Study On The Anti-tumor Effect Of Polypeptide-modified Liposome Loaded With Gambogic Acid

Cancer is one of the leading causes of death worldwide.Among female patients,breast cancer is the most frequently diagnosed and the leading invasive cancer in terms of incidence;among all cancer patients,breast cancer is the second leading cause of death lung cancer.Gambogic acid(GA)is a natural anti-tumor compound obtained from gamboge resin,which has been shown to perform anti-proliferative effects on several tumor cells(e.g.,breast cancer,prostate cancer,lung cancer,and pancreatic cancer),and able to block the cell cycle,induce apoptosis,and prevent the development of cancer cells.However,poor solubility,short half-life and low bioavailability in vivo have greatly limited its clinical application.liposome is a drug delivery system widely used in tumor therapy,which can alter the biodistribution and clearance rate of drug molecules in vivo.In this study,we constructed a liposome system loaded with GA modified with cell-penetrating and tumor-targeting peptide,and performed a comprehensive physicochemical characterization,in vivo and in vitro evaluation of this nano-delivery vehicle.Hydrogenated soybean phospholipids(HSPC),cholesterol and DSPE-m PEG2000 were used as membrane materials and gambogic acid(GA)was the model drug to fabricate the liposome loaded with gambogic acid(GA-Lip)by thin film hydration method.Evaluated the encapsulation rate,particle size,PDI and Zeta potential.The results showed that the fabricated nanocarriers were yellow opalescent,with small particle size and uniform distribution,and high encapsulation capacity.Established the analytical method to check the encapsulation rate of GA-Lip,which showed good linearity in the linear range,good method reproducibility and sample stability within 12 h.The particle size,PDI and zeta potential of R9 d GR/CB5005N-GA-Lip were determined using a Malvern laser particle size tester,and the results showed that the nanocarriers had small particle size,uniform distribution and positive surface charge.The appearance and morphology of the nanoformulations were examined by transmission electron microscopy(TEM),and the TEM images showed that the formulations were all rounded and spherical in shape,with good dispersion and uniform size distribution.The presence sate of GA in the liposome was investigated by differential scanning calorimetry(DSC),the results showed that GA was encapsulated in the liposome in a non-crystalline state.The blood safety of the nanocarriers was investigated using hemolysis test,and the results showed that the nanocarriers were non-hemolytic and had good biocompatibility and blood safety.Coumarin-6 loaded liposome(Cou-6-Lip)was prepared by using Cou-6 as a fluor escent probe instead of GA encapsulated in the liposome.The fabricated Lip labeled with cou-6(Cou-6-Lip,CB5005N-Cou-6-Lip,R9 d GR-Cou-6-Lip,R9 d GR/CB5005N-Cou-6-Lip)was co-incubated with human breast cancer cells(MDA-MB-231)and mouse br east cancer cells(4T1),and the fluorescence intensity in each group of cancer cell lin es was quantitatively analyzed using cell high connotation imaging system.The resultsof co-incubation for 4h showed that,compared with free Cou-6,Cou-6 liposome perf ormed a significantly increased cell uptake on MDA-MB-231 and 4T1 cells,and the f luorescence intensity of the peptide-modified Lip group was significantly stronger thanthat of the unmodified grou-p.The results indicated that peptide-modified Lip could promote the uptake of drug and perform good tumor targeting in vitro.Cytotoxic study of gambogic acid solution(GA-Sol),gambogic acid loaded liposome(GA-Lip),and peptide-modified gambogic acid loaded liposome(CB5005N-GA-Lip,R9 d GR-GA-Lip,R9 d GR/CB5005N-GA-Lip)was performed on MDA-MB-231 cells and 4T1 cells.The CCK-8 was used to detect the cytotoxic effects on two cell lines.The cytotoxicity results showed that the toxicity of GA on tumor cells showed concentration dependence,the survival rate of MDA-MB-231 cells and 4T1 cells decreased with the increase of GA concentration,and the cytotoxicity of peptide-modified Lip was greater than that of unmodified Lip and GA solution groups.The breast cancer mouse model was constructed by subcutaneously injecting mouse tumor 4T1 cells into BALB/c nude mice,and the In-vivo Imaging System(IVIS)for small animals was used to examine the distribution within the tumor of Di R solution(Di R-Sol),Di R loaded liposome(Di R-Lip),and peptide-modified Di R loaded liposome(CB5005N-Di R-Lip,R9 d GR-Di R-Lip,R9 d GR/CB5005N-Di R-Lip)in BALB/c nude mice,to evaluate the tumor targeting of the peptide-modified agents.The results showed that no obvious fluorescence signal at the tumor site of mice in the Di R-Sol group throughout the imaging process,and the fluorescence intensity at the tumor site of the peptide modified Lip group was higher than that of the unmodified group,and the fluorescence signal was still clearly visible after 24 h.This indicates that Lip can improve the circulation time of Di R in vivo,and peptide R9 d GR/CB5005 N perform some potential in improving the tumor targeting of drug.