制备异甘草素壳聚糖纳米粒抑制人肺癌细胞A549的增殖
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摘要
背景:异甘草素是一种异黄酮化合物,有着很强的药理活性如抗癌、抗病毒、抗糖尿病等,但水溶性差,这极大地限制了其临床应用。目的:制备异甘草素壳聚糖纳米粒,探讨其在体外对人肺癌细胞A549生长的抑制作用。方法:以壳聚糖为载体材料,异甘草素为模型药物,三聚磷酸钠为离子交联剂,利用离子交联法制备异甘草素壳聚糖纳米粒,测定其粒径、分散度和Zeta电位,通过透射电镜观察纳米粒形态,通过离心测定其包封率和载药量。将异甘草素与甘草素壳聚糖纳米粒分别置于透析袋内,以PBS为释放介质,动态观察其体外释放。分别以含不同质量浓度(1,5,25 mg/L)异甘草素、甘草素壳聚糖纳米粒及壳聚糖纳米粒的培养液培养人肺癌细胞A549,培养48 h后,采用MTT法检测细胞增殖,计算细胞生长抑制率。结果与结论:(1)异甘草素壳聚糖纳米粒为球形或类球形,结构完整,大小较为均一,纳米粒平均粒径为(159±20)nm,Zeta电位为+17.2m V,分散度为0.243,包封率和载药量分别为(85.28±1.31)%和(13.28±0.53)%;(2)游离异甘草素在8 h内释放完毕;异甘草素壳聚糖纳米粒具有缓慢释放特性,72 h累积释放量达83.98%,体外释药过程符合一级动力学方程;(3)不同质量浓度的壳聚糖纳米粒对A549细胞生长无抑制作用;异甘草素、异甘草素壳聚糖纳米粒均呈浓度依赖性抑制A549细胞的生长,并且相同质量浓度下,异甘草素壳聚糖纳米粒对A549细胞生长的抑制作用强于异甘草素(P <0.05);(4)结果表明采用离子交联法制备的异甘草素壳聚糖纳米粒,具有良好的缓释性能,提高了异甘草素在体外对A549细胞增殖的抑制作用。
BACKGROUND: Isoliquiritigenin, one kind of isoflavone compounds, has a wide range of biological activities, such as anti-cancer, anti-virus,and anti-diabetes, but its clinical application is limited by its poor water solubility.OBJECTIVE: To prepare chitosan/isoliquiritigenin nanoparticles and investigate their inhibitory effects on human lung cancer cell line A549 in vitro.METHODS: The chitosan/isoliquiritigenin nanoparticles were prepared by ionic gelation method with chitosan as the carrier material,isoliquiritigenin as the model drug, and sodium tripolyphosphate as the ionic crosslinking agent. The nanoparticle size, dispersion, and Zeta potential were investigated by malvern laser particle size analyzer. The morphology of the nanoparticles was observed by transmission electron microscopy. The encapsulation efficiency and cumulative release rate of the nanoparticles were measured by centrifugation.Isoliquiritigenin and chitosan/isoliquiritigenin nanoparticles were placed in dialysis bags and their dynamic release was determined in PBS buffer. Human lung cancer cell line A549 was cultured in different concentrations(1, 5, 25 mg/L) of isoliquiritigenin, chitosan/isoliquiritigenin nanoparticles, and chitosan nanoparticles. MTT assay was used to investigate the inhibitory effects of chitosan/isoliquiritigenin nanoparticles on human lung cancer cell A549 in vitro after 48 hours of culture. The growth-inhibitory rate was calculated.RESULTS AND CONCLUSION: The chitosan/isoliquiritigenin nanoparticles were spherical or quasi spherical in shape with complete structure and of relatively uniform size. The average particle size, dispersion and Zeta potential of chitosan/isoliquiritigenin nanoparticles were(159±20) nm, 0.243 and +17.2 mV, respectively. The encapsulation efficiency and cumulative release rate of the nanoparticles were(85.28±1.31)% and(13.28±0.53)% respectively.The release of free isoliquiritigenin was completed within 8 hours, while the chitosan/isoliquiritigenin nanoparticles had sustained release property in vitro, with 72-hour cumulative release amount of 83.98%. This well fitted the first-order kinetic model. Chitosan nanoparticles at different concentrations had no inhibitory effects on the growth of A549 cells. Both isoliquiritigenin and chitosan/isoliquiritigenin nanoparticles inhibited the growth of A549 cells in a concentration-dependent manner, and the inhibitory effect of chitosan/isoliquiritigenin nanoparticles was stronger than that of isoliquiritigenin when used at the same concentration(P < 0.05). The results suggest that the chitosan/isoliquiritigenin nanoparticles prepared by ionic crosslinking method had encouraging sustained release performance and enhanced the inhibitory effects of isoliquiritigenin on the proliferation of A549 cells in vitro.
