氰基丙烯酸烷氧基酯新纳米基因载体的制备及评价
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摘要
目的制备新化学结构的氰基丙烯酸烷氧基酯脑靶向纳米微球,研究负载和递送转化生长因子-β2(TGF-β2)反义寡核苷酸(ASON)的能力,评价体外抗肿瘤活性。方法氰基丙烯酸(甲氧基二乙二醇)酯单体,以二乙氨乙基-葡聚糖(DEAE-Dextran)为阳离子稳定剂,采用乳化聚合法制备空白纳米微球(NS),负载ASON后以聚山梨醇-80进行脑靶向修饰。透射电镜观察形态;动态光散射粒度仪(DLS)测定粒径、Zeta电位;紫外分光光度法测定包封率及载药量;琼脂糖凝胶电泳分析其最佳负载比例以及在DNA酶Ⅰ(DNaseⅠ)及血清环境下对ASON的保护能力;透析法测定ASON的释放速率;激光共聚焦考察细胞摄取情况;MTS法评价对肿瘤细胞A172的体外抑制活性。结果微球外形圆整且无粘连,平均粒径为(79.04±4.33)nm,Zeta电位为(33.60±0.60)m V,负载ASON的包封率为(83.14±1.90)%,载药量为(11.59±0.56)%。可有效延缓ASON在DNaseⅠ及血清环境下的降解。能有效递送ASON进入A172肿瘤细胞并产生抑制活性。结论成功制备出新氰基丙烯酸烷氧基酯脑靶向纳米微球,具有良好的ASON负载及递送能力,为核酸药物提供了新的载体。
Objective To prepare a new(alcoxyle cyanoacrylate)-based nanosphere for brain targeting gene delivery and evaluate its physicochemical properties,capability of delivery of transforming growth factor beta 2(TGF-β_2)antisense oligonucleotides(ASON),and its potential use on tumor cell suppression in vitro. Methods The cationic nanospheres(NS)were prepared by emulsion polymerization method with DEAE-dextran as cationic stabilizer. The ASON were adsorbed by charge interaction,and polysorbate-80 was used as brain-targeting modification. The morphology was observed by transmission electron microscopy(TEM). The average particle size and Zeta potential were determined by dynamic light scattering(DLS). The ultraviolet spectrophotometry was used to determine the entrapment efficiency and drug loading. Agarose gel electrophoresis was used to analyze the optimal loading ratio of ASON-NS,and also the protection of ASON in DNaseⅠand serum containing environment. The release rate of ASON was determined by dialysis. The cytotoxicity on L929 cells and the anti-tumor activity on A172 cells were evaluated by MTS. Results The TEM showed a typical round nanospheres morphology,and no adhesion was detected. The particle size was(79.04±4.33)nm,the dispersion coefficient was 0.04±0.03,the Zeta potential was(33.60±0.60)m V. The encapsulation efficiency of ASON-NS was(83.14±1.90)%,and the drug loading of ASON-NS was(11.59±0.56)%. The NS provided ASON protection against the Dnase I and serum containing environment. The NS-ASON could effectively deliver ASON into A172 cells and show anti-tumor activity. Besides,little L929 cytotoxicity was detected. Conclusion A new cyanoacrylate nanosphere with alcoxyle side group for brain targeting gene delivery was prepared successfully. It had good ASON loading and delivery capability,providing new carrier materials for nucleic acid drugs.
