Reduction Responsive Nanomicelles For Diagnosis And Treatment Of Liver Cancer

Object1.In this project,we attempt to construct a reduction responsive diagnostic and therapeutic nanomicelle with the functions of MRI diagnosis,antitumor drug release and liver active targeting: β-CD(PCL22)14-(-SS-PGlu Gla30)7@SPIO/DOX and β-CD(PCL22)14-(-SS-PGlu Lac30)7@SPIO/DOX.2.Through in vitro cell experiments,we demonstrated the active targeting and uptake mechanism of the micelle on Hep G2 cells,studied its killing ability on cancer cells,and proved that the micelle has the potential to become a new multi-functional imaging probe through MRI imaging.Methods First of all,seven(6-azido-6-deoxy)-β-cyclodextrin was produced by water bath method.Then,β-CD-(N3)7 was synthesized by ring opening polymerization with ε-CL as monomer,β-CD-(N3)7 as initiator and Sn(OCT)2 as catalyst.After a series of chemical reactions,we converted β-CD(PCL)14-(N3)7 into AELG-NCA.After deionization dialysis,collection of dialysate and freeze-drying,AELG-NCA became star polymer β-CD(PCL)14-(-SS-PAELG)7 and β-CD(PCL)14-(PCAELG)7.Finally,β-CD(PCL)14-(-SS-PAELG)7 and β-CD(PCL)14-(PCAELG)7 were added with propargy lactose and propargy galactose respectively to produceβ-CD(PCL)14-(-SS-PGlu Gla)7,β-CD(PCL)14-(PGlu Gla)7,β-CD(PCL)14-(-SS-PGlu La c)7,β-CD(PCL)14-(PGlu Lac)7.β-CD(PCL)14-(-SS-PGlu Gla)7,β-CD(PCL)14-(PGlu Gla)7,β-CD(PCL)14-(-SS-PGlu Lac)7,β-CD(PCL)14-(PGlu Lac)7 was characterized by 1H NMR and FTIR.The surface morphology,particle size and particle size distribution of micelles were measured by DLS and TEM respectively;SPIO and DOX loading rate of micelles were also studied.In vitro,CCK-8 method was used to evaluate the biocompatibility of four micelles to human hepatoma cells(Hep G2).We used the same method to evaluate the killing effect of DOX loaded micelles on Hep G2 cells.Prussian blue staining was used to verify the targeting of the micelles on Hep G2 cells.The uptake and distribution of micelles in cells were studied by DOX fluorescence microscopy,DAPI staining and flow cytometry.Results1.The results of 1H NMR and FTIR confirmed that the above four kinds of nanomicelles were successfully constructed.The particle size of β-CD(PCL22)14-(-SS-PGlu Gla30)7 is about 102.4 nm,that of β-CD(PCL22)14-(PGlu Gla30)7 is about 128.1 nm,that of β-CD(PCL22)14-(-SS-PGlu Lac30)7 is about96.5 nm,that of β-CD(PCL22)14-(PGlu Lac30)7 is about 109.6 nm.2.The results of in vitro MRI showed that the T2 WI signal of four kinds of micelles decreased with the increase of Fe concentration.β-CD(PCL)14-(-SSPGlu Gla)7@SPIO,β-CD(PCL)14-(-SS-PGlu Lac)7@SPIO,β-CD(PCL)14-(PGlu Gla)7@SPIO,β-CD(PCL)14-(PGlu Lac)7@SPIO.Their corresponding T2 relaxation rates are 191.5 femm-1 · s-1,196.5 femm-1 · s-1,185.7femm-1 and 239.4 femm-1s-1,respectively,which proves the excellent T2 contrast effect of the above micelles.3.Cytotoxic test results: after incubation with Hep G2 cells for 48 hours,the cell survival rates of four kinds of blank micelles measured by CCK-8 method were all maintained above 80%.Four kinds of micelles containing DOX and free DOX were respectively incubated with Hep G2 for 48 hours,we found that the cell survival rate was inversely related to the drug concentration.With the increase of DOX concentration,the cell growth inhibition was more significant.Among them,free DOX has the strongest killing effect on Hep G2.The cell inhibitory effect ofreduction responsive micelles containing disulfide bondβ-CD(PCL)14-(-SS-PGlu Gla)7@DOX,β-CD(PCL)14-(-SS-PGlu Lac)7@DOX was higher than that of non reduced micelles without disulfide bondβ-CD(PCL)14-(PGlu Gla)7@DOX,β-CD(PCL)14-(PGlu Lac)7@DOX.4.The results of Prussian blue staining showed that SPIO particles could be effectively introduced into the cells by four micelles,which confirmed the Hep G2 targeting of the above four micelles.5.The results of fluorescence observation showed that the four drug loaded micelles could be quickly absorbed by cells and mainly distributed in the cytoplasm,while the free DOX mainly entered the nucleus.6.Flow cytometry showed that free DOX showed the strongest fluorescence intensity.Compared with the non reduced micelles without disulfide bond β-CD(PCL)14-(-SS-PGlu Gla)7@DOX,β-CD(PCL)14-(-SS-PGlu Lac)7@DOX,the reduction responsive micelles with disulfide bond β-CD(PCL)14-(PGlu Gla)7@DOX,β-CD(PCL)14-(PGlu Lac)7@DOX showed a slightly stronger DOX fluorescence intensity.The difference of DOX fluorescence intensity between lactose and galactose micelles was not significant.Conclusion1.In this study,a novel glycosylation reduction responsive nano diagnostic micelle was successfully prepared,which has many functions such as MRI diagnosis,intelligent release of anti-tumor drugs,active targeting of liver cancer and so on.2.β-CD(PCL)14-(-SS-PGlu Gla)7@SPIO/DOX andβ-CD(PCL)14-(-SS-PGlu Lac)7@SPIO/DOX reduction responsive micelles prepared in this project have uniform spherical shape,small particle size ratio,good biocompatibility,colloidal stability,environmental response and antitumor effect,which can be used in MRI imaging,and have certain potential application prospects.