Construction And Anti-Tumor In Vitro Of Nano-Drug Delivery Systerm Of Arsenic Trioxide-Tetrandrine

[Object]:Prepared "arsenic trioxide-tetrandrine bifuncnationl double combination of molecular" on targeted drug delivery system by PLGA and MPEG modified PLGA as carrier.Established As₂O₃ and TET detection method,the drug loading capacity,particle size and morphology were compared before and after modification by surface MPEG.Studied the safety evaluation of nanoparticles and the release ability of nanoparticles in vitro and the comparison of the drug release ability before and after modification.Evaluated the pharmacokinetics of the administration of rat tail vein.Investigated the effects of nanoparticles on apoptosis and cycle of acute promyelocytic leukemia（HL-60）cells in vitro.[Method]:Screend the effective concentration range of As₂O₃ and TET against tumor cells in vitro by MTT,and to determine the optimal proportion of HL-60.Determined As2o3 by hydride generation atomic absorption spectrometry and the TET by UV Spectrophotometry.Prepared As₂O₃-TET-PLGA-NPS and As₂O₃-TET-MPEG-PLGA-NPS,according to L9（34）orthogonal test to optimize the formulation and compared two kinds of nanoparticles morphology,particle size,entrapment efficiency;in appearance,color,dispersion,after re-dissolution encapsulation efficiency,particle size index to determine the best freeze-drying process.Studied the stability of freeze dried powder.The morphology of nanoparticles was observed by transmission electron microscope.The particle size and potential distribution were measured by particle size analyzer.Analysed the release profiles of As₂O₃-TET-PLGA-NPS or As₂O₃-TET-MPEG-PLGA-NPS in vitro by dynamic dialysis method and mathematical parameter fitting to analyze the possible release patterns.The pharmacokinetic study of the nanoparticles in rat plasma was carried out by DAS.The fitting parameters of the non-compartmental model were obtained.The apoptosis of HL-60 cells was detected by Annexin V-FITC/PI double staining method and Hoechst 33342 staining method.The apoptosis of HL-60 cells was evaluated by MTT.The cell cycle arrest of HL-60 cells was detected by flow cytometry with PI single staining.[Result]:When the TET concentration is 2μg·ml^-1 and As₂O₃ was 0.5-0.6 μg·ml^-1 in the concentration range,they had a synergistic effect in combination with two（Q>1.15）;when the TET concentration is 3 μg·ml^-1 and As₂O₃ was in 0.8-1.0μg·ml^-1 concentration range,they combined with a synergistic effect（Q>1.15）.As₂O₃-TET-PLGA-NPS lyophilization injection with an average diameter of 84.66nm,PDI was 0.132,Zeta potential was-7.5mV.As₂O₃-TET-MPEG-PLGA-NPS lyophilization injection with an average diameter of 66.21nm,PDI is 0.178,Zeta potential is-0.701mV.Transmission electron microscope observed two kinds of nanoparticles were spherical.As₂O₃-TET-MPEG-PLGA-NPS had a smaller particle size.The particle size of As₂O₃-TET-MPEG-PLGA-NPS was smaller and the encapsulation efficiency was higher.The average encapsulation efficiency was 86.18%and the drug loading was about 10.49%.When thiourea cryoprotector was 5%,then lyophilized nanoparticles well dispersed,clear liquid solution was transparent with light blue milk.There was no precipitation and particle stability study of As₂O₃-TET-PLGA-NPS freeze dry powder for 6 months at room temperature,particle size of 7.34nm decreased,the encapsulation rate decreased 4.23%.In the case of 4℃for 6 months,particle size decreased 6.58nm,encapsulation rate decreased by 2.63%.As₂O₃-TET-MPEG-PLGA-NPS at room temperature for 6 months,particle size decreased 8.68nm,encapsulation rate decreased by 5.70%.In the case of 4℃ for about 6 months,the particle size decreased by 8.93nm,the encapsulation efficiency decreased by 3.80%.The initial stability of freeze-dried powder at room temperature and room temperature was better than that of the other two kinds of nanoparticles under the condition of 4℃.The cumulative release parameters As₂O₃-TET-PLGA-NPS and As₂O₃-TET-MPEG-PLGA-NPS met the pharmacopoeia in the release of 2h was less than 30%,the total length of the 96h release in the middle 48h cumulative release rate of more than 50%,reflecting the good sustained-release effect.Two kinds of nanoparticles in drug release overall was a biphasic process analysis by mathematical fitting parameters.Solution permeated ceaseless,then the crystalline TET released.With the release of TET,nanoparticle matrix relaxation.As₂O₃ released in the inner layer,hesitation block copolymer skeleton relaxation.There was a sudden release of the phenomenon,but with the release of the skeleton,the situation of sudden release gradually disappeared.The release of the drug were synchronized to the rendering and stable condition.According to the analysis of the experimental results of dynamic agents,we found that the release effect of As₂O₃-TET-MPEG-PLGA-NPS compared to As₂O₃-TET-PLGA-NPS had higher bioavailability,longer half-life and residence time in vivo.Study on the anti-tumor effect of in vitro showed that As₂O₃-TET emulsion and As₂O₃-TET-MPEG-PLGA-NPS could effectively inhibit the growth of tum or cells,the apoptosis rate in a certain range of concentration and time depend ence,and As₂O₃-TET-MPEG-PLGA-NPS group,the early apoptosis rate was sig nificantly higher than that of As₂O₃-TET emulsion for injection group;The cell cycle were blocked in G2/M phase by As₂O₃-TET emulsion and As₂O₃-TETMPEG-PLGA-NPS,and nanoparticles group was better.[Conclusion]:Prepared "arsenic trioxide-tetrandrine bifuncnationl double combination of molecular" on targeted drug delivery system,nano particle size less than 100nm and high encapsulation efficiency.After comparing the two kinds of nanoparticles,the As₂O₃-TET-MPEG-PLGA-NPS showed a smaller particle size,higher encapsulation efficiency.The pharmacokinetic study in rats showed that the bioavailability was higher,and the release effect was better.In vitro anti-tumor studies showed that As₂O₃-TET-MPEG-PLGA-NPS could effectively increase the number of tumor cells in the early stage of apoptosis,induce the non programmed apoptosis of cells,and block the growth of cells in G2/M phase.