BACKGROUND: Isoliquiritigenin, one kind of isoflavone compounds, has a wide range of biological activities, such as anti-cancer, anti-virus,and anti-diabetes, but its clinical application is limited by its poor water solubility.OBJECTIVE: To prepare chitosan/isoliquiritigenin nanoparticles and investigate their inhibitory effects on human lung cancer cell line A549 in vitro.METHODS: The chitosan/isoliquiritigenin nanoparticles were prepared by ionic gelation method with chitosan as the carrier material,isoliquiritigenin as the model drug, and sodium tripolyphosphate as the ionic crosslinking agent. The nanoparticle size, dispersion, and Zeta potential were investigated by malvern laser particle size analyzer. The morphology of the nanoparticles was observed by transmission electron microscopy. The encapsulation efficiency and cumulative release rate of the nanoparticles were measured by centrifugation.Isoliquiritigenin and chitosan/isoliquiritigenin nanoparticles were placed in dialysis bags and their dynamic release was determined in PBS buffer. Human lung cancer cell line A549 was cultured in different concentrations(1, 5, 25 mg/L) of isoliquiritigenin, chitosan/isoliquiritigenin nanoparticles, and chitosan nanoparticles. MTT assay was used to investigate the inhibitory effects of chitosan/isoliquiritigenin nanoparticles on human lung cancer cell A549 in vitro after 48 hours of culture. The growth-inhibitory rate was calculated.RESULTS AND CONCLUSION: The chitosan/isoliquiritigenin nanoparticles were spherical or quasi spherical in shape with complete structure and of relatively uniform size. The average particle size, dispersion and Zeta potential of chitosan/isoliquiritigenin nanoparticles were(159±20) nm, 0.243 and +17.2 mV, respectively. The encapsulation efficiency and cumulative release rate of the nanoparticles were(85.28±1.31)% and(13.28±0.53)% respectively.The release of free isoliquiritigenin was completed within 8 hours, while the chitosan/isoliquiritigenin nanoparticles had sustained release property in vitro, with 72-hour cumulative release amount of 83.98%. This well fitted the first-order kinetic model. Chitosan nanoparticles at different concentrations had no inhibitory effects on the growth of A549 cells. Both isoliquiritigenin and chitosan/isoliquiritigenin nanoparticles inhibited the growth of A549 cells in a concentration-dependent manner, and the inhibitory effect of chitosan/isoliquiritigenin nanoparticles was stronger than that of isoliquiritigenin when used at the same concentration(P < 0.05). The results suggest that the chitosan/isoliquiritigenin nanoparticles prepared by ionic crosslinking method had encouraging sustained release performance and enhanced the inhibitory effects of isoliquiritigenin on the proliferation of A549 cells in vitro.
引文
[1]Peng F,Du Q,Peng C,et al.A Review:The Pharmacology of Isoliquiritigenin.Phytother Res.2015;29(7):969-977.
[2]Chen G,Zhu L,Liu Y,et al.Isoliquiritigenin,a flavonoid from licorice,plays a dual role in regulating gastrointestinal motility in vitro and in vivo.Phytother Res.2009;23(4):498-506.
