CN111420073A - 携载amd070和icg的多模态靶向纳米泡及其制备方法 - Google Patents
携载amd070和icg的多模态靶向纳米泡及其制备方法 Download PDFInfo
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- CN111420073A CN111420073A CN202010306885.3A CN202010306885A CN111420073A CN 111420073 A CN111420073 A CN 111420073A CN 202010306885 A CN202010306885 A CN 202010306885A CN 111420073 A CN111420073 A CN 111420073A
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- polyethylene glycol
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Abstract
本发明属于生物医药技术领域,涉及纳米泡及其制备方法,具体涉及携载AMD070和ICG的多模态靶向纳米泡及其制备方法。携载AMD070和ICG的多模态靶向纳米泡,包括外壳,外壳外侧固定有AMD070,外壳内包裹有吲哚菁绿和生物惰性气体。本方案首次将AMD070、ICG、纳米泡三者结合构建一种新型的诊断和治疗一体化的多模态靶向纳米泡,它具备超声、光声、荧光多模态成像的能力,同时能在超声辐照下对乳腺癌细胞具有明显的抑制生长作用。
Description
技术领域
本发明属于生物医药技术领域,涉及超声纳米泡及其制备方法,具体涉及携载AMD070和ICG的多模态靶向纳米泡及其制备方法。
背景技术
自1967年首次发现可增强超声显像的微小空气泡后,超声造影剂(UCAs)就得到了迅速发展,由原先简单的空气微泡到目前临床应用的脂质包裹六氟化硫的微泡,有效提高了超声造影检查技术诊断肿瘤的准确性。用于肿瘤超声显像的超声造影剂(超声微泡)主要有两个不足:目前临床上使用的超声微泡(如SonoVue、Sonozoid等)并不具备与肿瘤及其血管特异性结合的能力,随血液流动并不能主动识别肿瘤组织并与之结合,在肿瘤部位滞留时间短,无论是增强显影还是靶向释药都得不到预期的效果;现有的超声微泡不具有多模态显像的能力,超声和光声、荧光等多种成像技术的结合有利于提高肿瘤的诊断准确性和治疗后随访,需要开发一种集多种成像能力为一体的超声造影剂;现有的超声造影剂尚无法实现恶性肿瘤实质细胞靶向显像和靶向治疗的诊疗一体化。
发明内容
本发明的目的在于提供携载AMD070和ICG的多模态靶向纳米泡,该纳米泡可靶向结合CXCR4阳性的乳腺癌细胞,且具备超声、光声、荧光多模态成像的能力。
为解决上述技术问题,本发明技术方案如下:
携载AMD070和ICG的多模态靶向纳米泡,所述纳米泡包括外壳,所述外壳外侧连接有AMD070,所述外壳内包裹有吲哚菁绿和生物惰性气体。
采用上述技术方案,技术原理以及有益效果如下:
为实现对恶性肿瘤的靶向超声显像及靶向治疗,需将与肿瘤细胞膜表达的靶点有高特异性、高亲和力的配体连接到超声造影剂表面。CXC型趋化因子受体4(CXCR4)是多种肿瘤特异性显像和靶向治疗中的一个高特异性靶点。CXCR4在大多数肿瘤细胞膜上均异常高表达,在乳腺癌细胞上同样存在过表达,并且在其细胞膜上广泛分布,它的配体为趋化因子基质细胞衍生因子-1(SDF-1),在肿瘤的生长、增殖、转移、侵袭过程中发挥重要作用,阻断SDF-1/CXCR4信号转导轴能有效抑制肿瘤的生长和转移。
AMD070(Mavorixafor)是一种已进入临床前研究的针对CXC型趋化因子受体4(CXCR4)的小分子拮抗剂,它拥有结构简单、分子量小、特异性高、渗透性强和免疫反应低的优点。CXCR4是多种肿瘤特异性显像和靶向治疗中的一个高特异性靶点。CXCR4在大多数肿瘤细胞膜上均异常高表达,并且在其细胞膜上广泛分布,它的配体为趋化因子基质细胞衍生因子-1(SDF-1),在肿瘤的生长、增殖、转移、侵袭过程中发挥重要作用,阻断SDF-1/CXCR4信号转导轴能有效抑制肿瘤的生长和转移。AMD070具备良好的与CXCR4结合的能力,能阻断SDF-1/CXCR4信号转导轴,有效抑制肿瘤的生长和转移。将AMD070固定在纳米泡上可实现对肿瘤组织的靶向,并同时起到抑制肿瘤的作用。
