八臂聚乙二醇纳米结合物脑靶向传递光疗剂的研究
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摘要
以八臂聚乙二醇为载体、cRGD为靶头制备用于脑癌靶向光疗的纳米结合物,探讨其抗肿瘤作用与机制。通过紫外可见光谱对合成的纳米结合物进行表征,借助激光共聚焦显微镜对纳米结合物的细胞摄取行为进行观察,采用Alamar Blue法与Calcein AM/PI染色考察细胞毒作用,借助肿瘤球生长曲线评估肿瘤抑制效果,通过观察细胞内活性氧的生成、细胞凋亡与肿瘤球穿透性探讨纳米结合物的作用机制。结果表明,cRGD-8PEG-IR700能够被整合素受体高表达的U87MG细胞高效摄取,而整合素受体不表达的NIH/3T3细胞对其几乎无摄取。在考察浓度范围内,仅cRGD-8PEG-IR700光照组的U87MG细胞呈现明显毒性作用;在3D细胞模型上仅cRGD-8PEG-IR700光照组的肿瘤球生长受到明显抑制,这是由于光照诱导细胞内活性氧的产生并引起细胞凋亡和靶向纳米结合物较强的肿瘤穿透性能。因此,该聚乙二醇纳米结合物具有肿瘤靶向性,有望为肿瘤的光疗提供一个有前景的药物递送系统。
The aim of this study was to prepare the nanoconjugates for targeted photodynamic therapy of brain cancer by using eight-arm polyethylene glycol(8 PEG) as the carrier and cRGD as the targeting ligand,and to investigate the antitumor effect and its mechanism.UV-Vis spectra and confocal microscopy were used for characterization and cellular uptake behavior of nanoconjugates respectively.Alamar Blue assay and Calcein AM/PI staining were applied to investigate the cytotoxocity of nanoconjugates against tumor cells,and tumor spheroid growth curve was used to assess the tumor growth suppression effect.In addition,the generation of reactive oxygen species(ROS),apoptosis and spheroid permeability test was used to reveal the antitumor mechanism of nanoconjugates.The results showed that cRGD-8 PEG-IR700 was taken up efficiently by integrin overexpressed U87 MG cells,while almost no uptake was found in integrin free NIH/3 T3 cells.Remarkable photokilling effect against U87 MG cells was only shown in cRGD-8 PEG-IR700 group due to the light-induced ROS generation and apoptosis,whereas growth suppression effect was also observed in U87 MG spheroids treated with cRGD-8 PEG-IR700 plus light owing to the superior penetration ability of targeted nanoconjugates.Hence,tumor-targeted PEG nanoconjugates may provide a promising drug delivery system for photodynamic therapy of cancers.
The aim of this study was to prepare the nanoconjugates for targeted photodynamic therapy of brain cancer by using eight-arm polyethylene glycol(8 PEG) as the carrier and cRGD as the targeting ligand,and to investigate the antitumor effect and its mechanism.UV-Vis spectra and confocal microscopy were used for characterization and cellular uptake behavior of nanoconjugates respectively.Alamar Blue assay and Calcein AM/PI staining were applied to investigate the cytotoxocity of nanoconjugates against tumor cells,and tumor spheroid growth curve was used to assess the tumor growth suppression effect.In addition,the generation of reactive oxygen species(ROS),apoptosis and spheroid permeability test was used to reveal the antitumor mechanism of nanoconjugates.The results showed that cRGD-8 PEG-IR700 was taken up efficiently by integrin overexpressed U87 MG cells,while almost no uptake was found in integrin free NIH/3 T3 cells.Remarkable photokilling effect against U87 MG cells was only shown in cRGD-8 PEG-IR700 group due to the light-induced ROS generation and apoptosis,whereas growth suppression effect was also observed in U87 MG spheroids treated with cRGD-8 PEG-IR700 plus light owing to the superior penetration ability of targeted nanoconjugates.Hence,tumor-targeted PEG nanoconjugates may provide a promising drug delivery system for photodynamic therapy of cancers.
引文
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[2] Lim CK,Heo J,Shin S,et al.Nanophotosensitizers toward advanced photodynamic therapy of Cancer[J].Cancer Lett,2013,334(2):176-187.
[3] Duse L,Pinnapireddy SR,Strehlow B,et al.Low level LED photodynamic therapy using curcumin loaded tetraether liposomes[J].Eur J Pharm Biopharm,2018,126:233-241.
