| Objective: To prepare perillyl alcohol(POH)-Distearoyl Phosphoethanolamine(DSPE)-polyethylene glycol(PEG)-triphenyl phosphate(TPP)-liposomes(DSPE-PEG-TPP-POH-Lip),and investigate its physicochemical characteristics,mitochondrial targeting and the inhibitory effect on breast cancer MDA-MB-231 cells.Methods: The POH content was quantitatively determined in vitro through High Performance Liquid Chromatogram(HPLC);DSPE-PEG-TPP-POH-Lip was prepared by thin film dispersion method,and the encapsulation efficiency(EE%)was used as the index to conduct single factor investigation,orthogonal design experiments to optimize the formulation,and analyzed the physicochemical characteristics.In the in vitro antitumor experiments,to evaluate the mitochondrial targeting and tumor-inhibiting effect of the drug-loading system through the CCK-8,cloning assay,cell scratch assay,Transwell,apoptosis assay,JC-1 staining assay and Western blot assay.Results: The HPLC system in vitro content detection conditions of perillyl alcohol were established as follows: the mobile phase/water:acetonitrile(60:40,v/v)and with a flow rate of 1m L/min.The detector wavelength of 210 nm and injection volume is 20μL was used to analyse POH.The optimal prescription process of DSPE-PEG-TPP-POH-Lip was as follows: the phospholipid concentration was 20 mg/m L,the mass ratio of phospholipid-drug was1:10,the mass ratio of phospholipid-cholesterol was 4:1.The DSPE-PEG-TPP-POH-Lip was spherical in shap with no aggration or fusion.The average particle size of liposomes was(111.0±0.8)nm,the PDI was(0.185±0.006),and the EE% was(80.6±1.8)%;The released of two liposomes were slower than the free POH solution,and exhibited a certain delayed-release effect;After 30 d of 4℃,The particle size of DSPE-PEG-TPP-POH-Lip increased from 95.7 nm becomes 98.0 nm,the encapsulation efficiency decreased from 80.9% becomes 78.1%.The particle size of DSPE-PEG-POH-Lip increased from 101.3 nm becomes106.7 nm,and the encapsulation efficiency decreased from 81.2%becomes 78.2%.The particle size and encapsulation efficiency of the two liposomes changed little.The results of CCK-8 cytotoxicity test showed that the IC50 of DSPE-PEG-TPP-POH-Lip was 63.32 μg/m L,which had a good effect on inhibiting the growth of MDA-MB-231 cells;In the colony formation experiment,the number of cell colonies in the experimental group was reduced and scattered,compared with the control group,the formation of cell colonies in DSPE-PEG-TPP-POH-Lip group was significantly reduced and has significant statistical significance;The scratch test results showed that the migration ability of MDA-MB-231 cells in DSPE-PEG-TPP-POH-Lip group was significantly weakened;In the invasion experiment,the DSPE-PEG-TPP-POH-Lip group had obvious inhibitory effect on cell invasion;In the JC-1 staining experiment,The results of DSPE-PEG-TPP-POH-Lip group showed that the green fluorescence was significantly enhanced and the red fluorescence almost completely disappeared.The mitochondrial membrane potential was greatly depolarized,and the targeting effect of TPP on the mitochondria increases the accumulation concentration of POH in the mitochondria,which enhances the cytotoxic effect;In Western blot results,DSPE-PEG-TPP-POH-Lip can significantly increase the expression of pro-apoptotic P53,Bax,Caspase-3 and reduce the expression of anti-apoptotic Bcl-2.Conclusion: In the current investigation,the DSPE-PEG-TPP-POH-Lip was prepared by thin film dispersion method,and the properties of liposomes,including particle size,EE,release profile and stability,can meet the potential requirement of breast cancer therapy based on the in-vitro experiment.DSPE-PEG-TPP-POH-Lip can aggregate POH into mitochondria,activate mitochondrial apoptosis pathway,trigger apoptosis,effectively inhibit the proliferation of MDA-MB-231 breast cancer cells,and finally enhance the anti-tumor effect. |
PDF Full Text Request