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光动力治疗(PDT)基于光辐照聚集光敏剂的肿瘤组织,由光敏剂诱发光动力反应形成单线态氧(1O2)等活性氧(ROS),通过对肿瘤细胞和肿瘤血管的直接杀伤及激活机体系统免疫反应等多种机制发挥抗肿瘤作用[1-3]。二氢卟吩及菌绿素类光敏剂是PDT新药研究的热点[4-8]。其中,已获批上市的代表药物有他拉泊芬(talaporfin)和帕利泊芬(padeliporfin)等[9, 10]。
光敏剂作为结构非特异性药物,存在缺乏肿瘤靶向性摄入和明确的作用药靶等缺陷。此外,PDT受制于局部治疗,对浸润较深的肿瘤组织,及已发生转移的肿瘤疗效有限。目前,PDT和化疗联用是克服上述缺陷,提高PDT疗效最为普遍和有效的策略之一。研究表明,抗代谢化疗药物氟尿嘧啶(5-Fu)与PDT联用具有协同抗肿瘤作用[11-13]。据此,我们设想利用在肿瘤微环境下能响应性断裂的连接基团(linker)将光敏剂与化疗药物偶联,希望实现二者在肿瘤组织的靶向释放,从而发挥其PDT和化疗协同抗肿瘤作用。酰腙键是酸敏感化学键,常被用来连接载体,以药物制备智能药物载体。这种药物载体到达肿瘤细胞的内涵体或溶酶体中时,会发生酸性水解将药物有效释放出来。因此,本文针对肿瘤微环境呈弱酸性的特点,采用药物化学最经典的前药设计策略,以脱镁叶绿素a(Phephorbide a)粗提物经酸碱降解制得的二氢卟吩e6(3)[14]为先导光敏剂,通过其152-羧基与抗肿瘤药物5-Fu以酸敏感酰腙键连接,设计合成pH响应型光化疗协同抗肿瘤光敏剂二氢卟吩e6-偕氟尿嘧啶(1),并考察其体外PDT抗肿瘤活性和pH响应性5-Fu释放,及其对黑色素瘤B16-F10和肝癌HepG2细胞的光动力抗癌活性及其作用机制,以期获得高效、低毒的PDT治癌药物候选药物,合成路线见图1。
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分别测定目标化合物1及其先导化合物二氢卟吩e6(3)的甲醇溶液(10 μmol/L)在300~800 nm处的紫外吸收谱和激发波长为400 nm的荧光发射光谱,结果见图2。
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分别配制浓度为50 μmol/L的化合物1的HOAc-NaOAc缓冲液(pH 5.0)和PBS溶液(10 ml),并于0.5、1.0、3.0、6.0、12、24 h时分别取样(500 μl)。其中,HOAc-NaOAc缓冲液(pH 5.0)组取样液用0.1 mol/L氢氧化钠水溶液迅速调节pH值至7.4。每份取样液加PBS稀释至原溶液1/3浓度,微孔滤膜(孔径0.22 μm)过滤,HPLC进样检测;实验重复3次。根据5-Fu的HPLC峰面积-浓度标准曲线分析计算,绘制目标化合物1于弱酸(pH 5.0)中的5-Fu体外释放量-时间曲线,结果见图3。
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参照文献[6-8]的方法,每孔5×103个B16-F10细胞或HepG2细胞悬液(100 μl)接种于96孔板上,加入等体积上述细胞培养液孵育24 h;更换含不同浓度待测物的培养液(DMSO浓度小于1%,100 μl),继续避光孵育48 h;再更换含10%(V/V)CCK-8(Beyotime,中国)的RPMI 1640基础培养基(100 μl),继续培养1.5 h,然后用Varioskan Flash全波长酶标仪(Thermo)于波长450 nm处测定每孔的吸光度值,计算各浓度对应的细胞存活率,并拟合得到待测物的肿瘤细胞半数抑制浓度即IC50值。
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每孔5×103个B16-F10细胞或HepG2细胞悬液(100 μl)接种于96孔板上,加入等体积细胞培养液孵育24 h;更换含不同浓度待测物的细胞培养液(DMSO浓度小于1%,100 μl),继续避光孵育24 h;再更换新鲜培养液(100 μl),以波长为660 nm的激光辐照受试细胞样品(光照剂量为10 J/cm2),继续孵育24 h。最后按“2.3.2”项下CCK-8法测定各待测物的肿瘤细胞IC50值。
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以临床光敏药物他拉泊芬为阳性对照,化合物1及其先导化合物3对肿瘤细胞株的体外PDT抗癌活性结果见表1。
表 1 目标化合物1的体外光动力抗癌活性(IC50,μmol/L)
化合物 B16-F10细胞 暗毒/光毒比 HepG2细胞 暗毒/光毒比 暗毒性 光毒性 暗毒性 光毒性 化合物 1 46.84±8.46*, ΔΔΔ 0.73±0.16**, ΔΔΔ 64.2 50.80±6.45**, #, ΔΔΔ 0.90±0.22**, ΔΔΔ 56.4 二氢卟吩e6 69.72±4.69 3.36±0.59 20.8 70.38±10.9 2.75±0.41 25.6 他拉泊芬 254.8±18.8 11.31±3.88 22.5 176.4±28.4 15.47±5.07 11.4 5-Fu 35.80±6.68 NTa − 39.16±2.7 NTa − NTa:未测定;*P < 0.05,**P < 0.01,与二氢卟吩 e6组比较;#P < 0.05,与5-Fu组比较;ΔΔΔP < 0.001,与他拉泊芬组比较。 -
