近红外光响应的光热敏感型脂质体载药系统在肿瘤靶向治疗和缓控药物释放中的应用

韩智豪，吕丽伟，顾月清

（1.中国药科大学 生物医学工程教研室，江苏 南京 210009）

近红外光响应的光热敏感型脂质体载药系统在肿瘤靶向治疗和缓控药物释放中的应用

韩智豪，吕丽伟，顾月清1

（1.中国药科大学 生物医学工程教研室，江苏 南京 210009）

药物递送系统如能通过光热触控将包裹的药物释放，同时进行癌症成像和治疗，这将是一个新兴的探索焦点。我们合成了一种新型多功能的纳米脂质体药物递送系统，即所述的光热敏感脂质体（PTSL），它的水溶性层掺杂了阿霉素作为治疗剂，同时它的疏水层包裹了cypate作为诊断剂。我们测试了PTSL的物理性质，稳定性以及脂质体的装载性能，合成的纳米复合材料具有针对性，稳定性和精准性，具有载药量9±1.5%和包封率82.7±2.1%。其Tm值落在43.1℃，提高脂质体的Tm温度达到热敏脂质体的热敏温度，脂质体崩解同时包裹的化疗药物释放。这表明该PTSL可用于肿瘤的诊断和靶向治疗。

Cypate；光热敏感脂质体；EPR效应触发释放；光热转换；光化学内化；光热治疗

1 简介

截至到目前，临床上的化疗药物总是低水溶性，缺乏稳定性，代谢快速和非选择性的分布，这可能将导致高器官毒性和剂量限制性的副作用[1,2]。纳米脂质体递送系统正引起研究者的关注，它作为一种替代品可以克服现有的抗癌药的局限性[3-6]。 我们设计并合成了一种新型多官能的纳米脂质体药物递送系统，即所述的光热敏感脂质体（PTSL）。多柔比星（DOX）被包裹在亲水层和cypate被掺杂在热敏感脂质体（TSL）的疏水层，以得到DOX@PTSL复合物。这里的cypate可实现多种功能[7-13]，包括近红外成像，光热转化和诱导产生单线态氧。PTSL，粒子直径为100-200nm，一旦静脉注射到体内，则通过EPR效应聚集在肿瘤部位。当暴露在780nm近红外光下[14]，cypate可以用于辅助成像，将激光能量转化为热能，并产生单线态氧。综合以上各种因素，DOX将被释放到细胞质中，然后进入细胞核[15]，发挥它们的药理作用。

2 实验方法

图1 （A）-（C）的流体动力学直径和PTSL，DOX @ PTSL和DOX @ PTSL纳米粒子的TEM照片（D）的荧光光谱（Ex=780 nm）和cypate和PTSL的紫外吸收光谱。内部：cypate和PTSL的近红外图像780纳米激光辐射下（E）DOX的紫外吸收光谱，CYP以及DOX @ PTSL和DOX @ PTSL作了妥善处理1%TX100Fig. 1. (A)-(C) The hydrodynamic diameter and TEM images of PTSL, DOX@PTSL and DOX@PTSL nanoparticles. (D) The fluorescence spectrum (Ex=780 nm) and UV absorbance spectrum of cypate and PTSL. Insert: the near infrared image of cypate and PTSL under 780 nm laser irradiation. (E) The UV absorbance spectrum of DOX, CYP as well as DOX@PTSL and DOX@PTSL dealed with 1% TX100.

图2 （A）游离DOX和DOX @ PTSL的CLSM图片与U87细胞系的定位性能研究培养后DOX @PTSL与U87细胞系加780近红外激光照射培养，DOX @ PTSL的动力学孵育后U87细胞系和近红外激光（1.5W）5分钟，在不同的孵育时间点，20分钟（B）和40分钟（C）照射两个不同信道的荧光强度的半定量的结果,红色为阿霉素信号，绿色为溶酶体信号（D）在U87脑胶质瘤细胞中的细胞毒性试验Fig. 2. (A) The CLSM pictures of free DOX and DOX@PTSL after incubated with U87 cell lines for the locating properties study and that of DOX@PTSL incubated with U87 cell lines plus 780 NIR laser irradiation. Dynamics of DOX@PTSL after incubated with U87 cell lines and irradiated with NIR laser (1.5W) for 5 minutes, at different incubation time points, the semi-quantitative results 20 minutes (B) and 40 minutes (C)for flurescence intensity of two different channels. In vitro cytotoxicity studies of five kinds of treatments performed on U87 cell lines(D) by MTT assays.