外壳内包裹生物惰性气体,较其周围生物介质易于压缩,其在超声场中具有较强的回波反射性能,能显著增强超声信号,使得纳米泡具有超声成像的功能。吲哚菁绿(Indocyanine green,ICG)是一种在临床上广泛应用的荧光染料,具备安全无毒、不影响人体正常生理活动的优点,同时ICG作为一种良好的光吸收材料,能实现光声成像,纳米泡携载ICG将同时具备超声成像、光声成像和荧光成像的能力。由于超声成像技术具有实时性好、但分辨率低、容易受干扰等缺点,光声成像具有较高的图像分辨率和图像对比度,特别是光声和荧光显像可对肿瘤的发生、发展及转移进行直接快速的定位监测和跟踪,同时ICG的荧光成像具有高敏感度,易于观察的优点,已在临床上广泛应用。超声成像和光声成像、荧光成像的结合有利于提高肿瘤的诊断准确性和治疗后随访。
本方案首次将AMD070、ICG、纳米泡(含生物惰性气体的脂质球)三者结合构建一种新型的诊断和治疗一体化的多功能靶向纳米泡,它具备超声、光声、荧光多模态成像的能力,同时能在超声辐照下对乳腺癌细胞具有明显的抑制生长作用。其中,生物惰性气体是指不参与生物体的生命活动和生化反应的且对生物体无毒性作用的气体。
进一步,所述生物惰性气体为全氟丙烷。
采用上述技术方案,全氟丙烷是惰性气体,在水中溶解度低,扩散速率低,生物相容性高,故有利于维持多模态靶向纳米泡的稳定性。
进一步,所述外壳为脂质单分子层外壳。
采用上述技术方案,脂质外壳生物相容性好,安全性高。
进一步,所述纳米泡的粒径为400-600nm。
采用上述技术方案,本多模态靶向纳米泡可穿过肿瘤血管壁进入肿瘤组织间隙,能够在肿瘤组织中实现精确成像和治疗。超声造影增强显像的原因是微泡内含有气体,较其周围生物介质易于压缩,其在超声场中具有较强的回波反射性能,能显著增强超声信号,且其含有的气体越多,增强信号能力越大。但肿瘤新生血管壁的间隙在380-780nm之间,只允许粒径小于700nm的颗粒穿过肿瘤血管壁进入肿瘤组织间隙。靶向纳米泡特异性增强显像不仅需要其内含有气体,而且需要进入肿瘤组织间隙,故综合考虑其增强显像能力和穿透性,靶向纳米泡的粒径应在400-600nm。
进一步,携载AMD070和ICG的多模态靶向纳米泡的制备方法,包括以下依次进行的步骤:
(1)将AMD070连接到二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基上,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070;
(2)将二棕榈酰磷脂酸、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺、二棕榈磷脂酰甘油、二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070和吲哚菁绿分散于溶剂中,获得悬浊液;所述溶剂为磷酸盐缓冲液和甘油组成的混合物;
(3)将步骤(2)获得的悬浊液移入可密封容器中,再用全氟丙烷气体置换可密封容器中的空气,获得置换后的可密封容器,将置换后的可密封容器震荡后4℃静置,获得携载AMD070和ICG的多模态靶向纳米泡。
采用上述技术方案,可制备获得本方案的多模态靶向纳米泡。该纳米泡具有脂质单分子外壳,CXCR4小分子拮抗剂AMD070固定在脂质单分子层外壳外侧,吲哚菁绿和全氟丙烷包裹在脂质单分子层外壳的内部。
进一步,在(1)中,使用碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺在溶剂二甲基亚砜中活化二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺;再使二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺与AMD070发生酰胺反应,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070。
采用上述技术方案,通过其携带的二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基可与携带游离氨基的AMD070发生化学反应,从而多模态靶向荧光纳米泡表面可连接AMD070。并且聚乙二醇可有效避免体内内皮网状系统对其摄取,提高其在体内的稳定性。