[3]Wu TY,Khor TO,Saw CL,et al.Anti-inflammatory/Anti-oxidative stress activities and differential regulation of Nrf2-mediated genes by non-polar fractions of tea Chrysanthemum zawadskii and licorice Glycyrrhiza uralensis.AAPS J.2011;13(1):1-13.
[4]Jin XY,Sohn DH,Lee SH.Isoliquiritigenin suppresses tumor necrosis factor-α-induced inflammation via peroxisome proliferator-activated receptor-γin intestinal epithelial cells.Arch Pharm Res.2016;39(10):1465-1471.
[5]Traboulsi H,Cloutier A,Boyapelly K,et al.he Flavonoid Isoliquiritigenin Reduces Lung Inflammation and Mouse Morbidity during Influenza Virus Infection.Antimicrob Agents Chemother.2015;59(10):6317-6327.
[6]Cronin H,Draelos ZD.Top 10 botanical ingredients in 2010anti-aging creams.J Cosmet Dermatol.2010;9(3):218-225.
[7]Yerra VG,Kalvala AK,Kumar A.Isoliquiritigenin reduces oxidative damage and alleviates mitochondrial impairment by SIRT1 activation in experimental diabetic neuropathy.J Nutr Biochem.2017;47:41-52.
[8]覃月穆,姜泽群,马艳霞,等.异甘草素对人肺癌细胞株H460增殖、凋亡及NF-κB信号通路的影响[J].天然产物研究与开发,2018,30(5):847-855.
[9]邓云秋.异甘草素对人胶质瘤细胞系U251增殖、凋亡的影响及相关机制研究[J].医学理论与实践,2018,31(9):1260-1261,1270.
[10]Wang JR,Luo YH,Piao XJ,et al.Mechanisms underlying isoliquiritigenin-induced apoptosis and cell cycle arrest via ROS-mediated MAPK/STAT3/NF-κB pathways in human hepatocellular carcinoma cells.Drug Dev Res.2019.[Epub ahead of print]
[11]哈尼克孜·吐尔逊,古再丽努尔·麦麦提图尔荪,木合布力·阿布力孜,等.异甘草素诱导人宫颈癌细胞增殖的影响及作用机制的研究[J].重庆医学,2017,46(24):3324-3327,3331.
[12]Zhang XR,Wang SY,Sun W,et al.Isoliquiritigenin inhibits proliferation and metastasis of MKN28 gastric cancer cells by suppressing the PI3K/AKT/mTOR signaling pathway.Mol Med Rep.2018.[Epub ahead of print]
[13]Li N,Yang L,Deng X,et al.Effects of isoliquiritigenin on ovarian cancer cells.Onco Targets Ther.2018;11:1633-1642.
[14]Jin H,Seo GS,Lee SH.Isoliquiritigenin-mediated p62/SQSTM1 induction regulates apoptotic potential through attenuation of caspase-8 activation in colorectal cancer cells.Eur J Pharmacol.2018;841:90-97.
[15]Hu FW,Yu CC,Hsieh PL,et al.Targeting oral cancer stemness and chemoresistance by isoliquiritigenin-mediated GRP78regulation.Oncotarget.2017;8(55):93912-93923.
[16]Peng F,Tang H,Liu P,et al.Isoliquiritigenin modulates miR-374a/PTEN/Akt axis to suppress breast cancer tumorigenesis and metastasis.Sci Rep.2017;7(1):9022.
[17]Si L,Yang X,Yan X,et al.Isoliquiritigenin induces apoptosis of human bladder cancer T24 cells via a cyclin-dependent kinase-independent mechanism.Oncol Lett.2017;14(1):241-249.
[18]周锦霞,伍林,王建国.异甘草素微胶囊的制备及释放性的研究[J].现代食品科技,2010,26(10):1132-1135,1159.
[19]龚又明,覃军,邓广海,等.异甘草素羟丙基-β-环糊精包合物的透皮吸收研究[J].食品与药品,2017,19(4):291-294.
