HPLC法测定DNA纳米运输系统载药阿霉素的含量

目的：建立反相高效液相色谱法测定DNA四面体纳米运输系统中阿霉素的含量。方法：采用Agilent Extend C₁₈色谱柱，以含0.05%三氟乙酸的水溶液-含0.05%三氟乙酸的乙腈溶液（72∶28）为流动相，流速1.0 mL&#183;min^-1，检测波长260 nm，柱温30℃。结果：阿霉素浓度在5~150 μmol&#183;L^-1范围内与峰面积呈现良好的线性关系（r=0.999 5）；加样回收率为97.6%~104.2%（RSD<1.5%，n=6）。优化得到DNase Ⅰ脱氧核糖核酸酶酶解DNA四面体释放阿霉素的最佳酶解浓度和最佳酶解时间分别为0.3 mg&#183;mL^-1和30 min。3批样品中DNA四面体结合阿霉素含量分别为131.6、131.4、132.1 μmol&#183;L^-1。结论：该方法为定量分析DNA载体系统中阿霉素的含量提供了有效方法。

Objective:To establish a RP-HPLC analysis method for the determination of doxorubicin in the DNA tetrahedrons.Methods:An Agilent Extend C₁₈ column was used with a mobile phase of a mixture containing 0.05% trifluoroacetic acid in an aqueous solution and 0.05% trifluoroacetic acid in acetonitrile(72:28).The flow rate was 1.0 mL&#183;min^-1,the detection wavelength was 260 nm and the column temperature was 30℃.Results:The concentration of doxorubicin showed a good linear relationship within a range of 5-150 μmol&#183;L^-1(r=0.999 5).Recovery rates were ranged from 97.6% to 104.2%(RSD<1.5%,n=6).The optimal enzymolysis concentration and the enzymolysis time for DNaseⅠ hydrolysis of DNA tetrahedron to release doxorubicin were 0.3 mg&#183;mL^-1and 30 min,respectively.The contents of 3 samples were 131.6,131.4,132.1 μmol&#183;L^-1, respectively.Conclusion:This method provides an effective way to quantify doxorubicin in the DNA carrier system.

[1] DENG B,WANG Z,SONG J,et al.Analysis of doxorubicin uptake in single human leukemia K562 cells using capillary electrophoresis coupled with laser-induced fluorescence detection[J].Anal Bioanal Chem,2011,401(7):21432
[2] 陈召红,刘皈阳,魏亚超.微柱离心高效液相色谱法测定盐酸阿霉素磁性热敏脂质体包封率[J].解放军药学学报,2010,26(6):496 CHEN ZH,LIU GY,WEI YC.Determination of entrapment efficiency of doxorubicin magnetic thermosensitive liposomes by mini-column centrifugation-reversed-phase high performance liquid chromatography[J].Pharm J Chin PLA,2010,26(6):496
[3] MULTANI P,WHITE CA,GRILLO-L&#211;PEZ A.Non-Hodgkins lymphoma:review of conventional treatments[J].Curr Pharm Biotechnol,2001,2(4):279
[4] WALSH AS,YIN H,ERBEN CM,et al.DNA cage delivery to mammalian cells[J].Acs Nano,2011,5(7):5427
[5] CHAROENPHOL P,BERMUDEZ H.Aptamer-targeted DNA nanostructures for therapeutic delivery[J].Mol Pharm,2014,11(5):1721
[6] PEI H,LU N,WEN YL,et al.A DNA nanostructure-based biomolecular probe carrier platform for electrochemical biosensing[J].Adv Mater,2010,22(42):4754
[7] TAY CY,YUAN L,LEONG DT.Nature-inspired DNA nanosensor forreal-time in situ detection of mRNA in living cells[J].ACS Nano,2015,9(5),5609
[8] LI ZH,ZHAO B,WANG DF,et al.DNA nanostructure-based universal microarray platform for high-efficiency multiplex bioanalysis in biofluids[J].Acs Appl Mater Interfaces,2014,6(20):17944
[9] GE ZL,LIN MH,WANG P,et al.Hybridization chain reaction amplification of MicroRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor[J].Anal Chem,2014,86(4):2124
[10] KIM KR,LEE YD,LEE T,et al.Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron[J].Biomaterials,2013,34(21):5226
[11] LI J,PEI H,ZHU B,et al.Self-assembled multivalent DNA nanostructures for noninvasive intracellular delivery of immunostimulatory CpG oligonucleotides[J].Acs Nano,2011,5(11):8783
[12] KIM KR,KIM DR,LEE T,et al.Drug delivery by a self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells[J].Chem Commun,2013,49(20):2010
[13] LEE H,LYTTONJEAN AK,CHEN Y,et al.Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery[J].Nat Nanotechnol,2012,7(6):389
[14] ZHANG YM,JIANG SX,ZHANG DD,et al.DNA-affibody nanoparticles for inhibiting breast cancer cells overexpressing HER2[J].Chem Commun,2017,53(3):573
[15] 吴李娜.适体共轭的DNA四面体用于乳腺癌细胞的检测及阿霉素靶向输送初步研究[D].济南:山东大学,2015 WU LN.Aptamer-conjugated DNA Tetrahedron for the Detection of Breast Cancer and Target Delivery of Doxrubincin[D].Jinan:Shandong University,2015
[16] GOODMAN RP,SCHAAP IAT,TARDIN CF,et al.Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication[J].Science,2005,310(5754):1661
[17] GOODMAN RP,BERRY RM,TURBERFIELD AJ.The single-step synthesis of a DNA tetrahedron[J].Chem Commun,2004,12(12):1372