CN108578696B - 一种脂质体微泡载金属-icg自组装复合体系 - Google Patents
一种脂质体微泡载金属-icg自组装复合体系 Download PDFInfo
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Abstract
本发明公开了一种脂质体微泡载金属‑ICG自组装复合体系。该自组装复合体系具有多模态成像/声动力治疗(SDT)功能。该体系展示出了优于单纯ICG分子的声动力性能,同时具备脂质体微泡的定点爆破和药物定点释放并增加肿瘤、感染及动脉粥样硬化等动物疾病模型的声动力治疗效果。本发明还涉及所述金属离子/ICG@Microbubbless超声成像,荧光成像和光声成像手段指导下的声动力治疗,该治疗体系制备简单、价格便宜以及疗效显著的优点。
Description
技术领域
本发明涉及一种脂质体微泡载金属离子-ICG自组装体系,还涉及其制备方法及所属脂质体微泡用于皮下肿瘤、原位肿瘤、动脉粥样硬化等动物疾病模型的声动力治疗中诊疗一体化的应用,属于生物医药材料以及纳米医学领域。
背景技术
恶性肿瘤和心血管疾病是21世纪威胁全球人类生命健康的最大杀手之一,对其有效的诊断和治疗是当前生物医学研究领域所面临的重大挑战。临床上传统的治疗方法主要是有手术治疗、放射性疗法以及化学疗法。但是传统疗法有着方法风险高,缺少特异性,会破坏免疫系统,且化疗药物耐药性的产生也极大地限制了其应用。光学治疗作为是一种新兴的治疗方法,包括有光热治疗(PTT)和光动力治疗(PDT)。而光热治疗是光热药物在近红外光照射以后产生过高热杀死肿瘤细胞或者动脉粥样硬化部位的巨噬细胞。光动力治疗是在特定波长光的照射下,通过光敏化作用将肿瘤部位氧气转化为活性氧(ROS)从而杀死肿瘤细胞和巨噬细胞。光学疗法可以在特定肿瘤组织或者心血管病变部位进行定位照射,可以重复给药,操作相对方便。但是由于所使用的特定波长的激光多为630nm或808nm等,其组织穿透深度有限,限制了其用于更深部肿瘤和所有心血管疾病治疗的可能。而本发明使用的SDT(多模态成像/声动力治疗)的主要原理是利用超声激活在细胞中富集并长时间滞留的声敏剂分子产生一系列活性氧簇(reactive oxygen species,ROS) 来杀伤细胞并抑制细胞的增殖。此处使用的声波频率为1MHz,能够穿透更深的组织,可实现深部病变的定点治疗并且对正常组织无创伤性。
FDA已批准的临床用药吲哚菁绿(ICG)超声照射下可产生ROS,以及在肿瘤、感染病变区域和动脉粥样硬化部位等的富集的趋向性而可被用于声动力治疗中。近些年,利用功能分子自组装成纳米结构已经在材料科学领域引起的极大的兴趣。如CN107029236A公开了一种吲哚菁绿自组装纳米疫苗的制备方法,其包括以下步骤:(1)将抗原蛋白水溶液与吲哚菁绿琥珀酰亚胺酯二甲基亚砜溶液等体积混合,所述抗原蛋白与吲哚菁绿琥珀酰亚胺酯的摩尔比为1:1~50,在20~30℃下反应18~48小时,使吲哚菁绿通过酰胺键连接在所述抗原蛋白上;(2)将步骤(1)制备的产物加入到水中,制成质量百分比浓度为0.001%~1%的溶液,在20~30℃下孵育18~48小时;调节温度至4~10℃,以15000~30000rpm转速离心20~40min,收集纳米粒,冷冻干燥后得到吲哚菁绿自组装纳米疫苗。而CN105193831A公开了一种负载吲哚菁绿的自组装多功能纳米靶向系统的制备方法及其应用,其中阿霉素通过二硫键作为连接臂与羟基氯喹相连形成自组装纳米粒,然后将吲哚菁绿物理吸附到此纳米粒中,再用磷脂聚乙二醇叶酸包裹纳米粒形成负载吲哚菁绿的自组装多功能纳米靶向系统。此外,“基于吲哚菁绿近红外荧光特性手术影像导航系统的设计”,吕铁军等,中国组织工程研究,2012,16(52):9802-9806,基于吲哚氰绿因为其独特的物理性质和近红外光谱特性,研究用来进行医学成像,设计了基于吲哚氰绿近红外荧光特性的手术影像导航系统,依据原理设计光学系统和硬件系统,开发出相应的软件系统,并利用前期实验进行系统成像效果验证。
然而ICG本身代谢周期短、超声波照射下ROS产量有限的缺点也限制了其在SDT中的应用。
发明内容