操作步骤如下:a. 每孔3 × 105个B16-F10细胞悬液(2 ml)接种6孔板上,按“2.3.1”项条件避光孵育24 h;b. 分别更换含一定浓度化合物1或他拉泊芬的新鲜培养液(DMSO浓度小于1%,2 ml),继续避光孵育24 h;c. 加入10 mmol/L DCFH-DAROS荧光检测探针(Beyotime,1.5 μl),吹打混匀,继续避光孵育20 min;d. PBS洗涤3次,再加新鲜培养液(2 ml),以660 nm波长的激光辐照(光剂量10 J/cm2)细胞样品,继续避光孵育20 min;e. 收集每孔细胞样品,用流式细胞仪检测各孔细胞ROS水平,结果见图4。
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按“2.4”项下操作方法,仅从步骤c开始,更换新鲜培养液(2 ml),用660 nm波长的激光辐照(光剂量10 J/cm2)细胞样品,继续避光孵育20 min;d. 以1 500 r/min离心(5 min)细胞样品,PBS洗涤,再以1 000 r/min离心(5 min)后获取细胞样品;e. 按Annexin V-FITC细胞凋亡检测试剂盒(Beyotime)操作流程操作,结果见图5。
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按“2.5”项下操作方法,仅在e步骤中,换以细胞周期阻滞检测试剂盒(Beyotime)的操作流程,每份细胞样品中分别加入染色缓冲液(300 µl)、RNase A(6 µl)和碘化丙啶染色液(15 µl),轻轻混匀,避光孵育20 min后,用流式细胞仪进行细胞周期阻滞检测,结果见图6。
Synthesis and biological activities of chlorin e6-based conjugate of fluorouracil as dual-mode antitumor photosensitizer
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摘要:
目的 文献报道氟尿嘧啶(5-Fu)与光敏剂联用具有协同抗肿瘤作用,笔者设计合成二氢卟吩e6(化合物 3 )与5-Fu经酰腙键偶联的pH响应性、光化疗双模抗肿瘤光敏剂(化合物 1 ),研究其初步体外光动力抗癌活性及作用机制。 方法 首先,5-Fu与五硫化二磷于吡啶中回流反应形成4-硫代-5-氟尿嘧啶,再和水合肼于甲醇中反应制得5-氟尿嘧啶-4-腙(化合物 2 );然后,将脱镁叶绿素a(化合物 4 )酸碱降解产物 3 经EDC·HCl催化缩合形成二氢卟吩e6-131,152-酸酐中间体后,直接与 2 发生选择性酰化反应,制得目标化合物 1 ,并考察其体外pH响应性5-Fu释放及对黑素瘤B16-F10和肝癌HepG2细胞的光动力抗癌活性和作用机制。 结果 化合物 1 在微酸(pH 5.0)环境中能有效释放5-Fu,24 h累积释放率可达60.3%;其在光照下对黑素瘤B16-F10和肝癌HepG2细胞的半数抑制浓度(IC50)分别为0.73 μmol/L和0.90 μmol/L,均显著优于先导物 3 和上市药物他拉泊芬(talaporfin),且能显著提升肿瘤细胞内活性氧(ROS)水平和诱导肿瘤细胞凋亡,并阻滞肿瘤细胞周期于S期。其结构经紫外、电喷雾质谱、氢谱和元素分析确证。 结论 新型双模抗肿瘤光敏剂化合物 1 具有光动力抗癌活性强、治疗指数(暗毒/光毒比)高,且可在微酸(pH 5.0)环境响应性释放5-Fu等优点,从而实现“单分子”光化疗双重抗肿瘤作用,值得进一步开发研究。 Abstract:Objective To design and synthesize the conjugate (compound 1 ) of chlorin e6 (compound 3 ) with fluorouracil (5-Fu) as novel pH-responsive dual-mode antitumor photosensitizer by acyl hydrazone bond coupling, based on literature reports that combination of 5-Fu and photosensitizer possess synergistic anti-tumor effect, and investigate its photodynamic antitumor activity and mechanism. Methods Lead compound 3 was obtained by alkali degradation with 25% KOH-CH3OH on pheophorbide a (compound 4 ) which was prepared through