Objective To prepare a new(alcoxyle cyanoacrylate)-based nanosphere for brain targeting gene delivery and evaluate its physicochemical properties,capability of delivery of transforming growth factor beta 2(TGF-β_2)antisense oligonucleotides(ASON),and its potential use on tumor cell suppression in vitro. Methods The cationic nanospheres(NS)were prepared by emulsion polymerization method with DEAE-dextran as cationic stabilizer. The ASON were adsorbed by charge interaction,and polysorbate-80 was used as brain-targeting modification. The morphology was observed by transmission electron microscopy(TEM). The average particle size and Zeta potential were determined by dynamic light scattering(DLS). The ultraviolet spectrophotometry was used to determine the entrapment efficiency and drug loading. Agarose gel electrophoresis was used to analyze the optimal loading ratio of ASON-NS,and also the protection of ASON in DNaseⅠand serum containing environment. The release rate of ASON was determined by dialysis. The cytotoxicity on L929 cells and the anti-tumor activity on A172 cells were evaluated by MTS. Results The TEM showed a typical round nanospheres morphology,and no adhesion was detected. The particle size was(79.04±4.33)nm,the dispersion coefficient was 0.04±0.03,the Zeta potential was(33.60±0.60)m V. The encapsulation efficiency of ASON-NS was(83.14±1.90)%,and the drug loading of ASON-NS was(11.59±0.56)%. The NS provided ASON protection against the Dnase I and serum containing environment. The NS-ASON could effectively deliver ASON into A172 cells and show anti-tumor activity. Besides,little L929 cytotoxicity was detected. Conclusion A new cyanoacrylate nanosphere with alcoxyle side group for brain targeting gene delivery was prepared successfully. It had good ASON loading and delivery capability,providing new carrier materials for nucleic acid drugs.
引文
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[27]Ambruosi A,Khalansky AS,Yamamoto H,et al.Biodistribution of polysorbate 80-coated doxorubicin-loaded[14C]-poly(butyl cyanoacrylate)nanoparticles after intravenous administration to glioblastoma-bearing rats[J].J Drug Target,2006,14(2):97-105.
[28]沈丽萍,颜冬雪,赵善科,等.重复注射掺入不同形式PEG的大黄素脂质体对ABC现象的影响[J].中国新药杂志,2017(7):818-824.
[29]牛玉洁,庞文浩,于敏之,等.PEG修饰在基因递送系统中的应用:优势、不足及改善策略[J].中国新药杂志,2016(2):157-162.
[30]Wang CX,Huang LS,Hou LB,et al.Antitumor effects of polysorbate-80 coated gemcitabine polybutylcyanoacrylate nanoparticles in vitro and its pharmacodynamics in vivo on C6 glioma cells of a brain tumor model[J].Brain Res,2009,1261:91-99.
[31]Gao K,Jiang X.Influence of particle size on transport of methotrexate across blood brain barrier by polysorbate 80-coated polybutylcyanoacrylate nanoparticles[J].Int J Pharm,2006,310(1/2):213-219.
[2]Milano MT,Johnson MD,Sul J,et al.Primary spinal cord glioma:a surveillance,epidemiology,and end results database study[J].J Neuro-Oncol,2010,98(1):83-92.
[3]Bush NAO,Chang SM,Berger MS.Current and future strategies for treatment of glioma[J].Neurosurg Rev,2017,40(1):1-14.
[4]Bishwajitsutradhar K,Amin ML.Nanotechnology in cancer drug delivery and selective targeting[J].ISRN Nanotechnol,2014,2014(2014):1-12.
[5]Srivastava A,Yadav T,Sharma S,et al.Polymers in drug delivery:a review[J].JBM,2016,04(1):69-84.
[6]C′uri c′A,M?schwitzer JP,Fricker G.Development and characterization of novel highly-loaded itraconazole poly(butyl cyanoacrylate)polymeric nanoparticles[J].Eur J Pharm Biopharm,2017,114:175-185.
[7]Yordanov G,Skrobanska R,Evangelatov A.Entrapment of epirubicin in poly(butyl cyanoacrylate)colloidal nanospheres by nanoprecipitation:formulation development and in vitro studies on cancer cell lines[J].Colloids Surf B,2012,92(1):98-105.
[8]C′uric′A,Keller BL,Reul R,et al.Development and lyophilization of itraconazole loaded poly(butyl cyanoacrylate)nanospheres as a drug delivery system[J].Eur J Pharm Sci,2015,78(14):121-131.
[9]Wohlfart S,Gelperina S,Kreuter J.Transport of drugs across the blood-brain barrier by nanoparticles[J].J Control Release,2012,161(2):264-273.
[10]Karim R,Palazzo C,Evrard B,et al.Nanocarriers for the treatment of glioblastoma multiforme:current state-of-the-art[J].J Control Release,2016,227:23-37.
[11]Joseph JV,Balasubramaniyan V,Walenkamp A,et al.TGF-βas a therapeutic target in high grade gliomas-promises and challenges[J].Biochem Pharmacol,2013,85(4):478-485.
