CN113827742A - 一种乳腺癌前哨淋巴结示踪靶向分子探针及其制备方法 - Google Patents
一种乳腺癌前哨淋巴结示踪靶向分子探针及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种乳腺癌前哨淋巴结示踪靶向分子探针及其制备方法，该方法旨在将靶向M2巨噬细胞标志物CD206多肽与DSPE‑PEG2000偶联，装载ICG后形成纳米胶束，构建一款能够靶向识别淋巴结癌转移灶肿瘤微环境的荧光探针，可通过NIR‑Ⅱ荧光活体成像技术实现乳腺癌淋巴结癌转移无创靶向性识别；由于CD206靶向多肽荷载ICG的自组装纳米胶束易于制备，具有出色的近红外荧光成像质量以及良好的生物安全性，该探针具有很大的临床翻译潜力。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种乳腺癌前哨淋巴结示踪靶向分子探针及其制备方法。
背景技术
恶性肿瘤相关性死亡主要由肿瘤远处转移引起。淋巴转移是癌转移的关键一环，癌细胞可以通过淋巴管进入淋巴结，随后通过淋巴结中的血管向远处器官转移。前哨淋巴结(Sentinel lymph node，SLN)是肿瘤原发灶淋巴引流到达的第一个淋巴结或第一组淋巴结，因其与肿瘤转移有密切关系，也叫做肿瘤引流淋巴结(Tumor-draining lymph nodes，TDLN)。早期淋巴结癌转移因其体积小、位置分散，检出十分困难。术中前哨淋巴结示踪和病理活检(Sentinel lymph node bioposy,SLNB)是判断淋巴结癌转移的主要方法，该技术作为一种有创的检查方式，需要经验丰富的外科医生和病理医生协作完成。
然而，术中往往难以凭借肉眼确认SLN，需要在乳晕或肿瘤周围注射SLN示踪剂，在腋窝部位经特殊设备探测或者可视化示踪剂的分布情况以此识别SLN，并予以切除进行病理检查。目前，乳腺癌的前哨淋巴结活检方法主要分为蓝染料示踪法、放射性核素示踪法和吲哚菁绿荧光成像法。蓝染料示踪法缺乏专用的成像设备，具有一定的盲目性，导致前哨淋巴结检出成功率较低。而放射性核素示踪法具有放射性，需要核医学配合，导致其应用收到极大限制。应用荧光染料吲哚氰绿(Indocyanine green，ICG)结合近红外荧光成像技术在肿瘤前哨淋巴结活检方面展现了极大的应用潜力，该技术具有实时、动态、直视、灵敏度高优点，有望弥补其他方法不足之处，其该方法提高了SLN检测的敏感性和特异性、减少了盲目性、提高了SLNB成功率，同时避免不必要的手术创伤，减少了上肢淋巴水肿等并发症的发生，有助于提高患者的生活质量。
巨噬细胞是肿瘤微环境中的成员之一，它能够因细胞外环境的不同而被诱导成两种不同的表型，分别是经典激活型(M1型)和替代激活型(M2型)。M2型巨噬细胞又叫肿瘤相关性巨噬细胞(Tumor associated macrophage，TAM)，在肿瘤生长过程中，癌细胞诱导外周血中单核细胞在癌灶聚集并使之分化为M2型，TAM反之通过释放促肿瘤因子和促生长因子作用于癌细胞促进其增值。此外，TAM通过抑制T细胞功能协助癌细胞免疫逃逸、促进肿瘤转移。已有研究证实，肿瘤间质中TAM浸润性高的恶性肿瘤进展更快，患者的生存率更低。所有这些特性使得巨噬细胞活性成为诊断和治疗的相关靶点，这促使了近年来靶向TAM成像探针的发展。
发明内容
本发明提供了一种乳腺癌前哨淋巴结示踪靶向分子探针及其制备方法，该方法制备了一种肿瘤相关性巨噬细胞靶向ICG纳米胶束，该纳米材料作为探针在近红外区具有较强的光吸收，可利用其光学特性作为分子探针用于乳腺癌前哨淋巴结癌转移诊断。
为实现上述目的，本发明采用以下技术方案：
一种乳腺癌前哨淋巴结示踪靶向分子探针的制备方法，该方法包括以下步骤：
S1、将3mg磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺固体粉末和1mg CD206binding peptide固体粉末，溶解于2ml N,N-二甲基甲酰胺中，得到A溶液；
S2、将A溶液搅拌均匀，充分反应后，得到含有二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物的B溶液；
S3、将1ml B溶液和520ul浓度为1mg/ml的溶于N,N-二甲基甲酰胺的吲哚氰绿溶液混合均匀，得到C溶液；
S4、采用1ml注射器将C溶液逐滴滴加于7ml去离子水中，并用磁力搅拌器搅拌去离子水，C溶液在去离子水中充分散开后，得到含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的D溶液；
S5、将D溶液转移至5kDa透析袋中，将该透析袋置于含有2L去离子水的容器中，再将该容器置于4℃冰箱中充分透析24小时，去除未被二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物包载的游离吲哚氰绿，获得纯化的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的溶液。
优选地，还包括步骤S6、将纯化的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的溶液转移到10kDa超滤离心管中，在4℃条件下，经5000rpm离心30分钟，得到浓缩后的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的纯化液。
