Preliminary Study On Dual-targeted Liposomes Load Gambogic Acid For Treating Breast Cancer

Breast cancer is one of the most common malignant tumors in women.According to the expression of the receptor,breast cancer is divided into four types:ductal type A,ductal type B,HER-2 type,and triple-negative.Among them,triple-negative breast cancer has problems such as has a high incidence rate,small age of onset,a young age,and Problems such as high recurrence rate,short survival period,unobvious disease characteristics and large metastasis potential have brought certain difficulties to its treatment.Gambogic acid（Gambogic acid,GA）has a variety of biological activities and can cause apoptosis through depolarization of the mitochondrial membrane.It is a commonly used drug for breast cancer,especially triple-negative breast cancer.However,GA also has some problems in its application,such as poor water solubility,poor selectivity,and large toxic and side effects,which limits its application.Therefore,it is urgent to solve the above problems and enhance the application value of GA.Compared with drug carriers such as polymer nanoparticles and polymer micelles,liposomes（Liposome,LP）can keep insoluble drugs in a lipid environment through their phospholipid bilayer structure to solve the problem of low water solubility of drugs.In addition,liposomes have the advantages of low toxicity,good biocompatibility,and non-immunogenicity because their phospholipid structure is similar to the structure of cell membranes.At the same time,liposomes can also improve the distribution of drugs in the body through passive targeting and increase efficiency.Therefore,choosing liposomes as the carrier of GA can improve the solubility of GA and partially reduce the toxicity of GA.However,the passive targeting effect of liposomes lacks selectivity,cannot accurately deliver drugs to the lesion site,and still has a certain toxic effect on normal cells and tissues.Compared with passive targeting active targeting has higher selectivity,can more accurately deliver the drug to the lesion site,and reduce the toxicity of the drug.In this study,A7RC（ATWLPPRC,A）polypeptide was used as a tumor cell targeting peptide,and Triphenylphosphine（TPP）was used as a targeting molecule for intracellular mitochondria to prepare a dual targeting system in order to better inhibit the growth of triple-negative breast cancer cells.In this project,we designed and prepared dual-targeting gambogic acid liposomes that can simultaneously target cancer cells and mitochondria,and investigated its inhibitory effect on breast cancer cells and the inhibitory effect of HUVEC cell formation.The specific research contents of the inhibitory effect of breast cancer cells are as follows:1.Synthesis and methodology establishment of GA derivatives and carrier materialsIn order to synthesize dual-targeted liposomes,this study synthesized A7RC-PEG₂₀₀₀-DSPE and GP by addition reaction and condensation reaction respectively,and characterized their structures by NMR H spectroscopy,infrared spectroscopy,and flight mass spectrometry.In order to detect the content of GA and GP in liposomes,an in vitro analysis method of GA and GP was established.2.Preparation,prescription screening and structural characterization of liposomesIn this study,four liposomes,GA-LP,GA-A-LP,GP-LP,and GP-A-LP were prepared by ethanol injection.The optimal formulation and preparation method of liposomes were screened out based on the indicators of particle size,PDI,potential,and encapsulation rate.3.Evaluation of the inhibitory effect of GA liposomes on breast cancer cells and the inhibition of angiogenesis in vitroMDA-MB-231 cells and MCF-7 cells were selected as model cells.After cytotoxicity experiment,laser confocal experiment,flow cytometry uptake experiment,mitochondrial membrane potential,and reactive oxygen species detection,investigated the inhibitory effect of GA-LP and GA-A-LP,GP-LP,GP-A-LP on breast cancer cells.HUVEC cells were selected as model cells to evaluate the in vitro angiogenesis inhibitory effects of the four liposomes.In the cytotoxicity experiment,the A7RC polypeptide modification group showed anenhanced inhibitory effect in MDA-MB-231 cells;but it did not show an enhanced inhibitory effect in MCF-7 cells.The TPP modified group showed an enhanced inhibitory effect in both types of cells.In the laser confocal and flow cytometric uptake experiments,the A7RC polypeptidemodification group showed higher fluorescence intensity in MDA-MB-231 cells;but it did not show a significant increase in fluorescence intensity in MCF-7 cells.Both cells in the TPP modified group showed an increase in fluorescence intensity.In the detection experiment of mitochondrial membrane potential and intracellular ROS level,the depolarization tendency of the mitochondrial membrane potential of MDA-MB-231 cells affected by the A7RC polypeptide modification group was more obvious,and the ROS level was higher;but the change was not obvious in MCF-7 cells.The TPP-modified group showed more obvious depolarization tendency of mitochondrial membrane potential and higher ROS levels in MDA-MB-231 cells and MCF-7 cells.The results of anti-HUVEC cell angiogenesis show that the number of nodes in GA-LP is 3.23 times that of GA-A-LP,and the number of nodes in GP-LP is 3.03 times that of GP-A-LP,which indicates that A7RC polypeptide can inhibit HUVEC blood vessels generate.In summary,in order to solve the problems of poor water solubility,poor selectivity,and large side effects of GA,the A7RC polypeptide and TPP co-modified GA liposomes（GP-A-LP）with dual targeting properties were prepared,and its physical and chemical properties and effects were investigated,And at the same time examined anti-HUVEC cell angiogenesis effect in vitro,It adds more options for gambogic acid in the treatment of breast cancer,especially triple-negative breast cancer.