[12]Stein CA,Castanotto D.FDA approved oligonucleotide therapies in 2017[J].Mol Ther,2017,25(5):1069-1075.
[13]Bennett CF,Baker BF,Pham N,et al.Pharmacology of antisense drugs[J].Annu Rev Pharmacol Toxicol,2017,57(1):81.
[14]Zhou Z,Singh R,Souweidane MM.Convection-enhanced delivery for diffuse intrinsic pontine glioma treatment[J].Curr Neuropharmacol,2017,15(1):116-128.
[15]Mehta AM,Sonabend AM,Bruce JN.Convection-enhanced delivery[J].Neurotherapeutics,2017,14(2):358-371.
[16]张振中.PBCA纳米粒介导的h TERT反义寡核苷酸对A549细胞的抑制作用[J].药学学报,2006,41(5):446-451.
[17]Weyermann J,Lochmann D,Georgens C,et al.Physicochemical characterisation of cationic polybutylcyanoacrylate-nanoparticles by fluorescence correlation spectroscopy[J].Eur J Pharm Biopharm,2004,58(1):25-35.
[18]李春梅,张林,侯艳红,等.抗EGFR单抗偶联吉西他滨聚氰基丙烯酸正丁酯纳米粒对胰腺癌的靶向治疗[J].世界华人消化杂志,2015,23(12):1890-1896.
[19]Chung CY,Lin MH,Lee IN,et al.Brain-derived neurotrophic factor loaded PS80 PBCA nanocarrier for in vitro neural differentiation of mouse induced pluripotent stem cells[J].Int J Mol Sci,2017,18(3):663.
[20]Kolter M,Ott M,Hauer C,et al.Nanotoxicity of poly(n-butylcyano-acrylate)nanoparticles at the blood-brain barrier,in human whole blood and in vivo[J].J Control Release,2015,197:165-179.
[21]Gelperina SE,Khalansky AS,Skidan IN,et al.Toxicological studies of doxorubicin bound to polysorbate 80-coated poly(butyl cyanoacrylate)nanoparticles in healthy rats and rats with intracranial glioblastoma[J].Toxicol Lett,2002,126(2):131.
[22]Ramge P,Unger RE,Oltrogge JB,et al.Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate(PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells[J].Eur J Neurosci,2000,12(6):1931-1940.
[23]Li J,Cai P,Shalviri A,et al.A multifunctional polymeric nanotheranostic system delivers doxorubicin and imaging agents across the blood-brain barrier targeting brain metastases of breast cancer[J].Acs Nano,2014,8(10):9925.
[24]Maeda H.Tumor-selective delivery of macromolecular drugs via the EPR effect:background and future prospects[J].Bioconjugate Chem,2010,21(5):797-802.
[25]Jin SE,Bae JW,Hong S.Multiscale observation of biological interactions of nanocarriers:from nano to macro[J].Microsc Res Tech,2010,73(9):813-823.
[26]Chaudhari KR,Ukawala M,Manjappa AS,et al.Opsonization,biodistribution,cellular uptake and apoptosis study of PEGylated PBCA nanoparticle as potential drug delivery carrier[J].Pharm Res,2012,29(1):53-68.
[27]Ambruosi A,Khalansky AS,Yamamoto H,et al.Biodistribution of polysorbate 80-coated doxorubicin-loaded[14C]-poly(butyl cyanoacrylate)nanoparticles after intravenous administration to glioblastoma-bearing rats[J].J Drug Target,2006,14(2):97-105.
[28]沈丽萍,颜冬雪,赵善科,等.重复注射掺入不同形式PEG的大黄素脂质体对ABC现象的影响[J].中国新药杂志,2017(7):818-824.
[29]牛玉洁,庞文浩,于敏之,等.PEG修饰在基因递送系统中的应用:优势、不足及改善策略[J].中国新药杂志,2016(2):157-162.
[30]Wang CX,Huang LS,Hou LB,et al.Antitumor effects of polysorbate-80 coated gemcitabine polybutylcyanoacrylate nanoparticles in vitro and its pharmacodynamics in vivo on C6 glioma cells of a brain tumor model[J].Brain Res,2009,1261:91-99.
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