CN108743980B - 一种叶酸靶向可视化光热-化疗治疗剂及其制备方法 - Google Patents
一种叶酸靶向可视化光热-化疗治疗剂及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种叶酸靶向可视化光热‑化疗治疗剂及其制备方法。先采用溶剂挥发‑乳化法制备载多西他赛的磷脂液态氟碳纳米球，然后在纳米球表明包裹一层聚吡咯，最后在聚吡咯表面修饰具有肿瘤主动靶向功能的叶酸分子，得到叶酸靶向可视化光热‑化疗治疗剂。该叶酸靶向可视化光热‑化疗治疗剂大小均一，粒径为209.1±7.2nm，具有良好的体外光热效应，光声成像效果和超声成像效果。细胞学实验表明该叶酸靶向可视化光热‑化疗治疗剂空白载体具有良好的生物相容性。体内实验结果表明该叶酸靶向可视化光热‑化疗治疗剂具有良好的体内超声成像以及光声成像效果，同时，其光热‑化疗联合作用具有良好的体内抑瘤效果。该叶酸靶向多功能纳米有望成为一种新型的可视化光热‑化疗治疗剂。
Description
技术领域
本发明涉及医疗药物领域，具体涉及一种叶酸靶向可视化光热-化疗治疗剂及其制备方法。
背景技术
癌症现如今已经成为人类危险最大的疾病，目前，全世界有几百万人死于癌症，癌症死亡率也在逐年上升。目前癌症治疗的主要手段是手术切除，化疗以及放射性治疗。虽然这些治疗手段在临床上发挥了重要的治疗效果，但也不能彻底治疗癌症。比如，手术切除虽然能切除大部分的肿瘤组织，但是没有切除的少部分的肿瘤组织依然可能复发，化疗药物在治疗癌症的同时，也对人体的正常细胞产生毒副作用，比如肝肾损伤，以及心脏毒性，并且在长期应用化疗药物后，肿瘤也会对化疗药物产生耐药性，从而降低药物的治疗效果。
癌症热治疗是一种新型的肿瘤治疗方法，现在也已经成为肿瘤治疗的研究热点。癌症热治疗是使肿瘤温度升高，从而达到肿瘤治疗的效果。肿瘤热治疗的原理就是利用高热(40-45℃)损伤肿瘤细胞，而不会对周围正常组织造成影响。当温度高于45℃时，可以直接消融肿瘤细胞。传统的热疗方法包括局部水浴加热，外部电磁和超声照射等方式。虽然这些加热方法能够有效地加热肿瘤组织，但是也存在风险，比如，水浴加热时可能会加热到其他部位，有些需要手术，或者由于没有针对性地加热会导致其他正常组织受到损伤。基于此，光热治疗(Photothermal therapy,PTT)成为肿瘤热疗的新方式。光热治疗作为一种新型的肿瘤热治疗手段，近年来受到广泛的关注。光热治疗的基本原理是在近红外激光照射下，光热转化剂将激光的能量转换成热能，使肿瘤温度升高，从而杀死肿瘤细胞，达到肿瘤治疗效果。将光热治疗与化疗相结合起来可以发挥更好的肿瘤治疗效果，这主要是因为光热治疗产生的合适的温度能与化疗药物产生协同作用，产生协同作用的机制主要包括：适合的高温能够增加血管通透性，增强肿瘤细胞对化疗药物的敏感性，减少肿瘤细胞耐药性等。具有光热特性的材料可以被设计成光热转化剂，当光热治疗剂达到肿瘤部位，在激光的照射下能够形成高热，可以局部加热损伤肿瘤组织。波长在700-1100nm的近红外激光能够有效地穿透人体组织，达到光热转化剂富集的肿瘤组织，从而加热肿瘤组织。作为光热治疗的光热治疗剂应该具有以下特点：在红外区具有具有较强的吸收，能够选择性地聚集于肿瘤组织，具有较强的光热转换性能，良好的生物相容性以及生物可降解性。许多具有光热转换性能的材料用于光热治疗剂，比如无机金纳米材料(金纳米壳层，金纳米棒，金纳米笼)，碳纳米结构等，以及具有光吸收特性的有机材料，比如吲哚菁绿等。有机聚吡咯纳米材料由于其固有的特点近年来受到了生物电子和生物医疗领域的广泛的关注，比如，聚吡咯材料具有较高的导电性，稳定性以及良好的生物相容性。由于聚吡咯纳米在近红外区域具有良好的光热转换性能，近年来也被应用于光热治疗剂。
