CN112494665A - 一种荧光NP-Au靶向造影剂及其制备方法和应用 - Google Patents
一种荧光NP-Au靶向造影剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明属生物医用材料和造影剂制备的技术领域，涉及一种荧光NP‑Au靶向造影剂及其制备方法和应用。一种荧光NP‑Au靶向造影剂，该靶向造影剂以TAMs标志物CD163/MMR/Arg‑1为靶点，所述荧光NP‑Au靶向造影剂在制备肿瘤靶向造影剂中的应用。本发明采用多孔纳米新材料荧光NP‑Au，利用分子生物学等技术深入研究荧光NP‑Au靶向造影剂在组织细胞中的成像能力，该造影剂能够对肿瘤中的TAMs进行靶向显影。
Description
技术领域
本发明属生物医用材料和造影剂制备的技术领域，涉及一种荧光NP-Au靶向造影剂及其制备方法和应用。
背景技术
在肿瘤发生机制的研究中，其发生发展不但是由于肿瘤细胞自身的恶变，同时与肿瘤微环境关系密切。肿瘤相关巨噬细胞(tumor-associated macrophages，TAMs)是肿瘤微环境中最丰富的免疫细胞，是免疫抑制细胞中重要的一员。TAMs通过创造促变异的炎性环境，分泌免疫抑制因子、细胞因子和生长因子，抑制T细胞的增殖和活化，调节并促进Th2型免疫应答，促进肿瘤细胞侵袭、参与肿瘤血管新生和促进肿瘤的浸润和转移。研究表明，TAMs存在于肿瘤发展的所有阶段，其数量与肿瘤的恶性程度和不良预后相关，在肿瘤的侵袭和转移过程中起到了促进作用，而靶向抑制TAMs可减少肿瘤生长、增殖和转移。在结肠癌小鼠模型中，使用CSF-1R抗体靶向调控TAMs极化导致小鼠肿瘤体积显著减少和长期生存，表明TAMs可以作为肿瘤治疗研究中强有力的靶标。
发明内容
鉴于现有技术存在的问题，本发明目的在于提供一种荧光NP-Au靶向造影剂及其制备方法和应用，本发明采用多孔纳米新材料荧光NP-Au，利用分子生物学等技术深入研究荧光NP-Au靶向造影剂在组织细胞中的成像能力，该造影剂能够对肿瘤中的TAMs进行靶向显影。
为实现上述目的，本发明采用以下技术方案。
一种荧光NP-Au靶向造影剂，该靶向造影剂以TAMs标志物CD163/MMR/Arg-1为靶点。
进一步地，所述荧光NP-Au靶向造影剂的制备方法，具体包括以下步骤。
步骤1、将1M HEPES用dd水稀释为20mmol缓冲液，分别取5μl、6μl、7μl、8μl、9μlCD163抗体溶于20mmolHEPES缓冲液中，得到50μl混合液。
步骤2、将不同浓度的CD163抗体溶液分别与1ml金纳米棒溶液作用，30分钟后测量各溶液的吸光度；当金纳米棒溶液最大吸收峰值不变时，进入吸收峰平台的初始浓度即为所需最佳CD163抗体浓度。
步骤3、取该浓度混合物溶液1ml加入50μl聚乙二醇作用10分钟后，在4℃下以15000rmb的转速离心15分钟，得到mAb-CD163/Au共轭物；然后弃去上清液，加入PBS缓冲液，得到分散良好稳定的溶液，4℃下保存。
进一步地，所述荧光NP-Au靶向造影剂在制备肿瘤靶向造影剂中的应用。
与现有技术相比，本发明的有益效果如下。
本发明提供的荧光NP-Au靶向造影剂，采用荧光NP-Au利用分子生物学等技术，深入研究靶向性NP-Au在组织细胞中的成像能力。向微环境中非肿瘤细胞的优势是荧光NP-Au靶向造影剂的基因更稳定，更不容易形成耐药。从肿瘤微环境相关的巨噬细胞着手，研究TAMs在肿瘤中的靶向显像。实验证明mAb-CD163/Au在M2型巨噬细胞内的聚集明显多于M1型巨噬细胞，说明M2型巨噬细胞表面高表达CD163分子，因此可以吞噬更多的mAb-CD163/Au；随着纳米颗粒浓度的增加，存活细胞越少。当浓度相同时，mAb-CD163/Au+NIR组细胞被杀伤的情况最明显；通过靶向杀伤瘤周组织内CD163(+)巨噬细胞，可以抑制肿瘤的生长。
附图说明
图1是金纳米棒溶液的流式图像。
图2是mAb-CD163/Au的流式图像。流式图像峰值明显右移，说明制备的mAb-CD163/Au表明含有CD163抗体。
图3是金纳米棒溶液吸收峰。
图4是经CD163抗体包裹后，mAb-CD163/Au的吸收峰略右移。经CD163抗体包裹的金纳米颗粒，光学性质没有明显改变，其在1064nm附近仍具有吸收峰。
