Doxorubicin-triggered Self-assembly Of Human ?-Helical Peptides Into Nanoparticles

Objective:In this study,the C-terminal?-hecial peptide of human cartilage matrix protein was used as the nanomaterial and fused RGD peptide at the N-terminal to form amphiphilic peptide(P45)which can target integrin ?v?3.P45 were self-assembled into Dox/P45 nanoparticles triggered by doxorubicin.In vitro cytology studies was performed to investigate targeted drug delivery,pH-sensitive release and the effective cytotoxicity.Methods:1.Preparation and characterization of Dox/P45 nanoparticles:pyrene as a fluorescent probe was used to detect the critical micelle concentration of P45.The secondary structure of P45 was analyzed by circular dichroism.P45 and Dox were prepared by different molar ratios,and the morphology and size of nanoparticles were observed by transmission electron microscopy(TEM)to determine the best binding ratio of P45 and Dox.The particle size and morphology of Dox/P45 nanoparticles were observed by TEM.The particle size,PDI and Zeta potential of Dox/P45 nanoparticles were measured.The cumulative drug release rate of Dox/P45 nanoparticles was detected by dialysis method.The particle size of Dox/P45 nanoparticles was measured by laser particle size analyzer to evaluate its stability.To reduce the pH of P45 solution or eight amino acid residues(EEDPCACE)of P45 removed(P32)and RGD peptide removal(P41),nanoparticles were prepared in the same way,and TEM was used to observe the presence of nanoparticle formation and to explore the importance of negative charge regions in self-assembly behavior.3.In vitro cytology studies of Dox/P45 nanoparticles:including cell uptake test(flow cytometry,laser confocal scanning microscopy)and cytotoxicity test(CCK-8 test).A549,MCF-7 and HEK293 cell lines represent the cell models of high expression,low expression and negative expression of integrin ?v?3,respectively.Flow cytometry(FCM)was used to detect the fluorescence intensity of Dox/P45 nanoparticles,Dox/P41 nanoparticles and free Dox.Laser confocal scanning microscopy(CLSM)was used to observe the uptake of Dox/P45 nanoparticles,Dox/P41 nm Granules and free Dox in the cells.CCK-8 test was used to detect the effects of Dox/P45 nanoparticles,Dox/P41 nanoparticles and free Dox on the activity of three cell lines.Results:1.Preparation and characterization of Dox/P45 nanoparticles:a sudden slip appeared at a concentration of 1.25 mM,which was assumed the CMC of P45.CD demonstrated maximum absorption levels at 208 and 221 nm;these peaks are usually considered typical of ?-helix.According to Dox and P45 molar ratio(16:1,10:1,8:1,4:1,2:1,1:1),the particle size of nanoparticles was 371.6±8.9,209.3±5.5,167.1±3.3,217.8±7.5,291.8±9.1 and 279.4±4.5 nm,respectively,and the PDI was 0.223,0.277,0.189,0.208,0.303 and 0.229,respectively.The encapsulation efficiency was 9.0±2.2%,21.1±1.3%,28.2±2.8%,38.6±2.9%,36.8±1.1%and 35.9±2.3%,respectively.The drug loading was 14.4±2.1%,35.1±1.2%,49.2±1.7%,48.6±2.3%and 49.0±1.3%,respectively.The particle size,PDI and Zeta potential of Dox/P45 nanoparticles were 167.1±3.3 nm,0.189,0 mV.TEM was used to observe the morphology and size of Dox/P45 nanoparticles:a uniform mulberry-like spherical shape with a diameter of 63 nm,excellent polydispersity.In acid buffer(pH 5.5,pH 6.5),Dox released from Dox/P45 nanoparticles rapidly in the first hour for 44.8±1.4%and 25.2±1.5%,respectively,and then slowly released wiht the cumulative release rates reaching 60.8±2.1%and 39.9±1.2%at 6 h,respectively.Significant release was not observed after 6 h in buffer at pH 7.4(6 h cumulative release rate was 5.9± 1.4%),and the difference was statistically significant(p<0.001),The particle size of Dox/P45 nanoparticles fluctuated between 167 and 171 nm within 6 h,and the change of particle size did not exceed 10 nm.The pH of P45 solution was adjusted to 6.0 or 4.0.As a result,the nanoparticles were not observed.The negetive charge region EADPCACE of P45 removal(P32)could not form spherical nanoparticles,while the P41 without RGD peptide could be tirggered by Dox into nanoparticles which are similar to Dox/P45 nanoparticles.2.In vitro cytology studies of Dox/P45 nanoparticles:FCM results showed that Dox/P45 nanoparticles had stronger fluorescence intensity in A549 and MCF-7 cell lines than Dox/P41 nanoparticles,while free Dox showed the strongest fluorescence intensity in three cell lines.No fluorescence intensity was observed for Dox/P45 and Dox/P41 nanoparticles in HEK293 cells.CLSM results showed that Dox/P45 nanoparticles had strong fluorescence in the cytoplasm of A549 cells,and showed weak fluorescence in MCF-7 cells,but little fluorescence in HEK293 cells.The fluorescence of free Dox was mainly present in the nuclei of A549,MCF-7 and HEK293 cells,while Dox/P41 nanoparticles did not show intracellular fluorescence in these cell lines.CCK-8 test showed that the IC50 values of free Dox and Dox/P45 nanoparticles on A549 cells were 0.39±0.03 ?g/mL and 0.57±0.05 gg/mL,respectively.For MCF-7 cells,the IC50 of Dox/P45 nanoparticles was 2.2±0.04 ?g/mL and the IC50 of free Dox was 0.42±0.01 ?g/mL.Dox/P45 nanoparticles did not show significant cytotoxicity to HEK293 cells,while free Dox showed a strong cytotoxicity with IC50 of 0.24±0.02 ?g/mL,and the difference was statistically significant(p<0.01).Dox/P41 nanoparticles showed low cytotoxicity to A549,MCF-7 and HEK293 cells,and the difference was statistically significant(p<0.01)compared with Dox/P45 nanoparticles and free Dox.Conclusions:P45 self-assembled into a spherical nanoparticle triggered by Dox and its negative charge region played an important role in self-assembly behavior.As a smart,efficient and safe nanocarrier,Dox/P45 nanoparticles can be used for targeted drug delivery,pH-sensitive release and effective killing tumor cells.Native amphiphilic peptide can be used as a carrier of nano drugs to achieve targeted delivery of anti-tumor drugs and controlled release,and is a powerful and promising tool.