠单抗的超声靶向微泡的显微镜图,其中a为明场图片,b为荧光图。由图2可知,微泡粒径均匀,在1-3微米左右。同时显示声敏剂的红色荧光,说明声敏剂装载到了微泡上。
图3为实施例1所得同时递送声敏剂和曲妥珠单抗的超声靶向微泡经超声靶向微泡爆破后形成的纳米粒子的透射电镜图和采用动态光散射法测试所得的粒径分布图。由图3可知,同时递送声敏剂和曲妥珠单抗的超声靶向微泡经靶向微泡爆破后形成的纳米粒子的平均粒径为115.87±11.23纳米,且粒径分布集中,有利于该超声靶向微泡经超声靶向微泡爆破后形成的纳米粒子通过肿瘤组织的渗透与滞留效应(EPR)富集在肿瘤组织。
实施例2、偶联曲妥珠单抗后电位变化
将递送声敏剂的微泡和同时递送声敏剂和曲妥珠单抗的超声靶向微泡经超声靶向微泡爆破后形成的纳米粒子进行电位测量。
结果如图4所示,与曲妥珠单抗偶联后,纳米粒子的表面电荷由-37.17±0.21mV变化为-16.33±1.44mV,这与曲妥珠单抗的正电荷有关,这一结果表明曲妥珠单抗成功地偶联到了微泡上。
实施例3、体外产生活性氧性能评估
为了考察实施例1所得同时递送声敏剂和曲妥珠单抗的超声靶向微泡在超声处理下活性氧的生成情况。实验中使用单线态氧荧光探针SOSG检测单线态氧的生成,将SOSG探针加到同时递送声敏剂和曲妥珠单抗的超声靶向微泡中,然后进行超声处理,超声参数为1.0MHz,50%占空比,1.0W·cm-2,每超声一分钟进行一次荧光光谱扫描(激发波长:504nm,发射波长:510-600nm)
结果如图5所示,超声照射后,可以观察到SOSG在525nm左右最大荧光发射峰强度有明显的增加,且具有时间依耐性,随着超声时间增加,荧光强度增强,说明在超声辐照下同时递送声敏剂和曲妥珠单抗的超声靶向微泡具有显著的生成单线态氧的能力。
实施例4、超声造影能力测试
将实施例1所得同时递送声敏剂和曲妥珠单抗的超声靶向微泡稀释成不同浓度(1×108,5×107,2.5×107/毫升)的溶液放入橡胶管中,以生理盐水为对照,然后使用超声成像仪观察其造影能力。
结果如图6所示,在超声造影模式下,靶向微泡呈现出均匀密集的点状强回声,且呈浓度依赖性,证明同时递送声敏剂和曲妥珠单抗的超声靶向微泡具有较好的超声成像效果。
实施例5、靶向微泡提高细胞对声敏剂摄取评估
取处于对数生长期的NCI-N87细胞接种于共聚焦小皿中,每孔细胞密度约为6×104个,于37℃,5%CO2条件下培养24小时。将原培养基换成含有只递送声敏剂的超声微泡、同时递送声敏剂和曲妥珠单抗的超声靶向微泡以及同时递送声敏剂和曲妥珠单抗的纳米粒子的三组培养基,经超声靶向爆破处理后培养8h,最后用4%多聚甲醛固定细胞,DAPI染色3分钟,然后在激光共聚焦显微镜下观察声敏剂荧光(λex=405nm,λem=650-720nm)和DAPI(λex=405nm,λem=410-490nm)。
结果如图7所示,同时递送声敏剂和曲妥珠单抗的超声靶向微泡组NCI-N87细胞的荧光强度明显强于同时递送声敏剂和曲妥珠单抗的纳米粒子组和只递送声敏剂的超声微泡组,说明超声靶向微泡爆破和曲妥珠单抗的靶向作用使NCI-N87细胞对声敏剂的摄取增加。
实施例6、细胞毒性测试
取处于对数生长期的NCI-N87细胞接种于96孔板中,每孔细胞密度约为1×104个,于37℃,5%CO2条件下培养24小时。然后将细胞分为7组:对照(Control)组,超声(US)组,曲妥珠单抗(Tra)组,同时递送声敏剂和曲妥珠单抗的超声靶向微泡(TP MBs)组,只递送声敏剂的超声微泡加超声(P MBs+US)组,同时递送声敏剂和曲妥珠单抗的纳米粒子加超声(TPNPs+US)组,同时递送声敏剂和曲妥珠单抗的超声靶向微泡加超声(TP MBs+US)组。将培养基按照分组进行替换,其中声敏剂的浓度为20微克/毫升。超声靶向微泡爆破后在37℃,5%CO2条件下培养8小时,进行超声处理后继续培养16小时,通过标准的MTT法检测细胞活性。
