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

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

目的制备装载氨基酮戊酸（ALA）的聚乙交酯丙交酯纳米粒（PLGANP），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24h内0．1mmol／LALA组、1mmol／LALA组、ALAPLGANP组（含0．1mmol／LALA）、PLGANP组生成的原卟啉IX（PplX）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、0．1mm01／LALA避光组、1mmol／LALA避光组、ALAPLGANP避光组、PLGANP避光组、单纯照光组、0．1mmol／LALAPDT组、1mmol／LALAPDT组、ALAPLGANPPDT组、PLGANPPDT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、ALAPDT组、ALAPLGA NP PDT组，对照组避光，PDT组采用He—Ne激光照射，12、24h后用流式细胞仪分析细胞凋亡情况。结果AIJA 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）％。AIA 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24h内ALA组、ALAPLGANP组荧光强度随时间增加而上升。当孵育6h、24h时，ALAPLGANP组生成的PplX荧光强度均高于0．Immol／LALA（均P〈0．01）。ALA PLGA NP PDT组对A431细胞的杀伤效应也明显强于0．1mmol／LALAPDT（6h：t=35．685，P〈0．01；24h：t=5．262，P〈0．01）。ALAPLGA NP PDT组细胞的凋亡率也高于ALAPDT组（12h：t=9．074，P〈0．01；24h：t=9．095，P〈0．01）。结论ALA PLGA NP能提高PpIX生成量，增强ALAPDT对A43l细胞的体外杀伤效应以及ALAPDT诱导肿瘤细胞凋亡的效应。

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 laetie-co-glycolie 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 （MTF） assay was conducted to estimate the survival rate of cells. To study the effect of ALA PLGA NPs on cell apuptosis, snme A431 cells were divided into three groups： control group receiving neither treatment nor irradiation, ALA-PI）T group and ALA PLGA NP PDT group receiving PDT after ineubation with ALA and AIA PI,GA NPs respectively. Flow cytometry was performed to detect the apoptosis of A431 cells at 12 and 24 hours, separately, after the photodynamie therapy. Data were statistieally analyzed using SPSS 13.0 soliware package by means of a t tesh 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 dntg loading capacity of （0.62 ±0.27 ）%. ALA PLGA NPs could be uptaken by A431 cells and gathered in the cytoplasm. The PplX 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 inculmtion, the A431 cells ineuhated with 2.7 g/L ALA PLGA NPs showed a significant increase in the protoporphyrin IX fluorescence intensity compared with those ineuhated 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 PI）T gruup than in the 0.1 rnmoH, ALA PDT group at 6 and 24 hours （t = 35.685, 5.262, respectively, both P 〈 0.01 ）. Elevated apnptnsis 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 tbrnmtion of protopotT, hyrin IX, strengthen the killing effect of ALA-PDT on A431 cells in vitro, and enhance the apoptosis induced by ALA-PDT in tumor cells.