[4] Wang Y,Xie Y,Li J,et al.Tumor-Penetrating nanoparticles for enhanced anticancer activity of combined photodynamic and hypoxia-activated therapy[J].ACS Nano,2017,11(2):2227-2238.
[5] Wang Y,Zheng K,Xuan G,et al.Novel pH-sensitive zinc phthalocyanine assembled with albumin for tumor targeting and treatment[J].Int J Nanomed,2018,13:7681-7695.
[6] Park W,Park SJ,Cho S,et al.Intermolecular structural change for thermoswitchable polymeric photosensitizer[J].J Am Chem Soc,2016,138(34):10734-10737.
[7] Dai L,Yu Y,Luo Z,et al.Photosensitizer enhanced disassembly of amphiphilic micelle for ROS-response targeted tumor therapy in vivo[J].Biomaterials,2016,104:1-17.
[8] Gao M,Fan F,Li D,et al.Tumor acidity-activatable TAT targeted nanomedicine for enlarged fluorescence/magnetic resonance imaging-guided photodynamic therapy[J].Biomaterials,2017,133:165-175.
[9] Yuan A,Yang B,Wu J,et al.Dendritic nanoconjugates of photosensitizer for targeted photodynamic therapy[J].Acta Biomater,2015,21:63-73.
[10] Perry JL,Reuter KG,Luft JC,et al.Mediating passive tumor accumulation through particle size,tumor type,and location[J].Nano Lett,2017,17(5):2879-2886.
[11] Liu Y,Zhou JP,Wang W.Advances in PEGylated targeted nano-preparation[J].J China Pharm Univ(中国药科大学学报),2017,48(3):268-275.
[12] Wang J,Zhang F,Tsang WP,et al.Fabrication of injectable high strength hydrogel based on 4-arm star PEG for cartilage tissue engineering[J].Biomaterials,2017,120:11-21.
[13] Gok O,Erturk P,Sumer Bolu B,et al.Dendrons and multiarm polymers with thiol-exchangeable cores:a reversible conjugation platform for delivery[J].Biomacromolecules,2017,18(8):2463-2477.
[14] Zhao Y,Li F,Mao C,et al.Multiarm nanoconjugates for cancer cell-targeted delivery of photosensitizers[J].Mol Pharm,2018,15(7):2559-2569.
[15] Hagemann J,Jacobi C,Hahn M,et al.Spheroid-based 3D cell cultures enable personalized therapy testing and drug discovery in head and neck cancer[J].Anticancer Res,2017,37(5):2201-2210.
[16] Ming X,Carver K,Wu L.Albumin-based nanoconjugates for targeted delivery of therapeutic oligonucleotides[J].Biomaterials,2013,34(32):7939-7949.
[17] Huang BW,Gao JQ.Application of 3D cultured multicellular spheroid tumor models in tumor-targeted drug delivery system research[J].J Control Release,2018,270:246-259.
[18] Sant S,Johnston PA.The production of 3D tumor spheroids for cancer drug discovery[J].Drug Discovery Today:Technol,2017,23:27-36.
[19] Ramaiahgari SC,Waidyanatha S,Dixon D,et al.From the cover:three-dimensional (3D) HepaRG spheroid model with physiologically relevant xenobiotic metabolism competence and hepatocyte functionality for liver toxicity screening[J].Toxicol Sci,2017,159(1):124-136.
[20] Lu C,Zahedi P,Forman A,et al.Multi-arm PEG/silica hydrogel for sustained ocular drug delivery[J].J Pharm Sci,2014,103(1):216-226.
[21] Lv L,Shen Y,Li M,et al.Novel 4-arm poly(ethylene glycol)-block-poly(anhydride-esters) amphiphilic copolymer micelles loading curcumin:preparation,characterization,and in vitro evaluation[J].Biomed Res Int,2013,2013:507103.
[22] Ma G,Zhang C,Zhang L,et al.Doxorubicin-loaded micelles based on multiarm star-shaped PLGA-PEG block copolymers:influence of arm numbers on drug delivery[J].J Mater Sci Mater Med,2016,27(1):17.
[23] Montet X,Funovics M,Montet-Abou K,et al.Multivalent effects of RGD peptides obtained by nanoparticle display[J].J Med Chem,2006,49(20):6087-6093.