CN111888487B - 肿瘤靶向型光声成像引导多阶段治疗纳米探针及制备方法 - Google Patents

肿瘤靶向型光声成像引导多阶段治疗纳米探针及制备方法 Download PDF

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CN111888487B
CN111888487B CN202010921912.8A CN202010921912A CN111888487B CN 111888487 B CN111888487 B CN 111888487B CN 202010921912 A CN202010921912 A CN 202010921912A CN 111888487 B CN111888487 B CN 111888487B
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CN111888487A (zh
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张瑞平
郑子良
陈琪
秦宇飞
段艾林
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Shanxi Medical University
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Abstract

本发明涉及生物医学纳米材料技术领域，具体涉及一种肿瘤靶向型光声成像引导多阶段治疗纳米探针及制备方法。该探针以牛血清白蛋白生物矿化法合成的分散均匀、表面电荷为负的牛血清白蛋白包裹硫化铜纳米颗粒为基底，通过静电吸附层层包裹的方式，在硫化铜纳米颗粒外部依次包裹具有正电荷的左旋多聚赖氨酸和电性为负的葡萄糖氧化酶混合透明质酸层，以提供一种具备光声成像及多阶段治疗的纳米探针，该材料在肿瘤微环境中可依靠透明质酸主动靶向三阴性乳腺癌细胞膜表面丰富的CD44受体，释放葡萄糖氧化酶进行饥饿治疗，纳米颗粒表面电荷翻转，增加其细胞摄取率，进一步在肿瘤细胞内进行光热治疗和化学动力学治疗，实现和提高纳米探针的成像和治疗性能。

Description

肿瘤靶向型光声成像引导多阶段治疗纳米探针及制备方法
技术领域
本发明涉及生物医学纳米材料领域，更具体而言，涉及一种肿瘤靶向型光声成像引导多阶段治疗纳米探针及制备方法。
背景技术
乳腺癌是世界范围内女性死亡率较高的肿瘤，尤其是三阴性乳腺癌(TNBC)是一种具有高度侵袭性和转移性的乳腺癌亚型。随着纳米材料日益发展，开发针对TNBC的靶向、诊断和治疗的纳米平台，是有效治疗的迫切需要。
由于严重的缺氧和低pH微环境，癌细胞比正常细胞对温度升高更敏感。光热疗法（PTT）作为一种新型的物理疗法，在癌症治疗领域引起了越来越多的关注。纳米颗粒介导的PTT使用光热剂，通过将吸收的近红外（NIR）激光能量转化为热能，以精准的肿瘤特异性定位热杀伤达到有效地消融癌细胞和组织的效果。与传统的一区近红外光相比，二区近红外光（NIR-II，900-1700 nm）具有许多优点，如对生物组织的穿透更深，组织散射和皮肤吸收更少。为了增加肿瘤的可见性和治疗效果，需要提高癌细胞区域多功能药物的聚集量。而纳米颗粒本身具有在实体瘤区域的高通透性和滞留效应（EPR效应）。然而，单一EPR效应驱动的纳米药物传递有累积不足和肿瘤保留不足的缺点。为了解决这些问题，肿瘤微环境响应型纳米药物已经被开发出来，以达到精确的抗肿瘤效果并减少不良反应，这些纳米颗粒只有在肿瘤微环境中被特异性激活后才能产生治疗作用，而在非肿瘤病变中保持“沉默状态”。因此，合理设计刺激响应型纳米药物对于提高肿瘤细胞的有效摄取，从而获得优异的抗肿瘤性能至关重要。
