Preparation And Anti-Breast Cancer Study Of Gambogic Acid And Paclitaxel Coloaded Folic Acid Modified Albumin Nanoparticles

Breast cancer is the most common malignant tumor in women,which seriously threatens women’s health.The increasing incidence and younger trend make the research on breast cancer treatment more urgent.Usually,chemotherapy is the main method for breast cancer therapy,but its severe side effects and low solubility of chemotherapy drugs seriously affect the therapeutic effect.Therefore,it is of great significance to reduce the toxic side effects of chemotherapy drugs on normal tissues and improve the solubility of chemotherapy drugs in breast cancer therapy.Paclitaxel（PTX）,an active ingredient extracted from the bark of Pacific yew,is one of the most widely used drugs for cancer therapy in clinical practice.However,although paclitaxel has good anticancer activity,the poor water solubility,and the severe toxic side effects by the addition of solubilizers such as polyoxyethylene castor oil limited its further application.Gambogic acid（GA）is a component extracted from the dry resin of the natural Garcinia cambogia.It also has defects such as poor water solubility high toxic and side effects,which limit its application.At the same time,studies have shown that GA can enhance the sensitivity of PTX-resistant breast cancer cells.Therefore,we envisaged that the combination therapy of PTX and GA could improve the therapeutic effect on breast cancer.Combination therapy can make multiple drugs work together,thereby reducing drug dosage,alleviating side effects,overcoming drug resistance of tumor cells,and enhancing curative effect.However,due to the different physiochemical properties and distribution properties of drugs in combination therapy,and the lack of targeting,it is difficult to gather chemotherapeutic drugs at the diseased site.Therefore,it is very important to choose an appropriate carrier for drug delivery.Folate receptor（FR）is overexpressed in cancer cells such as human breast cancer and absent in most normal tissues.Thus,folic acid（FA）is often used as a targeting ligand for tumor cells to improve the active targeting of drug delivery systems.In view of this,a FA-targeted albumin nanoparticle co-loaded with PTX and GA was designed to effectively deliver two poorly water-soluble drugs to the tumor site.The research mainly includes the following four parts:1.Establishment of in vitro analysis methods for PTX and GAIn this study,high performance liquid chromatography was used to quantitatively analyze PTX and GA,and the methodlogical validation was validated.Experimental results showed that the R² of the standard curves of PTX and GA were both greater than0.9990,indicating that PTX and GA had a good linear relationship in the range of1.0～250.0μg/m L.The methodological results showed that the specificity,repeatability,stability,precision,accuracy and sample recovery rate of PTX and GA detected by this method met the relevant requirements,which indicated that this method could be used to detect the contents of PTX and GA.2.Preparation and characterization of FA-PTX/GA-NPsIn this study,anti-solvent method was used to prepare nanoparticles co-loaded with PTX and GA（PTX/GA-NPs）.The particle size of PTX/GA-NPs was 120.6±2.1 nm,and the surface potential was-10.7±0.8 m V.Then,folic acid（FA）was linked to the surface of human serum albumin（HSA）through the transesterification reaction between NHS and protein-NH₂ groups to prepare folic acid-targeted nanoparticles（FA-PTX/GA-NPs）.After the targeting modification of FA,the particle size of FA-PTX/GA-NPs was 130.0±1.4 nm,and the surface potential was-14.3±1.7 m V.The encapsulation efficiencies of FA-PTX/GA-NPs for PTX and GA were 84.22±0.55%and 97.41±0.32%,respectively.Overall,experimental results showed that PTX/GA-NPs and FA-PTX/GA-NPs had uniform particle size,high encapsulation efficiency and drug loading capacity.3.In vitro evaluation of FA-PTX/GA-NPsThe anti-tumor of both PTX/GA-NPs and FA-PTX/GA-NPs were evaluated in vitro.Compared with free drugs,PTX/GA-NPs and FA-PTX/GA-NPs showed higher inhibitory effect on tumor cells;In the cell uptake experiment,the experimental results observed by laser confocal microscopy showed that FA-PTX/GA-NPs had the highest fluorescence intensity for MDA-MB-231 cells with high FR expression,while there was no significant difference for MCF-7 cells with low FR expression.Flow cytometry was used for quantitative analysis of cell uptake.Experimental results demonstrated that MDA-MB-231 cells had the strongest uptake for FA-PTX/GA-NPs.The fluorescence intensity of FA-PTX/GA-NPs-FITC uptake by MDA-MB-231 cells was1.64,1.83,1.88 times that of PTX/GA-NPs-FITC and FA+FA-PTX/GA-NPs respectively.Meanwhile,the uptake of FA+FA-PTX/GA-NPs was weaker than that of FA-PTX/GA-NPs.There was no significant difference for the uptake of PTX/GA-NPs and FA-PTX/GA-NPs in MCF-7 cells.The results showed that FA-modified nanoparticles could be significantly uptaked by cells with high FR expression;In the hemolysis experiment,PTX/GA-NPs and FA-PTX/GA-NPs could reduce the amount of co-solvent,thereby significantly reducing the hemolysis rate,which showed that the toxic and side effects of drugs could be reduced by the encapsulation of nanoparticles.4.In vivo evaluation of FA-PTX/GA-NPsThe in vivo anti-tumor effect of FA-PTX/GA-NPs was evaluated by established heterotopic MDA-MB-231 tumor-bearing nude mice,Experimental results indicated that FA-PTX/GA-NPs had the strongest anti-tumor effect.Compared with the saline group,FA-PTX/GA-NPs group inhibited 74.8%of the growth of tumor.Moreover,in vivo imaging experiments results showed that FA-PTX/GA-NPs could effectively accumulate in the tumor site with good targeting.Furthermore,the results of tissue section showed that PTX/GA-NPs and FA-PTX/GA-NPs had no obvious damage to the tissues and major organs in nude mice,indicating the good biocompatibility of FA-PTX/GA-NPs.In summary,anti-solvent method was used to prepare FA-PTX/GA-NPs in this paper,and demonstrated good tumor inhibition effect and good biosafety both in vivo and in vitro,which indicated that FA-PTX/GA-NPs was a potential drug delivery system for the treatment of breast cancer.