acid hydrolysis of chlorophyll a in crude chlorophyll extracts from silkworm excrement. Reflux reaction of 5-Fu with P2S5 in pyridine formed crude 4-thio-5-fluorouracil which was followed to react with hydrazine hydrate (N2H4·H2O) in CH3OH to give 5-fluorouracil-4-hydrazone (compound 2 ). Then, treatment of compound 3 i.e. acid alkali degradation product of chlorophyll a in silkworm excrement with EDC·HCl generated its 171- and 152 cyclic anhydride which was followed to directly react with intermediate compound 2 to successfully get title compound 1 . In addition, its pH-responsive 5-Fu release and photodynamic antitumor activity and their mechanisms in vitro were investigated. Results Compound 1 could responsively release 5-Fu at pH 5.0, with a cumulative release rate of 60.3% within 24 h. It exhibited much higher phototoxicity against melanoma B16-F10 and liver cancer HepG2 cells than talaporfin and its precursor compound 3 , with IC50 value being 0.73 μmol/L for B16-F10 cells and 0.90 μmol/L for HepG2 cells, respectively. Upon light irradiation, it also could significantly induce cell apoptosis and intracellular ROS level and block cell cycle in S phase. Its structure was confirmed by UV, 1H-NMR, ESI-MS and elemental analysis data. Conclusion The conjugate compound 1 of compound 3 and 5-Fu has the advantages of strong PDT anticancer activity, high therapeutic index (i.e. dark toxicity/phototoxicity ratio) and responsively release 5-Fu at pH 5.0 etc. which shows “unimolecular” dual antitumor effects of PDT and chemotherapy and is worthy of further research and development. -
Key words:
- synthesis /
- photodynamic therapy /
- photosensitizer /
- chlorin e6 /
- fluorouracil (5-Fu) /
- antitumor
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表 1 目标化合物1的体外光动力抗癌活性(IC50,μmol/L)
化合物 B16-F10细胞 暗毒/光毒比 HepG2细胞 暗毒/光毒比 暗毒性 光毒性 暗毒性 光毒性 化合物 1 46.84±8.46*, ΔΔΔ 0.73±0.16**, ΔΔΔ 64.2 50.80±6.45**, #, ΔΔΔ 0.90±0.22**, ΔΔΔ 56.4 二氢卟吩e6 69.72±4.69 3.36±0.59 20.8 70.38±10.9 2.75±0.41 25.6 他拉泊芬 254.8±18.8 11.31±3.88 22.5 176.4±28.4 15.47±5.07 11.4 5-Fu 35.80±6.68 NTa − 39.16±2.7 NTa − NTa:未测定;*P < 0.05,**P < 0.01,与二氢卟吩 e6组比较;#P < 0.05,与5-Fu组比较;ΔΔΔP < 0.001,与他拉泊芬组比较。 -
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