Long Term Release System Self-assembled From Functional Peptide Drug Conjugate For Research

The development of nanostructured drug has been progressed for more than 10 years,but few listed or clinical research can be found due to many reasons.However,the lack of good biocompatibility,strong controllability,cheap building materials is an important bottleneck restricting its development.Based on the tumor microenvironment and the physiological characteristics of mitochondria,the functional oligomeric peptide is rationally designed functional oligomeric peptide through regulating the types and sequences of amino acids with safe amino acids as the basic material.The first-line antitumor drug doxorubicin is covalently attached to functional peptide to prepare a functional peptide drug conjugate which can self-assemble to nanofibers(FPD-NF)with the function of lysosomal esacping and mitochondrial targeting,and further to form Gel(FPD-Gel).The FPD is used for in situ tumor injection to reach sustained-releasing requirement in order to overcome exist limitations of construction materials,such as low security and poor control.In the second chapter,the condensation reaction to generate amide bond was caused by a reaction of the lysine with amino group through the succinic anhydride to prepare FPD.The product was separated and purified by preparation HPLC and the result showed that purification rate of the purified product was higher than 95%.The structure of product was characterized by MALDI-TOF-MS,showed that the molecular weight of FPD was 1696.8,proved target product was successful preparedIn the third chapter,the physicochemical properties and self-assembly of FPD were investigated.When the pH is about 10,positive FPD turn to negative charge,so the isoelectric point(PI)of FPD is about 10.The oil-water partition coefficient(logP)results show that with the increase of pH value,the logP ranges from-0.3 to 0.1,when the pH of 7,the logP is zero.The critical aggregation concentration of FPD was monitored by pyrene fluorescence method and the result of its critical aggregation concentration is 50μM.The results show that hydrophobicity and π-π stacking governs the early stages of the FPD self-assembly process of forming a sphere or bilayer.Furthermore,hydrogen bonding and electrostatics plays a critical role in the lateral growth and eventually determining the morphology of the self-assembled nanofiber.When increase the concentration of nanofiber,it can intertwine to form gel(FPD-Gel).According to the result of drug release behavior in vitro,the FPD-Gel can slowly release FPD and the effect is long termThe cytotoxicity,the mechanism of entering cells and the function of lysosomal esacping and mitochondrial targeting were systematic studied based on the SMMC7721 cell model.The result showed that the inhibition rate of FPD was obviously dose-dependent with a 50%inhibitory concentration of 48.91 μg/mL.The mechanism of entering cells and the function of lysosomal esacping and mitochondrial targeting was monitored by confocal microscope.The result showed that the mechanism of FPD entering cells was different from passive diffusion of DOX.FPD was uptaking by cells through endocytosis to form endosome,then many kinds of enzymes were all involved in the formation of lysosomes to complete the process of entering.Arginine residues in FPD with strong proton buffering capacity can effectively escape lysosomes and the mitochondrial targeting is by mean of its surficial positive charges.The result showed that FPD achieves preliminary design idea.In vitro release study results indicate that the FPD-Gel can slow dissociation to release FPD,but it is need to verify if FPD also has good slow release behavior after in situ tumor injection.In the fifth chapter,the pharmacokinetics of FPD-Gel and DOX solution in rats was investigated using DOX solution as reference.A sensitive,specific and rapid ultra high performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS)method has been established to determine FPD-Gel and DOX solution in rats plasma to compare two kinds of preparation in vivo pharmacokinetics.It is found that the MRT and T1/2 of FPD-Gel is improved markedly,which is 11.35 and 3.55 times than DOX solution.The experiment showed that FPD-Gel had long-term effect.The functional peptide drug conjugate was successfully synthesized through regulating the types and sequences of amino acids with safe amino acids as the basic material,which is coupled with doxorubicin to prepare a self-assembly,lysosome escape,mitochondria targeting system.The results showed that FPD had good assembly function,lysosome escape and mitochondrial targeting and long-term effect in vivo and vitro.Because of the constraints of time,this thesis need to research in the future,such as the assembly mechanism of FPD in vitro,the transport of FPD in vivo especially the mechanism of lymph circulation and metabolism,the relationship between distribution of the FPD within the tumor and the overall,safety and efficacy of FPD and so on.All in all,the thesis laid a solid foundation for further research of FPD.It provides a new direction and some useful information for in situ treatment of tumor and the choosing of boating material.