Study On Cathepsin-responsive And Liver-targeting GA Modified PEG-GFLG-PEI-PLGA Nanoparticles

A liver-targeting and cathepsin B–responsive nano-carrier,glycyrrhetic acid(GA)modified PEG-GFLG-PEI-PLGA nano-carrier was constructed,which could achieve target-controlled release of the drug in hepatocellular carcinoma(HCC)by the liver-targeting effect of GA and the response of GFLG to cathepsin B over-expressed in the tumor tissue.It could enhance the selectivity,reduce toxicity and side effects,and increase the therapeutic effect of the chemotherapeutic agents.Based on our group's earlier research work about the synthesis of glycyrrhetinic acid(GA)modified PEI-PLGA(GA-PEI-PLGA,GPP),here,PEG was connected to GPP with cathepsin B-responsive peptide GFLG as a linker,to sythesize GA modified PEG-GELG-PEI-PLGA(PGPP)with different molar ratio of PEG to GPP(1:2 and 1:3).PGPP was confirmed by FTIR and 1H-NMR.The ninhydrin colorimetric method was used to determine the degree of substitution(DS)of PLGA,GA and PEG in PEI.The DS of PLGA,GA and PEG in PGPP(1:3)were 1.01 mol,6.84 mol and 3.70 mol,respectively;which in PGPP(1:2)were 1.06 mol,5.82 mol and 10.95 mol,respectively.PGPP(1:2)and PGPP(1:3)were readily soluble in anhydrous ethanol,chloroform,dimethyl sulfoxide and methanol.They could self-assemble in water,phosphate buffer solution(PH7.4 PBS)and methylene chloride to form nanoparticles.The particle size and Zeta potential of PGPP(1:2)nanoparticles were(254.27±2.82)nm and +(31.73±3.85)m V,respectively;which of PGPP(1:3)nanoparticles were(202.13±3.09)nm and +(32.33±1.18)mV,respectively.PGPP nanoparticles were uniform particles with hydrophilic surface,keeping them stable in aqueous solution,and not easy to aggregate and precipitate.Both PGPP(1:2)and PGPP(1:3)had low critical aggregate concentration(CAC),which could maintain their high stability during dilution.The response of PGPP nanoparticles to cathepsin B was investigated by determining the change of particle size.The results showed that PGPP nanoparticles responsed well to cathepsin B,and their responsiveness were attributed to GFLG.The safety of PGPP was evaluated by hemolysis experiment,MTT assay and serum stability.The hemolysis rate of PGPP(1:2)and PGPP(1:3)at the concentration of 0.5 mg/m L were less than 2%,which met the requirements of biomaterials,indicating their good hemocompatibility.They showed slight toxic on L929,HepG2,and LO2 cell lines,indicating their good safety.PGPP(1:2)and PGPP(1:3)nanoparticles showed good stability in serum without obvious protein adsorption.Using hydroxycamptothecin(HCPT)as a model drug,HCPT/PGPP nanoparticles were prepared by emulsifying-solvent evaporation method.The drug-loaded nanoparticles were uniform,had high entrapment efficiency,drug loading capacity and stability,which could be easily resuspended in aqueous solution after freeze-drying.The release behavior of HCPT/PGPP nanoparticles was investigated in different pH media with or without cathepsin B.The results showed that HCPT/PGPP nanoparticles could effectively prolong the release of HCPT.The presence of cathepsin B incereased the release rate of HCPT from the drug-loaded nanoparticles,indicating that PGPP nanoparticles had good cathepsin B-responsiveness.The inhibitory effects of HCPT/PGPP nanoparticles on HepG2 hepatocellular carcinoma cells,BEL-7402 hepatocellular carcinoma cells and normal liver LO2 cells were investigated by MTT method.The results showed that,compared with the HCPT solution group,the inhibitory effects of HCPT/GPP nanoparticles and HCPT/PGPP nanoparticles on HepG2 cells and BEL-7402 cells were stronger than that of LO2 cells,indicating that HCPT/GPP nanoparticles and HCPT/PGPP nanoparticles had a certain targeting effect on liver cancer.Cathepsin B inhibitors could attenuate the cytostatic effects of HCPT/PGPP nanoparticles,suggesting that cathepsin B is one of the key factors that ensured HCPT/PGPP exert its strong cytostatic activity.Laser confocal microscopy was used to observe the uptake of HCPT solution,HCPT/GPP nanoparticles and HCPT/PGPP nanoparticles by HepG2 cells.The results showed that HCPT was more easily taken up by HepG2 cells after being loaded into PGPP nanoparticles.In addition,liver targeting effect of drug-loaded PGPP nanoparticles was also evaluated by in vivo distribution experiment.The results confirmed that the encapsulation of HCPT into PGPP nanoparticles could prolong the blood circulation time,improve the bioavailability and avoid the release of drugs before reaching the target.HCPT/PGPP nanoparticles had good liver targeting effect,could reduce the toxicity of other tissues and prolong the time of drug action.PGPP nanoparticles are ideal liver-targeting nanocarriers.