虽然光热治疗是一种新型肿瘤治疗技术，可以将激光照射在肿瘤部位，在光热转化剂的作用下产生高热，达到肿瘤治疗的效果，但是在光热治疗前，需要预先判断肿瘤的形态大小，深度，以及光热治疗剂能否有效地达到和聚集于肿瘤部位，在治疗过程中也需要监测肿瘤的局部温度是否达到光热治疗的温度，在光热治疗结束后需要判断在光热治疗以后肿瘤是否减小，是否达到了光热治疗的效果，因此，可视化的影像导航在光热治疗过程中显得尤为重要。作为一种辅助性的手段，可视化的影像导航技术能够帮助研究人员对肿瘤做出及时和准确的诊断，对治疗方案做出及时调整，因此，可视化的光热治疗可以实现诊疗一体化。可视化的影响导航技术主要包括超声成像技术，光声成像技术，红外热成像技术以及荧光成像技术等。
超声成像技术是一种使用广泛的成像技术，其基本原理是频率为1～40MHz的高频声波可以穿透皮肤，可以被体内深部组织反射回来，体内深部组织的结构信息反馈给超声波探测器，重建扫描区域的图像。超声造影剂是一种能够增强声波背向散射能力，增强超声信号的制剂。微泡是一种良好的超声造影剂，但是其固有的缺点比如粒径较大，稳定性差等限制了其在超声成像领域的应用。基于液-气相变的液态氟碳的纳米制剂成为超声造影剂领域的研究热点，液态氟碳纳米超声造影剂具有稳定性好，粒径小等优点，能够通过EPR效应或者主动靶向作用聚集于肿瘤部位，在聚焦超声，激光等外部条件的触发下发生液-气相变，成为具有超声造影效果的微泡。聚焦超声是一种常用的触发液-气相变的手段，但是其也存在缺点，比如超声波负压可能对周围的正常组织造成损伤，超声波可能不能有效地穿透含气体丰富的组织，比如肺组织。因此，激光是一种更可选的手段，近红外激光比较适合成像和医疗领域，在这个波段的激光能够有效地穿透组织。将具有光热效应功能的物质与液态氟碳相结合能够更容易触发液-气相变，光热物质在吸收激光后能够将光能转换成热能，其热量传递给液态氟碳，加速其相变。
光声成像(Photoacoustic，PA)技术是一种复合超声和光学的成像技术。具有分辨率和高灵敏度高，非侵入性，实时成像等优点。光声成像技术已经逐渐成为早期诊断，药物递送，深部组织探测的一种重要的手段。其原理为：脉冲激光的光能量被光声造影剂吸收并转换成热，瞬时热弹性膨胀形成超声波，其可以被超声传感器探测到并形成超声图像。最近一些研究发现，聚吡咯具有良好的光声成像性能，可以作为一种良好的光声造影剂。
本研究设计制备了一种新型的具有叶酸靶向功能的可视化光热治疗剂。首先，将具有超声成像功能的全氟己烷和具有抗肿瘤效果的药物多西他赛制备成磷脂液态氟碳纳米球，然后在纳米球的外面包裹一层具有光热效应和光声成像功能的聚吡咯壳层，最后在聚吡咯壳层外面修饰具有肿瘤主动靶向功能的叶酸分子。得到的叶酸靶向可视化光热-化疗治疗剂：光热治疗，化疗，叶酸介导的主动靶向，光声成像功能和超声成像功能。该叶酸靶向多功能纳米制备工艺简单，经济，为肿瘤的可视化光热治疗以及肿瘤的光热-化疗联合应用提供了新的策略。
发明内容
本发明的目的在于针对目前肿瘤化疗存在的问题，设计和制备了一种叶酸靶向可视化光热-化疗治疗剂。先采用溶剂挥发-乳化法制备载多西他赛的磷脂液态氟碳纳米球，然后在纳米球表明包裹一层聚吡咯，最后在聚吡咯表面修饰具有肿瘤主动靶向功能的叶酸分子，得到叶酸靶向可视化光热-化疗治疗剂。该叶酸靶向可视化光热-化疗治疗剂大小均一，粒径为209.1±7.2nm，具有良好的体外光热效应，光声成像效果和超声成像效果。细胞学实验表明该叶酸靶向可视化光热-化疗治疗剂空白载体具有良好的生物相容性。体内实验结果表明该叶酸靶向可视化光热-化疗治疗剂具有良好的体内超声成像以及光声成像效果，同时，其光热-化疗联合作用具有良好的体内抑瘤效果。该叶酸靶向多功能纳米有望成为一种新型的肿瘤可视化光热治疗剂。
