CN110025576A - 一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法及其应用 - Google Patents
一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法及其应用 Download PDFInfo
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- CN110025576A CN110025576A CN201910326194.7A CN201910326194A CN110025576A CN 110025576 A CN110025576 A CN 110025576A CN 201910326194 A CN201910326194 A CN 201910326194A CN 110025576 A CN110025576 A CN 110025576A
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法及其应用,该方法包括:将二棕榈酰基卵磷脂、氢化大豆磷脂、高纯胆固醇、二硬脂酰基磷脂酰乙醇胺‑聚乙二醇2000、吲哚菁绿置于氯仿和甲醇混合溶液中溶解,制备得到薄脂质膜;将薄脂质膜在含有碳点的硫酸铵溶液中水化、超声、挤出、除去游离的吲哚菁绿和碳点,并在含有碳点的硫酸铵溶液中水化、超声、挤出、透析,制得热敏性脂质体‑吲哚菁绿‑碳点复合纳米颗粒。本发明所述的复合纳米颗粒在可见光到近红外区均有明显的吸收,其在激光照射下具有明显的细胞毒性,其能有效靶向并长时间停留在肿瘤处,从而有效治疗肿瘤,且对各主要器官均无明显的病理性损失,更具安全性和高效性。
Description
技术领域
本发明属于光热试剂制备技术领域,尤其涉及一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法及其应用。
背景技术
近红外光热治疗剂是指利用在近红外光区具有较高光热转换效率的试剂,将其通过血管注射入生物内部,利用靶向性识别技术聚集在肿瘤组织附近,并在近红外光的照射下将光能转化为热能来杀死癌细胞的一种治疗试剂。常见的光热治疗试剂有贵金属纳米颗粒、碳类材料、金属与非金属化合物和有机染料物质。但是目前的光热治疗剂的靶向性差、近红外区吸收能力差等特点,限制了光热治疗剂在临床中的应用。
目前临床上对肿瘤的主要诊断方式主要有核磁共振、计算机断层扫描等,这些检测手段不仅在特异性和灵敏度方面有很大的不足,而且具有难以避免的放射性风险。光学分子影像学是一种发展迅速的生物医学影像技术,具有无辐射、无创伤、成本低、检测方便和实时监控等优点。然而,目前绝大部分与光学分子影像学相关的研究工作使用的造影剂都未获得美国食品药品监督管理局的批准,容易引起难以预知的生物安全性问题。吲哚菁绿(ICG)是唯一被美国食品药品监督管理局(FDA)批准,可用于临床使用的近红外荧光造影剂。在临床上,ICG被广泛应用于对肝功能、心输出量、视网膜的脉管系统的辅助诊断。它还能作为荧光探针发射波长为820nm的近红外荧光,同时能作为感光剂,吸收光能并将其转化成热能或产生单线态氧,进而杀伤肿瘤细胞。然而,ICG在水溶液中很不稳定、在血液循环中容易被快速清除,分子间容易形成二聚体导致荧光淬灭。这些不足严重限制了ICG在肿瘤诊断及治疗方面的应用。研究人员近年来尝试利用各类纳米载体搭载ICG,以期提高其稳定性、延长其血液循环时间并赋予其肿瘤靶向性。
脂质体作为药物载体,具有可生物降解、无免疫原性、无毒性和特异性肿瘤靶向等优点,利用脂质体搭载药物可提高药物的治疗指数、降低药物剂量并降低毒副作用。近年来脂质体作为药物载体的研究已取得长足的进步,已有研究发现将HSA包覆于脂质体表面可降低微粒对巨噬细胞的亲和力,从而延长循环时间提高靶向性。
碳点的特点为易溶于水、毒性低、绿色环保、研制成本低、可生物相容等优点,是一种新型碳基零维材料,也是一种制备新型光热治疗剂的理想材料,但由于碳点尺寸小,不能通过静脉注射有效的进入到肿瘤部位。
但目前并未涉及将碳点和荧光染料(ICG)双重靶向近红外光热治疗剂的报道。