图3 （A）用激光照射或不不同的药剂注射后荷瘤小鼠的红外图像（B）肿瘤解剖图片，15天以来的治疗情况（C）用激光照射或不不同的药剂注射后荷瘤小鼠的肿瘤组织切片的显微HE染色照片Fig.3. (A) The infrared images of tumor-bearing mice after injected with different agents with laser irradiated or not. (B) the dissected tumor pictures, 15 days since treatment. (C) the microscopic pictures of tumor histological section（HE）

首先，我们用薄膜水化和浴超声法合成了DSPEPEG2000的不同比例的TSL，然后将脂质体4℃储存三周，研究其稳定性和分散性。为了研究药物装载能力和稳定性，通过在4℃进行三周和在37℃进行60分钟HBS透析。当确认DSPE-PEG2000的最佳比例，DOX和cypate分别掺入亲水和疏水层以得到DOX@ PTSL。DOX@ PTSL的基本物理性质进行了研究，包括粒径，TEM电镜成像，荧光光谱，Tm值，载药量，体内红外热成像和包封率。在细胞实验中，我们研究了细胞毒性，细胞定位和细胞内cypate介导的溶酶体逃逸。（图2）生物分布，光热成像，治疗效果和功能脂质体的组织病理学研究是在荷瘤小鼠中进行。

3 结果与讨论

首先，5%的DSPE-PEG2000被确认为合成的最佳比例，因其良好的稳定性以及高包裹效率。所有的表征结果显示DOX@ PTSL是结构完整性，水和粒径约160纳米（图1），并且能够产生较好的EPR肿瘤富集效果。通过体外温度依赖的释放研究确认Tm值为43.1℃。另一方面，无论是体外光热转化研究和体内光热成像，都表明掺入DOX@ PTSL的cypate可以在1.5W近红外激光照射5分钟下到达TSL的Tm温度。在体内动力学及生物分布研究表明，注入体内后1.5小时后，脂质体开始在肿瘤部位积聚并呈现明显的EPR效应。细胞研究表明DOX@ PTSL进行激光光照，在U87细胞系呈现出良好的治疗效果。40μmol/ L的DOX@ PTSL可以达到60%致死率。高肿瘤抑制活性在荷瘤小鼠上也得以证实。（图3）

4 结论

总之，本文建立了多功能光热敏感脂质体载药系统。包裹了阿霉素的纳米复合材料具有各种良好的物理化学性质，稳定性和生物活性。总而言之，DOX@PTSL这里展现了吸引人的功能，在癌症治疗中表现了较广阔的前景。

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NIR-responsive Thermosensitive Nanoliposomes for Targeted, Prolonged and Controlled-release of Drugs

Han Zhihao, Lv Liwei, Gu Yueqing1
(1.School of Engineering, China Pharmaceutical University, Nanjing, Jiangsu 210009, China)

It is an emerging focus to explore a drug delivery system displayed a photothermal release of entrapped drug for simultaneous cancer imaging and therapy. We synthesized a novel multifunctional nano liposome drug delivery system, the photothermal sensitive liposome(PTSL), with doxorubicin wrapped in the hydropholic layer as the therapeutical agents and cypate dopped in the hydrophobic layer as the diagnostic agents. The physical properties, stabilities and loading properties of the liposomes were tested, the nano composites we synthesized were targeted, stable and delicate, with a drug loading (DL) of 9±1.5% and encapsulation efficiency (EE) of 82.7±2.1%. The Tm value falls on 43.1℃, and it can raise the temperature of liposome to the Tm of thermal sensitive liposome, the liposome cracks and the wrapped chemotherapy drugs release. It indicates that the PTSL is a kind of promising drug delivery system for tumor diagnosis and targeting therapy.

Cypate; photothermal sensitive liposome; ERP effect; Triggered release; Photothermal conversion

R730.5

A

10. 11967/ 2017150111

R730.5

ADOI:10. 11967/ 2017150111

973国家重点基础研究发展计划(2015CB755504)，国家自然科学基金项目资助（81220108012）。

顾月清（1963—），女，江苏靖江，博士生导师, 研究方向为纳米靶向药物，邮箱：guyueqingsubmission@hotmail.com.