中华皮肤科杂志 ›› 2013, Vol. 46 ›› Issue (10): 702-706.

• 论著 • 上一篇    下一篇

聚乙交酯丙交酯纳米粒装载氨基酮戊酸光动力疗法对A431细胞的杀伤效应

石磊1,王秀丽1,涂庆峰赵锋3,栾瀚森4,王浩4,王宏伟1   

  • 收稿日期:2012-10-07 修回日期:2013-03-20 出版日期:2013-10-15 发布日期:2013-10-01
  • 通讯作者: 王宏伟 E-mail:hongweiwang@yahoo.cn
  • 基金资助:
    上海市自然基金项目;上海市级医院适宜技术联合开发推广应用项目;上海市卫生系统新百人计划培养

Killing effect of aminolevulinic acid?鄄loaded poly lactic-co-glycolic acid nanoparticle-based photodynamic therapy on a human skin squamous cell carcinoma cell line A431

  • Received:2012-10-07 Revised:2013-03-20 Online:2013-10-15 Published:2013-10-01

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摘要： 【摘要】 目的 制备装载氨基酮戊酸（ALA）的聚乙交酯丙交酯纳米粒（PLGA NP），增强ALA光动力体外杀伤人皮肤鳞癌A431细胞的效应。方法 改良复乳溶剂挥发法制备ALA PLGA NP，并对其粒径、包封率、载药量和形态进行表征。用透射电镜观察体外培养的A431细胞吸收ALA PLGA NP后的形态；用多功能酶标仪测定24 h内0.1 mmol/L ALA组、1 mmol/L ALA组、ALA PLGA NP组（含0.1 mmol/L ALA）、PLGA NP组生成的原卟啉IX（PpIX）荧光强度变化以确定最佳孵育时间。将培养A431细胞分为对照组、0.1 mmol/L ALA避光组、1 mmol/L ALA避光组、ALA PLGA NP避光组、PLGA NP避光组、单纯照光组、0.1 mmol/L ALA PDT组、1 mmol/L ALA PDT组、ALA PLGA NP PDT组、PLGA NP PDT组，对照组及各避光组严格避光，PDT组及单纯照光组用He-Ne激光照射，用噻唑蓝法测定其细胞杀伤效应。将A431细胞分为对照组、ALA PDT组、ALA PLGA NP PDT组，对照组避光，PDT组采用He-Ne激光照射，12、24 h后用流式细胞仪分析细胞凋亡情况。 结果 ALA PLGA NP呈球形，平均粒径为（65.6 ± 26.0） nm，包封率为（65.8 ± 7.2）%，载药量为（0.62 ± 0.27）%。ALA PLGA NP能被A431细胞吸收并聚集于细胞质中。PpIX荧光动力学检测显示24 h内ALA组、ALA PLGA NP组荧光强度随时间增加而上升。当孵育6 h、24 h时，ALA PLGA NP组生成的PpIX荧光强度均高于0.1 mmol/L ALA（均P < 0.01）。ALA PLGA NP PDT组对A431细胞的杀伤效应也明显强于0.1 mmol/L ALA PDT（6 h：t = 35.685，P < 0.01；24 h：t = 5.262，P < 0.01）。ALA PLGA NP PDT组细胞的凋亡率也高于ALA PDT组（12 h：t = 9.074，P < 0.01；24 h：t = 9.095，P < 0.01）。 结论 ALA PLGA NP能提高PpIX生成量，增强ALA PDT对A431细胞的体外杀伤效应以及ALA PDT诱导肿瘤细胞凋亡的效应。 【关键词】 肿瘤，鳞状细胞； 光化学疗法； 氨基酮戊酸； 纳米粒子