进一步,在(1)中,二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基、碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺的摩尔比为1:2:2;在室温震荡的条件下,对使用碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺对二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基进行活化2h;二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺与AMD070的摩尔比为1:1.2。
采用上述技术方案,可实现AMD070和二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺的充分反应,保证足量反应。
进一步,在(2)中,二棕榈酰磷脂酸、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺、二棕榈磷脂酰甘油、二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070和吲哚菁绿的质量比为1:3:3:3:1:1。
采用上述技术方案,可以使得纳米泡将吲哚菁绿充分包裹在脂质单分子层外壳中,保证纳米泡中有足够的吲哚菁绿,并在外壳外部负载足量AMD070。
进一步,在(1)和(2)之间还包括纯化操作:先通过透析除去(1)中的酰胺反应后的未反应的底物,再通过冻干除去二甲基亚砜和水,获得固体状的二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070。
采用上述技术方案,通过透析和冻干两个操作步骤除去杂质和溶剂,实现对纳米泡的纯化,可保证纳米泡体内使用的安全性,以及减少杂质对纳米泡性能的影响。其中,未反应的底物包括未反应的碳酰二亚胺盐酸盐、N-羟基丁二酰亚胺和AMD070等。
进一步,在(3)中,将置换后的可密封容器置于ST-银汞胶囊调和器中震荡60-100s,再将可密封容器置于2-8℃环境中静置2-12h。
采用上述技术方案,将置换后的可密封容器置于ST-银汞胶囊调和器中震荡一定时间,可使得各组分充分混合,在后续的低温静置步骤中,脂质部分(二棕榈酰磷脂酸、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺、二棕榈磷脂酰甘油和二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070)自组装成纳米泡形态,并将全氟丙烷和吲哚菁绿包裹到脂质单分子层外壳中。
附图说明
图1为实施例1制备的纳米泡的结构示意图;
图2为实验例1的核磁波谱检测结果图;
图3为实验例2的纳米泡粒径检测结果图;
图4为实验例3的激光共聚焦显微镜观察纳米泡的图像(展示ICG的红色荧光);
图5为实验例3的激光共聚焦显微镜观察纳米泡的图像(展示DiO的绿色荧光);
图6为实验例3的激光共聚焦显微镜观察纳米泡的图像(展示ICG和DiO的重合荧光);
图7为实验例4的纳米泡的超声成像结果;
图8为实验例4的纳米泡的超声成像的定量分析结果;
图9为实验例4的纳米泡的光声成像结果;
图10为实验例4的纳米泡的光声成像的定量分析结果;
图11为实验例4的纳米泡的荧光成像结果;
图12为实验例4的纳米泡的荧光成像的定量分析结果;
图13为实验例4的纳米泡爆破前后超声成像结果;
图14为实验例4的纳米泡爆破前后超声成像的定量分析结果;
图15为实验例5的多模态靶向荧光纳米泡与乳腺癌细胞的结合实验结果;
图16为实验例6的CCK-8检测两种肿瘤细胞的生长情况结果;
图17为实验例6的流式细胞术检测各处理组对MCF-7细胞的促凋亡作用的实验结果;
图18为实验例6的流式细胞术检测各处理组对MDA-MB-468细胞的促凋亡作用的实验结果。
具体实施方式
下面通过具体实施方式进一步详细说明:
说明书附图中的附图标记包括:全氟丙烷气体1、吲哚菁绿2、外壳3、AMD070 4。