优选地，将浓缩后的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的纯化液放置在4℃冰箱保存备用。
优选地，步骤S2的具体过程为：将A溶液置于4℃冰箱中，用磁力搅拌器搅拌12小时以上，得到含有二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物的B溶液。
一种乳腺癌前哨淋巴结示踪靶向分子探针，所述乳腺癌前哨淋巴结示踪靶向分子探针采用如权利要求1～4任一所述的乳腺癌前哨淋巴结示踪靶向分子探针的制备方法制得。
采用上述技术方案后，本发明与背景技术相比，具有如下优点：
本发明提供一种乳腺癌前哨淋巴结示踪靶向分子探针及其制备方法，该方法旨在将靶向M2巨噬细胞标志物CD206多肽与DSPE-PEG2000偶联，装载ICG后形成纳米胶束，构建一款能够靶向识别淋巴结癌转移灶肿瘤微环境的荧光探针(简称探针或Probe)，可通过NIR-Ⅱ荧光活体成像技术实现乳腺癌淋巴结癌转移无创靶向性识别；由于CD206靶向多肽荷载ICG的自组装纳米胶束易于制备，出色的近红外荧光成像质量以及良好的生物安全性，可利用其光学特性作为分子探针用于乳腺癌前哨淋巴结癌转移诊断，该探针具有很大的临床翻译潜力。
附图说明
图1为本发明的CD206-ICG nanoparticle探针的电镜图和光谱特征图；
图2为本发明的CD206-ICG nanoparticle探针的细胞靶向性检测图；
图3为本发明的CD206-ICG nanoparticle探针的细胞毒性检测图；
图4为本发明的CD206-ICG nanoparticle探针的动物靶向性检测图；
图5为本发明的CD206-ICG nanoparticle探针的生化检测验证动物安全性的检测图；
图6为本发明的CD206-ICG nanoparticle探针的HE染色检测验证动物安全性的检测图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
在本发明中需要说明的是，术语“上”“下”“左”“右”“竖直”“水平”“内”“外”等均为基于附图所示的方位或位置关系，仅仅是为了便于描述本发明和简化描述，而不是指示或暗示本发明的装置或元件必须具有特定的方位，因此不能理解为对本发明的限制。
实施例
如图1至图6所示，本发明公开了一种乳腺癌前哨淋巴结示踪靶向分子探针的制备方法，其特征在于，该方法包括以下步骤：
S1、将3mg磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺(DSPE-PEG(2000)-Mal)固体粉末和1mg CD206 binding peptide固体粉末，溶解于2mlN,N-二甲基甲酰胺(DMF)中，得到A溶液；
S2、将A溶液置于4℃冰箱中，用磁力搅拌器搅拌12小时以上，充分反应后，得到含有二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物(DSPE-PEG(2000)-Mal-CD206 binding peptide)的B溶液；
S3、将1ml B溶液和520ul浓度为1mg/ml的溶于N,N-二甲基甲酰胺(DMF)的吲哚氰绿(ICG)溶液混合均匀，得到C溶液；
S4、采用1ml注射器将C溶液逐滴滴加于7ml去离子水中，并用磁力搅拌器搅拌去离子水，C溶液在去离子水中充分散开后，得到含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒(DSPE-PEG(2000)-Mal-CD206binding peptide loaded ICG，简称CD206-ICG nanoparticle)的D溶液；
S5、将D溶液转移至5kDa透析袋中，将该透析袋置于含有2L去离子水的容器中，再将该容器置于4℃冰箱中充分透析24小时，去除未被二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物(DSPE-PEG(2000)-Mal-CD206 binding peptide)包载的游离吲哚氰绿(ICG)，获得纯化的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒(DSPE-PEG(2000)-Mal-CD206 bindingpeptide loaded ICG，简称CD206-ICG nanoparticle)的溶液；
S6、将纯化的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒(DSPE-PEG(2000)-Mal-CD206binding peptide loadedICG，简称CD206-ICG nanoparticle)的溶液转移到10kDa超滤离心管中，在4℃条件下，经5000rpm离心30分钟，得到浓缩后的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒(DSPE-PEG(2000)-Mal-CD206 bindingpeptide loaded ICG，简称CD206-ICG nanoparticle)的纯化液，并将其放置在4℃冰箱保存备用。