本发明的目的可以通过以下技术方案实现：
步骤1：称取适量的大豆卵磷脂和药物溶解于二氯甲烷，在减压旋转蒸发除去二氯甲烷，得到均匀的脂膜，然后加入一定量的水，震荡或超声使薄膜脱落，得到脂质混悬液。
步骤2：将步骤1得到的脂质混悬液用超声波细胞破碎仪超声，得到脂质体，将该脂质体于4℃冷却。
步骤3：向步骤2得到的脂质体加入一定量全氟己烷，冰浴条件下，用超声波细胞破碎仪进行超声，即得到载药磷脂液态氟碳纳米球。
步骤4：将步骤3得到的载药磷脂液态氟碳纳米球转移至烧瓶中，加入三氯化铁溶液和聚乙烯吡咯烷酮溶液，于冰浴中搅拌，然后加入吡咯和吡咯-1丙酸。将整个反应体系于冰浴且避光条件下搅拌反应3h，得到外观为黑色的纳米混悬液。将此混悬液离心，弃去上清液，沉淀用去离子水洗涤三次，除去三氯化铁以及聚乙烯吡咯烷酮，并将沉淀重悬于去离子水中，得到表面羧基化的聚吡咯包裹载药磷脂液态氟碳纳米混悬液。
步骤5：称0.5g叶酸，0.47g二环己基碳二亚胺，0.26g N-N-羟基琥珀酰亚胺和0.25mL三乙胺溶于无水二甲基亚砜(DMSO)，于25℃条件下避光搅拌反应24h，过滤，用无水乙醚洗涤，干燥，得到叶酸活性酯。称量适量叶酸活性酯和聚氧乙烯二胺溶于5mL无水二甲基亚砜，加入适量三乙胺，于25℃条件下避光搅拌反应24h，加入无水乙醚萃取除去二甲基亚砜，将固体沉淀过滤，真空干燥，得到淡黄色的H2N-PEG-FA。
步骤6：向步骤4制备得到的表面羧基化的聚吡咯包裹载药磷脂液态氟碳纳米混悬液中分别加入适量N-N-羟基琥珀酰亚胺和1-乙基-(3-二甲基氨基丙基)碳二亚胺溶液，于室温搅拌。精密称量适量步骤5制备的H2N-PEG-FA，加入到反应体系中。室温搅拌反应6h，离心，弃去上清液，沉淀用去离子水洗涤三次，得到叶酸靶向可视化光热-化疗治疗剂。
该叶酸靶向可视化光热-化疗治疗剂制备工艺简单，经济，具有如下功能：光热治疗，化疗，叶酸介导的肿瘤主动靶向，光声成像功能和超声成像功能
附图说明
图1为本发明实例1中叶酸靶向可视化光热-化疗治疗剂粒径电位图。
图2为本发明实例1中叶酸靶向可视化光热-化疗治疗剂红外图谱图。
图3为本发明实例2中叶酸靶向可视化光热-化疗治疗剂体外光热效应图。
图4为本发明实例2中叶酸靶向可视化光热-化疗治疗剂体外超声造影图。
图5为本发明实例2中叶酸靶向可视化光热-化疗治疗剂体外光声显影图。
图6为本发明实例2中叶酸靶向可视化光热-化疗治疗剂空白载体对4T1和Hela细胞活力抑制作用图。
图7为本发明实例2中叶酸靶向可视化光热-化疗治疗剂的载激光作用下对细胞活力的抑制作用图。
图8为本发明实例2中叶酸靶向可视化光热-化疗治疗剂的体内超声成像图。
图9为本发明实例2中各种制剂对肿瘤生长抑制效果图。
具体实施方式
以下结合附图实施例对本发明进行详细描述，但本发明并不仅限于下述实施例。
实施例1
1磷脂液态氟碳纳米球的制备
精密称量30mg大豆磷脂和2mg药物并用二氯甲烷溶解，转移至圆底烧瓶中，旋转蒸发除去二氯甲烷成膜。向圆底烧瓶中加入2mL去离子水，水浴超声5min，制备成初乳。将初乳转移至离心管中，探头超声5min，制备成脂质体纳米混悬液。将脂质体纳米混悬液于4℃冰箱冷却，加入30μL全氟己烷，冰浴探头超声5min，制备得到乳白色的载多西他赛磷脂液态氟碳纳米乳。
