尼罗红示踪纳米制剂在荷瘤裸鼠体内活体成像的应用
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摘要
目的拟将尼罗红包裹入纳米制剂中,利用其荧光特性来考察纳米制剂在荷瘤鼠体内的肿瘤靶向性与组织分布情况。方法采用CCK-8法检测尼罗红纳米乳[NRNE(O)]对H1688的细胞毒性。建立BALB/c裸鼠肺癌模型,灌胃给予尼罗红混悬液(NRS)及NRNE(O),通过小动物活体成像系统观察其在荷瘤裸鼠中荧光强度的动态分布情况。结果 NRNE(O)具有较低的细胞毒性和良好的生物相容性,并且在体内的吸收量及肿瘤部位的蓄积量均高于NRS;NR在纳米制剂中能快速、稳定、清晰地反映纳米制剂在小鼠体内的动态分布情况。结论本研究中制备的NRNE(O)的稳定性好,为尼罗红作为示踪剂应用于纳米制剂在动物体内的研究提供了重要的研究手段。
Objective The fluorescent dye Nile red(NR) is commonly used in quantitative analysis of lipids in bacteria, fungi, and microalgae. In this study, NR was encapsulated in a nanoemulsion to investigate the tumor targeting and tissue distribution of the nanoemulsion in BALB/c nude mice bearing H1688 tumors. Methods To evaluate the prospects of NR as a bioimaging marker for nanoemulsions, its cytotoxicity was investigated in H1688 cells with a CCK-8 assay. An NR suspension(NRS) and NR nanoemulsion NRNE(O) were given by gavage. The dynamic fluorescence intensity distribution of NR in mice was observed with an in vivo fluorescence imaging system. Results The H1688 cells exhibited over 85% viability after incubation with a range of NRNE(O) concentrations for 24 h. The results demonstrated that NRNE(O) absorption in mice and the amount accumulated in tumor tissue were both higher than in the NRS group. Conclusions NRNE(O) has a low cytotoxicity and good biocompatibility. NR in a nanoemulsion combined with an in vivo fluorescence imaging system can reveal the dynamic distribution of the nanoemulsion in mice vividly and reliably in real time. The results of this study indicate that NR has significant application potential as a tracer for the bioimaging of nanoparticles in vivo.
Objective The fluorescent dye Nile red(NR) is commonly used in quantitative analysis of lipids in bacteria, fungi, and microalgae. In this study, NR was encapsulated in a nanoemulsion to investigate the tumor targeting and tissue distribution of the nanoemulsion in BALB/c nude mice bearing H1688 tumors. Methods To evaluate the prospects of NR as a bioimaging marker for nanoemulsions, its cytotoxicity was investigated in H1688 cells with a CCK-8 assay. An NR suspension(NRS) and NR nanoemulsion NRNE(O) were given by gavage. The dynamic fluorescence intensity distribution of NR in mice was observed with an in vivo fluorescence imaging system. Results The H1688 cells exhibited over 85% viability after incubation with a range of NRNE(O) concentrations for 24 h. The results demonstrated that NRNE(O) absorption in mice and the amount accumulated in tumor tissue were both higher than in the NRS group. Conclusions NRNE(O) has a low cytotoxicity and good biocompatibility. NR in a nanoemulsion combined with an in vivo fluorescence imaging system can reveal the dynamic distribution of the nanoemulsion in mice vividly and reliably in real time. The results of this study indicate that NR has significant application potential as a tracer for the bioimaging of nanoparticles in vivo.
引文
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[15] 邰文, 孙敏敏, 刘楠, 等. 应用动物活体生物发光技术观察紫杉醇混合胶束的抑瘤效果[J].药学学报, 2010, 45(4): 530-534.Tai W, Sun MM, Liu N, et al. Study on the anti-tumor effect of paclitaxel mixed micelle by using in vivo optical imaging technique [J]. Acta Pharm Sin, 2010, 45: 530-534.
[16] 沈锦秋, 甘勇, 甘莉, 等. 氟比洛芬酯眼用纳米乳-离子敏感型原位凝胶的研究 [J]. 药学学报, 2010, 45(1): 120-125.Shen JQ, Gan Y, Gan L, et al. Ion-sensitive nanoemulsion-in situ gel system for ophthalmic delivery of flurbiprofen axetil [J]. Acta Pharm Sin, 2010, 45: 120-125.
