Construction Of Smart Drug Delivery System Based On Human Alpha-helix Peptide And Its Preliminary Application

Background: Peptide self-assembly has attracted extensive attention because its morphological structure can be precisely controlled,and meanwhile it also has bioactivity,biodegradability,biocompatibility,sequence designability and so on.Therefore,peptide self-assembly has great potential in biomedical field,especially in drug delivery system.Non-covalent bond forces,such as hydrogen bond,hydrophobic,electrostatic,van der Waals force,and π-π stacking,trigger peptide self-assembly and when these forces reach balance in a certain environment,self-assembled peptides can form a specific ordered spatial structure.Therefore,the changes in the external environment would naturally trigger the ordered structure changes.From this,these different environments responsive triggers can be arranged in the peptides to smartly control peptide self-assembly and drug accurate release.Abnormal metabolism in tumor tissues and cells results in specific tumor microenvironments,such as acidic p H,high reducibility,and specific enzymes.Therefore,construction of tumor microenvironment-responsive peptide selfassembly drug delivery systems can realize the targeted delivery and smart controlled release of drugs.However,more accurate and efficient control release of drugs to enhance anti-tumor therapy and reduce the toxic and side effects of anti-tumor drugs is still a great challenge.Objectives: Two human α-helical peptides with different sequences were designed to encapsulate antitumor drugs to form an intelligent nano-drug delivery system based on peptide self-assembly,in order to achieve efficient anti-tumor and reduce the toxic and side effects of anti-tumor drugs.1)A human α-helix peptide responsive multiple tumor microenvironments is designed and an antitumor drug is selected to trigger self-assembly of this peptide to form a smart nanodrug delivery system.After this drug delivery system are targeted to tumor tissues,multiple tumor microenvironments can trigger the more accurate and efficient control release of drugs to enhance anti-tumor therapy and reduce the toxic and side effects of anti-tumor drugs.2)A human α-helix peptide carrying two different types of anti-tumor drugs is designed and these two drugs are orderly loaded based α-helix peptide self-assembly to form a multidrug delivery system.After this drug delivery system are targeted to tumor tissues,tumor acidic microenvironment in situ can trigger the precise release of two drugs at the subcellular level in order to combine multiple antitumor therapies.The ultimate aim is to enhance anti-tumor therapy and reduce the toxic and side effects of anti-tumor drugs.Part Ⅰ: Construction of multi-tumor microenvironment responsive nano-drug delivery system based on matrilin-1 peptides and its preliminary applicationMethod: 1.To design a human α-helical peptides responsive to multiple tumor microenvironment and theoretically analyze the feasibility of loading pirubicin In order to more accurately and smartly control antitumor drug release,multiple tumor microenvironment,such as the acidic p H,high reducibility and specificity of enzymes responsive human α-helical peptides(P51,sequence: RGDSEEDPC-s-sCESPVGLIGGLVKFQAKVEGLLQALTRKLEAVSKRLAILENT,α-helical from matrilin-1)was designed.Homology modeling and molecular docking methods were used to verify the capability of the peptide to carry anti-tumor drug pirubicin(THP),and to explore the threedimensional structure of pirubicin triggered human α-helical peptide self-assembly.2.Pirarubicin triggers human α-helical peptides self-assembly form nano-drug delivery system(P51-THP NPS)and its characterization The method of human α-helical peptides self-assembly triggered by THP was investigated.The morphology and size of P51-THP NPs were analyzed by transmission electron microscopy(TEM)and atomic force microscopy(AFM).Meanwhile,the particle size and distribution of P51-THP NPs in solution were determined by dynamic light scattering particle size analyzer(DLS).The main components of P51-THP NPS and their spatial distances were analyzed by infrared spectroscopy(FTIR)and fluorescence spectroscopy,respectively.3.In vitro simulation that multiple tumor microenvironments trigger the release of THP from P51-THP NPs and evaluate its release efficiency Multiple tumor microenvironments,such as acidic p H,high reducibility(DTT)and specific enzyme(MMP-2),were simulated in vitro.Gel electrophoresis,fluorescence spectrometer and dialysis method were used to analyze the release of THP from P51-THP NPS in different tumor microenvironments and their release efficiency