发明内容
为了克服现有技术中存在的缺陷,本发明提供一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法,其将碳点和荧光染料(ICG)包封于热敏性脂质体中,借助脂质体的较大尺寸被动靶向到肿瘤部位,随后通过近红外激光辐照产生光热转换,达到杀死肿瘤的作用,实现双重靶向近红外光热治疗剂的制备;具体为用于荧光成像介导的光热肿瘤治疗的热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒的制备,并对其荧光成像和光热治疗性能进行相应的研究。
为了实现上述目的,本发明采取的技术方案包括:
本发明的第一个目的是提供一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法,其特征在于,所述光热试剂为热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒,所述制备方法包括:
步骤a)称量二棕榈酰基卵磷脂(DPPC)、氢化大豆磷脂(HSPC)、高纯胆固醇(CHO)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)、吲哚菁绿,于氯仿和甲醇混合溶液中溶解,旋转蒸发除去有机溶剂,并进一步干燥得到薄脂质膜;
步骤b)将步骤a)得到的薄脂质膜在含有碳点的硫酸铵溶液中水化,对水化溶液进行超声处理以形成均匀的悬浮液,将所述悬浮液通过脂质体挤出器挤出,使用凝胶层析柱除去游离的吲哚菁绿和碳点,获得中间液;
步骤c)向步骤b)获得的中间液中加入含有碳点的硫酸铵溶液在旋转蒸发仪里水化,水化结束后超声处理,使其充分分散,用脂质体挤出器将其挤出,将挤出后的脂质体溶液以磷酸盐缓冲液为透析液透析,获得热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒。
为了进一步优化上述制备方法,本发明采取的技术措施还包括:
进一步地,所述二棕榈酰基卵磷脂、氢化大豆磷脂、高纯胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿的摩尔比为40~60:20~30:10~20:2~4:0.5~2,混合后总磷脂浓度为5~60mg/mL;所述氯仿和甲醇的体积比为3~5:1。
进一步地,所述步骤b)和所述步骤c)中,所述旋转蒸发温度、水化温度、超声处理温度均为40~60℃,所述水化时间均为1~4小时,所述超声处理时间均为3~8分钟。
进一步地,所述步骤b)中,所述碳点在硫酸铵溶液中的浓度为200~800μg/mL;所述步骤c)中,所述碳点在硫酸铵溶液中的浓度为1~4mg/mL。
进一步地,所述步骤b)和所述步骤c)中,所述脂质体挤出器为滤膜为100nm的聚碳酸酯滤器。
进一步地,所述步骤b)中,所述凝胶层析柱为sephadex G-150柱;所述步骤c)中,所述透析包括采用pH=7.4的磷酸盐缓冲液(PBS)作为透析液透析0.5~2天,脂质体挤出器挤出的脂质体溶液与磷酸盐缓冲液的体积比为1:150~300,透析液更换2~4次。
进一步地,所述中间液和热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒均需低温保存,保存温度为2~8℃。
进一步地,所述制备方法具体包括以下步骤:
步骤1)称量二棕榈酰基卵磷脂、氢化大豆磷脂、高纯胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿摩尔比为50:25:15:3:1,总磷脂浓度为10~40mg/mL,加入4mL的氯仿和1mL的甲醇混合溶液中溶解,在55℃下旋转蒸发除去有机溶剂,并进一步干燥得到薄脂质膜;
步骤2)将步骤1)得到的薄脂质膜在10ml含有浓度为500μg/mL碳点和200mM的硫酸铵溶液中于55℃水化2小时,在55℃下超声处理该水化溶液5分钟以形成均匀的悬浮液,并通过滤膜为100nm的聚碳酸酯滤器挤出60次,使用sephadex G-150柱除去除游离的吲哚菁绿和碳点,获得中间液,并于4℃保存;