研究表明，三阴性乳腺癌细胞表面有CD44受体高表达，肿瘤细胞膜表面电性为负，肿瘤细胞微环境因肿瘤细胞过度生长导致营养缺乏，营养物质葡萄糖浓度低，Fenton反应产生的·OH可以有效抑制癌细胞的生长。我们可以利用上述特征，使纳米材料可以主动靶向到CD44受体，在肿瘤微环境中响应后使材料表面电荷及释放其他具有治疗效果的成分，从而实现肿瘤部位纳米颗粒的高效聚集和光热治疗、饥饿疗法和化学动力学治疗。从毒性、降解性、稳定性以及合成工艺等因素出发，设计开发以白蛋白包裹金属硫化物为核心的层层包裹结构的纳米颗粒，不仅材料本身毒性低，特异性聚集效率高，还可以充分利用层层分解的响应过程，达到多种肿瘤治疗方法在治疗的各个阶段发挥作用。到目前为止，国内外有关利用白蛋白生物矿化法，集合NIR-II的光声成像和光热治疗功能为一体的纳米探针，并且有饥饿治疗和化学动力治疗辅助的肿瘤靶向性纳米诊疗一体化探针的研究较少，且主要局限于穿透能力较差的近红外一区的功能的纳米探针研究。因此，这项工作为实现高效的肿瘤治疗提供了一种新的多阶段治疗策略。
发明内容
为了克服现有技术中所存在的不足，本发明提供一种肿瘤靶向型光声成像引导多阶段治疗纳米探针及制备方法，该材料在三阴性乳腺癌细胞的微环境中可以特异性靶向CD44受体，在肿瘤微环境中响应发生电荷翻转，提升材料在肿瘤部位的富集程度。在光声成像和光热治疗中，由于该材料的吸收光谱位于近红外二区，故具有更深的穿透深度，可以达到更好的成像及治疗效果。同时以饥饿治疗和化学动力学治疗作为辅助疗法，可以全面提高该材料在肿瘤治疗各阶段发挥的作用。
为了解决上述技术问题，本发明所采用的技术方案为：
一种肿瘤靶向型光声成像引导多阶段治疗一体化纳米探针，其特征在于：该纳米探针为CuS基纳米，通过静电层层包覆左旋多聚赖氨酸（poly(L-lysine)， PLL）、葡萄糖氧化酶（GOx）和透明质酸（HA），其其中CuS占纳米探针的13.2wt%，牛血清白蛋白占纳米探针的57.3wt%，左旋多聚赖氨酸占纳米探针的13.4wt%，葡萄糖氧化酶占纳米探针的5.6wt%，透明质酸占纳米探针的10.5wt%。
进一步地，所述纳米探针的晶粒直径为35~65nm。
一种肿瘤靶向型光声成像引导多阶段治疗一体化纳米探针制备方法，包括以下步骤：
S1、按照牛血清白蛋白：去离子水=100：3的质量比，将牛血清白蛋白加入去离子水中，超声分散15min，室温搅拌15min，形成透明的牛血清白蛋白水溶液；
S2、按照无机铜源：硝酸=48.32mg:1mL的摩尔比，将无机铜源固体加入2mol/L的硝酸溶液中，超声分散5min后快速加至S1中的牛血清白蛋白水溶液，强力搅拌10min；
S3、向S2中混合溶液滴加浓度为2mol/L的氢氧化钠溶液，调整混合溶液pH值为12；
S4、取0.5mL浓度为0.1mol/L的硫化钠水溶液匀速滴加入S3中混合溶液，90℃下搅拌0.5~3h；溶液由棕黑色变成墨绿色；
S5、将S4中墨绿色产物经去离子水洗涤和超滤离心3次，冷冻干燥，得到牛血清白蛋白包裹的硫化铜（CuS@BSA）纳米颗粒；
S6、将上述CuS@BSA纳米颗粒配制成浓度为1mg/mL的水溶液，通过超声分散将PLL粉末配制成浓度为1mg/mL的水溶液，按照CuS@BSA水溶液：PLL水溶液=1:1的体积比，将CuS@BSA水溶液缓慢滴加入PLL水溶液中，充分搅拌15min，经去离子水洗涤和离心1次，冷冻干燥，得到CuS@BSA-PLL纳米颗粒；
S7、将上述CuS@BSA-PLL纳米颗粒配制成浓度为1mg/mL的CuS@BSA-PLL水溶液，将GOx与HA按照质量比为1:1的比例配制混合溶液使GOx浓度为0.5mg/mL、HA浓度为0.5mg/mL，按照混合溶液：CuS@BSA-PLL水溶液体积比为1:2的比例缓慢将混合溶液滴加入CuS@BSA-PLL水溶液中，充分搅拌15min，经去离子水洗涤和离心1次，冷冻干燥，得到光声成像引导多阶段治疗一体化纳米探针。