2表面羧基化修饰的聚吡咯包裹载多西他赛磷脂液态氟碳纳米粒的制备
将载药磷脂液态氟碳纳米乳转移至西林瓶中，加入1mL的三氯化铁溶液(100mg/mL)和1mL聚乙烯吡咯烷酮溶液(100mg/mL)，于冰浴中搅拌，然后加入吡咯(Py)和吡咯-1-丙酸(Py-COOH),吡咯和羧基吡咯的摩尔比为7:3。将整个反应体系于冰浴且避光条件下搅拌反应3h，得到外观为黑色的纳米混悬液。将此混悬液离心，弃去上清液，沉淀用去离子水洗涤三次，除去三氯化铁以及聚乙烯吡咯烷酮，并将沉淀重悬于去离子水中，得到表面羧基化的聚吡咯包裹载药磷脂液态氟碳纳米粒混悬液。
3H2N-PEG-FA的制备
精密称0.5g叶酸(FA)，0.47g二环己基碳二亚胺(DCC)，0.26g N-N-羟基琥珀酰亚胺(NHS)和0.25mL三乙胺溶于10mL无水二甲基亚砜(DMSO)，于25℃条件下避光搅拌反应24h，过滤，用无水乙醚洗涤，干燥，得到叶酸活性酯(FA-NHS)。精密称量8mg叶酸活性酯和112.5mg聚氧乙烯二胺(H2N-PEG-NH2)，并溶于5mL无水二甲基亚砜，加入4μL三乙胺，于25℃条件下避光搅拌反应24h，加入无水乙醚萃取除去二甲基亚砜，将固体沉淀过滤，真空干燥，得到淡黄色的H2N-PEG-FA。
4叶酸靶向可视化光热-化疗治疗剂
向2项下制备得到的表面羧基化的聚吡咯包裹载药磷脂液态氟碳纳米粒混悬液中分别加入150μL，浓度为150mg/mL的N-N-羟基琥珀酰亚胺(NHS)和1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)溶液，于室温搅拌。精密称量8mg 3项下制备的H2N-PEG-FA，加入到反应体系中。室温搅拌反应6h，离心，弃去上清液，沉淀用去离子水洗涤三次，得到叶酸靶向可视化光热-化疗治疗剂。
将DTX/PFH@PPy-FA分散于超纯水中，用马尔文粒径仪分别对粒径和电位进行分析，如图1所示，DTX/PFH@PPy-FA的粒径分布均匀，平均粒径为209.1±7.2nm，电位为8.95±1.08mv。通过傅里叶红外分析验证叶酸的成功修饰，如图2所示，(a)为表面羧基化的聚吡咯包裹载药磷脂液态氟碳纳米粒，(b)为H2N-PEG-FA，(c)为叶酸靶向可视化光热-化疗治疗剂。H2N-PEG-FA在2875-2917cm-1处出现PEG的C-H伸缩振动峰，而表面羧基化的聚吡咯包裹载药磷脂液态氟碳纳米粒在这个波段内并未出现此峰，也没有检测到酰胺键的红外吸收峰。在修饰H2N-PEG-FA得到叶酸靶向可视化光热-化疗治疗剂后，在2858-2825cm-1处出现了C-H伸缩振动峰，并且在1563cm-1处出现酰胺键的红外吸收峰，以上结果表明H2N-PEG-FA成功修饰到表面羧基化的聚吡咯包裹载药磷脂液态氟碳纳米粒的表面上得到叶酸靶向可视化光热-化疗治疗剂。
实施例2
1.叶酸靶向可视化光热-化疗治疗剂体外光热效应考察
将叶酸靶向可视化光热-化疗治疗剂分散于RPMI-1640细胞培养液中配成以下浓度：0.5,0.25，0.125，0.0625,0.03125mg/mL.分别取上述混悬液1mL于石英比色皿中,用808激光仪照射样品，用温度探测器监测温度(测量每份样品时，将温度探测探头伸到比色皿的底部的同一位置)，照射时间为10min，每隔10s记录一次温度，以温度对时间作图。结果如图3所示，空白RPMI-1640无明显光热效应，激光照射10min，温度从24.4升高至29.7℃，温度仅升高了5.3℃。DTX/PFH@PPy-FA纳米具有明显的光热效应，且呈浓度依耐性，浓度越高，温度升高得越高。当浓度为0.5mg/mL时，温度从24.8升高至71.6℃，升高了46.8℃，而当浓度为0.03125mg/mL时，温度从24.7升高至47.8℃，温度升高了23.1℃。