步骤3)向步骤2)得到的中间液中加入5mL含有浓度为2mg/mL的碳点和200mM的硫酸铵溶液在旋转蒸发仪里水化1h,转速为100rpm,温度55~60℃;水化结束后超声3~5min,使其充分分散,并用滤膜为100nm的聚碳酸酯滤器将其挤压20次,将挤压后的脂质体溶液以pH=7.4的磷酸盐缓冲液为透析液透析1天,脂质体溶液与磷酸盐缓冲液的体积比为1:200,透析液更换3次,获得热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒,并于4℃保存。
本发明的第二个目的是提供一种由任一上述的制备方法制得的热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒。
本发明的第三个目的是提供一种任一上述的热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒在制备用于肿瘤治疗的药物中的应用。
进一步地,所述肿瘤包括但不限于:肺癌,胃癌,结肠癌,食管癌,肝癌,乳腺癌,宫颈癌,口腔癌、淋巴癌、前列腺癌、恶性淋巴瘤等。
可理解的是,上述碳点为本领域中任一合适的近红外一区和近红外二区响应的碳点。
本发明采用上述技术方案,与现有技术相比,具有如下技术效果:
发明人经过广泛而深入地研究,提供一种快速高效的制备方法,制得一种具有荧光成像功能和光热治疗肿瘤功能的复合纳米颗粒,该纳米颗粒包含用于荧光成像的小分子染料吲哚菁绿、用于近红外区光热治疗的碳点和纳米载体热敏性脂质体。该纳米颗粒对在可见光到近红外区均有明显的吸收,其在激光照射下具有明显的细胞毒性,其能有效靶向肿瘤并长时间停留在肿瘤处,能有效治疗肿瘤,且其对各主要器官均无明显的病理性损失,更具安全性和高效性。
附图说明
图1为本发明一实施例中a)游离的碳点和吲哚菁绿的吸收光谱图;b)热敏性脂质体、热敏性脂质体-吲哚菁绿、热敏性脂质体-吲哚菁绿-碳点的吸收光谱图;
图2为本发明一实施例中a)不同浓度热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒在功率密度为0.8W/cm2的1064nm激光照射下的温度变化的示意图;b)不同浓度热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒在功率密度为0.8W/cm2的808nm激光照射下的温度升高值的示意图;c)500μg/mL的热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒在不同功率的1064nm激光照射下的温度变化的示意图;d)热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒的光热稳定性的示意图;
图3为本发明一实施例中在不同的1064nm激光功率照射5分钟的条件下,光热治疗试剂碳点和热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒的红外热成像图。
图4为本发明一实施例中a)热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒对Hela细胞存活率的影响的示意图;b)热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒在0.8W/cm2光照5分钟条件下对Hela细胞存活率的影响的示意图;
图5为本发明一实施例中热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒通过静脉注射后被动靶向肿瘤的体内成像的示意图;
图6为本发明一实施例中热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒静脉注射给药后在体内器官中的分布情况的示意图。