优选地，S1、S2、S6中超声分散频率50KHz。
优选地，所述无机铜源为水合硝酸铜、五水合硫酸铜或二水合氯化铜。
优选地，S6中超声分散频率为45KHz。
优选地，所用透明质酸（HA）分子量为3000Mw。
优选地，所述S5离心分离采用超滤分离管，转速为3000r/min，时间为15min。
优选地，所述S6、S7离心分离采用超滤分离管，转速为1000r/min，时间为10min。
与现有技术相比，本发明所具有的有益效果为：
本发明提供了一种肿瘤靶向型光声成像引导多阶段治疗纳米探针及制备方法，该纳米探针以白蛋白生物矿化法合成的牛血清白蛋白包裹的硫化铜为基础，通过静电吸附的方式在外包裹正电荷层PLL和负电荷层的GOx和HA，以提供一种具备光声成像及多阶段治疗功能的纳米探针，该材料在肿瘤微环境中与CD44受体响应，释放出GOx达到饥饿治疗的效果，使该材料表面电荷翻转，增加肿瘤细胞的摄取量，从而提高其在近红外二区光声成像和光热治疗的效果，该材料分解后可以产生化学动力学治疗的效果。本工艺过程是一种简单、快速、可控的肿瘤靶向型光声成像引导多阶段治疗一体化纳米探针，本发明制备的纳米探针晶粒直径为35~65nm，产品纯度达到99.9％。
附图说明
图1为本发明实施例1所合成的诊疗一体化纳米探针的透射电子显微镜图。
图2为本发明实施例1所合成的诊疗一体化纳米探针的粒度及Zeta电位变化分析。
图3为本发明实施例1所合成的诊疗一体化纳米探针的XPS谱图。图中（a）为Cu元素的X射线光电子能谱，图中（b）为S元素的X射线光电子能谱。
图4为本发明实施例1所合成的诊疗一体化纳米探针不同浓度的光热性能。
图5为本发明实施例1所合成的诊疗一体化纳米探针的光热稳定性研究。
图6为本发明实施例1所合成的诊疗一体化纳米探针的细胞毒性CCK-8研究。
图7为本发明实施例1所合成的诊疗一体化纳米探针不同浓度细胞细胞光热作用后细胞存活率图。
图8为本发明实施例1所合成的诊疗一体化纳米探针的裸鼠活体光声成像图。
图9为本发明实施例1所合成的诊疗一体化纳米探针的裸鼠活体光热治疗效果图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一种肿瘤靶向型光声成像引导多阶段治疗一体化纳米探针，其特征在于：该纳米探针为CuS基纳米，通过静电层层包覆左旋多聚赖氨酸（poly(L-lysine) (PLL)、葡萄糖氧化酶（GOx）和透明质酸（HA），其中CuS占纳米探针的13.2wt%，牛血清白蛋白占纳米探针的57.3wt%，PLL占纳米探针的13.4wt%，GOx占纳米探针的5.6wt%，HA占纳米探针的10.5wt%。所述纳米探针的晶粒直径为35~65nm。
一种肿瘤靶向型光声成像引导多阶段治疗一体化纳米探针制备方法，包括以下步骤：
S1、按照牛血清白蛋白：去离子水=100：3的质量比，将牛血清白蛋白加入去离子水中，超声分散15min，室温搅拌15min，形成透明的牛血清白蛋白水溶液；
S2、按照无机铜源：硝酸=48.32mg:1mL的摩尔比，将无机铜源固体加入2mol/L的硝酸溶液中，超声分散5min后快速加至S1中的牛血清白蛋白水溶液，强力搅拌10min；
S3、向S2中混合溶液滴加浓度为2mol/L的氢氧化钠溶液，调整混合溶液pH值为12；
S4、取0.5mL浓度为0.1mol/L的硫化钠水溶液匀速滴加入S3中混合溶液，90℃下搅拌0.5~3h；溶液由棕黑色变成墨绿色；
S5、将S4中墨绿色产物经去离子水洗涤和超滤离心3次，冷冻干燥，得到牛血清白蛋白包裹的硫化铜（CuS@BSA）纳米颗粒；
S6、将上述CuS@BSA纳米颗粒配制成浓度为1mg/mL的水溶液，通过超声分散将PLL粉末配制成浓度为1mg/mL的水溶液，按照CuS@BSA水溶液：PLL水溶液=1:1的体积比，将CuS@BSA水溶液缓慢滴加入PLL水溶液中，充分搅拌15min，经去离子水洗涤和离心1次，冷冻干燥，得到CuS@BSA-PLL纳米颗粒；
