Design And Functional Verfication Of A Stimuli-responsive Biomacromolecular Targeted Delivery System

The most abundant apolipoprotein in high density lipoprotein（HDL）is apo A-Ⅰ protein,which can guide HDL to deliver cholesterol to tumor cells by binding to the SR-BI receptor on tumor cell membrane surface.ApoA-Ⅰ mimetic peptide 4F has been reported to have similar biological functions to ApoA-Ⅰ protein and has been proved to have anti-tumor effects.Studies have found that the HDL receptor SR-BI is not only highly expressed on a variety of tumor cells,but also expressed in normal tissues such as liver,breast,and prostate.The application of the target properties of ApoA-Ⅰ may lead to off-target behavior and damage to liver and other organs.Therefore,this study designed a series of tumor microenvironment stimulus-responsive mimic peptides based on the characteristics of low p H and high reactive oxygen species（ROS）of tumor microenvironment,combined with the structural characteristics of 4F mimic peptides,and screened them by molecular dynamics simulation technology.A number of mimetic peptides with conformational stimulus response capabilities have been experimentally verified on in vitro cells and in vivo animal models.This dissertation is divided into the following five parts:PartⅠ.ReviewThis chapter mainly focused on the tumor-targeted delivery of biological macromolecules and the research progress of apo A-Ⅰ mimic peptides,as well as the application of molecular dynamics simulation technology.Firstly,the multi-level biological barriers that bioactive macromolecules face during the tumor targeted delivery and the related targeted delivery strategies are summarized;then the research progress of apo A-Ⅰ mimic peptides is reviewed,including the design strategy of mimic peptides and it’s biological function.Finally,the research progress of molecular dynamics simulation technology is also discussed,including a brief overview,basic processes,common force fields and other related content,as well as the application of MD in medicine.The content summarized in this chapter provides theoretical and technical support for this research.PartⅡ.Design and screening of stimulus-responsive ApoA-Ⅰ mimic peptides based on molecular dynamics technologyThis chapter aimed to design a stimulus-responsive apo A-Ⅰ mimic peptide that would respond to the tumor microenvironment.Studies have shown that apo A-Ⅰ protein has an Class A amphiphilicα-helix secondary structure,and its specific conformation determines its biological activity.By mimicking the conformation of Class A amphiphilicα-helix,various peptides could exert similar protein functions or even better one.Therefore,in this study,a stimulus-responsive amino acid derivative was designed to control the conformational transformation of the mimicking peptide under the tumor microenvironment,such as slightly acidic p H and high ROS.Meanwhile,considering the influence of the side chain length,two special amino acid derivatives were designed.Then,with regard to the sequence characteristics of the mimicking peptide 4F,a series of mimicking peptides were designed by selectively replacing hydrophobic residues with special amino acid derivatives while maintaining the hydrophilic-hydrophobic interface.Subsequently,molecular dynamics simulation technology was used to screen the mimicking peptides.Firstly,the corresponding force field files of the designed stimulus-responsive amino acid derivatives were built according to the protonation state of amino acid residues in different environments.Then,PDB files of polypeptides with idealαhelical conformation were constructed,and traction simulation was performed on the designed peptides to screen out the random conformational states.After 60 ns molecular dynamics simulation,RMSD values of these peptides were calculated to prove the stable conformation of the polypeptide.DSSP program was used to detect the helix content of the designed peptides in different environments,and the designed peptide 5-A with good amphiphilic conformation was identified.Theαhelicity of 5-A was 71.56%in the tumor simulation environment,while the average helicity of this peptide was only 18.37%in the normal simulation environment after 20 ns,indicating a good stimuli-response ability.PartⅢ.Synthesis of stimulus-responsive ApoA-Ⅰ mimic peptideIn this chapter,the synthesis of amino acid derivatives was conducted firstly,and the structure of theses compounds were confirmed using various technology.Next,the basic peptides were synthesized through solid phase synthesis,followed by the conjugation of peptides 5-A,2-A and 4-A with designed amino acid derivatives were synthesized by thiol-ene click reaction.The obtained peptides were identified by high performance liquid chromatography（HPLC）and matrix assisted laser desorption tandem time-of-flight mass spectrometry（MALDI-TOF）.Part Ⅳ.Study the in vitro activity of stimulus-responsive ApoA-Ⅰ mimicking peptidesIn this chapter,we investigated the tumor microenvironment response ability and anti-tumor ability of simulated peptides through APOA-Ⅰ activity simulation experiment and cellular cytotoxicity experiment.Firstly,the stimulation response ability of the simulated peptide was detected by fluorescence spectrophotometer and high-performance liquid chromatography under the condition of tumor microenvironment.The results showed that the amino acid derivatives could change their physical and chemical properties rapidly under the tumor microenvironment conditions,showing the required stimuli responsiveness.Subsequently,liposomes encapsulating rhodamine B were prepared to simulate tumor cell membranes,and the effects of the mimicking peptides on tumor cell membranes were investigated under different environments.The results showed that the accumulative release rate of rhodamine B was 13.5%within 24h under normal physiological conditions;however,the release rate of rhodamine B was as high as83%within 24h in the simulated tumor microenvironment,confirming that the designed mimicking peptides could simulate the biological activity of ApoA-Ⅰ protein.Finally,the antitumor activities of the mimicking peptides were investigated by cellular cytotoxicity assay.The results showed that these peptide had no cytotoxicity under normal physiological environment,while under slightly acidic condition,the mimicking peptides showed different degree of cytotoxic effects on two cell lines.5-A peptide had the best result,followed by 2-A;however,the performance of 4-A was the worst(IC₅₀value of 5-A was 2.446μg/m L,the IC₅₀value of 2-A 3.686μg/m L,and IC₅₀value of 4-A 7.930μg/m L).The cytotoxicity results were consistent with the dynamic simulation results,and the antitumor effect of 5-A was far better than that of the positive control 4F,indicating that the mimicking peptide had more sensitive stimulus response ability and better anti-tumor effect.Part Ⅴ.Study the in vivo activity of stimulus-responsive ApoA-Ⅰ mimicking peptideIn this chapter,A 4T1 tumor-bearing mouse model was established to investigate the in vivo antitumor activity of peptide 5-A.The results showed that the tumor volumes of 5-A high-dose group were significantly smaller than that of normal saline group（p<0.0001）,and was also smaller than that of positive control 4F high-dose group,4F low-dose group and 5-Fu group,respectively（p<0.0001）.The tumor inhibitory rate of 5-A（6 mg/kg）group was 71.43%,4F（15mg/kg）group was57.14%,4F（5mg/kg）group was 28.57%,and 5-FU（25mg/kg）group was 60.57%,respectively.These results revealed that peptide 5-A had a best anti-tumor effect compared with all other groups.Furthermore,in vivo imaging results showed that the5-A group drugs could be accumulated in the tumor after 24h administration,and also observed in the liver and kidney;on the contrary,the control 4F peptides were widely distributed in many tissues,including heart,liver,spleen,lung,kidney,and tumor,indicating that the stimulus-responsive peptide 5-A designed in this study had better tumor targeted ability.In summary,a novel stimulus-responsive peptide was designed and identified in this paper,which had excellent response ability to the tumor microenvironment.Taking together,it was verified that the mimicking peptide can mimic the activity of apo A-Ⅰ protein,and our peptides showed sensitive stimulus response and significant anti-tumor effect through in vitro cellular and in vivo animal models.In addition,the reliability of molecular dynamics simulation method was confirmed and could be applied in the further design of tumor-specific biomacromolecule drugs.