| Breast cancer is one of the most common malignant tumors,and the mortality rate of female patients suffering breast cancer in China is arising.Conventional chemotherapy and radiotherapy are hard to eliminate all cancer cells,which will eventually lead to drug resistance,tumor metastasis and relapse of disease.In recent years,it has been found that salinomycin(SAL)is capable of killing tumor cells efficiently,but its poor water solubility and strong toxic side effects limit the clinical application.In this study,liposome was used as a carrier for SAL,which can target tumor tissues with increased drug delivery efficiency and decreased nonspecific toxicity.Furthermore,the liposomes were modified with a targeting peptide iRGD,which can enhance the targeting ability of the drug towards breast cancer cells through integrin/neuropilin-1(NRP-1)mediated endocytosis.This paper includes the following parts:In the first part of our study,we established a method for the determination of salinomycin in solution by high performance liquid chromatography(HPLC),and verified specificity,precision and accuracy of the method through the standard curve.The linear equation is A=2429c+35214,R~2=0.999,and the fitting degree is high;the intra-day precision and inter-day precision at high,medium and low concentration are less than 5%respectively.The accuracy range is 96.16%-103.14%,and the extraction recovery rate is between 95.19 and 99.54%.Compared with pre-column or post-column derivating method,this method is easy,fast and highly reproducible for the determination of SAL content in vitro.In the second part,iRGD-PEG2000-DSPE was successfully synthesized and deterimined by high-resolution mass spectrometry,and iRGD-Lip-SAL was prepared with soybean phospholipids and cholesterol by thin film dispersion method.In order to evaluate the potential role of iRGD-PEG2000-DSPE,we also synthesized Lip-SAL,an untargeted liposome.The particle size,surface charge,morphology,encapsulation efficiency and drug release profile of nanoformulations were determined.The results showed that the particle size of liposomes was about 100 nm,and the encapsulation efficiency was higher than 67%,and it also had a relatively sustained release profile.These studies established the fundamental basis for further experiments.In the third part,we examined the cytotoxicity of iRGD-Lip-SAL in vitro.After determining that Nrp-1 receptors were expressed on the surface of MDA-MB231 and MCF-7 cell membranes,we measured the uptake efficiency of MDA-MB231 cells towards iRGD-Lip-SAL and Lip-SAL.The results showed that iRGD could increase the uptake efficiency of cells and enhance the anti-tumor effects.In the MTT assay,we selected three cell lines,MDA-MB231,MCF-7 and MCF-7/ADR.The results showed that the inhibition rate of iRGD-Lip-SAL was higher than that of SAL and Lip-SAL.In the apoptosis experiment,even at the concentration of 10?mol/L,the inhibition of tumor cells by iRGD-Lip-SAL was apparently higher than free SAL at a concentration of 20?mol/L.We also used Transwell chamber as an experimental model to test the penetration ability of iRGD.The experiment proved that the liposomes modified with iRGD could not only improve the targeting ability of drugs,but also enhance the permeability of drugs into tumor tissues and cells.In the fourth part,a breast cancer animal model was established by subcutaneous injection of MCF-7 cells in the back of Balb/c mice.The mice were randomly divided into iRGD-Lip-SAL,Lip-SAL,SAL and saline.The body weight,tumor volume,and survival time of all animals were examined.The results showed that the average volume of iRGD-Lip-SAL group was 698.91±253.36 mm~3 and that of Lip-SAL group was 969.59±449.39 mm~3,normal saline group was 1289.03±384.67 mm~3 with significant statistic difference(P<0.05).The iRGD-Lip-SAL group could reduce the toxicity of SAL,improve the efficiency of treatment,and have promising anti-tumor effect in vivo. |
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