Enzyme-responsive Peptide Dendrimer Self-Assembly With Nanoscale For Drug Delivery

In the treatment of cancer,chemotherapy drugs are widely used.However,immune suppression,bone marrow suppression,liver and kidney damage and other toxic and side effects widely exixted which caused by poor selectivity.In addition,poor solubility and rapid removal by RES should also be considered.EPR effect is the gold standard for targeted delivery of antitumor drugs.Peptide dendrimer can deliver drugs through covalent linkage or(and)internal void.PEGlated peptide dendrimer also can self-assembly with nanoscale.PEG modification can prolong the circulation time,enhance the EPR effect,hydrophilic,and reduce the toxicity of the delivery materials,but disadv antage endocytosis.CB responsive GFLG can achieve the precise release of drug in cells,MMP-2 responsive PVGLIG can detach PEG.In order to solve this problem,we design an enzyme-responsive peptide dendrimer self-assembly with nanoscale for anti-tumor drugs delivery:Methods1.Cathepsin B responsive peptide dendrimer drug delivery system1)The synthesis of N3-GFLG-GEM Boc-GF-OMe was obtained through dehydration condensation reaction of Boc-Gly-OH and the H-Phe-OMe·HCl,deprotection of OMe,Boc-GF-OH was obtained.Boc-LG-OMe was obtained as the same method by Boc-Leu-OH and H-Gly-OMe·HCl,deprotection of Boc,H-LG-OMe was obtained.Boc-GFLG-OMe was obtained through dehydration condensation reaction of Boc-GF-OH and H-LG-OMe,deprotection of Boc,then reaction with 4-azidobenzoic acid,N3-GFLG-OMe was obtained.Deprotection of OMe,then reaction with GEM·HCl,N3-GFLG-GEM was obtained.2)The synthesis of Boc-G3(Lys-Glu)-OMe Boc-Lys(Boc)-OH reaction with 2,2',2''-Tria Minotriethyla Mine,Boc-G1(Lys)was obtained;Deprotection of Boc,then reaction with Boc-Lys(Boc)-OH,Boc-G2(Lys)was obtained;Deprotection of Boc,then reaction with Boc-Glu(OMe)-OH,Boc-G3(Lys-Glu)-OMe was obtained.3)The synthesis of PEG-G3(Lys-Glu)-Boc Deprotection of OMe,then reaction with 2-Propynylamine,Boc-G3(Lys-Glu)-Alkyne was obtained;Rreaction with N3-PEG through the Cu+ catalytic click-reaction to get PEG-G3(Lys-Glu)-Boc.4)The synthesis of PEG-G3-GFLG-GEM Deprotection of Boc,then reaction with 5-Hexynoicacid,PEG-G3(Lys-Glu)-Alkyne was obtained;Reaction with N3-GFLG-GEM through the Cu+ catalytic click-reaction to get PEG-G3-GFLG-GEM.5)PEG-G3-GFLG-GEM co-loading DOX Dissolve PEG-G3-GFLG-GEM and DOX with DMSO,added 2ml water into the centrifuge tube,drop DMSO solution under ultrasonic probe,the PEG-G3-GFLG-GEM:DOX mass ratio was 2:0?2:0.2?2:0.5?2:1?2:2?2:4,shaking 12 h,5000rpm centrifugation for 5 min,1.8 m L from the supernatant,lyophilization,dissolve with 1.8 m L water.6)The preparation and characterization of nanoscale co-loading materials Preparation co-loading materials with previous method as mass ratio 1:1.The particle size and Zeta potential were measured by Malvin zen3690 and TEM.7)Measurement of drug-loading rate Weighed co-loading materials,with the concentration of 1mg/ml,Abs was measured after diluted 5 times.8)Drug release test Co-loading materials buffer solution was added to the dialysis membrane in p H=5.4?37??papaya protease added.Sampling at different time,test with HPLC.9)Cytotoxicity experiment 96 well plates of 4T1 cells was added different concentrations of GEM?DOX?and co-loading materials.Cell viability was measured by CCK-8 and ELIASA.2.MMP-2 and Cathepsin B dual responsive peptide