图7为本发明一实施例中a)肿瘤治疗期间肿瘤体积的变化的示意图,通过静脉注射生理盐水、热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒,在功率密度为0.8W/cm2的条件下用1064nm激光照射5min。肿瘤体积每隔一天测量一次,静脉注射生理盐水、游离的阿霉素和用相同功率激光照射肿瘤(无光热治疗碳点)作为阴性对照;b)肿瘤治疗期间裸鼠的体重变化的示意图;
图8为本发明一实施例中光热治疗碳点的长期毒性评估的的示意图,各实验组后在治疗结束后取出主要器官进行组织学分析。
具体实施方式
本发明涉及一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法,所述光热试剂为热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒,所述制备方法包括:步骤a)称量二棕榈酰基卵磷脂、氢化大豆磷脂、高纯胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿,于氯仿和甲醇混合溶液中溶解,旋转蒸发除去有机溶剂,并进一步干燥得到薄脂质膜;步骤b)将步骤a)得到的薄脂质膜在含有碳点的硫酸铵溶液中水化,对水化溶液进行超声处理以形成均匀的悬浮液,将所述悬浮液通过脂质体挤出器挤出,使用凝胶层析柱除去游离的吲哚菁绿和碳点,获得中间液;步骤c)向步骤b)获得的中间液中加入含有碳点的硫酸铵溶液在旋转蒸发仪里水化,水化结束后超声处理,使其充分分散,用脂质体挤出器将其挤出,将挤出后的脂质体溶液以磷酸盐缓冲液为透析液透析,获得热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒。本发明还涉及上述复合纳米颗粒的应用。
下面结合附图和实施例,对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案,而不能以此来限制本发明的保护范围。
实施例1
本实施例为碳点、热敏性脂质体-碳点复合纳米颗粒以及热敏性脂质体-吲哚菁绿-光热治疗碳点复合纳米颗粒的较佳制备方法。
本实施例制备的碳点为近红二区响应碳点,其包括如下制备步骤:
1)将4g芘加入到320mL浓硝酸中,80℃冷凝回流32h,用去离子水不断稀释得到的混合物洗涤溶液中的酸,最后用220nm的滤膜除去大的颗粒物,然后烘干得到三硝基芘粉末。
2)取第一步所得到的三硝基芘0.1g,将1.6g聚乙烯亚胺溶于10mL去离子水并且与三硝基芘搅拌混匀,放入微波反应器,200℃反应1.5min。
3)将步骤2得到的黑色液体通过220nm滤膜,将滤液转到二氯甲烷相,用中性氧化铝层析柱进行过滤去除为反应的小分子有机物,然后烘干得到黑色粉末,即近红二区响应碳点,备用。
本实施例制备的含有上述近红二区响应碳点的热敏性脂质体-碳点复合纳米颗粒的制备步骤包括:
(一)称量二棕榈酰基卵磷脂(DPPC)、氢化大豆磷脂(HSPC)、高纯胆固醇(CHO)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000),摩尔比为50:25:15:3,加入4mL的氯仿和1mL的甲醇混合溶液中溶解,总磷脂浓度为10~40mg/mL。将溶液转入到100mL的烧瓶中,放入55~60℃的旋转蒸发仪中旋蒸,以150rpm的转速蒸发有机溶剂。溶剂蒸干之后保持1个小时,烧瓶底部形成半透明的膜。
(二)向步骤(a)的烧瓶里加入5mL的浓度为2mg/mL的碳点溶液在旋转蒸发仪里水化1h,转速为100rpm,温度55~60℃。水化结束后超声3~5min,使其充分分散。然后用滤膜为100nm的脂质体挤出器将其挤压20次,将挤压后的脂质体放入4℃冰箱里保存,得到装载有光热治疗试剂碳点的热敏性脂质体。
本实施例制备的含有上述近红二区响应碳点的热敏性脂质体-吲哚菁绿-光热治疗碳点复合纳米颗粒的制备步骤包括:
a)称量二棕榈酰基卵磷脂(DPPC)、氢化大豆磷脂(HSPC)、高纯胆固醇(CHO)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿摩尔比为50:25:15:3:1,总磷脂浓度为10~40mg/mL,加入4mL的氯仿和1mL的甲醇混合溶液中溶解。然后在55℃下旋转蒸发除去有机溶剂,并进一步干燥所得的薄脂质膜。
