| Breast cancer is the most common malignant tumor in women,which seriously threatens women's life and health.Triple-negative breast cancer(TNBC)is a type of breast cancer that lacks the expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2).TNBC is prone to early recurrence and distant metastasis,showing the worst survival rate in all breast cancer subtypes.TNBC is not sensitive to endocrine therapy and lacks effective targeted drugs.Therefore,chemotherapy is the most effective systemic treatment for TNBC.It is especially important to find effective ways to reduce early recurrence and distant metastasis of TNBC.Doxorubicin(DOX)is an anthracycline-type chemotherapeutic drug with broadspectrum anti-tumor activity,which commonly used as the second-line chemotherapy of breast cancer.However,DOX has shortcomings such as short half-life,poor targetability,and serious side effects,which limit its further application.The tumorigenic ability and migration ability of triple negative breast cancer cells are highly correlated with the overexpression of aldose reductase AKR1B1.Epalrestat(EPS),an aldose reductase AKR1B1 inhibitor,can inhibit epithelial-mesenchymal transition(EMT)in TNBC cells,thereby reducing tumor cell tumorigenic capacity and inhibiting tumor migration.However,the application of EPS is limited by its poor water solubility,low bioavailability of oral administration,and non-selectivity to tumor cells.Therefore,it needs a reliable drug delivery system to tumor cells when EPS treats the migration of TNBC.In this paper,a targeted liposome was designed for simultaneously delivering EPS and DOX with different physical and chemical properties to the tumor site,establishing a drug delivery system with anti-tumor activity and anti-tumor migration activity.And its inhibition of growth and migration of TNBC were also investigated.The research thesis mainly includes the following four parts: 1.Establishment of in vitro analytical methods for EPS and DOXIn this study,high-performance liquid chromatography was used to quantitatively analyze EPS and DOX.And the methodological verification was performed.The R2 of EPS and DOX standard curve was greater than 0.9990,indicating the good linear relationship of EPS at 2.0~120.0 ?g/m L and DOX at 10.0~400.0 ?g/m L.The methodological verification results showed the good specificity of EPS and DOX.The RSD of the precision and the recovery rate was both less than 5.00%,which met the relevant requirements.Therefore,the method satisfied the requirement of detecting the concentration of the EPS and DOX.2.Preparation and characterization of HA-LP-EPS/DOXIn this study,Egg PC,Chol,and DOTAP were used to prepare LP-EPS by ethanol injection.EPS was incorporated in the hydrophobic region of liposome,and subsequently DOX in lipid via active drug loading,preparing the EPS and DOX-loaded liposomes(LP-EPS/DOX).The LP-EPS/DOX had a particle size of 98.2±2.3 nm and a surface potential of 26.3±0.41 m V.HA-LP-EPS/DOX was prepared by hyaluronic acid modification of LP-EPS/DOX by electrostatic adsorption.The surface potential of HALP-EPS/DOX was reversed,and the particle size increased slightly.HA-LP-EPS/DOX had a particle size of 116.4±3.4 nm and a surface potential of-10.9±0.51 m V.The encapsulation efficiency of EPS and DOX was 97.33% and 90.01%.The results show that LP-EPS/DOX,HA-LP-EPS/DOX have suitable particle diameters,which are suitable for in vivo research.3.In vitro evaluation of HA-LP-EPS/DOXIn this study,the in vitro activity of LP-EPS/DOX,HA-LP-EPS/DOX were evaluated.In the carrier toxicity assay,the empty liposome LP and HA-LP showed almost no cytotoxicity,which proved that the two liposomes were safe drug delivery vehicles and could be used for subsequent research.In the cytotoxicity assay,HA-LPEPS/DOX showed stronger growth inhibition on MDA-MB-231 cells and BT-549 cells,compared with LP-EPS/DOX.In the cellular uptake assay,the fluorescence intensity of cells was increased with the prolonged incubation time,which were observed by laser confocal microscopy.The HA-LP-EPS/DOX had the highest fluorescence intensity under the same incubation time.The cellular uptake was quantified by flow cytometry.HA-LP-EPS/DOX had the highest cellular uptake.The fluorescence intensity of HA-LP-EPS/DOX was 1.79,1.19 times of EPS/DOX,LP-EPS/DOX(MDA-MB-231 cells),and 1.43,1.19 times of EPS/DOX,LP-EPS/DOX(BT-549 cells),respectively,after incubation for 4 h.In the in vitro anti-migration assay,HA-LP-EPS/DOX had the strongest anti-cell migration effect,which inhibited 71.1%,68.6%,52.9%(MDA-MB-231 cells),and 77.5%,74.2%,55.8%(BT-549 cells)of cell migration,compared with the control group,EPS/DOX group,LP-EPS/DOX group,respectively.4.In vivo evaluation of HA-LP-EPS/DOXTo evaluate the in vivo anti-tumor effect of HA-LP-EPS/DOX,the MDA-MB-231 cell,the nude mice tumor-bearing model was established.Results indicated that HALP-EPS/DOX had the strongest anti-tumor activity,which inhibited 81.1%,65.5% and 40.5% of tumor volume growth,compared with the control group,EPS/DOX group and LP-EPS/DOX group,respectively.The in vivo imaging results showed that HA-LPEPS/DOX could effectively accumulate in the tumor site and had higher targeting efficiency after hyaluronic acid modification.The histological sections showed that the package of liposomes significantly reduced the toxicity of free drugs.HA-LPEPS/DOX showed no obvious damage to the main organs of nude mice.The immunohistochemistry results showed that HA-LP-EPS/DOX could up-regulate the expression of E Cadherin,while down-regulating the expression of Vimentin in tumor tissues.HA-LP-EPS/DOX inhibited cell epithelial-mesenchymal transition and had in vivo antitumor migration activity.In summary,the HA-LP-EPS/DOX delivery system was established in this paper.The in vivo and in vitro experiments showed that HA-LP-EPS/DOX had both antitumor activity and anti-tumor migration activity.The simultaneous delivery of EPS and DOX by hyaluronic acid-modified liposomes is a potential strategy for the treatment of TNBC. |
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