Preparation And Evaluation Of Tumor Microenvironment Response Multistage Nanoparticles For Epirubicin Delivery And Deep Tumor Penetration

Tumor targeting nano-drug delivery system,one of the methods for the treatment of solid tumors,can improve the distribution of drugs in tumor tissue and tumor cells.However,extracellular matrix(ECM)and high fluid pressure(IFP)within the solid tumor limits the penetration of drugs into the solid tumor.To further enhance the tumor penetration efficiency,we developed a tumor-microenvironment-responsive multistage drug delivery system which was formed layer by layer via electrostatic interaction with cationic drug-loaded nanoparticles,hyaluronidase(HAase)and iRGD-modified gelatin(G-iRGD).Amphiphilic triblock polymer mPEG-b-poly(2-diisopropylaminoethyl methacrylate)-b-poly(2-guanidinoethyl methacrylate)(PEDG,mPEG-PDPA-PG)was synthesized by RAFT polymerization.The EPI-loaded nanoparticles(NPs-EPI)was formed by self-assembling PEDG and encapsulation with epirubicin(EPI).Polymer was identified by ~1HNMR and gel permeation chromatograph(GPC);the morphology and structure of NPs-EPI was observed by transmission electron microscope(TEM);the particle size and potential of NPs-EPI were measured by Malvern Zetasizer Nano instrument;The loading efficiency and encapsulation efficiency of EPI were determined by microplate reader;Drug release from NPs-EPI under different pH conditions was investigated by dialysis;MTT method was used to investigate the cytotoxicity of NPs/HAase/G,NPs-EPI,EPI with HepG2 cells.The penetration efficiency of NPs/EPI and NPs-EPI/HAase to HepG2 tumor sphoreid were investigated by confocal microscopy.The distribution of the NPs-EPI in HepG2 cells was investigated by fluorescence labeling.The cellular uptake of NPs-EPI/G and NPs-EPI/G-iRGD in HepG2cells were investigated by flow cytometry.In vivo penetration efficiency of NPs-EPI and NPs-EPI/HAase were investigated by frozen section.Living animal imaging system was used to observe the in vivo distribution of NPs-EPI、NPs-EPI/G and NPs-EPI/G-iRGD.The results showed that the Polymer was synthesized successfully.NPs-EPI was regular spherical particles which the average particle size was 85.1±0.2nm and the average potential was 39.9±0.6mV;the loading efficiency and encapsulation efficiency of EPI were 4.4%and 31.7%respectively.The 72h cumulative release ratio of EPI fromNPs-EPI was 50%under pH 7.4,whereas that under pH 5.5 was 82%which showing a certain pH dependence.This NPs/HAase/G-iRGD delivery system show great biocompatibility in vitro,confirmed by MTT method.In vitro sphericaltumorpenetration investigation and in vivo tumor permeability investigation show NPs-EPI/HAase could significantly enhance its penetrating efficiency.The flow cytometry studies show that NPs-EPI/G-iRGD was more easily taken up by HepG2 cells than NPs-EPI/G,which reveal the active targeting ability of iRGD.Intracellular distribution studies show that NPs-EPI could escape from the lysosomal more as the incubated time went on.NPs-EPI/G-iRGD was more likely to be concentrated in the liver than NPs-EPI,NPs-EPI/G and have a certain enrichment in tumor as time went on.