实验：将上述制备的CD206-ICG nanoparticle进行乳腺癌前哨淋巴结癌转移检测
实验1、构建淋巴结癌转移小鼠模型，待模型小鼠患侧腹股沟淋巴结出现生物发光信号后，将模型小鼠随机分成两组，一组尾静脉注射CD206-ICG nanoparticle，另一组尾静脉注射DSPE-ICG nanoparticle，注射剂量以ICG浓度进行定量，为15mg/kg；分别于尾静脉注射前以及注射后第2、6、12、18、24小时用小动物荧光成像系统进行观察，观察模型小鼠体内荧光信号分布和强弱的变化，观察模型小鼠双侧腹股沟淋巴结在体荧光强度的变化，在观察终点将模型小鼠脱颈处死，取出模型小鼠双侧腹股沟淋巴结，离体观察模型小鼠腹股沟淋巴结荧光信号强度，分析和比较两组差异。
实验2、将取出的腹股沟淋巴结用福尔马林固定，制作成石蜡组织块，用HE染色的方法评估淋巴结癌转移情况，用免疫组化的方法观察淋巴结中CD206阳性的M2型巨噬细胞浸润情况。
实验测试结果：图1显示本实施例成功构建了CD206-ICG nanoparticle探针，其在电镜下粒径较为均一，酶标仪检测吸收光谱和发射光谱与ICG一致。图2显示本实施例所合成的探针用于孵育不同CD206表达水平的巨噬细胞RAW264.7细胞，制作成细胞爬片于荧光显微镜下观察，发现探针具有良好的靶向性。图3显示不同浓度的探针孵育4T1细胞24h后，CCK8实验分析细胞活性，证明探针的细胞毒性较低。图4显示种植4T1-luc细胞的乳腺癌前哨淋巴结癌转移模型小鼠尾静脉注射探针后，荧光成像发现CD206-ICG nanoparticle相较于DSPE-ICG nanoparticle具有良好的肿瘤富集效果。图5显示两组正常小鼠分别尾静脉注射CD206-ICG nanoparticle探针和磷酸缓冲盐溶液(PBS)，分别在2天、14天、28天眼眶取血收集小鼠外周血血清，检测血清中ALT、AST、BUN、Cr，两组间无明显差异。图6显示两组正常小鼠分别尾静脉注射CD206-ICG nanoparticle探针和磷酸缓冲盐溶液(PBS)，分别在2天、14天、28天处死小鼠，取出心、肝、脾、肺、肾，HE染色观察两组均无明显损伤。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。
Claims (5)
1.一种乳腺癌前哨淋巴结示踪靶向分子探针的制备方法，其特征在于，该方法包括以下步骤：
S1、将3mg磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺固体粉末和1mg CD206 bindingpeptide固体粉末，溶解于2ml N,N-二甲基甲酰胺中，得到A溶液；
S2、将A溶液搅拌均匀，充分反应后，得到含有二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物的B溶液；
S3、将1ml B溶液和520ul浓度为1mg/ml的溶于N,N-二甲基甲酰胺的吲哚氰绿溶液混合均匀，得到C溶液；
S4、采用1ml注射器将C溶液逐滴滴加于7ml去离子水中，并用磁力搅拌器搅拌去离子水，C溶液在去离子水中充分散开后，得到含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的D溶液；
S5、将D溶液转移至5kDa透析袋中，将该透析袋置于含有2L去离子水的容器中，再将该容器置于4℃冰箱中充分透析24小时，去除未被二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物包载的游离吲哚氰绿，获得纯化的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的溶液。
2.如权利要求1所述的乳腺癌前哨淋巴结示踪靶向分子探针的制备方法，其特征在于：还包括步骤S6、将纯化的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的溶液转移到10kDa超滤离心管中，在4℃条件下，经5000rpm离心30分钟，得到浓缩后的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的纯化液。
3.如权利要求2所述的乳腺癌前哨淋巴结示踪靶向分子探针的制备方法，其特征在于：将浓缩后的含有装载吲哚菁绿的二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽纳米颗粒的纯化液放置在4℃冰箱保存备用。
4.如权利要求1所述的乳腺癌前哨淋巴结示踪靶向分子探针的制备方法，其特征在于，步骤S2的具体过程为：将A溶液置于4℃冰箱中，用磁力搅拌器搅拌12小时以上，得到含有二硬脂酰基磷脂酰乙醇胺-聚乙二醇(2000)-马来酰亚胺-CD206靶向肽化合物的B溶液。
5.一种乳腺癌前哨淋巴结示踪靶向分子探针，其特征在于：所述乳腺癌前哨淋巴结示踪靶向分子探针采用如权利要求1～4任一所述的乳腺癌前哨淋巴结示踪靶向分子探针的制备方法制得。
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