新型~(99m)Tc-DTPA-GGGRDN-IF7靶向肿瘤血管Anxa1分子探针的制备及SPECT显像
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摘要
目的制备针对肿瘤血管表面受体Annexin A1(Anxa1)分子探针~(99m)Tc-二乙三胺五乙酸(DTPA)-甘氨酸-甘氨酸-甘氨酸-精氨酸-天冬氨酸-天冬酰胺(GGGRDN)-异亮氨酸-苯丙氨酸-亮氨酸-亮氨酸-色氨酸-谷氨酰胺-精氨酸(IFLLWQR,IF7),进行体外肿瘤细胞(人脑星形胶质细胞瘤U87细胞)实验;探讨~(99m)Tc-DTPA-GGGRDN-IF7在荷瘤小鼠体内的生物学分布规律,并利用单光子发射计算机体层摄像术(SPECT)进行显像研究。方法将100μg DTPA-GGGRDN-IF7溶于10μL二甲基亚砜(DMSO)中,充入氮气保护。加入10μL 1 mg/mL氯化亚锡(SnCl2)溶液(pH 4.0),再加入约111 MBq(3 mCi)Na~(99m)TcO_4,37℃水浴反应30 min,经C_(18)柱淋洗后制备~(99m)Tc-DTPA-GGGRDN-IF7。取样注入分析型高效液相色谱(HPLC)仪分析~(99m)Tc-DTPA-GGGRDN-IF7的放射化学纯度及体外血清稳定性。将U87细胞与~(99m)Tc-DTPA-GGGRDN-IF7共同孵育后,测定放射性计数并计算摄取率。取30只4～5周龄18～20 g荷瘤裸鼠,建立U87荷瘤裸鼠模型,进行~(99m)Tc-DTPA-GGGRDN-IF7的体内分布实验及SPECT显像。结果 ~(99m)Tc-DTPA-GGGRDN-IF7标记产率大于95%,放射化学纯度大于95%。在大鼠血清中37℃保温2 h后性状稳定,放射化学纯度大于92.3%。U87细胞在体外对~(99m)Tc-DTPA-GGGRDN-IF7摄取在60 min时达峰值(6.85%±0.97%),过量的GGGRDN-IF7可以明显降低细胞的摄取。体内分布实验结果显示,药物均在血浆清除较快,肝肾摄取高,主要从肝肾排泄。SPECT显像结果表明,肿瘤部位呈明显放射性浓聚态,注射2 h后肿瘤对~(99m)Tc-DTPA-GGGRDN-IF7摄取值为(3.56±0.44)%ID/g。结论 ~(99m)Tc-DTPA-GGGRDN-IF7合成简便,放射化学纯度高,易于推广;U87细胞对~(99m)Tc-DTPA-GGGRDN-IF7具有较强亲和力;SPECT显像表明肿瘤明显浓聚~(99m)Tc-DTPA-GGGRDN-IF7,体内生物分布理想,靶向性强,有望用于肿瘤显像。
Objective To prepare the molecular probes for tumor vascular surface receptor Annexin A1(Anxal), conduct in vitro tumor cell(human brain astrocytoma U87 cells) experiments and investigate the in vitro biodistribution of ~(99m)Tc-DTPAGGGRDN-IF7 in tumor bearing mice, and perform imaging study by single-photon emission computed tomography(SPECT).Methods The 100 μg of DTPA-GGGRDN-IF7 was dissolved in 10 μL dimethyl sulfoxide(DMSO) and filled with nitrogen. A solution of 10 μL 1 mg/mL SnCl_2 in hydrochloric acid(pH 4.0) was added, then 111 MBq(3 mCi) Na ~(99m)TcO_4 was added and reacted at 37 ℃ for 30-minute to elute with C_(18) column and prepare ~(99m)Tc-DTPA-GGGRDN-IF7. The radiochemical purity and in vitro serum stability of ~(99m)Tc-DTPA-GGGRDN-IF7 was analyzed by high performance liquid chromatography(HPLC). After U87 cells were incubated with ~(99m)Tc-DTPA-GGGRDN-IF7, the radioactivity count was measured and uptake rate was calculated. Thirty nude mice aged 5-week old with body weight of 18-20 g were used to establish U87 tumor-bearing nude model,the in vivo distribution experiment and SPECT imaging of ~(99m)Tc-DTPA-GGGRDN-IF7 were performed. Results The labeling yield of ~(99m)Tc-DTPA-GGGRDN-IF7 was more than 95 %, and radiochemical purity was more than 95 %. It was stable in mouse serum after heat preservation at 37 ℃ for 2-hour, and radiochemical purity was more than 92.3 %. The in vitro uptake of U87 cells for ~(99m)Tc-DTPA-GGGRDN-IF7 reached peak value of(6.85 ± 0.97) % at 60-minute, and excessive GGGRDNIF7 significantly reduced cell uptake. In vivo distribution experiment results showed that drug eliminated faster in plasma, intake in liver and kidney, and mainly excreted from liver and kidney. The SPECT imaging showed that the tumor was markedly radioactive, and uptake value of ~(99m)Tc-DTPA-GGGRDN-IF7 in tumor was(3.56 ± 0.44) %ID/g at 2-hour after injection. Conclusion It is demonstrated that ~(99m)Tc-DTPA-GGGRDN-IF7 has the advantages of simple synthesis, high radiochemical purity and easy for promotion. U87 cell has strong affinity for ~(99m)Tc-DTPA-GGGRDN-IF7. The SPECT imaging showed that ~(99m)Tc-DTPA-GGGRDN-IF7 concentrate obviously in tumor, which has ideal in vivo biodistribution with strong targeting, and is expected to be used for tumor imaging.