were compared.4.To conduct in vitro cell experiments to verify and evaluate the cellular uptake pathway,targeting and antitumor efficacy of p51-THP NPS Firstly,lysosomal probes were used to investigate whether p51-THP NPs were absorbed by cells through lysosomal pathways.Secondly,laser confocal microscopy and flow cytometry were used to investigate the uptake efficiency of p51-THP NPS by different types of cells at different times to prove its targeting.Thirdly,the intracellular release efficiency of FITC-P51-THP NPs and FICT-P45-THP NPs(P45 containing no highly reductive and specific enzymeresponsive peptide sequences)was compared to verify that multiple tumor microenvironment responses could improve drug release efficiency.Finally,CCK-8 was used to determine the viability of different types of cells treated with p51-THP NPS to evaluate its anti-tumor effect.5.To establish tumor model verify in vivo targeting,safety and antitumor effect of P51-THP NPS Firstly,P51-THP NPs were injected into the tumor model of MDA-MB-231 through tail vein.After 48 hours,the tumor tissues and organs such as heart,liver,spleen,lung and kidney were collected.The content of P51-THP NPs in each organ and tumor tissue was analyzed by in vivo Imaging System.Secondly,Cy5.5-P51-THP NPs were injected into the tumor model of MDA-MB-231 through tail vein.The tissues and tumors were collected for the preparation of tissue sections and immunofluorescence assays.The cell nuclei were stained with DAPI,and vascular endothelial cells were stained with FITC-CD31.,and laser confocal microscopy was used to collect fluorescence image and analyze the distribution of P51-THP NPs in the tissue.Finally,the tumor models of MDA-MB-231 were divided into five groups and injected with normal saline,free THP,P51,p45-THP NPS and P51-THP NPS,respectively.Tumor size and body weight of the mice were measured every day.After the end of the treatment,visceral tissue sections were stained with HE to evaluate the toxic effects of the drugs.Results: 1.Design and synthesize human α-helical peptide P51 with the following sequence: RGDSEEDPC-s-s-CESPVGLIGGLVKFQAKVEGLLQALTRKLEAVSKRLAILENT,from the N end to end C contains targeted tumor markers in turn the sequence of integrin(Arg-Gly-Asp,RGD),the sequence of negatively charged(Ser-Glu-Glu-Asp-Pro)and three kinds of tumor microenvironment response,the sequence of amino acids were high reducibility cracking sites(Cys-s-s-Cys,disulfide bond).Tumor specific enzyme MMP-2 restriction site(Pro-Val-Gly-Leu-Ile-Gly)and tumor acidic environment response site(Ahelix 41 residue peptide).Homology modeling and molecular docking showed that the peptide self-assembled could load THP through electrostatic and hydrophobic interactions to form stable nano-spherical particles(P51-THP NPS).2.TEM and AFM images of P51-THP NPS showed about 40 nm-spherical nanoparticles,constructed from about 5 nm particles which were mainly THP.The size distribution of P51-THP NPS in solution measured by DLS was 39 ± 10 nm.The main components of P51-THP NPS measured by FTIR were THPand peptide.FITC was labeled on P51,and the resonance transfer of fluorescent energy of FITC and THP in FITC-P51-THP NPS was analyzed by fluorescence spectrometer,which indicated that the distance between P51 and THP was less than 10 nm,and it proved that the self-assembly occurred.3.The results of gel electrophoresis,fluorescence spectrometer and dialysis method showed that the acid p H,DTT and MMP-2 could trigger the release of THP from P51-THP NPS,and the acid p H had the best release effect.When the three tumor microenvironments exist at the same time,the release efficiency can be superimposed to further improve the efficiency.4.P51-THP NPs can enter cells through the endocytosis pathway,and release THP under the action of tumor microenvironment and deliver it to the nucleus.With the assistance of RGD,the efficiency of tumor cells uptake of P51-THP NPs was significantly higher than that of normal cells.It was also found that improving the release efficiency of THP in tumor cells was more beneficial to the separation of THP and peptide.CCK-8 results also indicated that P51-THP NPs with the highest drug release efficiency had the strongest anti-tumor effect.5.In animal experiments,P51-THP NPS with more efficient targeting,can deliver the largest amount of HP to the tumor nucleus and exert the strongest anti-tumor effect compared with the control group.P51-THP NPS can reduce the cytotoxic side effects of THP by analyzing the body weight and tissue HE staining of mice.Conclusion: 1.Based on peptide self-assembly,THP can be assembled by human α-helical peptide group to form a stable structure of