2.叶酸靶向可视化光热-化疗治疗剂体外显影研究
首先在体外用琼脂模型考察了叶酸靶向可视化光热-化疗治疗剂在激光作用下的超声造影功能，如图4所示，相比于没有光热性质的载药磷脂液态氟碳纳米球，叶酸靶向可视化光热-化疗治疗剂在激光的作用下展现出优良的超声成像信号。同时也以生理盐水作为空白对照，考察了其体外光声成像效果，如图5所示，叶酸靶向可视化光热-化疗治疗剂具有良好的体外光声成像效果。
3.叶酸靶向可视化光热-化疗治疗剂细胞毒性研究
分别采用4T1细胞和Hela细胞考察酸靶向叶酸靶向可视化光热-化疗治疗剂空白载体的生物相容性，这两种细胞均是叶酸受体高表达细胞。实验结果如图6所示，叶酸靶向靶向可视化光热-化疗治疗剂空白载体对两种细胞都展现出低毒性，而没有叶酸修饰的聚吡咯包裹磷脂液态氟碳纳米粒空白载体在高浓度对细胞显现出一定的毒性。同时也考察了叶酸靶向可视化光热-化疗治疗剂的激光作用下对细胞的抑制作用。如图7所示，单纯的激光照射对细胞几乎没有杀伤作用。对于叶酸靶向可视化光热-化疗治疗剂空白载体+激光组，当没有激光照射时，几乎对细胞没有杀伤作用，表明单独的空白载体对细胞没有杀伤作用。当给予激光照射后，细胞存活率降低，且随照射时间增加逐渐降低，表明叶酸靶向可视化光热-化疗治疗剂空白载体在被激光照射后产生光热效应，对细胞产生杀伤作用。对于模型药物原料药多西他赛，其细胞活力不随激光照射时间的延长而降低，表明激光并不能增强原料药对细胞的杀伤作用。而对于叶酸靶向可视化光热-化疗治疗剂+激光组，没有激光照射时，其就显示出明显的细胞杀伤作用，而当激光照射后，其细胞存活率明显降低，且随着照射时间的增加其细胞存活率逐渐降低，其细胞存活率明显低于叶酸靶向可视化光热-化疗治疗剂空白载体+激光和原料药的叠加作用，显示出光热治疗和化疗的协同作用。
4.叶酸靶向可视化光热-化疗治疗剂体内超声造影和光声显影研究
构建BALB/c小鼠异位4T1乳腺癌动物模型，将荷瘤成功的BALB/c小鼠的肿瘤部位脱毛，采集肿瘤部位在注射不同制剂前的超声成像图，然后通过尾静脉将生理盐水以及叶酸靶向可视化光热-化疗治疗剂注射进入小鼠体内，用激光仪照射肿瘤部位(功率为1W/cm)，时间分别为0和5min。结果如图8，在尾静脉注射制剂前，肿瘤部位没有超声成像信号，在注射叶酸靶向可视化光热-化疗治疗剂后,BALB/c小鼠肿瘤部位能检测到超声成像信号，在用激光照射5min后，肿瘤部位超声信号明显增强，而生理盐水组的BALB/c小鼠肿瘤部位在任何时刻都没有检测到明显的超声成像信号。此结果表明，叶酸靶向可视化光热-化疗治疗剂具有体内超声成像效果。对于考察体内光声显影，将生理盐水以及叶酸靶向可视化光热-化疗治疗剂尾静脉注射进BALB/c小鼠体内，24h后，用光声成像仪探测肿瘤部位的光声成像信号，在尾静脉注射生理盐水24h后，于肿瘤部位不能检测到光声信号，而在注射叶酸靶向可视化光热-化疗治疗剂24h后，于肿瘤部位检测到光声信号。此结果表明，叶酸靶向可视化光热-化疗治疗剂具有体内光声成像效果。
5.叶酸靶向可视化光热-化疗治疗剂对肿瘤抑制实验