[20]车小琼,孙庆申,赵凯.甲壳素和壳聚糖作为天然生物高分子材料的研究进展[J].高分子通报,2008,21(2):45-49.
[21]马茜,范娟.壳聚糖纳米粒载体的应用研究进展[J].西北药学杂志,2015,30(2):213-215.
[22]吴立明,习温瑜,管正红.壳聚糖纳米粒制备的研究进展[J].齐鲁药事,2008,27(11):682-685.
[23]Fangueiro JF,Parra A,Silva AM,et al.Validation of a high performance liquid chromatography method for the stabilization of epigallocatechin gallate.Int J Pharm.2014;475(1/2):181-190;
[24]高奋,杨慧宇,边云飞,等.载血管紧张素转换酶短发卡RNA-聚乙二醇-壳聚糖纳米粒制备以及生物学特性[J].中国药物与临床,2015,15(1):29-33.
[25]Ahmed A,Zhang C,Guo J,et al.Fabrication and characterization of Gd-DTPA-Loaded Chitosan-Poly(acrylic acid)nanoparticles for magnetic resonance imaging.Macromol Biosci.2015;15(8):1105-1114.
[26]何媛,张雅溶,李秋霞,等.透明质酸修饰姜黄素壳聚糖纳米粒的制备及初步细胞毒性考察[J].中国医药工业杂志,2015,46(2):162-167.
[27]Tian XX,Groves MJ.Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccaein chitosan nanoparticles.J Pharm Pharmacol.1999;51(2):151-157.
[28]Nagamoto T,Hattori Y,Takayama K,et al.Novel chitosan particles and chitosan-coated emulsion inducing immune response via intranasal vaccine delivery.Pharm Res.2004;21(4):671.
[29]Bodmeier R,Chen H,Paeratakul O.A novel approach to the delivery of microparticles or nanoparticles.Pharm Res.1989;6(5):413-417.
[30]Calvo P,Remunan-Lopez C,Vila-Jato JL,et al.Novel hydrophilic chitosan/polyethylene oxide nanoparticles as proteins carriers.J Appl Polym Sci.1997;63:125-132.
[31]张亚会,李喜香,包强,等.甘草次酸-壳聚糖纳米粒的制备及其质量评价[J].中草药,2015,46(15):2232-2237.
[32]欧歌,廖德华,张凯,等.采用离子交联法制备壳聚糖胰岛素纳米粒的研究进展[J].中南药学,2013,11(2):116-119,156.
[33]奚宏伟,魏长征,王文斌,等.壳聚糖-三聚磷酸钠纳米粒对于肝组织严重出血模型的止血效果[J].现代生物医学进展,2014,14(15):2831-2834,2885.
[34]刘占军,张卫国,于九皋,等.负载紫杉醇壳聚糖纳米粒的制备、表征与释药性能[J].中国组织工程研究与临床康复,2009,13(3):493-495.
[35]刘康,秦梦,杨婷婷,等.槲皮素/壳聚糖纳米粒的制备、表征及其体外抗氧化活性研究[J].中国生化药物杂志,2016,36(11):17-21.
[36]任振堃,高闻,刘畅,等.载柚皮素壳聚糖纳米粒的制备及其体外细胞评价[J].中国医院药学杂志,2017,37(20):2056-2060.
[37]吴振宇,陈钟,黄华,等.离子凝胶法制备壳聚糖纳米微粒[J].南通大学学报(医学版),2005,26(1):20-22.
[38]陈瑞,赵群,邵江娟,等.雷公藤甲素壳聚糖纳米粒的制备与初步评价[J].辽宁中医杂志,2016,43(4):806-808,895.
[39]彭琳,李利锋,韩刚,等.载姜黄素羧甲基壳聚糖纳米粒的制备和性能[J].中国医药工业杂志,2016,47(2):178-182
[2]Chen G,Zhu L,Liu Y,et al.Isoliquiritigenin,a flavonoid from licorice,plays a dual role in regulating gastrointestinal motility in vitro and in vivo.Phytother Res.2009;23(4):498-506.