实施例1:多模态靶向纳米泡的制备
1)将二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基与碳酰二亚胺盐酸盐、N-羟基丁二酰亚胺按照1:2:2的摩尔比例溶于二甲基亚砜中,在室温下震荡活化2h,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺。再加入AMD070(二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基与AMD070摩尔比为1:1.2)。二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺与AMD070发生酰胺反应,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070(待纯化)。反应结束后将溶液转移至4℃透析袋中(截留分子量MwCO:2000),除去未反应的碳酰二亚胺盐酸盐、N-羟基丁二酰亚胺和AMD070,将透析之后的溶液放于冻干机中去除二甲基亚砜和去离子水,得到二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070(固体状态)。
2)按质量份数配比,将二棕榈酰磷脂酸1mg、二棕榈酰磷脂酰胆碱3mg、二棕榈酰磷脂酰乙醇胺3mg、二棕榈磷脂酰甘油3mg、二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070 1mg、吲哚菁绿(ICG)1mg溶于1ml的磷酸盐缓冲液和甘油组成的溶剂中(磷酸盐缓冲液:甘油=9:1),摇床中振荡过夜,转速为210r/min,获得混悬液。
3)将上述混悬液移入西林瓶中,用全氟丙烷气体置换其内的空气,现有技术中的置换气体的具体结构为:西林瓶、注射针和全氟丙烷储气罐三者通过三通管相互连通,用注射针抽出西林瓶内的空气,并使用全氟丙烷储气罐向西林瓶中充入全氟丙烷气体。完成气体置换之后,在ST-银汞胶囊调和器中震荡西林瓶90s(震荡时间在60-100s均可使物料混合均匀),在4℃冰箱中静置12h(在温度2-8℃的条件下,静置2-12h均可实现纳米泡的自组装),获得含有携载AMD070和ICG的多模态靶向纳米泡的混合物。
4)通过离心的方法纯化获得携载AMD070和ICG的多模态靶向纳米泡(简写为:ICG-TNBs),离心条件为300r/min,离心时间为3min,离心后取固相即可获得ICG-TNBs。
AMD070的化学结构式如式Ⅰ所示。
本实施例制备的纳米泡如图1所示,包括外壳3,外壳3为磷脂单分子层,外壳3内包裹有全氟丙烷气体1和吲哚菁绿2,外壳外连接有AMD070 4。
实施例2:携带DiO的多模态靶向超声纳米泡的制备
本实施例基本同实施例1,不同点在于,用DiO染料(细胞膜绿色荧光探针)对靶向超声纳米泡进行了修饰。制备获得实施例1中的ICG-TNBs之后,使用DiO染料对ICG-TNBs进行荧光标记,将DiO染料加入实施例1制备的ICG-TNBs中孵育20min,即可完成荧光标记。
对比例1:空白纳米泡的制备
本实施例基本同实施例1,不同点在于,纳米泡中不加入ICG,也不连接固定AMD070,具体制备方法如下:
按质量份数配比,将二棕榈酰磷脂酸1mg、二棕榈酰磷脂酰胆碱3mg、二棕榈酰磷脂酰乙醇胺3mg、二棕榈磷脂酰甘油3mg、二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基1mg溶于1ml的磷酸盐缓冲液和甘油组成的溶剂中(磷酸盐缓冲液:甘油=9:1),摇床中振荡过夜,转速为210r/min,获得混悬液。将上述混悬液移入西林瓶中,用全氟丙烷气体置换其内的空气。完成气体置换之后,在ST-银汞胶囊调和器中震荡西林瓶90s,在4℃冰箱中静置12h,获得含有空白纳米泡的混合物。通过离心的方法纯化获得空白纳米泡(简写为:NBs),离心条件为300r/min,计离心时间为3min,离心后取固相即可获得NBs。
对比例2:携载无关配体纳米泡
本对比例基本同实施例1,不同点在于,使用无关配体谷氨酰胺替换AMD070,谷氨酰胺含有氨基,可以与二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基连接,但是谷氨酰胺没有结合CXCR4的功能。