为了克服现有技术中存在的上述缺陷,本发明人经过深入研究和大量实验,利用金属离子与ICG发生自组装成纳米材料的特性,提高ICG在1MHz 超声波照射下ROS的产量,从而增强声动力治疗效果。
本发明所述的金属离子为Fe3+,Mn2+,Ga3+或者Zn2+中的至少一种。
本发明人鉴于ICG本有优异的结构特点,设计利用ICG分子中磺酸根和金属离子作用组装成纳米结构,通过分子间的相互作用,团聚后的金属离子 /ICG纳米结构密度增大,使其声动力效果增强,当以脂质体微泡为模板可实现金属离子/ICG的完全装载。超声成像仪可指导微泡在肿瘤周边血管的定点爆破,光声成像仪及荧光成像仪则监测药物在肿瘤、感染病变区域及动脉粥样硬化等病变部位的富集情况,以期施以最佳的声动力治疗。综上,本发明设计的金属离子/ICG@脂质体微泡体系具备良好的声动力治疗的潜力。
为此,在本发明的一方面,提供了一种脂质体微泡载金属-ICG自组装复合体系,该体系包含作为载体的微泡形式的脂质体,离子形式的金属,和ICG 自组装形成的纳米结构(亦即纳米结构体)。
优选地,由所述金属离子诱导ICG组装成所述纳米结构。
优选地,所述金属离子为Mn2+、Fe3+、Ga3+、Zn2+中的至少一种。金属离子可以是硝酸盐、硫酸盐、卤素盐等方式存在。优选为氯化盐。
在所述自组装复合体系,其中金属离子与ICG的摩尔比优选为1:20-20:1,更优选为1:5-5:1,最优选为1:2。
就本发明的自组装复合体系而言,在形成该自组装复合体系时使用包含醇的溶剂,即该自组装复合体系使用包含醇的溶剂制得。
优选地,所述的醇包括甲醇和乙醇。溶剂为水。
优选地,包含甲醇的溶剂为甲醇水溶液,其中,甲醇和水的体积比为 10:1-1:10。更优选为5:1-1:5,最优为2:1。
优选地,就本发明的自组装复合体系而言,脂质体微泡将纳米结构部分包覆或全部包覆,优选基本全部包覆。
在本发明的另一方面,提供了制备所述自组装复合体系的方法,该方法包括以下步骤:(1)配置金属离子的醇溶液;(2)配置醇:水溶液,将ICG溶于其中,并在其中加入按步骤(1)中所得的金属离子溶液,室温下搅拌 0.5-10h;(3)使用步骤(2)中得到的金属离子-ICG混合物,称取脂质体且使用三氯甲烷溶解,而后加入到搅拌着的所述金属离子-ICG混合物中,然后除去三氯甲烷溶剂和甲醇溶剂,再在40-80℃水浴中,在密闭条件下边加入气体边使用超声探头间断式超声起泡,得到最终复合脂质体微泡。
在所述方法中,所述脂质体为大豆卵磷脂、胆固醇和DSPE-PEG2000中的至少一种。
脂质体在三氯甲烷中的浓度,优选为0.1-100mg/ml,更优选为0.5-50 mg/ml。
更优选地,本发明所述的金属离子/ICG@复合脂质体微泡可通过如下步骤制取:
(1)利用Isothermal titration calorimetry(等温滴定量热法,ITC)检测最佳Fe3+:ICG摩尔比例。具体地,在167mL Hepes buffer(10mM,pH=6.8)中加入70.5mL CH3OH、11.5mL CHCl3和1mL n-hexane(正己烷)并且静置 24h,将金属离子及ICG用上述预备的buffer分别配置为0.05mM和1mM 溶液,在20℃下将ICG滴入金属离子溶液中,最终金属离子与ICG的配比为 1:2;
(2)分别配置不同体积比的甲醇:水溶剂(如10:1,5:1,3:1,2:1,1:1,1:2, 1:3,1:5,1:10)。将1mg ICG分别用5mL不同甲醇:水溶剂溶解,并在其中滴入按(1)中所得最佳配比的金属离子摩尔量并用同种甲醇:水溶剂配制的金属离子溶液(5mL),室温下搅拌2h;使用粒径仪检测不同甲醇:水溶剂下所形成的自组装体系的纳米尺寸,甲醇:水溶剂比例为2:1的溶剂为最终使用溶剂。
(3)金属离子/ICG@Microbubbless复合微泡的制备:使用(2)中最终溶剂配制(1)中最佳金属离子-ICG混合物(共25ml)(ICG,2.5mg),并且称取5mg大豆卵磷脂,2.7mg胆固醇,0.7mg DSPE-PEG2000,分别使用1ml三氯甲烷溶解,而后加入到搅拌着的金属离子-ICG混合物中,使用旋蒸仪在程序性升温下去除三氯甲烷溶剂和甲醇溶剂,并将最终混合物定容至 10ml;65℃水浴中,在密闭条件下边加入C3H8气体边使用超声探头(脉冲: 10s/10s;750W,20kHz,33%)间断式超声起泡,得到最终复合脂质体微泡。