b)将得到的薄脂质膜在含有碳点(500μg/mL)的(NH4)2SO4溶液(10mL,200mM)中于55℃水化2小时。然后通过在55℃下超声处理该溶液5分钟以形成均匀的悬浮液,并通过聚碳酸酯滤器(100nm)挤出60次。最后,使用sephadex G-150柱除去游离的吲哚菁绿和碳点,并保存在4℃,得到中间液;
c)将得到的中间液在5mL含有浓度为2mg/mL的碳点和200mM的硫酸铵溶液在旋转蒸发仪里水化1h,转速为100rpm,温度55~60℃。水化结束后超声3~5min,使其充分分散。然后用滤膜为100nm的脂质体挤出器将其挤压20次。将挤压后的脂质体溶液以磷酸盐缓冲液(PBS,pH=7.4)为透析液透析1d,脂质体溶液与PBS的体积比为1:200,透析液更换3次。将透析好的脂质体溶液放入4℃冰箱里保存,得到热敏性脂质体-吲哚菁绿-光热治疗碳点复合纳米颗粒。
实施例2
本实施例为实施例1的热敏性脂质体-吲哚菁绿-光热治疗碳点复合纳米颗粒的另一较佳制备方法,其包括如下步骤:
a)称量二棕榈酰基卵磷脂(DPPC)、氢化大豆磷脂(HSPC)、高纯胆固醇(CHO)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿摩尔比为55:20:20:2:2,总磷脂浓度为10~40mg/mL,加入5mL的氯仿和1mL的甲醇混合溶液中溶解。然后在60℃下旋转蒸发除去有机溶剂,并进一步干燥所得的薄脂质膜。
b)将得到的薄脂质膜在含有碳点(800μg/mL)的(NH4)2SO4溶液(10mL,200mM)中于50℃水化1.5小时。然后通过在50℃下浴超声处理该溶液8分钟以形成均匀的悬浮液,并通过聚碳酸酯滤器(100nm)挤出60次。最后,使用sephadex G-150柱除去游离的吲哚菁绿和碳点,并保存在5℃,得到中间液;
c)将得到的薄脂质膜在5mL含有浓度为3mg/mL的碳点和200mM的硫酸铵溶液在旋转蒸发仪里水化1.5h,转速为100rpm,温度58℃。水化结束后超声4min,使其充分分散。然后用滤膜为100nm的脂质体挤出器将其挤压20次。将挤压后的脂质体溶液以磷酸盐缓冲液(PBS,pH=7.4)为透析液透析1.5天,脂质体溶液与PBS的体积比为1:300,透析液更换4次。将透析好的脂质体溶液放入5℃冰箱里保存,得到热敏性脂质体-吲哚菁绿-光热治疗碳点复合纳米颗粒。
实施例3
对实施例1中制备的碳点、热敏性脂质体-碳点复合纳米颗粒以及热敏性脂质体-吲哚菁绿-光热治疗碳点复合纳米颗粒的相关性能进行如下研究:
(1)碳点和热敏性脂质体-碳点复合纳米颗粒的体外光热特性:
将碳点(或热敏性脂质体-碳点复合纳米颗粒)稀释成不同的浓度(0-500μg/mL,然后转移至1.5mL的离心管,用1064nm的激光器以0.8W·cm-2的功率密度照射5分钟,每隔20s用红外热成像仪记录温度的变化。改变功率(0.4-1W·cm-2)用1064nm的激光器照射500μg/mL碳点(或热敏性脂质体-碳点复合纳米颗粒)以测定不同功率对碳点升温的影响。通过功率密度为0.8W/cm2的1064nm激光器重复照射5min下温度的变化,探究碳点的光稳定性。
(2)热敏性脂质体-碳点复合纳米颗粒的体外光热治疗:
通过MTT法对经过热敏性脂质体-碳点复合纳米颗粒处理后的细胞进行细胞活度的检测。将人宫颈癌细胞(Hela)种到96孔板中,每孔的密度为5000个,培养24小时,然后加入不同浓度的敏性脂质体-碳点复合纳米颗粒,培养4小时后,用0.8W/cm2的1064nm激光器照射5分钟,然后将细胞继续培养24或48小时,到达待测时间后加入20μL的MTT,然后继续在37℃下继续孵育4小时,最后将培养液吸出,每孔加入150μL的二甲基亚砜进行溶解,用酶标仪测定在490nm处的吸收。
(3)热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒的体内荧光成像:
4T1荷瘤小鼠模型建立后,待肿瘤体积达到1000mm3左右时,静脉注射热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒,用体内成像系统进行观察和拍照。设置激发波长为700nm,荧光收集波长为790nm。分别在0、12、24、48和72小时拍照。在静脉注射0、12、24、48和72小时后处死裸鼠,将主要器官取出用体内成像系统进行荧光拍照。
(4)热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒体内光热治疗治疗性能评估:
将100μL(100万)的4T1细胞皮下接种于3-5周的雌性裸鼠腋窝处,带肿瘤体积长到100-200mm3时,将裸鼠分为5组(每组5只):(1)生理盐水;(2)游离的碳点;(3)生理盐水+激光(0.8W/cm2、1064nm);(4)热敏性脂质体-吲哚菁绿-碳点+激光(0.3W/cm2、808nm);(5)热敏性脂质体-吲哚菁绿-碳点+激光(0.8W/cm2、1064nm)。通过静脉注射(200μL)到荷瘤小鼠体内,每隔一天测量肿瘤的体积大小每天记录裸鼠的体重。
(5)体内长期毒性的评估:
将Balb/c小鼠分为5组(每组5只),每组分别注射(1)生理盐水;(2)游离的碳点;(3)生理盐水+激光(0.8W/cm2、1064nm);(4)热敏性脂质体-吲哚菁绿-碳点+激光(0.3W/cm2、808nm);(5)热敏性脂质体-吲哚菁绿-碳点+激光(0.8W/cm2、1064nm)。在第18天处死小鼠,将心、肝、脾、肺、肾等器官取出,组织切片后染色、拍照,进行组织学分析。
对上述碳点、热敏性脂质体-碳点复合纳米颗粒、热敏性脂质体-吲哚菁绿-碳点光热试剂经仪器检测进行表征、MTT法测定其细胞毒性以及在体内通过静脉注射方式治疗肿瘤等相关实验,其结果如图1~图8所示,具体如下:
由图1可知,碳点、吲哚菁绿和热敏性脂质体-吲哚菁绿-碳点在可见光到近红外二区均有明显的吸收。
由图2可知,随着热敏性脂质体-碳点复合纳米颗粒浓度的增加,在相同功率照射下,热敏性脂质体-碳点复合纳米颗粒溶液温度的变化也随之增加;在相同浓度的热敏性脂质体-碳点复合纳米颗粒溶液条件下,当增加激光器功率时,热敏性脂质体-碳点复合纳米颗粒溶液温度的变化也随之增加。
由图3可知,热敏性脂质体-吲哚菁绿-碳点溶液在0.8W/cm2的功率照射下,5分钟内上升了23.6℃,热敏性脂质体-吲哚菁绿-碳点溶液在0.6W/cm2的功率照射下,5分钟内上升了18.5℃。
由图4可知,热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒没表现出明显的细胞毒性。激光照射条件下热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒表现出了明显的细胞毒性。
由图5可知,静脉注射热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒后,样品可以靶向肿瘤成像,并且长时间停留在肿瘤处。
由图6可知,静脉注射热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒后,样品主要在肝脏和肿瘤处富集。
由图7可知,静脉注射静脉注射热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒后,在0.8W/cm2功率下照射5分钟,肿瘤体积减小直至消失;在整个治疗过程中,治疗组裸鼠体重基本保持不变。
由图8可知,各治疗组对小鼠的主要器官均没有明显的病理性损伤。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (10)
1.一种用于荧光成像介导的光热肿瘤治疗的光热试剂的制备方法,其特征在于,所述光热试剂为热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒,所述制备方法包括:
步骤a)称量二棕榈酰基卵磷脂、氢化大豆磷脂、高纯胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿,于氯仿和甲醇混合溶液中溶解,旋转蒸发除去有机溶剂,并进一步干燥得到薄脂质膜;