实验例1:验证AMD070与纳米泡成功连接
将AMD070、二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基、二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070(固体形式)溶于氘代DMSO中,用600MHz核磁共振仪(Agilent,USA)检测,并绘制这三种样品的1H NMR氢谱图,如图2所示,图中展示了AMD070的核磁波谱,二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基的核磁波谱和二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070的核磁波谱。4.81ppm是AMD070的氨基特征峰,二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070中二棕榈酰磷脂酰乙醇胺的特征峰为0.81ppm和1.23ppm。二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070的核磁图谱显示,在4.81ppm处无氨基特征峰,但0.81ppm和1.23ppm处有二棕榈酰磷脂酰乙醇胺特征峰,即二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070中无氨基基团,但含有二棕榈酰磷脂酰乙醇胺成分,表明化合物二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070成功合成。
实验例2:粒径检测
用PBS稀释ICG-TNBs,稀释液加入比色皿中,用马尔文粒径检测仪检测ICG-TNBs的粒径及其分布,结果见图3,ICG-TNBs的粒径处于400-600nm之间,均一性良好。
实验例3:验证纳米泡携载ICG及其荧光特性
用DiO染料对ICG-TNBs的脂质壳膜进行标记(实施例2制备),然后置于Zeiss 780激光共聚焦显微镜(Carl Zeiss AG,Oberkochen,Germany)下观察ICG-TNBs的荧光图像。如图4-图6所示,纳米泡携载的ICG呈现红色荧光(图4),同时ICG-TNBs壳膜结合了DiO染料而呈现绿色(图5),二者的荧光能完全重合呈现黄色(图6),表明ICG-TNBs能携载ICG并显像。
实验例4:体外验证多模态靶向荧光纳米泡的超声、光声、荧光成像能力
用2%琼脂糖粉末制备体外成像模型,采用vevo2100小动物成像仪(FujiFilmVisualsonics)进行成像。将探头固定,调整成像模式(频率为7-18MHz),监测不同浓度的多模态靶向纳米泡(实施例1制备的ICG-TNBs)与空白纳米泡(对比例1制备的NBs)的超声成像强度及光声成像强度(1.0×108,2.0×107,4.0×106,8.0×105和1.6×105个/mL),并在活体成像仪中对不同浓度(1.0×108,2.0×107,4.0×106,8.0×105和1.6×105个/mL)的ICG-TNBs进行荧光成像(ICG的激发波长为780nm,发射波长为808nm).另外当多模态靶向纳米泡(ICG-TNBs)浓度为2.0×107/mL时采用高机械指数的超声波破坏,并进行破坏前后的超声成像强度比较。如图7和图8所示,在体外成像模型中,多模态靶向纳米泡与空白纳米泡在不同浓度下的超声信号均无显著性差异,都随着浓度的降低,超声信号平均灰度值越来越低。如图9、图10、图11和图12所示,光声信号和荧光信号的强度都随着ICG-TNBs浓度的下降而逐渐降低。在使用超声爆破后(图13和图14),ICG-TNBs的超声成像强度显著降低,表明ICG-TNBs可以被高机械指数的超声波破坏,有利于靶向释放小分子拮抗剂。
实验例5:体外验证多模态靶向纳米泡与CXCR4阳性表达的乳腺癌细胞特异性