在本发明的又一方面,提供了上文所述自组装复合体系的应用,其用于皮下肿瘤、原位肿瘤、或动脉粥样硬化的高分辨荧光成像和光声成像。
优选地,形成的脂质体微泡可实现原位肿瘤如原位肝脏肿瘤的超声成像,及成像指导下的微泡在病变部位的实时爆破和药物的实时释放。
本发明人研究发现,在甲醇溶剂介导下,利用脂质体微泡作为金属离子 /ICG自组装模板,便于实现疏水性增强的金属离子/ICG在机体内的有效递送。
本发明提供了一种新的具有多模态成像/声动力治疗性能的纳米材料@复合脂质体微泡的简单实用、可以大规模生产该微泡的制备方法;
本发明还提供了一种所述的复合微泡作为疾病的超声成像、光声成像、荧光成像诊断/声动力治疗的应用。
本发明中多模态成像/声动力治疗复合微泡其结构中含有用于声动力治疗的金属离子/ICG自组装,及作为此自组装模板的脂质体微泡载体。
其中,所述的声敏剂为FDA临床批准的吲哚菁绿(ICG)。
其中,所述的金属离子包括Fe3+,Mn2+,Ga3+或者Zn2+中的至少一种。
其中,所述的脂质体微泡载体的主要成分为DSPE-PEG2000、大豆卵磷脂和/或胆固醇中的至少一种。分子量单位为道尔顿。
本发明改进了具备声动力效果的ICG的用药形式,使其通过自组装形式形成稳定的纳米结构体系,从而促使其声动力效果得以提升,也不影响其在良好成像上的应用。
与现有技术相比,本发明取得的突出技术效果包括:
(1)利用金属离子与ICG发生自组装成纳米材料的特性,提高ICG在 1MHz超声波照射下ROS的产量,从而增强肿瘤及心血管疾病如动脉粥样硬化的声动力治疗效果。
(2)在制备中,于甲醇溶剂介导下,利用脂质体微泡作为金属离子/ICG 自组装模板,从而实现疏水性增强的金属离子/ICG在机体内的有效运输。
(3)提供一种新的具有多模态成像、声动力治疗性能的纳米材料 @Microbubbles复合微泡的简单实用可以大规模生产该微泡的制备方法。并且此合成的复合微泡可用于肿瘤等疾病的超声成像、光声成像、荧光成像诊断、声动力治疗的应用
附图说明
图1是根据本发明实施例1的利用Isothermal titration calorimetry(ITC)检测Fe3+与ICG结合比例的坐标图。
图2是根据本发明实施例1的水:甲醇溶剂分别在3:1,2:1,1:1,1:2,1:3 中时,Fe3 +/ICG自组装纳米材料的粒径变化坐标图。
图3中的(A)是根据本发明实施例1的在ICG中加入不同成分之后其吸收变化图;(B)是根据本发明实施例1的微泡粒径分布图,其中(B)中的右上为微泡的SEM图,比例尺为4μm。
图4中的(A)是根据本发明实施例1的不同浓度ICG及微泡(ICG,2μg, 5μg,10μg,20μg)在超声(1MHz,1.61W cm-2)下产生的ROS量用DCFH荧光探针来检测获得的坐标图,荧光强度越强代表产生ROS量越高;(B)是根据本发明实施例1的倒置荧光显微镜下,HepG2细胞在有超声(右边)和未超声(左边)下的DCFH荧光强度图。
具体实施方式
以下通过具体的制备例和实施例可使本发明得到更清楚的说明,但本发明的整体构思并不局限于该实施例,本领域的专业技术人员往往可以通过同样的思路对实验中的某一步进行替换,但该专利的整体思路仍受保护:
实施例1
该实施例为制备例,其选择金属离子Fe3+为制备例。
(1)利用Isothermal titration calorimetry(ITC)检测最佳Fe3+:ICG摩尔比例:首先配置缓冲溶液。在167mL Hepes buffer(10mM,pH=6.8)中加入 70.5mL CH3OH,11.5mLCHCl3和1mL n-hexane并且静置24h;将Fe3+(氯化铁)及ICG用上述预备的buffer分别配置为0.05mM和1mM溶液;在20℃下将ICG滴入金属离子溶液中,最终得知金属离子与ICG的配比情况为2个 ICG分子与1个Fe3+配比。