图5是双光子显微镜观察mAb-CD163/Au在巨噬细胞内的聚集情况结果，其中，图5a为经PMA诱导后，mAb-CD163/Au在M1型巨噬细胞内的聚集情况；图5b为经PMA、IL-4诱导后，mAb-CD163/Au在M2型巨噬细胞内的聚集情况。
图6是经1064nm激光照射后，分别于0min、1min、2min、3min、4min、5min时，各组溶液温度变化的情况。
图7是经近红外激光处理后各组细胞被杀伤的情况。
图8a-8f是荧光NP-Au靶向造影剂的体内细胞实验结果。其中，图8a为照射15天后，各组裸鼠成瘤情况；图8b为各组裸鼠肿瘤生长曲线；图8c、8d、8e分别为各组瘤周组织CD163(+)巨噬细胞免疫组化；图8f为显示各组瘤周组织CD163(+)巨噬细胞免疫组化具有统计学差异。
具体实施方式
下面结合具体实施例和附图详细介绍本发明的技术方案和技术效果。未注明具体条件的实验方法，通常按照常规条件，例如教科书和实验指南中所述的条件，或按照制造厂商所建议的条件，为本领域普通技术人员熟知或易于获知，以下实施例仅为本发明的优选实施例，并不限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例1荧光NP-Au靶向造影剂的制备方法。
1、材料和试剂。
HEPES：pH7.2-7.4；H1095(100ml)：1M，Solarbio；聚乙二醇(PEG200)；P8490：500ml，Solarbio；APC anti-human CD163(monoclonal)，Biolegend；金纳米棒，NR-9-1064，OD 2，上海羧菲生物医药科技有限公司。
2、制备方法。
所述荧光NP-Au靶向造影剂的制备方法包括以下具体步骤。
步骤1、将1M HEPES用双蒸水(dd水)稀释为20mmol缓冲液。分别取5μl、6μl、7μl、8μl、9μlCD163抗体溶于20mmolHEPES缓冲液中，得到50μl混合液。
步骤2、将不同浓度的CD163抗体溶液分别与1ml金纳米棒溶液作用，30分钟后测量各溶液的吸光度。当金纳米棒溶液最大吸收峰值不变时，进入吸收峰平台的初始浓度即为所需最佳CD163抗体浓度。
步骤3、取该浓度混合物溶液1ml加入50μl聚乙二醇作用10分钟后，在4℃下离心(15000x)15分钟，得到mAb-CD163/Au共轭物。然后弃去上清液，加入PBS缓冲液，得到分散良好稳定的溶液，4℃下保存。
实施例2荧光NP-Au靶向造影剂的表征及体外细胞实验。
1、金纳米颗粒表面CD163抗体的检测。
1)实验材料。
实施例1制备好的mAb-CD163/Au溶液。
2)实验方法。
使用紫外—可见光分光光度计测量溶液吸收峰；使用马尔文粒径仪测量溶液物理属性；使用流式细胞仪验证金纳米颗粒表面包裹CD163抗体。
3)实验结果。
实验结果如图1-4所示。图1为金纳米棒溶液的流式图像；图2为mAb-CD163/Au的流式图像，图2流式图像峰值明显右移，说明制备的mAb-CD163/Au表明含有CD163抗体。图3为金纳米棒溶液吸收峰，图4显示经CD163抗体包裹后，mAb-CD163/Au的吸收峰略右移。经CD163抗体包裹的金纳米颗粒，光学性质没有明显改变，其在1064nm附近仍具有吸收峰。
2、双光子显微镜观察mAb-CD163/Au在巨噬细胞内的聚集情况。
1)实验材料。
实施例1制备好的mAb-CD163/Au溶液，人THP-1单核细胞，PMA(佛波酯)，IL-4。
2)实验方法。
将细胞爬片玻片置入24孔板内，将1mlTHP-1细胞悬液移入孔板。经PMA及IL-4诱导后，THP-1细胞分化成M2型巨噬细胞。然后向每孔滴入10ulmAb-CD163/Au，孵育45min。使用双光子显微镜观察mAb-CD163/Au在巨噬细胞内的聚集情况。
3)实验结果。
实验结果如图5所示，图5a为经PMA诱导后，mAb-CD163/Au在M1型巨噬细胞内的聚集情况。图5b为经PMA、IL-4诱导后，mAb-CD163/Au在M2型巨噬细胞内的聚集情况。mAb-CD163/Au在M2型巨噬细胞内的聚集明显多于M1型巨噬细胞，说明M2型巨噬细胞表面高表达CD163分子，因此可以吞噬更多的mAb-CD163/Au。
3、mAb-CD163/Au在光热条件下对M2型巨噬细胞的杀伤。
1)实验材料。