将荷瘤成功的BALB/c小鼠分成8组，每组5只。分别为(1)生理盐水组，(2)生理盐水+激光组，(3)原料药组，(4)聚吡咯包裹载药磷脂液态氟碳纳米粒，(5)叶酸靶向可视化光热-化疗治疗剂空白载体，(6)叶酸靶向可视化光热-化疗治疗剂空白载体+激光，(7)叶酸靶向可视化光热-化疗治疗剂，(8)叶酸靶向可视化光热-化疗治疗剂+激光。每一组通过尾静脉注射给予不同的制剂，注射体积为0.2mL，给药剂量为10mg/kg(DTX的量)，每两天给一次药。对于需要激光照射的分组，在给予不同制剂后24h，于肿瘤部位给予激光照射，照射时间为5min，功率为1w/cm2，治疗时间为10天。每天测量肿瘤大小，计算体积，结果如图9。生理盐水+激光，叶酸靶向可视化光热-化疗治疗剂空白载体与对照组生理盐水组并没有显著性差异，表明单独的激光和叶酸靶向可视化光热-化疗治疗剂空白载体并不能抑制肿瘤的生长。实验组叶酸靶向可视化光热-化疗治疗剂组的肿瘤生长速度明显比聚吡咯包裹载药磷脂液态氟碳纳米粒缓慢，表明叶酸靶向可视化光热-化疗治疗剂具有良好的肿瘤靶向性，在叶酸受体介导的主动靶向作用机制下，叶酸靶向可视化光热-化疗治疗剂更容易达到和聚集于肿瘤部位，从而增加肿瘤治疗效果。叶酸靶向可视化光热-化疗治疗剂空白载体+激光也具有明显的肿瘤治疗效果，证明了叶酸靶向可视化光热-化疗治疗剂空白载体具有良好的光热治疗效果，能够有效地抑制肿瘤生长。而对于叶酸靶向可视化光热-化疗治疗剂+激光组，其肿瘤体积在治疗以后不仅没有增长，反而有所减小，该实验结果表明，在激光照射下的光热治疗和化疗的联合作用显示出更加优越的治疗效果。叶酸靶向可视化光热-化疗治疗剂在激光照射下使肿瘤部位的温度，该温度不仅能够使肿瘤损伤，而且能够增加肿瘤对药物的敏感性，两者协同发挥卓越的抗肿瘤效果。
Claims (2)
1.一种叶酸靶向可视化光热-化疗治疗剂的制备方法，其特征在于包括以下步骤：
(1)精密称量30mg大豆磷脂和2mg药物，用二氯甲烷溶解，转移至圆底烧瓶中，旋转蒸发除去二氯甲烷成膜，向圆底烧瓶中加入2mL去离子水，水浴超声5min，制备成初乳，将初乳转移至离心管中，探头超声5min，制备成脂质体纳米混悬液，将脂质体纳米混悬液于4℃冰箱冷却，加入30μL全氟己烷，冰浴探头超声5min，制备得到载多西他赛磷脂液态氟碳纳米乳；
(2)将载多西他赛磷脂液态氟碳纳米乳转移至西林瓶中，加入1mL浓度为100mg/mL的三氯化铁溶液和1mL浓度为100mg/mL聚乙烯吡咯烷酮溶液，于冰浴中搅拌，然后加入吡咯和吡咯-1-丙酸，吡咯和吡咯-1-丙酸的摩尔比为7:3，将整个反应体系于冰浴且避光条件下搅拌反应3h，得到外观为黑色的纳米混悬液，将此混悬液离心，弃去上清液，沉淀用去离子水洗涤三次，将沉淀重悬于去离子水中，得到表面羧基化的聚吡咯包裹载多西他赛磷脂液态氟碳纳米粒混悬液；
(3)精密称0.5g叶酸，0.47g二环己基碳二亚胺，0.26g N-N-羟基琥珀酰亚胺和0.25mL三乙胺溶于10mL无水二甲基亚砜，于25℃条件下避光搅拌反应24h，过滤，用无水乙醚洗涤，干燥，得到叶酸活性酯，精密称量8mg叶酸活性酯和112.5mg聚氧乙烯二胺，并溶于5mL无水二甲基亚砜，加入4μL三乙胺，于25℃条件下避光搅拌反应24h，加入无水乙醚萃取，将固体沉淀过滤，真空干燥，得到淡黄色的H2N-PEG-FA；
(4)向步骤(2)制备得到的表面羧基化的聚吡咯包裹载多西他赛磷脂液态氟碳纳米粒混悬液中分别加入150μL，浓度为150mg/mL的N-N-羟基琥珀酰亚胺和1-乙基-(3-二甲基氨基丙基)碳二亚胺溶液，于室温搅拌，精密称量8mg步骤(3)制备的H2N-PEG-FA，加入到反应体系中，室温搅拌反应6h，离心，弃去上清液，沉淀用去离子水洗涤三次，得到叶酸靶向可视化光热-化疗治疗剂。
2.一种由权利要求1所述方法得到的叶酸靶向可视化光热-化疗治疗剂。
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