dendrimer drug delivery s ystem1)The synthesis of PEG-PVGLIG-Alkyne Boc-PV-OMe was obtained through dehydration condensation reaction of Boc-Pro-OH and H-Val-OMe·HCl,deprotection of OMe,reaction with H-Gly-OMe·HCl,Boc-PVG-OMe was obtained.Boc-LIG-OMe was obtained as the same method by Boc-Leu-OH,H-Ile-OMe·HCl and H-Gly-OMe·HCl.Boc-PVG-OMe deprotection of OMe,Boc-LIG-OMe deprotection of Boc,Boc-PVGLIG-OMe was obtained through dehydration condensation reaction.Deprotection of OMe,reaction with Propargylamine,Boc-PVGLIG-Alkyne was obtained.Deprotection of Boc,then reaction with m PEG-SC,PEG-PVGLIG-Alkyne was obtained.2)The synthesis of PEG-PVGLIG-G3-OMe Boc-G3(Lys-Glu)-OMe deprotection of Boc,reaction with 4-azidobenzoic acid,N3-G3(Lys-Glu)-OMe was obtained.Reaction with PEG-PVGLIG-Alkyne through Cu+ catalytic click-reaction to get PEG-PVGLIG-G3-OMe.3)The synthesis of N3-GFLG-DOX N3-GFLG-DOX was obtained through dehydration condensation reaction of N3-GFLG-OH and DOX·HCl4)The synthesis of PEG-PVGLIG-G3-GFLG-DOX PEG-PVGLIG-G3-OMe deprotection of OMe,reaction with Propargylamine,PEG-PVGLIG-G3-Alkyne was obtained.Reaction with N3-GFLG-DOX through Cu+ catalytic click-reaction to get PEG-PVGLIG-G3-GFLG-DOX.5)Self assembly and characterization Preparation of PEG-PVGLIG-G3-GFLG-DOX aqueous solution,particle size and Zeta potential was measured by Malvin zen3690.6)Measurement of drug-loading rate Preparation 1mg/ml PEG-PVGLIG-G3-GFLG-DOX solution,diluted 6 times then measured Abs.Results1.Cathepsin B responsive peptide dendrimer drug delivery system1)ESI+,MALDI-TOF-MS and 1H NMR was used to characterize the molecular weight and hydrogen spectrum of the synthesized product.The results showed that PEG-G3-GFLG-GEM was successfully synthesized.2)Without DOX,the system was negative charge.After the addition of DOX,Zeta potential turn to positive,and Zeta potential increases with the increase of DOX.3)The size of PEG-G3-GFLG-GEM is about 10 nm,and the size is between 100-300 nm after co-loading DOX.4)Manufacture of the co-loading materials as 1:1 mass ratio with a particle size of 290 nm,PDI 0.187,GEM loading was 6.07%,DOX loading was 11.5%.5)Drug release results showed that was not released without enzyme.In the presence of papaya protease,the p H5.4 group released(20%)was more than p H7.4(13%).The release of DOX was p H5.4+ enzyme(65%)>p H5.4(50%)>p H7.4+ enzyme(30%)= p H7.4(30%).6)Co-loading IC50=0.0545ug/ml,was half of PEG-G3-GFLG-GEM,and quarter of GEM·HCl.2.MMP-2 and Cathepsin B dual responsive peptide dendrimer drug delivery system1)ESI+,MALDI-TOF-MS and 1H NMR was used to characterize the molecular weight and hydrogen spectrum of the synthesized product.The results showed that PEG-PVGLIG-G3-GFLG-DOX was successfully synthesized.2)PEG-PVGLIG-G3-GFLG-DOX aqueous solution could self assembly to nanoscale size with 128 nm,and Zeta potential was-6.39 m V.3)DOX loading was 9.24%.Conclusions1.Cathepsin B responsive peptide dendrimer drug delivery system1)PEG-G3-GFLG-GEM was successfully synthesized.2)PEG-G3-GFLG-GEM can be assembled to form a nanoscale delivery material by co-loading DOX.3)Co-loading material was Cathepsin B responsive.4)Co-loading material had better anti-tumor effect.2.MMP-2 and Cathepsin B dual responsive peptide dendrimer drug delivery system1)PEG-PVGLIG-G3-GFLG-DOX was successfully synthesized.2)PEG-PVGLIG-G3-GFLG-DOX can self assemble nanoscale for targeted drug delivery system.