Supramolecular Nanodrugs Based On Covalent Assembly Of Therapeutic Peptides And Genipin Toward Synergistic Anticancer Therapy In Vitro

Objective: Plan to construct photothermal supramolecular nanoparticles based on non pigment biomolecular iridoid genipin,tyroscopic bright peptide(YSL)and two glycine modified tyroscopic bright peptide(GGYSL)to verify their physicochemical properties,stability and biological safety,and to further explore the application of peptide in photothermal therapy of liver cancer whether the antitumor activity is retained.Methods: 1.Synthesis of GYSL-NPs and GGGYSL-NPs: genipin was covalently crosslinked with YSL and GGYSL by simple hydrothermal method and self-assembled to form GYSL-NPs and GGGYSL-NPs.1.1 Characterization of GYSL-NPs and GGGYSL-NPs: Scanning electron microscope(SEM)and transmission electron microscope(TEM)were used to observe the morphology and dimensional stability of GYSL-NPs and GGGYSL-NPs.The particle size distribution,surface charge and stability of GYSL-NPs and GGGYSL-NPs were measured by Malvern particle size analyzer.The molecular structure and composition of GYSL-NPs and GGGYSL-NPs were determined by Fourier transform infrared spectroscopy(FTIR)and mass spectrometry(MS).The UV-NIR absorption spectra and fluorescence spectra of GYSLNPs and GGGYSL-NPs were measured by UV spectrophotometer and fluorescence spectrometer respectively.The photothermal conversion performance and stability of GYSL-NPs and GGGYSL-NPs were measured by using 808 nm infrared laser and thermal probe.High performance liquid chromatography(HPLC)was used to verify that YSL and GGYSL had short half-life and could not maintain effective plasma concentration for a long time.1.2.Study on cell uptake of GYSL-NPs and GGGYSLNPs: Laser confocal microscope(GLSM)was used to observe whether GYSL-NPs and GGGYSL-NPs could be absorbed by tumor cells.Three experimental groups were set up: the first group was blank control group,the second group was given GYSL-NPs160 ug / ml,and the third group was given GGGYSL-NPs 160 ug / ml.1.3 Photothermal therapy of GYSL-NPs and GGGYSL-NPs on hepatoma cells: MTT was used to detect the cytotoxicity of different concentrations(0.02,0.04,0.08,0.16,0.32 mg / ml)of GYSL-NPs and GGGYSL-NPs in the presence or absence of light after incubating with cells for 24 hours.Furthermore,EVOS Fluorescence microscope was used to verify that 0.32 mg / ml GYSL-NPs and GGGYSL-NPs were incubated with cells for 24 hours by calcein staining.The cytotoxicity of the cells in the presence or absence of light.Results: Stable GYSL-NPs and GGGYSL-NPs were successfully prepared.The results of SEM and TEM showed that they were uniform and well dispersed particles.The diameters of GYSL-NPs and GGGYSL-NPs were 87 ± 24 nm and 63 ± 27 nm,respectively.The surface potentials were-32.7 ± 5.2 m V and-18.1 ± 4.8 m V,respectively.FTIR and MS results show that genipin can react with amino groups to form N-heterocyclic intermediates through nucleophilic interaction.YSL or GGYSL and genipin are covalently conjugated at a stoichiometric ratio of 1:1.Due to the consumption of amino groups in the reaction,the resulting molecules show better hydrophobicity,which leads to the in-situ self-assembly of GYSL and GGGYSL.GYSL-NPs and GGGYSL-NPs show a wide absorption band from ultraviolet(UV)to near infrared(NIR)region,especially a strong absorption band of 550-900 nm is found.At the same time,both of them have a good fluorescence intensity near 561 nm.The results of photothermal properties of GYSL-NPs and GGYSL NPs showed that the temperature of GYSL-NPs(0.5 mg / ml in water)increased rapidly from 25 ℃ to 59.2 ℃and that of GGGYSL-NPs(0.5 mg / ml in water)increased rapidly from 25 ℃ to55.6 ℃.The photothermal conversion efficiencies of GYSL-NPs and GGGYSL-NPs were 55.7% and 50.6%,respectively.The uptake of GYSL-NPs or GGGYSL-NPs by BEL-7402 cells was studied by laser confocal microscopy.There were three experimental groups.The first group was blank control group,the second group was given GYSL-NPs 160 ug / ml,the third group was given GGGYSL-NPs 160 ug / ml.After changing the laser wavelength,strong red fluorescence appeared near the blue fluorescence of the nucleus in the second and third groups,and there was no red fluorescence in the area far away from the nucleus.There was no red fluorescence in all areas of the first group,indicating that BEL-7402 cells had good absorption ability to GYSL-NPs or GGGYSL-NPs.The results of cytotoxicity test showed that the photothermal killing effect of GYSL-NPs or GGGYSL-NPs on tumor cells increased with the increase of concentration under light conditions,and both of them had good photothermal therapeutic effect.At the same time,it was found that 0.32mg/ml GGGYSL-NPs could still kill about 25% of liver cancer cells without light.Conclusion: Two photothermal nanodrugs GYSL-NPs he and GGGYSL-NPs constructed from pigment free biomolecules have been successfully prepared,and both of them have good physicochemical properties,stability,biological safety and photothermal transformation performance,and have good photothermal ablation ability for tumor cells.At the same time,GGGYSL-NPs retain the antitumor activity derived from YSL.This strategy of combining the antitumor effect of bioactive peptides with photothermal therapy suggests that we can construct more abundant and multifunctional tumor photothermal nanodrugs.