Nano-polypeptide targeting Pannexin-1 channel protein as well as synthesis method and application of nano-polypeptide

Abstract

The invention provides a nano polypeptide targeting Pannexin-1 channel protein and a synthesis method and application thereof, the nano polypeptide targeting Pannexin-1 channel protein is formed by modifying polypeptide QE20 through NBC and curcumin, and the amino acid sequence of the polypeptide QE20 is as shown in SEQ ID NO: 1; the small molecule polypeptide is more stable in an in-vivo oxidative stress acid environment due to the coating of the nano-particles; the micro-molecule polypeptide has liver targeting property in vivo due to the coating of the nano-particles; the polypeptide modification enables the small molecule polypeptide to have an ROS response function; curcumin modification enables the small-molecule polypeptide to have a stronger anti-oxidation effect, and compared with existing small-molecule polypeptide, the technical problems that the small-molecule polypeptide is easy to degrade and unstable in vivo and does not have liver targeting and ROS response functions are solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nanopeptide targeting Pannexin-1 channel protein, a synthesis method and an application thereof. Background Art

[0002] Hepatic ischemia reperfusion injury (HIRI) usually occurs during liver transplantation, liver resection, or in cases of liver trauma, shock or ischemic hepatitis. HIRI is one of the main causes of liver failure and poor prognosis after liver surgery. Its pathology is mainly manifested by hepatocyte death and cascade inflammatory response, including cellular adenosine triphosphate (ATP) depletion, intracellular calcium overload, increased oxygen free radicals, enhanced inflammatory response, mitochondrial dysfunction, oxidative stress injury, etc. Although there are currently many strategies aimed at preventing or alleviating HIRI, finding more precise and effective intervention targets remains a research hotspot.

[0003] Pannexin-1 (PANX1) channel protein is a transmembrane protein involved in the release of adenosine triphosphate (ATP) to the extracellular space. It has attracted much attention for its key role in mediating the release of extracellular ATP, regulating inflammatory responses, and participating in the IRI process. Studies have shown that the activation of PANX1 not only promotes the release of ATP, but also exacerbates the increase in intracellular calcium levels, thereby exacerbating tissue damage. The PANX1 inhibitor carbenoxolone has a protective effect on renal ischemia-reperfusion injury in mice, and can also significantly reduce lung dysfunction, edema, cytokine production, and neutrophil infiltration caused by lung ischemia-reperfusion in mice. These data provide strong evidence for preventing or alleviating ischemia-reperfusion injury by intervening in PANX1.

[0004] In the prior art, although small molecule compounds such as carbenoxolone can inhibit PANX1, most PANX1 antagonists will also bind to unrelated channel proteins, transport proteins and non-membrane proteins. Therefore, the prior art has designed a new type of analog peptide QE20 for PANX1 with reference to the PANX1 protein structure (refer to Chinese patent application number 202211496945.8). The effect of QE20 in inhibiting PANX1 is better than that of carbenoxolone and 10panx, but the polypeptide QE20 still has obvious limitations, such as instability in the body, easy degradation, and lack of liver targeting. Therefore, it is very necessary to develop highly stable and liver-targeted PANX1-specific therapeutic drugs.

[0005] Therefore, the related technologies in the prior art regarding polypeptide antagonists targeting Pannexin-1 channel proteins still need to be further improved. Summary of the invention

[0006] In view of this, the present invention proposes a nanopeptide targeting Pannexin-1 channel protein, a synthesis method and an application, which solves the technical problems in the prior art that small molecule peptides are easily degraded and unstable in the body and lack liver targeting and ROS response functions.

[0007] The technical solution of the present invention is achieved in this way:

[0008] On the one hand, the present invention provides a nanopeptide targeting Pannexin-1 channel protein, wherein the nanopeptide targeting Pannexin-1 channel protein is formed by modifying the polypeptide QE20 by 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonate (NBC) and curcumin, and the amino acid sequence of the polypeptide QE20 is shown in SEQ ID NO: 1.

[0009] FKHLWLPLNGSESQLSNKQE(SEQ ID NO:1)

[0010] In a second aspect, the present invention further provides a method for synthesizing the nanopeptide targeting the Pannexin-1 channel protein according to the first aspect, comprising the following steps:

[0011] The polypeptide QE20 is synthesized by a solid phase method, stearic acid is conjugated to the N-terminus of the polypeptide QE20, and NBC is synthesized simultaneously;

[0012] reacting the NBC with the polypeptide QE20 to obtain the NBC-modified polypeptide QE20;

[0013] Curcumin is reacted with the NBC-modified polypeptide QE20 to obtain a nanopeptide targeting the Pannexin-1 channel protein.

[0014] On the basis of this technical solution, it is further preferred that the NBC is prepared by reacting 4-(hydroxymethyl)phenylboronic acid dihydropentadienyl ester, 4-nitrophenyl chloroformate and a base, specifically comprising the following steps:

[0015] 4-(Hydroxymethyl)phenylboronic acid dihydropentadienyl ester and 4-nitrophenyl chloroformate are dissolved in solvents respectively, mixed, and then a base is added at 0-4°C, stirred and mixed, reacted at 20-25°C for 3-5h, the solvent is removed, diluted, washed, the organic layer is collected, concentrated, and purified to obtain NBC.

[0016] On the basis of this technical solution, further preferably, the NBC is reacted with the polypeptide QE20, comprising the following steps:

[0017] The NBC and the polypeptide QE20 are dissolved separately, mixed and stirred, and reacted at 20-25° C. overnight to obtain a mixed solution, and the mixed solution is dialyzed to obtain the NBC-modified polypeptide QE20.