[17] 张胜华, 程昕, 钟根深, 等. 活体成像分析异硫氰酸荧光素标记Rituximab 在荷淋巴瘤裸鼠体内的生物分布 [J]. 中华医学杂志, 2010, 90(33): 2367-2370.Zhang SH, Cheng X, Zhong GS, et al. In vivo imaging analysis of biodistribution of FITC-labeled Rituximab in lymphoma-bearing nude mice [J]. Natl Med J Chin, 2010, 90(33): 2367-2370.
[18] Liu S, Chen D, Yuan Y, et al. Efficient intracellular delivery makes cancer cells sensitive to nanoemulsive chemodrugs [J]. Oncotarget, 2017, 8(39): 65042-65055.
[19] Münch C, Harper JW. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation [J]. Nature, 2016, 534(7069): 710-713.
[20] 付奎, 杨晓峰, 汪海龙, 等. 活体荧光成像评估Ag85A和Ag85BDNA疫苗对小鼠膀胱癌移植瘤的疗效 [J]. 中国肿瘤生物学杂志, 2009, 16(6): 588-594.Fu K, Yang XF, Wang HL, et al. In vivo fluorescence image analysis system in assessing efficacies of pVAX1-Ag 85A and pVAX1-Ag 85B DNA vaccines in treatment of bladder cancer cell implanted tumors in mice [J]. Chin J Cancer Biother, 2009, 16(6): 588-594.
[21] 闫明霞, 刘蕾, 荚德水, 等. 人肺癌裸小鼠模型活体成像的动态观察 [J]. 肿瘤, 2008, 28(10): 833-836.Yan MX, Liu L, Jia DS, et al. Dynamic observation of in vivo biofluorescence imaging in nude mouse model with human lung carcinoma [J]. Tumor, 2008, 28(10): 833-836.
[22] Gao Y, Chen G, Weselake RJ. A rapid Nile red fluorescence-based method for triacylglycerol content in microspore-derived cell suspension cultures of brassica napus [J]. Lipids, 2014, 49(11): 1161-1168.
[2] Kimura K, Yamaoka M, Kamisaka Y. Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence [J]. J Microbiol Methods, 2004, 56(3): 331-338.
[3] Bertozzini E, Galluzzi L, Penna A, et al. Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red [J]. J Microbiol Methods, 2011, 87(1): 17-23.
[4] 胡小文, 马帅, 弓淑芬, 等. 荧光光谱法检测微藻中油脂[J]. 中国油脂, 2011, 36(4): 70-74.Hu XW, Ma Shuai, Gong SF, et al. Determination of microalgae lipids by fluorescent spectrometry [J]. Chin Oils Fats, 2011, 36(4): 70-74.
[5] 王海英, 符茹, 黄宝祥. 基于尼罗红荧光染色的小球藻脂质快速检测方法研究 [J]. 中国油脂, 2012, 37(3): 78-81.Wang HY, Fu R, Huang BX. Rapid determination of lipid in Chlorella based on Nile red fluorescence [J]. Chin Oils Fats, 2012, 37(3): 78-81.
[6] Su J, Chen F, Cryns VL, et al. Catechol polymers for pH-responsive, targeted drug delivery to cancer cells [J]. J Am Chem Soc, 2011, 133(31): 11850-11853.
[7] 高会乐, 蒋新国. 新型药物递释系统的研究进展 [J]. 药学学报, 2017, 52(2): 181-188.Gao HL, Jiang XG. The progress of novel drug delivery systems [J]. Acta Pharm Sin, 2016, 51: 272-280.
[8] Song G, Suzuki OT, Santos CM, et al. Gulp1 is associated with the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) in inbred mouse strains [J]. Nanomedicine, 2016, 12(7): 2007-2017.
[9] 李大伟, 武玉敏, 刘正平,等. 肺部吸入纳米制剂治疗肺癌的研究进展 [J]. 中国药房, 2016, 27(31): 4460-4462.Li DW, Wu YM, Liu ZP, et al. Research progress of lung inhalation nano-agents for treating lung cancer [J]. Chin Pharm, 2016, 27(31): 4460-4462.
[10] 孔晓龙, 郭梅红, 范颖, 等. 纳米靶向制剂的研究进展 [J]. 广西医科大学学报, 2015, 32(4): 682-685.Kong XL, Guo MH, Fan Y, et al. Research progress of targeting nanodrug delivery system [J]. J Guangxi Med Univ, 2015, 32(4):682-685.