近红外激光(1064nm，Ligenesis，武汉)，制备好的mAb-CD163/Au溶液，人THP-1单核细胞，PMA(佛波酯)，IL-4，CCK-8。
2)实验方法。
M2型巨噬细胞诱导同前。将mAb-CD163/Au溶液稀释成15％、25％、35％浓度，分别加入各组M2型巨噬细胞内。另设一对照组，不加入mAb-CD163/Au。然后用1064nm激光分别照射各组细胞溶液，观察各组溶液温度的变化。
将金纳米棒溶和mAb-CD163/Au溶液分别制成15％、25％、35％浓度，然后加入各组M2型巨噬细胞内。然后各组细胞分别经1064nm激光(6W/cm2)照射5min。NIR组细胞只接受激光照射。然后弃去上清液，加入CCK-8试剂，计算各组细胞被杀伤的情况。
3)实验结果。
实验结果如图6-7所示，图6表明经1064nm激光照射后，分别于0min、1min、2min、3min、4min、5min时，各组溶液温度变化的情况。在照射时间相同时，溶液温度随纳米颗粒浓度增加而增高。图7表明经近红外激光处理后各组细胞被杀伤的情况。OD值越小，存活细胞越少。随着纳米颗粒浓度的增加，存活细胞越少。当浓度相同时，mAb-CD163/Au+NIR组细胞被杀伤的情况最明显。
实施例3荧光NP-Au靶向造影剂的体内细胞实验。
1、人乳腺癌MCF-7细胞荷瘤裸鼠模型建立。
1)实验材料。
人乳腺癌MCF-7细胞，BALB/c裸鼠(雌性)。
2)实验方法。
人乳腺癌MCF-7细胞(5x106/ml)在无菌条件下于裸鼠背侧靠腋窝皮下接种0.1ml/只。裸鼠分成3组，每组6只。当肿瘤平均直径达到5mm时，于肿瘤上下左右4个部位分别注入mAb-CD163/Au及PBS溶液。每部位注射0.1mL。然后对mAb-CD163/Au组及对照组分别给予1064nm近红外激光(6W/cm2)1min照射；PBS组不接受激光照射。15天后处死各组裸鼠，测量各组肿瘤大小，并进行免疫组化分析。
3)实验结果。
实验结果如图8a-8f所示，图8a为照射15天后，各组裸鼠成瘤情况。图8b为各组裸鼠肿瘤生长曲线。mAb-CD163/Au组肿瘤平均体积最小。图8c、8d、8e分别为各组瘤周组织CD163(+)巨噬细胞免疫组化。图8f显示各组瘤周组织CD163(+)巨噬细胞免疫组化具有统计学差异。该实验说明通过靶向杀伤瘤周组织内CD163(+)巨噬细胞，可以抑制肿瘤的生长。
Claims (4)
1.一种荧光NP-Au靶向造影剂，其特征在于，所述荧光NP-Au靶向造影剂以TAMs标志物CD163/MMR/Arg-1为靶点。
2.如权利要求1所述的荧光NP-Au靶向造影剂的制备方法，其特征在于，具体包括以下步骤：
步骤1、将1M HEPES用水稀释为20mmol缓冲液，分别取5μl、6μl、7μl、8μl、9μlCD163抗体溶于20mmolHEPES缓冲液中，得到50μl混合液；
步骤2、将不同浓度的CD163抗体溶液分别与1ml金纳米棒溶液作用，30分钟后测量各溶液的吸光度；当金纳米棒溶液最大吸收峰值不变时，进入吸收峰平台的初始浓度即为所需最佳CD163抗体浓度；
步骤3、取该浓度混合物溶液1ml加入50μl聚乙二醇作用10分钟后，在4℃下以15000rmb的转速离心15分钟，得到mAb-CD163/Au共轭物；然后弃去上清液，加入PBS缓冲液，得到分散良好稳定的溶液，4℃下保存。
3.如权利要求1所述的荧光NP-Au靶向造影剂的制备方法，其特征在于，所述的水为双蒸水。
4.如权利要求1所述的荧光NP-Au靶向造影剂在制备肿瘤靶向造影剂中的应用。
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| US20180271788A1 (en) * | 2015-12-11 | 2018-09-27 | The United States of America, as represented by the Secretary, Department of Health and | Vesicle containing metallic nanoparticle and method for production thereof |
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