intelligent nano-drug delivery system.2.Peptide self-assembly nano-drug delivery system responsive to multi-tumor microenvironment can improve the efficiency of drug release and deliver drugs to the tumor site more accurately.3.Peptide self-assembly nano-drug delivery system responsive to multi-tumor microenvironment has more effective anti-tumor effects and can significantly reduce the toxic and side effects of anti-tumor drugs.Part Ⅱ: Construction of dual-drug delivery system for THP and ICG based on matrilin-1/PEAK-1 peptides and its preliminary applicationMethod: 1.To screen antitumor drugs that can trigger peptide self-assembly and study and analyze the factors affecting self-assembly.P60,composed of two human α-helical peptides from different proteins,was designed to load two different types of anti-tumor drugs in order to combine multiple antitumor therapies.Antitumor drugs can be divided into two categories: soluble and insoluble.Methods of two types of drug-triggered peptides self-assembly were established.Different antitumor drugs were classified according to the number of carbon atoms,anti-tumor mechanism and soluble cause.The peptide self-assembly was triggered in turn according to the established method,and the occurrence of self-assembly was determined by whether precipitation was generated and stable particles were formed.The factors affecting peptide self-assembly were studied via the change of the solvent,p H and volume of solvent.2.P60 was successively triggered by ICG and THP to form a dual-drug delivery system(P60-ICG-THP)and its characterization The self-assembly method of P60 triggered by ICG and THP was optimized.TEM and SEM were used to collect the morphology and morphology of P60-ICG-THP.The particle size and distribution of P60-ICG-THP in aqueous solution were measured by DLS.The position relationship between the two fluorescent molecules of P60-ICG-THP was tracked by LSCM.3.Acid p H triggers the change of the size and charge of P60-ICG-THP to achieve the precise delivery and controlled release of the two drugs at the subcellular level The charge and size changes of P60-ICG-THP under different p H conditions were analyzed by gel electrophoresis and DLS.At the same time,TEM and SEM were used to collect the morphology of P60-ICG-THP under different p H values.After the P60-ICG-THP and ICG were irradiated by 808 nm infrared laser at different p H values,the surface temperature changes of the P60-ICG-THP and ICG were collected by a near-infrared imager at different times.Fluorescence microscopy and LSCM were used to track the release and cellular uptake of P60-ICG-THP in different p H environments,the pathway of uptake,and the final intracellular localization of two of the drugs.4.To evaluate the in vitro antitumor effect combined chemotherapy and photothermal therapy and acidic p H triggers selective occlusion of tumor blood vessels in vitro CCK-8 was used to determine the cell activity of P60-ICG-THP treated with MDA-MB-231 in different p H environments,and the cell activity of P60-ICG-THP treated with 808 nm infrared laser was used to evaluate the anti-tumor effect of in vitro combined chemotherapyphotothermal therapy.A microfluidic vascular simulation model was established to verify whether P60-ICG-THP released by acidic microenvironment could block the capillary,and the changes of ICG and THP in the capillary interior were tracked by LSCM to verify whether the capillary was blocked.5.The acidic p H in situ of the tumor triggered P60-ICG-THP to selectively block tumor blood vessels in vivo to achieve tumor starvation therapy The subcutaneous tumor model of nude mice was established.P60-ICG-THP was injected into mice through caudal vein.After 24 mice were sacrificed,the organs and tumor tissues were collected and prepared into frozen tissue sections.LSCM was used to observe whether there was vascular obstruction in various organs and tumor tissues.At the same time,the organs and tumor tissues were lysed to determine the levels of tumor starvation related factors,such as ATP and HIF-1α.6.The subcutaneous tumor model of nude mice was established to verify the targeting,safety and anti-tumor effect of P60-ICG-THP First,P60-ICG-THP was injected into the subcutaneous tumor model of MDA-MB-231 nude mice through tail vein.Fluorescence images of THP distribution in vivo at different time points were collected by in vivo Imaging System,and surface temperature images of mice were collected by a near-infrared thermal imager after the tumor site was for 5 minutes irradiated by 808 nm infrared laser to analyze the concentration of ICG in tumor tissues.Finally,the tumor models was divided into groups and injected with