[11] 刘源, 周建平, 王伟. 聚乙二醇修饰靶向纳米制剂的研究进展 [J]. 中国药科大学学报, 2017, 48(3): 268 -275.Liu Y, Zhou JP, Wang W. Advances in PEGylated targeted nano-preparation [J]. J Chin Pharm Univ, 2017, 48(3): 268 -275.
[12] 史一杰, 程刚. 纳米制剂生物安全性评价研究进展 [J]. 沈阳药科大学学报, 2010, 27(12): 987-992.Shi YJ, Cheng G. Review on the research of biological safety evaluation for nanopreparations [J]. J Shenyang Pharm Univ, 2010, 27(12): 987-992.
[13] 胡秀丽, 王瑞, 岳军, 等. 叶酸高分子纳米胶束在小鼠体内的靶向分布 [J]. 中国科学:化学, 2012, 42(8): 1172-1178.Hu XL, Wang R, Yue J, et al. Folic acid mediated tumor targeting effect observed by fluorescent imaging [J]. Sci Sin (Chim), 2012, 42(8): 1172-1178.
[14] Ntziachristos V, Ripoll J, Wang LV, et al. Looking and listening to light: the evolution of whole-body photonic imaging [J]. Nat Biotechnol, 2005, 23(3): 313-320.
[15] 邰文, 孙敏敏, 刘楠, 等. 应用动物活体生物发光技术观察紫杉醇混合胶束的抑瘤效果[J].药学学报, 2010, 45(4): 530-534.Tai W, Sun MM, Liu N, et al. Study on the anti-tumor effect of paclitaxel mixed micelle by using in vivo optical imaging technique [J]. Acta Pharm Sin, 2010, 45: 530-534.
[16] 沈锦秋, 甘勇, 甘莉, 等. 氟比洛芬酯眼用纳米乳-离子敏感型原位凝胶的研究 [J]. 药学学报, 2010, 45(1): 120-125.Shen JQ, Gan Y, Gan L, et al. Ion-sensitive nanoemulsion-in situ gel system for ophthalmic delivery of flurbiprofen axetil [J]. Acta Pharm Sin, 2010, 45: 120-125.
[17] 张胜华, 程昕, 钟根深, 等. 活体成像分析异硫氰酸荧光素标记Rituximab 在荷淋巴瘤裸鼠体内的生物分布 [J]. 中华医学杂志, 2010, 90(33): 2367-2370.Zhang SH, Cheng X, Zhong GS, et al. In vivo imaging analysis of biodistribution of FITC-labeled Rituximab in lymphoma-bearing nude mice [J]. Natl Med J Chin, 2010, 90(33): 2367-2370.
[18] Liu S, Chen D, Yuan Y, et al. Efficient intracellular delivery makes cancer cells sensitive to nanoemulsive chemodrugs [J]. Oncotarget, 2017, 8(39): 65042-65055.
[19] Münch C, Harper JW. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation [J]. Nature, 2016, 534(7069): 710-713.
[20] 付奎, 杨晓峰, 汪海龙, 等. 活体荧光成像评估Ag85A和Ag85BDNA疫苗对小鼠膀胱癌移植瘤的疗效 [J]. 中国肿瘤生物学杂志, 2009, 16(6): 588-594.Fu K, Yang XF, Wang HL, et al. In vivo fluorescence image analysis system in assessing efficacies of pVAX1-Ag 85A and pVAX1-Ag 85B DNA vaccines in treatment of bladder cancer cell implanted tumors in mice [J]. Chin J Cancer Biother, 2009, 16(6): 588-594.
[21] 闫明霞, 刘蕾, 荚德水, 等. 人肺癌裸小鼠模型活体成像的动态观察 [J]. 肿瘤, 2008, 28(10): 833-836.Yan MX, Liu L, Jia DS, et al. Dynamic observation of in vivo biofluorescence imaging in nude mouse model with human lung carcinoma [J]. Tumor, 2008, 28(10): 833-836.
[22] Gao Y, Chen G, Weselake RJ. A rapid Nile red fluorescence-based method for triacylglycerol content in microspore-derived cell suspension cultures of brassica napus [J]. Lipids, 2014, 49(11): 1161-1168.
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