将实施例1制备的多模态靶向纳米泡(ICG-TNBs)和对比例1制备的空白纳米泡按1×107个/ml加入贴壁培养过夜的乳腺癌细胞(加入之前已用4%多聚甲醛对细胞进行固定),同时为深入研究多模态靶向纳米泡特异性,预先采用CXCR4抗体对其中一组肿瘤细胞封闭1h。在纳米泡与肿瘤细胞孵育2h后,用磷酸盐缓冲液进行漂洗,在倒置显微镜下观察多模态靶向纳米泡和空白纳米泡与阳性表达CXCR4和阴性表达CXCR4肿瘤细胞结合情况(如图15所示,ICG-TNBs代表实施例1制备的多模态靶向纳米泡,NBs代表空白纳米泡,“阻断”代表预先使用CXCR4抗体封闭阳性表达CXCR4的MCF-7肿瘤细胞)。阳性表达CXCR4的MCF-7肿瘤细胞周围聚集较多的多模态靶向纳米泡,而空白纳米泡不能与CXCR4阳性表达的肿瘤细胞结合。多模态靶向纳米泡和空白纳米泡均不能与CXCR4阴性表达的MDA-MB-468肿瘤细胞结合。在预先使用CXCR4抗体封闭后,MCF-7细胞结合的多模态靶向纳米泡数量明显减少,而MDA-MB-468肿瘤细胞结合的多模态靶向纳米泡数量无明显变化,表明携载AMD070的多模态靶向纳米泡特异性结合CXCR4阳性表达肿瘤细胞,未结合CXCR4阴性表达肿瘤细胞。
实验例6:检测多模态靶向纳米泡抑制乳腺癌细胞的生长
CCK-8检测细胞活性
将MCF-7细胞和MDA-MB-468细胞接种于96孔板上,将MCF-7细胞和MDA-MB-468细胞分为8组,其中加入PBS处理组为空白对照组,细胞数量为5X103个/孔,37℃培养过夜,次日对两种细胞根据表1的试剂和条件进行处理,其中超声辐照通过使用WED-100US超声治疗仪完成,超声功率为1.0W/cm2,时间10s,然后37℃培养48h,每孔加入10μL CCK-8试剂,37℃孵育2h,用多功能酶标仪测定波长在450nm时的吸光度,并计算细胞生长抑制率(cell growthinhibition rate,IR),公式为IR=1-(处理组OD450平均值/空白对照组OD450平均值)×100%。各处理组中纳米泡的浓度为2×107个/mL,第7组的AMD070的浓度为40ug/mL。
表1:实验分组以及使用试剂情况
结果如图16(*p<0.05指针对MCF-7细胞,多模态靶向纳米泡处理组分别与空白对照组的比较;#p<0.05指针对MCF-7细胞,AMD070处理组、多模态靶向纳米泡+超声辐照组分别与空白对照组的比较),在针对MCF-7细胞的8个处理组中,多模态靶向纳米泡(ICG-TNB)、AMD070、多模态靶向纳米泡+超声辐照(ICG-TNBs+US)三个处理组的吸光度与其他处理组有显著统计学差异(P<0.05),细胞吸光度明显下降,表明细胞生长明显受限,MCF-7细胞生长抑制率分别为44.5%、63.5%、73.3%,且多模态靶向纳米泡、AMD070、多模态靶向纳米泡+超声辐照三组之间的吸光度具有显著的统计学差异(P<0.05),即多模态靶向纳米泡+超声辐照组抑制MCF-7细胞生长的能力最强。而对于MDA-MB-468细胞,上述各个处理组对细胞的生长抑制效果不明显,无显著统计学差异(P>0.05)。
流式细胞术检测细胞凋亡
将MCF-7细胞和MDA-MB-468细胞接种于6孔板上,细胞数量为1.0X106个/孔,置于37℃细胞培养箱中过夜,按照上述表格(表1)的试剂与条件处理48h,收集细胞并重悬于200μL结合缓冲液中,然后加入5μL膜联蛋白V-异硫氰酸荧光素(AnnexinV-FITC),室温下温育20min,再加入10μL碘化丙啶(PI),在流式细胞仪(BD Accuri C6,USA)上检测各组的细胞凋亡水平。结果如图17和图18,结果显示多模态靶向纳米泡(ICG-TNB)、AMD070、多模态靶向纳米泡+超声辐照(ICG-TNBs+US)对MCF-7的早期凋亡率分别为(7.24±0.29)%、(21.7±4.9)%、(30.5±2.9)%,晚期凋亡率分别为(12.9±0.2)%、(15.4±1.6)%、(22.0±4.7)%,总凋亡率分别为(20.2±0.4)%、(37.1±3.3)%、(52.4±2.9)%,早期凋亡、晚期凋亡以及总凋亡率相比于其他处理组有显著的统计学差异(P<0.05),且三组之间的早期凋亡率、晚期凋亡率和总凋亡率也存在显著的统计学差异(P<0.05)(图17),表明多模态靶向纳米泡、AMD070、多模态靶向纳米泡+超声辐照对MCF-7的促凋亡作用依次增强。而对于MDA-MB-468细胞,各个处理组的促凋亡作用不明显,没有显著的统计学差异(P>0.05)(图18)。
以上所述的仅是本发明的实施例,方案中公知的具体结构及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,在不脱离本发明结构的前提下,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 (10)