normal saline,free ICG,free THP,P60-ICGTH and P60-ICG-TH,respectively,through the tail vein.The free ICG group and one of the P60-ICG-TH group were irradiated with 808 nm infrared laser for 5 minutes every day.Tumor size and body weight of the mice were measured every day.After the end of the treatment,blood samples were collected to detect biological indicators of blood cells and liver function,and HE staining of visceral tissue sections were taken to evaluate the toxicity of the drugs.Immunohistochemistry was performed to detect the expression of nuclear protein Ki67,which is related to cell proliferation,in tumor tissues,and the anti-tumor effect of combined chemotherapy-photothermal-starvation therapy was evaluated by tumor growth curve and survival curve analysis.Results: 1.Human α-helix peptides P60 including two segments from different human proteins,such as matrilin-1(ESLVKFQAKVEGLLQALTRKLEAVSKRLAILENT)and PEAK-1(VVGKIRSLHTDALKKLAVKCEDLFMA)was designed and synthesized.2.ICG and THP were successfully screened out to trigger P60 self-assembly to form a 500~1000 nm and negative charged dual drug delivery system(P60-ICG-THP)under certain conditions.TEM showed that several 50-100 nm spherical nanoparticles formed clusters.LSCM results showed that ICG and THP basically overlapped in P60-ICG-THP,and the size was about 1000 nm.3.The acidic environment can quickly trigger the change of the size and charge of P60-ICGTHP.At this point,P60-ICG-THP released two kinds of particles with different size and charge properties:(i)the positively charged nanoparticles with the size of about 50 nm,mainly carrying THP(P60-THP);(ii)heterogeneous and negatively charged particles,mainly carrying ICG(P60-ICG).4.Because of its large size and negative charge,P60-ICG-THP stayed outside the cell membrane and could avoid being phagocytic and cleared by blood cells and normal tissue cells.In acidic environment,after the size and charge properties of P60-ICG-THP change,P60-THP can immediately cross the cell membrane and deliver THP to the tumor nucleus.P60-ICG enters the cytoplasm through the endocytosis pathway,or accumulates in tumor blood vessels to selectively block the supply of nutrients and oxygen for the treatment of tumor starvation.Both of these conditions can avoid the rapid excretion of ICG in the liver,which is conducive to the storage of more ICG in the tumor tissue to achieve photothermal therapy.5.In the normal p H environment,the anti-tumor effect of P60-ICG-THP was inferior to that of free THP(P<0.001),while in the acidic environment the antitumor effect of P60-ICGTHP was much greater than that of free THP(P<0.001).At the same time,the antitumor effect of P60-ICG-THP was further increased when photothermal therapy was added,and the photothermal therapy was not affected by acidic environment.6.Occluded tumor blood vessels were found in frozen sections of tumor tissue,but not in normal tissue.In normal tissues,the amount of P60-ICG-THP is low and the structure of most P60-ICG-THP is intact.However,in tumor tissues,the content of P60-ICG-THP is high and the function structure of P60-ICG-THP is changed in the acidic microenvironment.ICG and THP are separated,ICG stays in the cytoplasm,tissue space or blood vessels,and THP is mainly enriched in the tumor nucleus.The detection of related factors after starvation treatment found that the concentration of ATP in tumor tissues was decreased and the concentration of HIF-α was increased,which proved that hypoxia occurred after tumor blood vessels were blocked.7.The results of tumor growth curve,survival curve and the expression of Ki67 confirmed that P60-ICG-THP combined with chemotherapy,photothermal therapy and starvation had the best anti-tumor effect in vivo.In the HE staining tissue,blood cell analysis and blood biochemical results,there were obvious toxic and side effects of free THP,but no obvious toxic and side effects were found in P60-ICG-THP.Conclusion: 1.Based on human α-helical peptide,ICG and THP were successfully screened to triggered P60 self-assembly to construct a dual-drug nano-delivery system(P60-ICG-THP).2.Acid p H triggers the change of charge and size of P60-ICG-THP to achieve precise delivery and release of the dual drugs at the subcellular level.3.Acidic p H triggers P60-ICG-THP to release heterogeneous and negatively charged particles,mainly carrying ICG(P60-ICG),which can selectively block tumor blood vessels to achieve tumor starvation therapy.4.P60-ICG-THP can be combined with chemotherapy,photothermal therapy and starvation therapy to increase the anti-tumor effect and reduce the toxic and side effects under the action of the in-situ acidic microenvironment of tumor.