Studies On The Anti-cancer Activity And Long-circulating Thermosensitive Liposomes Of Decitabine

Decitabine(5-Aza-2'-deoxycytidine,DAC)is a synthesized cytosine analog that is a potent inhibitor of DNA methylation,inducing re-expression silenced genes.On May 2,2006,the U.S.Food and Drug Administration(FDA)approved decitabine for the treatment of patients with myelodysplastic syndromes(MDS)for its excellent capability to reactivate the expression of several mehtylated genes.Therapeutic activity of decitabine in acute leukemias has also been tested in phase ? clinical trials.In the present study,we studied the effects on the proliferation of cancer cells in vitro determined its possible mechanisms of action in melanoma K1735M2 cell.Lastly,DAC-LTSL(long-circulating thermosensitive liposomes)was developed to deliver DAC.The scope of thesis research included:(1)the study of the anticancer activity of DAC in vitro and its possible mechanisms of action;(2)the study of preformulation of DAC-LTSL;(3)the design of the DAC-LTSL and the character of the DAC-LTSL;(4)the pharmacokinetics of the DAC-LTSL in vivo;(5)the study of the anticancer activity of DAC-LTSL in vitro and in vivo and its possible mechanisms of action;In this study MTT test showed that the proliferation of melanoma K1735M2 cell in vitro was inhibited by DAC in a concentration-and time-dependent manner.Decitabine could induce a G2/M cell cycle arrest in K1735M2 cells in a dose-dependent manner.Decitabine could induce K1735M2 cells to undergo terminal differentiation.Further experiment showed that the morphological changes were irreversible.In a sense,decitabine can suppresses the malignant phenotype of melanoma K1735M2 cells.Although decitabine could cause apoptosis on K1735M2 cells,the total apoptosis rates maintained at a relatively low level.In our research,decitabine treatment failed to regulate the expressions of p21 and p53 protein in K1735M2 cells using Western bolt analysis.Thus we provided experimental evidences that the anti-tumor effect of decitabine on murine melanoma K1735M2 cells was associated predominately with cell cycle arrest and cell differentiation rather than apoptosis.The DAC content was detected by HPLC method.It was also used as a sensitive and reproducible method to determine the content of DAC in DAC-LTSL.Minicolumn centrifugation-HPLC method was established to determine encapsulation efficiency of DAC-LTSL.Decitabine is unstable and easy to be hydrolyzed.Methanol can restard hydrolysis of DAC in water.Decitabine aqueous solution is more stable at lower temperature.DAC apparent solubility in PBS and octanol/water partition coefficient(logPoct)were determined for further experiment.DAC apparent solubility in PBS is 19.19mg/mL and logpoct is-0.81.The reverse evaporation method was used to prepare DAC-LTSL.Based on the test of single factor,the formulation and the preparation techniques of DAC-LTSL were optimized by the orthogonal design.Mean DAC-LTSL encapsulation efficiency was 44.5%.The average particle size of DAC-LTSL was 140.2±2.4nm and the zeta potential was-8.34 mv.The release behavior of the drug from liposomes was studied by dialysis method.Most of the drug was released from the liposomes over 42 ? in vitro.The results showed that phase-transition temperature of DAC-LTSL was between 42?43 ?.DAC-LTSL freeze-drying curve was studied.After freeze-drying,the particle size of DAC-LTSL was 150±15nm and encapsulation efficiency was about 40%.Stability of DAC-LTSL was studied.The results showed that the particle size and entrapment efficiency had changed insignificantly after stored for 30 days in a cool,dark area.After i.v.administration of a single dose of DAC and DAC-LTSL,the pharmacokinetics were determined by the DAS software.The MTT test showed that the proliferation of the tumor cells in vitro was inhibited DAC-LTSL in a concentration-and time-dependent manner.There was no distinct difference between DAC-LTSL and DAC group at 37 ?.Decitabine could induce a G2/M cell cycle arrest in K1735M2 cells in a dose-dependent manner.The DAC-LTSL induced long dendrite-like projections.The in vivo experiments indicated DAC-LTSL inhibited the growth of implanted B16 solid tumor proliferation and growth.The results indicated that DAC-LTSL could improve treatment efficacy effectively.