[3]Wu TY,Khor TO,Saw CL,et al.Anti-inflammatory/Anti-oxidative stress activities and differential regulation of Nrf2-mediated genes by non-polar fractions of tea Chrysanthemum zawadskii and licorice Glycyrrhiza uralensis.AAPS J.2011;13(1):1-13.
[4]Jin XY,Sohn DH,Lee SH.Isoliquiritigenin suppresses tumor necrosis factor-α-induced inflammation via peroxisome proliferator-activated receptor-γin intestinal epithelial cells.Arch Pharm Res.2016;39(10):1465-1471.
[5]Traboulsi H,Cloutier A,Boyapelly K,et al.he Flavonoid Isoliquiritigenin Reduces Lung Inflammation and Mouse Morbidity during Influenza Virus Infection.Antimicrob Agents Chemother.2015;59(10):6317-6327.
[6]Cronin H,Draelos ZD.Top 10 botanical ingredients in 2010anti-aging creams.J Cosmet Dermatol.2010;9(3):218-225.
[7]Yerra VG,Kalvala AK,Kumar A.Isoliquiritigenin reduces oxidative damage and alleviates mitochondrial impairment by SIRT1 activation in experimental diabetic neuropathy.J Nutr Biochem.2017;47:41-52.
[8]覃月穆,姜泽群,马艳霞,等.异甘草素对人肺癌细胞株H460增殖、凋亡及NF-κB信号通路的影响[J].天然产物研究与开发,2018,30(5):847-855.
[9]邓云秋.异甘草素对人胶质瘤细胞系U251增殖、凋亡的影响及相关机制研究[J].医学理论与实践,2018,31(9):1260-1261,1270.
[10]Wang JR,Luo YH,Piao XJ,et al.Mechanisms underlying isoliquiritigenin-induced apoptosis and cell cycle arrest via ROS-mediated MAPK/STAT3/NF-κB pathways in human hepatocellular carcinoma cells.Drug Dev Res.2019.[Epub ahead of print]
[11]哈尼克孜·吐尔逊,古再丽努尔·麦麦提图尔荪,木合布力·阿布力孜,等.异甘草素诱导人宫颈癌细胞增殖的影响及作用机制的研究[J].重庆医学,2017,46(24):3324-3327,3331.
[12]Zhang XR,Wang SY,Sun W,et al.Isoliquiritigenin inhibits proliferation and metastasis of MKN28 gastric cancer cells by suppressing the PI3K/AKT/mTOR signaling pathway.Mol Med Rep.2018.[Epub ahead of print]
[13]Li N,Yang L,Deng X,et al.Effects of isoliquiritigenin on ovarian cancer cells.Onco Targets Ther.2018;11:1633-1642.
[14]Jin H,Seo GS,Lee SH.Isoliquiritigenin-mediated p62/SQSTM1 induction regulates apoptotic potential through attenuation of caspase-8 activation in colorectal cancer cells.Eur J Pharmacol.2018;841:90-97.
[15]Hu FW,Yu CC,Hsieh PL,et al.Targeting oral cancer stemness and chemoresistance by isoliquiritigenin-mediated GRP78regulation.Oncotarget.2017;8(55):93912-93923.
[16]Peng F,Tang H,Liu P,et al.Isoliquiritigenin modulates miR-374a/PTEN/Akt axis to suppress breast cancer tumorigenesis and metastasis.Sci Rep.2017;7(1):9022.
[17]Si L,Yang X,Yan X,et al.Isoliquiritigenin induces apoptosis of human bladder cancer T24 cells via a cyclin-dependent kinase-independent mechanism.Oncol Lett.2017;14(1):241-249.
[18]周锦霞,伍林,王建国.异甘草素微胶囊的制备及释放性的研究[J].现代食品科技,2010,26(10):1132-1135,1159.
