The Research Of PH Sensitive Lipid-polymer Hybrid Nanoparticles In The Treatment Of Breast Cancer

Objective:The purpose of our work is to prepare a noval pH-sensitive lipid–polypeptides hybrid nanoparticles(LPNs)loaded Doxorubicin(DOX),and evaluate its pH-sensitivity and the therapeutic effect on breast cancer.Content:The LPNs were prepared by a certain ratio of egg yolk phosphatidylcholine(EPC),distearoyl-phosphatidylethanolamine-poly(ethylene glycol)(DSPE-PEG),and polyhistidine(PHIS),loaded with DOX and modified with CRNGRGPDC(iNGR).The size,size distributions,zeta potential,encapsulation efficiency(EE%)and loading efficiency(LE%)were measured.The pH-sensitivity of the LPNs was evaluated,including the changes of size,zeta potential,as well as the drug release profile under different p H.Then their antitumor activities in vitro and in vivo were evaluated.Methods:Preparation of the drug delivery system: The iNGR was conjugated to DSPE-PEG by amide reaction to obtain DSPE-PEG-i NGR.The LPNs,iNGR-LPNs,LPNs/DOX and iNGR-LPNs/DOX were prepared by a single-step nanoprecipitation method.The liposomes as the control group were prepared by thin-film dispersion method.Characterization of drug delivery system: The hydrodynamic size,size distribution and zeta potential of LPNs were detected by a laser particle size analyzer.The morphology and size of LPNs were observed bytransmission electron microscopy(TEM).EE% and LE% of LPNs were determined by high performance liquid chromatography(HPLC).The pH-sensitivity of LPNs was evaluated by the change of size,zeta,the fluorescence of pyene that loaded into the LPNs and drug release behavior at different pH.Evaluation of drug delivery system in vitro: The celluar uptake of LPNs/DOX,iNGR-LPNs/DOX,Lipo/DOX and free DOX at different pH values were assessed by flow cytometry.The inhibitory effect of different drug delivery system to rat breast cancer cell was investigated by MTT assay.The half inhibition concentration(IC50)in each case was calculated.Evaluation of drug delivery system in vivo: The tumor targeting of the drug delivery system was evaluated by an in vivo imaging system using high metastatic murine breast cancer 4T1 mice as model.The therapeutic effect of the drug delivery system was evaluated by pharmacodynamic experiment,including change of tumor size,Immunohistochemical CD 31 staining and Ki 67 staining.The change of mice's body weight and H&E staining of mice's heart were carried out for systemic toxicity and cardiotoxicity.Results:The average particle size of LPNs was 109.70 ± 7.6 nm with a polydispersity index of 0.13±0.01.Their average zeta potential was-4.57 ± 1.24 mV.The DOX loading capacity and encapsulation efficiency of LPNs were 9.08±0.33 % and 84.59±2.19 % respectively.The LPNs was in spherical shape according to the observation of TEM.The pH-sensitivity experiment demonstrated that with the decrease of pH,the size of LPNs gradually increased with PDI increase,the zeta potential reversed to positive,and the fluorescence intensity of prene entrapped in LPNs underwent a sharp decrease at about p H 6.5.The result of drug release indicated that 80% of the DOX was released within 24 h at pH 5.5,50% of the DOX was released at p H 6.5,while the DOX release was only 20% at pH 7.4.The modification of iNGR did not influence the drug release behavior.The size distribution and zeta potential of LPNs/DOX had no obvious change after storing at 4 oC for 15 days.In the in vitro cellular tests,the cellular uptake of LPNs/DOX was 4.90 to 7.22 folds compared to Lipo/DOX under pH 7.4 or 6.8,respectively.The cellular uptake of iNGR-LPNs/DOX was dramatically increased to 1.23 and 1.43 folds compared to that of LPNs/DOX under pH 7.4 and 6.8,respectively.The half maximal inhibitory concentration(IC50)value of LPNs/DOX was 1.926 ?g/mL,lower than that of Lipo/DOX(4.188 ?g/mL).The IC50 of iNGR-LPNs/DOX(1.140 ?g/mL)was lower than that of LPNs/DOX,and close to that of free DOX(1.127 ?g/mL).In the investigation of tumor targeting effect observed by a living imaging system,LPNs/DiR and iNGR-LPNs/DiR were both accumulated into tumors 3 h after administration.Also,the fluorescence intensity of the group treated by iNGR-LPNs/DiR was stronger than that of the group treated by LPNs/Di R at each time point.The fluorescence intensities of the heart,liver,spleen,lung and kidney in the mice treated by i NGR-LPNs/Di R were stronger than that in the mice treated by LPNs/DiR.In vivo antitumor efficacy tests demonstrated that the suppressive ability of tumor growth: iNGR-LPNs/DOX > LPNs/DOX > Lipo/DOX > DOX > PBS;the mean survival rate: i NGR-LPNs/DOX(49 days)> LPNs/DOX(45 days)> DOX(41 days)> PBS(39.5 days)> Lipo/DOX(35 days).The results of CD 31 and Ki 67 stianing showed that the brown color represented new vessels and proliferation of cell in the mice treated by LPNs/DOX and iNGR-LPNs/DOX were less than that in the mice treated by PBS,DOX and Lipo/DOX.The systemic toxicity evaluation showed that the body weight of the mice treated by Lipo/DOX changed a lot,as high as 26%.While the body weight of the mice in other groups had no obvious change.The results of H&E staining showed that the myocardial cell of the mice treated by Lipo/DOX and DOX was intumescentia,lytic and disordered arrangement,indicating a certain cardiotoxicity;while there was no obvious change in the mice treated by LPNs/DOX,iNGR-LPNs/DOX and PBS.Conclusions:The generated iNGR-LPNs/DOX displayed pH sensitivity and active targeting to tumor.This drug delivery system showed better tumor cell inhibition ability,cellular uptake ablity,tumor growth inhibition ability and lower toxicity compared to Lipo/DOXTherefore,DOX-loaded iNGR-LPNs had potential to be a promising vector in the treatment of breast cancer.