Objective To prepare the molecular probes for tumor vascular surface receptor Annexin A1(Anxal), conduct in vitro tumor cell(human brain astrocytoma U87 cells) experiments and investigate the in vitro biodistribution of ~(99m)Tc-DTPAGGGRDN-IF7 in tumor bearing mice, and perform imaging study by single-photon emission computed tomography(SPECT).Methods The 100 μg of DTPA-GGGRDN-IF7 was dissolved in 10 μL dimethyl sulfoxide(DMSO) and filled with nitrogen. A solution of 10 μL 1 mg/mL SnCl_2 in hydrochloric acid(pH 4.0) was added, then 111 MBq(3 mCi) Na ~(99m)TcO_4 was added and reacted at 37 ℃ for 30-minute to elute with C_(18) column and prepare ~(99m)Tc-DTPA-GGGRDN-IF7. The radiochemical purity and in vitro serum stability of ~(99m)Tc-DTPA-GGGRDN-IF7 was analyzed by high performance liquid chromatography(HPLC). After U87 cells were incubated with ~(99m)Tc-DTPA-GGGRDN-IF7, the radioactivity count was measured and uptake rate was calculated. Thirty nude mice aged 5-week old with body weight of 18-20 g were used to establish U87 tumor-bearing nude model,the in vivo distribution experiment and SPECT imaging of ~(99m)Tc-DTPA-GGGRDN-IF7 were performed. Results The labeling yield of ~(99m)Tc-DTPA-GGGRDN-IF7 was more than 95 %, and radiochemical purity was more than 95 %. It was stable in mouse serum after heat preservation at 37 ℃ for 2-hour, and radiochemical purity was more than 92.3 %. The in vitro uptake of U87 cells for ~(99m)Tc-DTPA-GGGRDN-IF7 reached peak value of(6.85 ± 0.97) % at 60-minute, and excessive GGGRDNIF7 significantly reduced cell uptake. In vivo distribution experiment results showed that drug eliminated faster in plasma, intake in liver and kidney, and mainly excreted from liver and kidney. The SPECT imaging showed that the tumor was markedly radioactive, and uptake value of ~(99m)Tc-DTPA-GGGRDN-IF7 in tumor was(3.56 ± 0.44) %ID/g at 2-hour after injection. Conclusion It is demonstrated that ~(99m)Tc-DTPA-GGGRDN-IF7 has the advantages of simple synthesis, high radiochemical purity and easy for promotion. U87 cell has strong affinity for ~(99m)Tc-DTPA-GGGRDN-IF7. The SPECT imaging showed that ~(99m)Tc-DTPA-GGGRDN-IF7 concentrate obviously in tumor, which has ideal in vivo biodistribution with strong targeting, and is expected to be used for tumor imaging.
引文
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[12]Raulf N,Lucarelli P,Thavaraj S,et al.Annexin A1 regulates EGFR activity and alters EGFR-containing tumour-derived exosomes in head and neck cancers[J].Eur J Cancer,2018,102:52-68.
[2]WANG Rong-fu.Development and situation of radionuclide imaging and therapy in oncology[J].Journal of Isotopes,200619(2):112-118.[王荣福.肿瘤核素诊治现状与进展[J].同位素,2006,19(2):112-118.]
[3]Khondee S,Wang TD.Progress in molecular imaging in endoscopy and endomicroscopy for cancer imaging[J].J Healthc Eng,2013,4(1):1-22.
[4]HUANG Gang,LIU Jian-jun.Molecular imaging:key carrier of translational medicine[J].Journal of Shanghai Jiaotong U-niversity:Medical Science,2010,30(9):1021-1023.[黄钢,刘建军.分子影像学:转化医学的重要载体[J].上海交通大学学报:医学版,2010,30(9):1021-1023.]
[5]Hatakeyama S,Sugihara K,Shibata TK,et al.Targeted drug delivery to tumor vasculature by a carbohydrate mimetic peptide[J].Proc Natl Sci USA,2011,108(49):19587-19592.
[6]Yi M,Schnitzer JE.Impaired tumor growth,metastasis,angiogenesis and wound healing in annexin A1-null mice[J].Proc Natl Acad Sci USA,2009,106(42):17886-17891.
[7]Li ZJ,Cho CH.Peptides as targeting probes against tumor vasculature for diagnosis and drug delivery[J].J Transl Med,2012,10(Suppl 1):S1-S1.
[8]Bao X,Wang MW,Luo JM,et al.Optimization of early response monitoring and prediction of cancer antiangiogenesis therapy via noninvasive PET molecular imaging strategies of multifactorial bioparameters[J].Theranostics,2016,6(12):2084-2098.
[9]Li ZJ,Wu WK,Ng SS,et al.A novel peptide specifically targeting the vasculature of orthotopic colorectal cancer for imaging detection and drug delivery[J].J Control Release,2010,148(3):292-302.
[10]Nakamura M,Zhang Y,Yang Y,et al.Off-tumor targets compromise antiangiogenic drug sensitivity by inducing kidney erythropoietin production[J].Proc Natl Acad Sci USA,2017,114(45):E9635-E9644.
[11]Jia C,Kong D,Guo Y,et al.Enhanced antitumor effect of combination of annexin A1 knockdown and bortezomib treatment in multiple myeloma in vitro and in vivo[J].Biochem Biophys Res Commun,2018,505(3):720-725.
[12]Raulf N,Lucarelli P,Thavaraj S,et al.Annexin A1 regulates EGFR activity and alters EGFR-containing tumour-derived exosomes in head and neck cancers[J].Eur J Cancer,2018,102:52-68.
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