结果如图8所示,对NCI-N87细胞只进行超声处理时细胞的存活率几乎没有影响,但是同时递送声敏剂和曲妥珠单抗的超声靶向微泡与细胞共同孵育后,细胞存活率略有下降,这可能是由于曲妥珠单抗对HER2阳性细胞生长有抑制作用。只递送声敏剂的超声微泡加超声组和同时递送声敏剂和曲妥珠单抗的纳米粒子加超声组细胞存活率分别为73.55%±3.97%和68.50%±2.03%,表明声动力治疗对细胞有明显的杀伤作用。值得注意的是,同时递送声敏剂和曲妥珠单抗的超声靶向微泡加超声组的细胞存活率降至44.49%±5.49,明显低于只递送声敏剂的超声微泡加超声组和同时递送声敏剂和曲妥珠单抗的纳米粒子加超声组,进一步表明超声靶向微泡爆破和抗体靶向在提高细胞对声敏剂的摄取后也能显著提高对肿瘤的杀伤作用。
实施例7、相关细胞信号通路表达情况
取处于对数生长期的NCI-N87细胞接种于6孔板中,每孔细胞密度约为3×105个,于37℃,5%CO2条件下培养24小时。然后将细胞分为7组:对照(Control)组,超声(US)组,曲妥珠单抗(Tra)组,同时递送声敏剂和曲妥珠单抗的超声靶向微泡(TP MBs)组,只递送声敏剂的超声微泡加超声(P MBs+US)组,同时递送声敏剂和曲妥珠单抗的纳米粒子加超声(TPNPs+US)组,同时递送声敏剂和曲妥珠单抗的超声靶向微泡加超声(TP MBs+US)组。将培养基按照分组进行替换,其中声敏剂的浓度为20微克/毫升。超声靶向微泡爆破后在37℃,5%CO2条件下培养8小时,进行超声处理后继续培养16小时,通过Western blot检测细胞内Cle-caspase3、AKT、P-AKT蛋白表达。
结果如图9所示,同时递送声敏剂和曲妥珠单抗的超声靶向微泡加超声组凋亡相关蛋白Cle-Caspase3的表达明显高于其他组,说明诱导肿瘤细胞凋亡是靶向声动力治疗的主要机制之一,P-AKT蛋白的表达也最低,说明靶向微泡联合声动力治疗可显著抑制AKT的磷酸化,从而抑制肿瘤细胞的生长。
实施例8、肿瘤抑制情况
用18-20g Balb/c裸鼠建立NCI-N87肿瘤模型,肿瘤达到约200mm3时,随机分为7组:对照(Control)组,超声(US)组,曲妥珠单抗(Tra)组,同时递送声敏剂和曲妥珠单抗的超声靶向微泡(TP MBs)组,只递送声敏剂的超声微泡加超声(P MBs+US)组,同时递送声敏剂和曲妥珠单抗的纳米粒子加超声(TP NPs+US)组,同时递送声敏剂和曲妥珠单抗的超声靶向微泡加超声(TP MBs+US)组。每种药物尾静脉注射200μL(声敏剂当量浓度:4mg·kg-1,曲妥珠单抗:8mg·kg-1),超声靶向微泡爆破后(1.0MHz,2 0%占空比,1.0W·cm-2,3min)进行超声照射(1.0MHz,5 0%占空比,2.0W·cm-2,5min)。不同处理后,每隔2天测量一次小鼠肿瘤体积和重量,一共21天。
结果如图10所示,单独超声(US)治疗组肿瘤体积增长趋势与生理盐水(Control)组基本一致,说明单独超声治疗对肿瘤生长无影响。而曲妥珠单抗(Tra)和同时递送声敏剂和曲妥珠单抗的超声靶向微泡(TP MBs)组肿瘤生长略慢于生理盐水组,这可能是由于曲妥珠单抗的治疗作用。而只递送声敏剂的超声微泡加超声(P MBs+US)组、同时递送声敏剂和曲妥珠单抗的纳米粒子加超声(TP NPs+US)组和同时递送声敏剂和曲妥珠单抗的超声靶向微泡加超声(TP MBs+US)组均有明显的肿瘤生长抑制作用,表明这些组均能有效积聚药物和产生有毒的活性氧。但同时递送声敏剂和曲妥珠单抗的超声靶向微泡加超声(TP MBs+US)组治疗效果最好,21天内肿瘤体积几乎无变化,这是由于超声靶向微泡爆破及曲妥珠单抗的靶向作用增强声动力治疗和抗体治疗所致。