S7、将上述CuS@BSA-PLL纳米颗粒配制成浓度为1mg/mL的CuS@BSA-PLL水溶液，将GOx与HA按照质量比为1:1的比例配制混合溶液使GOx浓度为0.5mg/mL、HA浓度为0.5mg/mL，按照混合溶液：CuS@BSA-PLL水溶液体积比为1:2的比例缓慢将混合溶液滴加入CuS@BSA-PLL水溶液中，充分搅拌15min，经去离子水洗涤和离心1次，冷冻干燥，得到光声成像引导多阶段治疗一体化纳米探针。
在本实施例中，S1、S2、S6中超声分散频率50KHz。所述无机铜源为水合硝酸铜、五水合硫酸铜或二水合氯化铜。S6中超声分散频率为45KHz。所用透明质酸（HA）分子量为3000Mw。
在本实施例中，所述S5离心分离采用超滤分离管，转速为3000r/min，时间为15min。所述S6、S7离心分离采用超滤分离管，转速为1000r/min，时间为10min。
实施例1
取浓度为65%的硝酸138.6mL，去离子水806mL，一起加入烧杯充分混合，将混合液移至容量瓶中，配成浓度为2moL/L的硝酸溶液备用；
取氢氧化钠83.33g，去离子水1L，一起加入烧杯充分混合，将混合液移至容量瓶中，配成浓度为2moL/L的氢氧化钠溶液备用；
取九水合硫化钠24.22g，去离子水1L，一起加入烧杯充分混合，将混合液移至容量瓶中，配成0.1mol/L的硫化钠溶液备用；
取去离子水105mL，牛血清白蛋白3.75g，一起加入至烧杯中，置于超声波分散仪内进行溶解分散，频率50KHz，分散时间15min，室温搅拌15min，形成透明溶液；称取水合硝酸铜0.725g，缓慢加入15mL预先配好的2moL/L的硝酸溶液中，超声分散5min（50KHz），将该溶液快速加至以上透明溶液中，强力搅拌10min，溶液变为浅蓝色；利用预先配好的氢氧化钠溶液将混合溶液的pH调节至12，强力搅拌10min，溶液变为深紫色；向上述混合溶液中加入7.5mL预先配好的硫化钠溶液，溶液变为深棕色，加热至90摄氏度恒温搅拌1h，溶液由深棕色变为墨绿色，产物经去离子水洗涤和3000r/min超滤离心管分离多余水分3次，冷冻干燥，得到牛血清白蛋白包裹硫化铜纳米颗粒（CuS@BSA）的固体粉末；
取左旋多聚赖氨酸（PLL）1g，去离子水1L，一起加入烧杯超声分散30min，频率为50KHz，配置成浓度为1g/L的PLL水溶液备用；取CuS@BSA纳米颗粒的固体粉末1g，去离子水1L，一起加入烧杯超声分散30min，频率为50KHz，配置成浓度为1g/L的CuS@BSA水溶液后缓慢滴加入1g/L的PLL水溶液中，充分搅拌15min，产物经1000r/min超滤离心管分离1次，取滤膜以上液体冷冻干燥，得到CuS@BSA-PLL纳米颗粒固体粉末；
取CuS@BSA-PLL纳米颗粒固体粉末1g，去离子水1L，一起加入烧杯充分搅拌溶解，配制成浓度为1g/L的CuS@BSA-PLL水溶液备用；取葡萄糖氧化酶固体粉末0.5g、透明质酸固体粉末0.5g，去离子水1L，一起加入烧杯充分搅拌溶解，配置成葡萄糖氧化酶与透明质酸浓度均为0.5g/L的混合溶液备用；取0.5L混合溶液，缓慢滴加入CuS@BSA-PLL水溶液中，充分搅拌15min，产物经1000r/min超滤离心管分离1次，取滤膜以上液体冷冻干燥，得到光声成像引导多阶段治疗一体化纳米探针。
本发明将制备得到的光声成像引导的多阶段治疗纳米探针溶于PBS缓冲液，通过鼠尾静脉注射进行给药，检测纳米探针的成像性能与治疗效果。
图1为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针的透射电子显微镜图。由图可知所合成光声成像引导多阶段治疗一体化纳米探针的形貌，其纳米颗粒分布均匀，形貌为葫芦形小颗粒。