(2)寻找最佳分散溶剂:分别配置不同体积比的甲醇:水溶剂(如10:1, 5:1,3:1,2:1,1:1,1:2,1:3,1:5,1:10);将1mg ICG分别用5ml不同甲醇:水溶剂溶解,并在其中滴入按1)中所得最佳配比的Fe3+摩尔量并用同种甲醇:水溶剂配制的金属离子溶液(5ml),室温下搅拌2h,而后使用粒径仪检测不同甲醇:水溶剂下所形成的自组装体系的纳米尺寸,以可使粒径接近为0nm下使用的最小(甲醇:水)比例下的溶剂为最终使用溶剂(2:1)。
(3)金属离子/ICG@Microbubbles复合脂质体微泡的制备:使用(2) 中最终溶剂配制(甲醇:水=2:1)1)中最佳Fe3+-ICG混合物(共25ml)(
ICG,2.5mg)。并且称取5mg大豆卵磷脂,2.7mg胆固醇,0.7mg DSPE-PEG2000,分别使用1ml三氯甲烷溶解。而后加入到搅拌着的金属离子 -ICG混合物中。使用旋蒸仪在程序性升温下去除三氯甲烷溶剂和甲醇溶剂,并将最终混合物定容至10ml。65℃水浴中,在密闭条件下边加入C3H8气体边使用超声探头((脉冲:10s/10s;750W,20kHz,33%))间断式超声起泡,得到最终复合脂质体微泡。
如图1所示,由ITC数据可看出,当在0.05mM Fe3+中缓慢滴入1mM 的ICG溶液时,其呈现标准的两点结合模式。即两个ICG分子与一个Fe3+结合。因此本发明人以ICG分子与Fe3+摩尔比2:1的配比做后续实验。
如图2,由粒径数据所示,溶剂中随着甲醇体积比增加,Fe3+与ICG自组装尺寸逐渐减小。但为了适应各溶质溶解性问题,本发明人最终选用溶剂为甲醇:水=2:1用作后续的分散试剂。
如图3(A),各成分加入ICG小分子后其吸收变化有所不同,单纯与Fe3+自组装后吸收峰位置并不移动,当加入其余成分后主吸收峰出现红移,当复合微泡形成时峰位置变化最明显,因此可以通过这种变化判断微泡合成状态。如图3(B),加入C3F8起泡后,本发明人最终合成出微泡尺寸在1-10μm。
如图4(A)所示,以ICG浓度计量,在超声波照射下,随着ICG浓度升高,游离ICG与Fe3+/ICG@Microbubbles其ROS产量逐渐上升,但 Fe3+/ICG@Microbubbles的ROS产量约为游离ICG的ROS产量的7倍。由图4(B)所示,在HepG2细胞中加入Fe3+/ICG@Microbubbles,超声过后,有超声处理的细胞中可产生大量杀伤性ROS,这证明了 Fe3+/ICG@Microbubbles在声动力治疗中的潜在应用可能。
Claims (6)
1.一种脂质体微泡载金属-ICG自组装复合体系,该体系包含作为载体的微泡形式的脂质体,金属离子,和ICG自组装形成的纳米结构;
由所述金属离子诱导ICG组装成所述纳米结构;
所述金属离子为Mn2+、Fe3+、Ga3+、Zn2+中的至少一种;
金属离子与ICG的摩尔比为1:20-20:1;
脂质体微泡将纳米结构部分包覆或全部包覆。
2.如权利要求1所述的自组装复合体系,其特征在于,在形成该自组装复合体系时使用包含甲醇的溶剂。
3.一种制备如权利要求1或2所述自组装复合体系的方法,该方法包括以下步骤:
(1)配置金属离子的醇溶液;
(2)配置醇水溶剂,将ICG 溶于其中,并在其中加入按步骤(1)中所得的金属离子醇溶液,室温下搅拌0.5-10h;
(3)使用步骤(2)中得到的金属离子-ICG 混合物,称取脂质体且使用三氯甲烷溶解,而后加入到搅拌着的所述金属离子-ICG 混合物中,然后除去三氯甲烷溶剂和甲醇溶剂,再在40-80℃水浴中,在密闭条件下边加入气体边起泡,得到最终复合脂质体微泡。
4.如权利要求3所述的自组装复合体系的方法,其特征在于,所述脂质体为大豆卵磷脂、胆固醇和DSPE-PEG2000中的至少一种。
5.如权利要求1或2所述的自组装复合体系或者如权利要求3所述的方法制备的自组装复合体系在制备荧光成像剂和/或光声成像剂中的应用。
6.如权利要求1或2所述的自组装复合体系或者如权利要求3所述的方法制备的自组装复合体系在制备声动力治疗剂或药物载体的应用。
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