步骤b)将步骤a)得到的薄脂质膜在含有碳点的硫酸铵溶液中水化,对水化溶液进行超声处理以形成均匀的悬浮液,将所述悬浮液通过脂质体挤出器挤出,使用凝胶层析柱除去游离的吲哚菁绿和碳点,获得中间液;
步骤c)向步骤b)获得的中间液中加入含有碳点的硫酸铵溶液在旋转蒸发仪里水化,水化结束后超声处理,使其充分分散,用脂质体挤出器将其挤出,将挤出后的脂质体溶液以磷酸盐缓冲液为透析液进行透析,获得热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒。
2.根据权利要求1所述的制备方法,其特征在于,所述步骤a)中,二棕榈酰基卵磷脂、氢化大豆磷脂、高纯胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿的摩尔比为40~60:20~30:10~20:2~4:0.5~2,混合后总磷脂浓度为5~60mg/mL;所述氯仿和甲醇的体积比为3~5:1。
3.根据权利要求1所述的制备方法,其特征在于,所述步骤b)和所述步骤c)中,所述旋转蒸发温度、水化温度、超声处理温度均为50~60℃,所述水化时间均为1~4小时,所述超声处理时间均为3~8分钟。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤b)中,所述碳点在硫酸铵溶液中的浓度为200~800μg/mL;所述步骤c)中,所述碳点在硫酸铵溶液中的浓度为1~4mg/mL。
5.根据权利要求1所述的制备方法,其特征在于,所述步骤b)和所述步骤c)中,所述脂质体挤出器为滤膜为100nm的聚碳酸酯滤器,所述挤出次数为20~60次。
6.根据权利要求1所述的制备方法,其特征在于,所述步骤b)中,所述凝胶层析柱为sephadex G-150柱;所述步骤c)中,所述透析为采用pH=7.4的磷酸盐缓冲液作为透析液透析0.5~2天,脂质体挤出器挤出的脂质体溶液与磷酸盐缓冲液的体积比为1:150~300,透析液更换2~4次。
7.根据权利要求1所述的制备方法,其特征在于,所述中间液和热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒均需低温保存,保存温度为2~8℃。
8.根据权利要求1所述的制备方法,其特征在于,所述制备方法具体包括以下步骤:
步骤1)称量二棕榈酰基卵磷脂、氢化大豆磷脂、高纯胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、吲哚菁绿摩尔比为50:25:15:3:1,总磷脂浓度为10~40mg/mL,加入4mL的氯仿和1mL的甲醇混合溶液中溶解,在55℃下旋转蒸发除去有机溶剂,并进一步干燥得到薄脂质膜;
步骤2)将步骤1)得到的薄脂质膜在10ml含有浓度为500μg/mL碳点和200mM的硫酸铵溶液中于55℃水化2小时,在55℃下超声处理该水化溶液5分钟以形成均匀的悬浮液,并通过滤膜为100nm的聚碳酸酯滤器挤出60次,使用sephadex G-150柱除去除游离的吲哚菁绿和碳点,获得中间液,并于4℃保存;
步骤3)向步骤2)得到的中间液中加入5mL含有浓度为2mg/mL的碳点和200mM的硫酸铵溶液在旋转蒸发仪里水化1h,转速为100rpm,温度55~60℃;水化结束后超声3~5min,使其充分分散,并用滤膜为100nm的聚碳酸酯滤器将其挤压20次,将挤压后的脂质体溶液以pH=7.4的磷酸盐缓冲液为透析液透析1天,脂质体溶液与磷酸盐缓冲液的体积比为1:200,透析液更换3次,获得热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒,并于4℃保存。
9.一种由权利要求1~8任一项所述的制备方法制得的热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒。
10.一种如权利要求9所述的热敏性脂质体-吲哚菁绿-碳点复合纳米颗粒在制备用于肿瘤治疗的药物中的应用。
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