A Tetrazine Bearing Hydrogel For Sustained Prodrug Activation And Controlled Release

Objective:To prepare low-cytotoxicity,high-stability peptide self-assembled hydrogels,it is used as drug release carriers in drug delivery system,combined with biological orthogonal reactions to achieve long-term and stable drug release,to achieve the effect of treating tumors.Methods:The peptide NapYpFFK-Tz was prepared and purified by solid phase peptide synthesis(SPPS)and semi-preparative high-performance liquid chromatography,and the compound TCO-coumarin(TCO-CMR)was successfully prepared by light conversion.The compound was characterized by ~1H proton nuclear magnetic resonance(~1H NMR)and mass spectrum(MS),and the drug release process was monitored by fluorescence spectrophotometer and high performance liquid chromatography(HPLC).The transmission electron microscope(TEM)and rotational rheometer were used to characterize the stability of the hydrogel.Used cervical cancer cells(HeLa)as the research object,the cytotoxicity of different concentrations of peptide NapYpFFK-Tz solution and the incubation cytotoxicity at different times were investigated.The cell viability of HeLa cells was detected by MTT method,and the safety of peptide NapYpFFK-Tz as a drug release carrier was investigated.Results:The ~1H NMR results of the peptide NapYpFFK-Tz was consistent with the references,and the[M-H]~-molecular ion peak of 1062.3917 was found in the mass spectrometry results;the ~1H NMR results of the TCO-CMR was also consistent with the references,it was found that the molecular ion peak of[M+H]~+was 328.20.The gelation experiment results showed that when the concentration of the peptide carrier was 3.0 mg/mL,it could quickly form a highly stable hydrogel under the action of ALP within 3 minutes;the drug release experiment results demonstrated that the peptide carrier under the pH of 7.4,the drug could be continuously released for 8 rounds for a total of 43 days,and the rate of drug release was stable during the drug release process,and no suddenly release phenomenon occurred;at lower pH and lower concentration,the drug release rate of this carrier can still remain stable.TEM results showed that dense and uniform diameter of nanofibers will appear after the peptide was assembled.During the release of the drug,the structure of the nanofibers had not changed significantly;the rheological results demonstrated that the stability of peptide carrier has not changed a lot in the process of drug release,but the strength was slightly reduced.The cytotoxicity experiments results in vitro showed that:NapYpFFK-Tz was less cytotoxic to HeLa.When its concentration was less than 30?mol/L,after 24 hours,48 hours and 72 hours of incubation,the survival rate of HeLa cells can reach more than 80%,it has good biological safety,and the cytotoxic effect of peptide on HeLa was toxic dose effect and toxic time effect.Conclusion:The peptide DDS carrier NapYpFFK-Tz was successfully prepared and purified by solid-phase synthesis and semi-preparative high performance liquid chromatography.The method was easy to operate,the yield was high and the purity of the obtained compound was high.Under the action of alkaline phosphatase,the peptide can self-assemble into a peptide hydrogel with higher stability and low cytotoxicity to HeLa.Compared with other DDS carriers,peptide carriers have good biocompatibility,high stability,longer drug release time and more stable rate.Therefore,the peptide NapYpFFK-Tz as a DDS carrier still has a large space for development.