关键词: 肿瘤，鳞状细胞, 光化学治疗, 氨基酮戊酸, 纳米粒子

Abstract: SHI Lei *, WANG Xiu-li, TU Qing-feng, ZHAO Feng, LUAN Han-sen, WANG Hao, WANG Hong-wei. *Shanghai Skin Disease Hospital, Shanghai 200050, China Corresponding author: WANG Hong-wei, Email: hongweiwang2005@aliyun.com 【Abstract】 Objective To increase the killing effect of aminolevulinic acid （ALA）-based photodynamic therapy （PDT） on a human skin squamous cell carcinoma cell line A431 by poly lactic-co-glycolic acid nanoparticles （PLGA NPs）. Methods ALA-loaded PLGA NPs （ALA PLGA NPs） were prepared using a modified double emulsion solvent evaporation method, and characterized in terms of size, encapsulation efficiency, loading capacity and morphology. Transmission electron microscopy was carried out to observe the morphology of A431 cells after uptake of ALA PLGA NPs. To optimize incubation time, multi-mode microplate reader was used to describe the fluorescence kinetics of protoporphyrin IX generated by A431 cells during 24 hours of incubation with 0.1 mmol/L ALA, 1 mmol/L ALA, 2.7 g/L ALA PLGA NPs containing about 0.1 mmol/L ALA, and PLGA NPs without ALA separately. Some A431 cells were divided into 10 groups: control group receiving neither treatment nor irradiation, 0.1 and 1 mmol/L ALA dark/PDT group incubated 0.1 and 1 mmol/L ALA respectively, ALA PLGA NP dark/PDT group incubated with ALA PLGA NPs of 2.7 g/L, PLGA NP dark/PDT group incubated with PLGA NPs of 2.7 g/L, simple irradiation group irradiated with a He-Ne laser （wavelength: 635 nm, power density: 8.6 mW/cm2, energy density: 8 J/cm2） only. The dark groups were kept in darkness strictly by wrapping in aluminum foil, and PDT groups were irradiated using a He-Ne laser. After another 24 hours of culture following irradiation, methyl thiazolyl tetrazolium （MTT） assay was conducted to estimate the survival rate of cells. To study the effect of ALA PLGA NPs on cell apoptosis, some A431 cells were divided into three groups: control group receiving neither treatment nor irradiation, ALA-PDT group and ALA PLGA NP PDT group receiving PDT after incubation with ALA and ALA PLGA NPs respectively. Flow cytometry was performed to detect the apoptosis of A431 cells at 12 and 24 hours, separately, after the photodynamic therapy. Data were statistically analyzed using SPSS 13.0 software package by means of a t test. Results The prepared ALA PLGA NPs were spherical with a mean particle size of （65.6 ± 26） nm, encapsulation efficiency of （65.8 ± 7.2） %, and drug loading capacity of （0.62 ± 0.27）%. ALA PLGA NPs could be uptaken by A431 cells and gathered in the cytoplasm. The PpIX fluorescence kinetic study showed that the fluorescence intensity increased with time within 24 hours in A431 cells incubated with ALA or ALA PLGA NPs. After 6 and 24 hours of incubation, the A431 cells incubated with 2.7 g/L ALA PLGA NPs showed a significant increase in the protoporphyrin IX fluorescence intensity compared with those incubated with 0.1 mmol/L ALA （both P < 0.01）. Further more, the survival rate of A431 cells was statistically lower in the ALA PLGA NP PDT group than in the 0.1 mmol/L ALA PDT group at 6 and 24 hours （t = 35.685, 5.262, respectively, both P < 0.01）. Elevated apoptosis rate was observed in the ALA PLGA NP PDT group compared with the ALA PDT group at 12 （（13.10 ± 0.50）% vs. （4.90 ± 0.13）%, t = 9.074, P < 0.01） and 24 （（30.17 ± 1.02）% vs. （11.6 ± 0.59）%, t = 9.095, P < 0.01） hours. Conclusions ALA PLGA NPs can promote the formation of protoporphyrin IX, strengthen the killing effect of ALA-PDT on A431 cells in vitro, and enhance the apoptosis induced by ALA-PDT in tumor cells. 【Key words】 Neoplasms, squamous cell; Photochemotherapy; Aminolevulinic acid; Nanoparticles

Key words: aminolevulinic acid, Nanoparticles