Studies On Long-acting Drug Delivery System Of Doxorubicin Peptide Conjugate

The construction of the targeted preparation can greatly improve the distribution of the drug at the tumor site and reduce the toxic side effects of the drug.However,the residence time of the drug at the site of the tumor lesion is short and may leak from the tumor tissue,resulting in the need for frequent administration.And there is a risk of causing vasculitis,which increases the suffering of the patient.Therefore,how to increase the residence time of the targeted delivery system at the tumor site and reduce the number of administrations is extremely challenging.Based on previous research,self-assembling functional peptides were designed and used to chemically couple with DOX to construct long-acting nano delivery systems capable of self-assembly.The novel self-assembled doxorubicin peptide conjugates can be assembled into a 50-100 nm nanoparticle by an directional assembly regulation strategy.After intravenous injection,the nanoparticles are targeted to the tumor site by the EPR effect.In the tumor microenvironment,the nanoparticles recombine and assemble again to form large aggregates,avoiding EPR leakage,and making the drug to reside in the tumor site.The formed aggregates slowly disassemble at the tumor site to release the anti-tumor drug DOX,which plays the role of killing the tumor permanently.The second chapter of the research mainly includes the design of the self-assembly sequence KIGLFRWR with step-by-step step assembly.The target sequence was synthesized by a fully automated peptide synthesizer,and its physical and chemical properties,self-assembly function and safety were evaluated.The results were as follows:The isoelectric point of the self-assembled peptide was 10.5,and the fluorescent probe method was used to determine the critical aggregation concentration,and the critical aggregation concentration was 43.2?M.The results of its assembly morphology show that during the self-assembly process,the peptides aggregate to form nano-particles of about 50 nm firstly,then the nanoparticles are further assembled and aggregated to form larger micro-sized cross-linked fibrous aggregates.As a carrier material for inj ection administration,the in vitro hemolysis safety of self-assembled peptides was tested.The results showed that the hemolysis rate of the peptide was much lower than 5%at the experimental concentration,indicating good safety.In the third chapter,the self-assembled peptide drug conjugate DPCs was synthesized by chemically coupling the self-assembled sequence with the anhydride as the linkage.The physicochemical properties of the optimal absorption wavelength and critical aggregation concentration of DPCs are systematically characterized.The self-assembly ability of the designed DPCs was investigated by measuring the growth curve of the DPCs solution particle size.The transmission electron microscope image was used to show the change of the morphology during the assembly process.The relationship between assembly force and assembly morphology in self-assembly system was further studied by using various blockers and full-wavelength scanning patterns.The results show that hydrogen bonding and ?-? stacking are the main driving forces for DPCs molecular assembly.If there is a large electrostatic repulsion between the DPCs molecules,it can inhibit the further self-assembly of the DPCs molecules,because the pH of the solution will affect the degree of protonation of the DPCs molecules,thereby affecting the electrostatic repulsion between the molecules.Therefore,the assembly of DPCs molecules can only be carried out under a suitable pH solution system.The experimental results show that DPCs molecules have good assembly ability in the range of pH 5-8.Based on the previous chapters on the research of DPCs self-assembly morphology and force,the fourth chapter successfully designed an acid responsive functional material(Functional polylysine grafts,FPG).FPG can achieve the purpose of regulating its self-assembly morphology by blocking the assembly force between DPC-NPs.This chapter first designed the synthetic route of FPG,and MALDI-TOF was used to confirm its molecular weight.Hemolysis safety and acid sensitivity were studied and evaluated.Next,FPG was used to control the self-assembly force of DPC-NPs to study the effect of FPG on DPCs's self-assembly morphologies.A functional doxorubicin self-assembling peptide conjugate nanoparticle(FDPC-NPs)was constructed by self-assembly to form a stable micelle structure with a particle size of 50-100 nm.FDPC-NPs have good safety and stability,in vitro.Simulated the weak acid conditions of tumors,FDPC-NPs can respond to morphological transformation:FDPC-NPs is transformed into DPC-NPs with assembly ability,and further self-assembly aggregates to form FDPC-NPs-NFs.In vitro release experiments were carried out to demonstrate that FDPC-NPs have a good acid-responsive sustained-release function.In addition,in vitro cell pharmacodynamic experiments show that FDPC-NPs have comparable anti-tumor activity to DOX,providing a good research l basis for exerting anti-tumor efficacy in vivo.The fifth chapter is the in vivo study of FDPC-NPs.The Hepatoma xenograft mouse model was established.UPLC-MS/MS was used to study the tissue distribution of FDPC-NPs in trmor mice.The concentration and duration of FDPC-NPs in tumor,liver,heart,kidney,spleen,lung,and brain were examined to evaluate the tumor targeting and long-acting drug release functions of FDPC-NPs.Compared with the original drug DOX solution,the results showed that the enrichment of DOX in tumor tissues was significantly enhanced after intravenous injection of FDPC-NPs in hepatoma xenograft mouse.The relative uptake rate of FDPC-NPs group was 30 times higher than that of DOX group.It was confirmed that the tumor targeting of DOX was improved,and DOX was able to stay in the tumor site for a longer time,demonstrating a long-term efficacy.What's more,the distribution in the heart and kidney was also lower than that in the DOX group,which was beneficial to reduce the cardio-renal toxicity of DOX.In summary,this project designed the self-assembled peptide KIGLFRWR to construct a DPCs molecule with assembly function by chemical drug loading.The relationship between the assembly forces and the morphologies of DPCs were studied.In vitro,stable FDPC-NPs were constructed by blocking the ?-? stacking between DPC-NPs with FPG regulation.It can achieve the long-acting anti-tumor effect by achieving the morphological transformation under the condition of weakly acidic tumors,forming long-retained fibers,and exerting long-acting and responsive drug release.FDPC-NPs can not only improve the tumor targeting of DOX in the hepatoma xenograft mouse model,but also prolong the duration of DOX in tumor tissues.The above research results provide a useful reference for the design of self-assembling peptide drug conjugate molecules and regulating their assembly morphology to construct a long-acting anti-tumor system for hepatoma treatment.