图2为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针的粒度及Zeta电位变化分析。由图可知，所合成多阶段治疗纳米探针的最终粒度为45.7nm，大小分布均匀，合成过程中纳米颗粒表面电荷翻转。
图3为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针的XPS谱图。图中（a）为Cu元素的X射线光电子能谱，图中（b）为S元素的X射线光电子能谱。由图（a）可知，结合能为932.5 eV(Cu2p3/2)和952.4 eV(Cu2p1/2)的强峰可归属于CuS的Cu2+。由图（b）可知，在S元素的xps谱中，S在纳米颗粒中有几种不同的价态(峰分别在163.7 eV和168.2eV)，是分别产生于CuS的S2-和牛血清白蛋白的二硫键(-S-S-)。
图4为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针不同浓度的光热性能。由图可知，不同浓度的诊疗一体化纳米探针在近红外辐照（1064nm，1W/cm2）下的升温曲线显示，浓度越高升温越快，温度随辐照时间增长的越高。
图5为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针的光热稳定性研究。由图可知，多阶段治疗纳米探针在水分散体在1064nm激光（1W /cm2）照射下的光热稳定性，激光照射5min后关闭并重复5个周期。
图6为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针的细胞毒性CCK-8研究。由图（a）可知，葡萄糖氧化酶在高糖条件下可以产生葡萄糖酸，使肿瘤细胞存活率降低。由图（b）可知，不同浓度的光声成像引导多阶段治疗一体化纳米探针对细胞的增值抑制率影像均不明显，表现为低毒性，在光热治疗之后，细胞存活率随着纳米颗粒浓度升高而减小。
图7为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针不同浓度细胞光热作用后细胞存活率图。四图从左到右依次为为对照组、光照组、给药组、给药+光照组；由图可知，与对照组相比，给药后有一定的细胞杀伤效果，光热治疗后细胞几乎都被杀死，细胞光热治疗效果好。
图8为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针的裸鼠活体光声成像图。由图可知，在裸鼠尾静脉给药后6h肿瘤部位药物聚集浓度最高，光声信号最强，六小时后药物逐渐代谢，肿瘤及全身其他部位药物浓度减小。
图9为本发明实施例1所合成的光声成像引导多阶段治疗一体化纳米探针的裸鼠活体光热治疗效果图。如图所示，对裸鼠肿瘤部位进行光热治疗，给裸鼠尾静脉注射诊疗一体化纳米探针与注射PBS相比，肿瘤部位治疗温度可升高20度，远高于注射PBS对照组，光热治疗效果好。
实施例2
取浓度为65%的硝酸138.6mL，去离子水806mL，一起加入烧杯充分混合，将混合液移至容量瓶中，配成浓度为2moL/L的硝酸溶液备用；
取氢氧化钠83.33g，去离子水1L，一起加入烧杯充分混合，将混合液移至容量瓶中，配成浓度为2moL/L的氢氧化钠溶液备用；
取九水合硫化钠24.22g，去离子水1L，一起加入烧杯充分混合，将混合液移至容量瓶中，配成0.1mol/L的硫化钠溶液备用；
取去离子水105mL，牛血清白蛋白3.75g，一起加入至烧杯中，置于超声波分散仪内进行溶解分散，频率50KHz，分散时间15min，室温搅拌15min，形成透明溶液；称取五水合硫酸铜0.85g，缓慢加入15mL预先配好的2moL/L的硝酸溶液中，超声分散5min（50KHz），将该溶液快速加至以上透明溶液中，强力搅拌10min，溶液变为浅蓝色；利用预先配好的氢氧化钠溶液将混合溶液的pH调节至12，强力搅拌10min，溶液变为深紫色；向上述混合溶液中加入7.5mL预先配好的硫化钠溶液，溶液变为深棕色，加热至90摄氏度恒温搅拌1h，溶液由深棕色变为墨绿色，产物经去离子水洗涤和3000r/min超滤离心管分离多余水分3次，冷冻干燥，得到牛血清白蛋白包裹硫化铜纳米颗粒（CuS@BSA）的固体粉末；