1.携载AMD070和ICG的多模态靶向纳米泡,其特征在于,所述纳米泡包括外壳,所述外壳外侧连接有AMD070,所述外壳内包裹有吲哚菁绿和生物惰性气体。
2.根据权利要求1所述的携载AMD070和ICG的多模态靶向纳米泡,其特征在于,所述生物惰性气体为全氟丙烷。
3.根据权利要求2所述的携载AMD070和ICG的多模态靶向纳米泡,其特征在于,所述外壳为脂质单分子层外壳。
4.根据权利要求3所述的携载AMD070和ICG的多模态靶向纳米泡,其特征在于,所述纳米泡的粒径为400-600nm。
5.根据权利要求1-4中任一项所述的携载AMD070和ICG的多模态靶向纳米泡的制备方法,其特征在于,包括以下依次进行的步骤:
(1)将AMD070连接到二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基上,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070;
(2)将二棕榈酰磷脂酸、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺、二棕榈磷脂酰甘油、二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070和吲哚菁绿分散于溶剂中,获得悬浊液;所述溶剂为磷酸盐缓冲液和甘油组成的混合物;
(3)将步骤(2)获得的悬浊液移入可密封容器中,再用全氟丙烷气体置换可密封容器中的空气,获得置换后的可密封容器,将置换后的可密封容器震荡后4℃静置,获得携载AMD070和ICG的多模态靶向纳米泡。
6.根据权利要求5所述的制备方法,其特征在于,在(1)中,使用碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺在溶剂二甲基亚砜中活化二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺;再使二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺与AMD070发生酰胺反应,获得二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070。
7.根据权利要求5所述的制备方法,其特征在于,在(1)中,二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基、碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺的摩尔比为1:2:2;在室温震荡的条件下,对使用碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺对二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基进行活化2h;二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺与AMD070的摩尔比为1:1.2。
8.根据权利要求7所述的制备方法,其特征在于,在(2)中,二棕榈酰磷脂酸、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酰乙醇胺、二棕榈磷脂酰甘油、二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070和吲哚菁绿的质量比为1:3:3:3:1:1。
9.根据权利要求8所述的制备方法,其特征在于,在(1)和(2)之间还包括纯化操作:先通过透析除去(1)中的酰胺反应后的未反应的底物,再通过冻干除去二甲基亚砜和水,获得固体状的二棕榈酰磷脂酰乙醇胺-聚乙二醇-AMD070。
10.根据权利要求9所述的制备方法,其特征在于,在(3)中,将置换后的可密封容器置于ST-银汞胶囊调和器中震荡60-100s,再将可密封容器置于2-8℃环境中静置2-12h。
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