[19]龚又明,覃军,邓广海,等.异甘草素羟丙基-β-环糊精包合物的透皮吸收研究[J].食品与药品,2017,19(4):291-294.
[20]车小琼,孙庆申,赵凯.甲壳素和壳聚糖作为天然生物高分子材料的研究进展[J].高分子通报,2008,21(2):45-49.
[21]马茜,范娟.壳聚糖纳米粒载体的应用研究进展[J].西北药学杂志,2015,30(2):213-215.
[22]吴立明,习温瑜,管正红.壳聚糖纳米粒制备的研究进展[J].齐鲁药事,2008,27(11):682-685.
[23]Fangueiro JF,Parra A,Silva AM,et al.Validation of a high performance liquid chromatography method for the stabilization of epigallocatechin gallate.Int J Pharm.2014;475(1/2):181-190;
[24]高奋,杨慧宇,边云飞,等.载血管紧张素转换酶短发卡RNA-聚乙二醇-壳聚糖纳米粒制备以及生物学特性[J].中国药物与临床,2015,15(1):29-33.
[25]Ahmed A,Zhang C,Guo J,et al.Fabrication and characterization of Gd-DTPA-Loaded Chitosan-Poly(acrylic acid)nanoparticles for magnetic resonance imaging.Macromol Biosci.2015;15(8):1105-1114.
[26]何媛,张雅溶,李秋霞,等.透明质酸修饰姜黄素壳聚糖纳米粒的制备及初步细胞毒性考察[J].中国医药工业杂志,2015,46(2):162-167.
[27]Tian XX,Groves MJ.Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccaein chitosan nanoparticles.J Pharm Pharmacol.1999;51(2):151-157.
[28]Nagamoto T,Hattori Y,Takayama K,et al.Novel chitosan particles and chitosan-coated emulsion inducing immune response via intranasal vaccine delivery.Pharm Res.2004;21(4):671.
[29]Bodmeier R,Chen H,Paeratakul O.A novel approach to the delivery of microparticles or nanoparticles.Pharm Res.1989;6(5):413-417.
[30]Calvo P,Remunan-Lopez C,Vila-Jato JL,et al.Novel hydrophilic chitosan/polyethylene oxide nanoparticles as proteins carriers.J Appl Polym Sci.1997;63:125-132.
[31]张亚会,李喜香,包强,等.甘草次酸-壳聚糖纳米粒的制备及其质量评价[J].中草药,2015,46(15):2232-2237.
[32]欧歌,廖德华,张凯,等.采用离子交联法制备壳聚糖胰岛素纳米粒的研究进展[J].中南药学,2013,11(2):116-119,156.
[33]奚宏伟,魏长征,王文斌,等.壳聚糖-三聚磷酸钠纳米粒对于肝组织严重出血模型的止血效果[J].现代生物医学进展,2014,14(15):2831-2834,2885.
[34]刘占军,张卫国,于九皋,等.负载紫杉醇壳聚糖纳米粒的制备、表征与释药性能[J].中国组织工程研究与临床康复,2009,13(3):493-495.
[35]刘康,秦梦,杨婷婷,等.槲皮素/壳聚糖纳米粒的制备、表征及其体外抗氧化活性研究[J].中国生化药物杂志,2016,36(11):17-21.
[36]任振堃,高闻,刘畅,等.载柚皮素壳聚糖纳米粒的制备及其体外细胞评价[J].中国医院药学杂志,2017,37(20):2056-2060.
[37]吴振宇,陈钟,黄华,等.离子凝胶法制备壳聚糖纳米微粒[J].南通大学学报(医学版),2005,26(1):20-22.
[38]陈瑞,赵群,邵江娟,等.雷公藤甲素壳聚糖纳米粒的制备与初步评价[J].辽宁中医杂志,2016,43(4):806-808,895.
[39]彭琳,李利锋,韩刚,等.载姜黄素羧甲基壳聚糖纳米粒的制备和性能[J].中国医药工业杂志,2016,47(2):178-182