取左旋多聚赖氨酸（PLL）1g，去离子水1L，一起加入烧杯超声分散30min，频率为50KHz，配置成浓度为1g/L的PLL水溶液备用；取CuS@BSA纳米颗粒的固体粉末1g，去离子水1L，一起加入烧杯超声分散30min，频率为50KHz，配置成浓度为1g/L的CuS@BSA水溶液后缓慢滴加入1g/L的PLL水溶液中，充分搅拌15min，产物经1000r/min超滤离心管分离1次，取滤膜以上液体冷冻干燥，得到CuS@BSA-PLL纳米颗粒固体粉末；
取CuS@BSA-PLL纳米颗粒固体粉末1g，去离子水1L，一起加入烧杯充分搅拌溶解，配制成浓度为1g/L的CuS@BSA-PLL水溶液备用；取葡萄糖氧化酶固体粉末0.5g、透明质酸固体粉末0.5g，去离子水1L，一起加入烧杯充分搅拌溶解，配置成葡萄糖氧化酶与透明质酸浓度均为0.5g/L的混合溶液备用；取0.5L混合溶液，缓慢滴加入CuS@BSA-PLL水溶液中，充分搅拌15min，产物经1000r/min超滤离心管分离1次，取滤膜以上液体冷冻干燥，得到光声成像引导多阶段治疗一体化纳米探针。
实施例3
取浓度为65%的硝酸138.6mL，去离子水806mL，一起加入烧杯充分混合，将混合液移至容量瓶中，配成浓度为2moL/L的硝酸溶液备用；
取氢氧化钠83.33g，去离子水1L，一起加入烧杯充分混合，将混合液移至容量瓶中，配成浓度为2moL/L的氢氧化钠溶液备用；
取九水合硫化钠24.22g，去离子水1L，一起加入烧杯充分混合，将混合液移至容量瓶中，配成0.1mol/L的硫化钠溶液备用；
取去离子水105mL，牛血清白蛋白3.75g，一起加入至烧杯中，置于超声波分散仪内进行溶解分散，频率50KHz，分散时间15min，室温搅拌15min，形成透明溶液；称取二水合氯化铜0.792g，缓慢加入15mL预先配好的2moL/L的硝酸溶液中，超声分散5min（50KHz），将该溶液快速加至以上透明溶液中，强力搅拌10min，溶液变为浅蓝色；利用预先配好的氢氧化钠溶液将混合溶液的pH调节至12，强力搅拌10min，溶液变为深紫色；向上述混合溶液中加入7.5mL预先配好的硫化钠溶液，溶液变为深棕色，加热至90摄氏度恒温搅拌1h，溶液由深棕色变为墨绿色，产物经去离子水洗涤和3000r/min超滤离心管分离多余水分3次，冷冻干燥，得到牛血清白蛋白包裹硫化铜纳米颗粒（CuS@BSA）的固体粉末；
取左旋多聚赖氨酸（PLL）1g，去离子水1L，一起加入烧杯超声分散30min，频率为50KHz，配置成浓度为1g/L的PLL水溶液备用；取CuS@BSA纳米颗粒的固体粉末1g，去离子水1L，一起加入烧杯超声分散30min，频率为50KHz，配置成浓度为1g/L的CuS@BSA水溶液后缓慢滴加入1g/L的PLL水溶液中，充分搅拌15min，产物经1000r/min超滤离心管分离1次，取滤膜以上液体冷冻干燥，得到CuS@BSA-PLL纳米颗粒固体粉末；
取CuS@BSA-PLL纳米颗粒固体粉末1g，去离子水1L，一起加入烧杯充分搅拌溶解，配制成浓度为1g/L的CuS@BSA-PLL水溶液备用；取葡萄糖氧化酶固体粉末0.5g、透明质酸固体粉末0.5g，去离子水1L，一起加入烧杯充分搅拌溶解，配置成葡萄糖氧化酶与透明质酸浓度均为0.5g/L的混合溶液备用；取0.5L混合溶液，缓慢滴加入CuS@BSA-PLL水溶液中，充分搅拌15min，产物经1000r/min超滤离心管分离1次，取滤膜以上液体冷冻干燥，得到光声成像引导多阶段治疗一体化纳米探针。