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 (8)
1.一种同时递送声敏剂和靶向抗体的超声靶向微泡,其中,所述微泡由二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯、胆固醇和脂质化声敏剂构成,在微泡表面通过共价键偶联靶向抗体,所述微泡和靶向抗体的质量比为1:0.25~2.0;所述脂质化声敏剂为焦脱镁叶绿酸a脂质,其结构式如下:
构成微泡的各脂类中,按总量为100摩尔计,所述二硬脂酰基磷脂酰胆碱的含量为60~70,所述二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000的含量为3~7,所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯的含量为3~10,所述胆固醇的含量为3~10,所述脂质化声敏剂的含量为10~30。
2.如权利要求1所述的超声靶向微泡,其特征在于,所述靶向抗体选自曲妥珠单抗、帕妥珠单抗。
3.如权利要求1所述的超声靶向微泡,其特征在于,所述微泡的平均粒径为1~3微米。
4.如权利要求1所述的超声靶向微泡,其特征在于,在超声作用下,所述微泡转化为粒径均匀的纳米粒子。
5.权利要求1~4任一所述的同时递送声敏剂和靶向抗体的超声靶向微泡的制备方法,包括以下步骤:
1)将二硬脂酰基磷脂酰胆碱溶解在无水乙醇或三氯甲烷中得到溶液A;
2)将二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶于无水乙醇或三氯甲烷中得到溶液B;
3)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯溶解到无水乙醇或三氯甲烷中得到溶液C;
4)将胆固醇溶解到无水乙醇或三氯甲烷中得到溶液D;
5)将脂质化声敏剂溶解到四氢呋喃中得到溶液E;
6)将溶液A、B、C、D、E混合,在水浴超声条件下注入到PBS溶液或者生理盐水中,使其形成纳米粒子,然后在PBS溶液或者生理盐水中透析,除去有机溶剂;
7)将步骤6)所得的纳米粒子与靶向抗体在PBS溶液或者生理盐水中混合,避光条件下搅拌数小时,然后在PBS溶液或者生理盐水中透析除去未键合的靶向抗体,得到靶向声敏剂粒子分散液;
8)将步骤7)得到的靶向声敏剂粒子分散液与丙二醇和丙三醇混合均匀,然后充入SF6气体,机械震荡,得到同时递送声敏剂和靶向抗体的超声靶向微泡。
6.如权利要求5所述的制备方法,其特征在于,步骤5)中所述脂质化声敏剂为焦脱镁叶绿酸a脂质,在步骤6)按二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、二硬脂酰磷脂酰乙醇胺-聚乙二醇-活性酯、胆固醇、焦脱镁叶绿酸a脂质之间的摩尔比为65:5:5:5:20进行混合;在步骤7)中,将步骤6)所得纳米粒子与曲妥珠单抗按质量比2:1混合;在步骤8)中,所述靶向声敏剂粒子分散液的浓度为2~5mg/mL,其与丙二醇、丙三醇按体积比8:1:1混合。
7.权利要求1~4任一所述的同时递送声敏剂和靶向抗体的超声靶向微泡在制备诊断和/或治疗癌症的药物中的应用。
8.如权利要求7所述的应用,其特征在于,所述靶向抗体为曲妥珠单抗,所述癌症为HER2阳性胃癌。
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