上面仅对本发明的较佳实施例作了详细说明，但是本发明并不限于上述实施例，在本领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化，各种变化均应包含在本发明的保护范围之内。

Claims (9)

1.一种肿瘤靶向型光声成像引导多阶段治疗纳米探针，其特征在于：该纳米探针为CuS基纳米颗粒，所述CuS基纳米颗粒为CuS@BSA，通过静电层层包覆左旋多聚赖氨酸、葡萄糖氧化酶和透明质酸，其中CuS占纳米探针的13.2wt％，牛血清白蛋白占纳米探针的57.3wt％，左旋多聚赖氨酸占纳米探针的13.4wt％，葡萄糖氧化酶占纳米探针的5.6wt％，透明质酸占纳米探针的10.5wt％。
2.根据权利要求1所述的一种肿瘤靶向型光声成像引导多阶段治疗纳米探针，其特征在于：所述纳米探针的晶粒直径为35～65nm。
3.一种肿瘤靶向型光声成像引导多阶段治疗纳米探针制备方法，其特征在于，包括以下步骤：
S1、按照牛血清白蛋白：去离子水＝3：84的质量比，将牛血清白蛋白加入去离子水中，超声分散15min，室温搅拌15min，形成透明的牛血清白蛋白水溶液；
S2、按照无机铜源：硝酸＝48.32mg:1mL的比例，将无机铜源固体加入2mol/L的硝酸溶液中，超声分散5min后快速加至S1中的牛血清白蛋白水溶液，强力搅拌10min；
S3、向S2中混合溶液滴加浓度为2mol/L的氢氧化钠溶液，调整混合溶液pH值为12；
S4、取0.5mL浓度为0.1mol/L的硫化钠水溶液匀速滴加入S3中混合溶液，90℃下搅拌0.5～3h；溶液由棕黑色变成墨绿色；
S5、将S4中墨绿色产物经去离子水洗涤和超滤离心3次，冷冻干燥，得到牛血清白蛋白包裹的硫化铜纳米颗粒CuS@BSA纳米颗粒；
S6、将上述CuS@BSA纳米颗粒配制成浓度为1mg/mL的水溶液，通过超声分散将左旋多聚赖氨酸粉末配制成浓度为1mg/mL的水溶液，按照CuS@BSA水溶液：左旋多聚赖氨酸水溶液＝1:1的体积比，将CuS@BSA水溶液缓慢滴加入PLL水溶液中，充分搅拌15min，经去离子水洗涤和离心1次，冷冻干燥，得到CuS@BSA-PLL纳米颗粒；
S7、将上述CuS@BSA-PLL纳米颗粒配制成浓度为1mg/mL的CuS@BSA-PLL水溶液，将葡萄糖氧化酶与透明质酸按照质量比为1:1的比例配制混合溶液使葡萄糖氧化酶浓度为0.5mg/mL、透明质酸浓度为0.5mg/mL，按照混合溶液：CuS@BSA-PLL水溶液体积比为1:2的比例缓慢将混合溶液滴加入CuS@BSA-PLL水溶液中，充分搅拌15min，经去离子水洗涤和离心1次，冷冻干燥，得到光声成像引导多阶段治疗一体化纳米探针。
4.根据权利要求3所述的一种肿瘤靶向型光声成像引导多阶段治疗纳米探针制备方法，其特征在于：步骤S1与步骤S2中所述超声分散的分散频率为50KHz。
5.根据权利要求3所述的一种肿瘤靶向型光声成像引导多阶段治疗纳米探针制备方法，其特征在于：所述无机铜源为水合硝酸铜、五水合硫酸铜或二水合氯化铜。
6.根据权利要求3所述的一种肿瘤靶向型光声成像引导多阶段治疗纳米探针制备方法，其特征在于：步骤S6中所述超声分散频率为45KHz。
7.根据权利要求3所述的一种肿瘤靶向型光声成像引导多阶段治疗纳米探针制备方法，其特征在于：所用透明质酸分子量为3000Mw。
8.根据权利要求3所述的一种肿瘤靶向型光声成像引导多阶段治疗纳米探针制备方法，其特征在于：步骤S5所述离心分离采用超滤分离管，转速为3000r/min，时间为15min。
9.根据权利要求3所述的一种肿瘤靶向型光声成像引导多阶段治疗纳米探针制备方法，其特征在于：步骤S6、步骤S7所述离心分离采用超滤分离管，转速为1000r/min，时间为10min。
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