[0018] On the basis of this technical solution, it is further preferred that the molar ratio of the NBC and the polypeptide QE20 after they are dissolved is 1.2-1.5:1.

[0019] On the basis of this technical solution, further preferably, the molecular weight cut-off of the dialysis is 1000Da.

[0020] On the basis of this technical solution, further preferably, the reaction of curcumin with the NBC-modified polypeptide QE20 comprises the following steps:

[0021] The curcumin and the NBC-modified polypeptide QE20 are mixed at 20-25° C., reacted for 20-30 minutes, added with PBS solution and stirred, reacted for 3-4 hours, and dialyzed to obtain the nanopeptide targeting Pannexin-1 channel protein.

[0022] On the basis of this technical solution, further preferably, the volume ratio of the curcumin to the NBC-modified polypeptide is 1:3-4.

[0023] On the basis of this technical solution, further preferably, the molecular weight cut-off of the dialysis is 3000-3500Da.

[0024] In a third aspect, the present invention also provides the use of the nanopeptide targeting Pannexin-1 channel protein described in the first aspect in liver ischemia-reperfusion.

[0025] The nanopeptide targeting Pannexin-1 channel protein, the synthesis method and the application provided by the present invention have the following beneficial effects compared with the prior art:

[0026] The nanopeptide targeting Pannexin-1 channel protein is self-assembled by modifying the polypeptide QE20 with NBC and curcumin. The presence of the nanostructure significantly improves the stability of the polypeptide QE20 in the circulatory system, effectively preventing it from being degraded by enzymes. At the same time, the nanostructure enables the drug to be specifically enriched in liver tissue, thereby having liver targeting. NBC, as a ROS-responsive linker, gives the nanomedicine an inflammatory response function and can achieve precise and traceless QE20 release without affecting the biological activity of the polypeptide. In addition, the released curcumin small molecules can also synergistically exert anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the nanopeptide described in Example 1 of the present invention;

[0029] Figure 2 This is the mass spectrum of the nanopeptide described in Example 1 of the present invention;

[0030] Figure 3 The toxicity results of polypeptide (QE20, QE20-M, QE20-N) and curcumin (Cur) at different concentrations on mouse liver cells AML12 of Application Example 1 of the present invention are shown in FIG.

[0031] Figure 4 The results of the polypeptides (QE20, QE20-M, QE20-N) and curcumin (Cur) in Application Example 1 of the present invention inhibiting ATP release in mouse liver cells AML12 in normal culture and hypoxia-reoxygenation model;

[0032] Figure 5 The results of the polypeptides (QE20, QE20-M, QE20-N) and curcumin (Cur) in Application Example 1 of the present invention in reducing apoptosis in mouse liver cells AML12 in normal culture and hypoxia-reoxygenation model;

[0033] Figure 6 This is a result diagram showing the degree of liver damage at different times after liver ischemia-reperfusion in mice in Application Example 2 of the present invention;

[0034] Figure 7 The result diagram of liver function indexes in the mouse liver ischemia 1h reperfusion 6h model in Application Example 2 of the present invention treated with different concentrations of QE20;

[0035] Figure 8 This is a result diagram of liver function indicators in the mouse liver ischemia 1h reperfusion 6h model treated with the optimal concentrations of QE20, QE20-M, QE20-N, and Cur in Application Example 2 of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The present invention provides a nanopeptide targeting Pannexin-1 channel protein, a synthesis method and an application thereof. The nanopeptide targeting Pannexin-1 channel protein is formed by modifying polypeptide QE20 by NBC and curcumin. Nanoparticle encapsulation makes the small molecule polypeptide more stable in an acidic environment of oxidative stress in vivo; and nanoparticle encapsulation makes the small molecule polypeptide have liver targeting in vivo.

[0038] Example 1

[0039] A nanopeptide targeting Pannexin-1 channel protein, wherein the nanopeptide targeting Pannexin-1 channel protein is formed by modifying polypeptide QE20 by NBC and curcumin, and the amino acid sequence of the polypeptide QE20 is shown in SEQ ID NO: 1.

[0040] FKHLWLPLNGSESQLSNKQE(SEQ ID NO:1);

[0041] In this embodiment, specifically, the method for synthesizing the nanopeptide targeting the Pannexin-1 channel protein comprises the following steps:

[0042] The polypeptide QE20 is synthesized by a solid phase method, stearic acid is conjugated to the N-terminus of the polypeptide QE20, and NBC is synthesized simultaneously;

[0043] reacting the NBC with the polypeptide QE20 to obtain the NBC-modified polypeptide QE20;

[0044] Curcumin is reacted with the NBC-modified polypeptide QE20 to obtain a nanopeptide targeting the Pannexin-1 channel protein.

[0045] The polypeptide QE20 was synthesized by a polypeptide synthesis company (Zhejiang Paipeptide Biological Co., Ltd.) through a solid phase method, specifically comprising the following steps:

[0046] First, the aminated peptide is synthesized by solid phase method, and then stearic acid is conjugated to the N-terminus of the peptide by chemical reaction. The stearic acid peptide is released from the solid phase by chemical reaction. The synthesized peptide is purified by reverse phase chromatography to a purity of >95%. The purity of the peptide is determined by high performance liquid chromatography (HPLC), and the molecular weight of the peptide is determined by mass spectrometry (MALDI-TOF).

[0047] In this embodiment, specifically, the NBC is prepared by reacting 4-(hydroxymethyl)phenylboronic acid dihydropentadienyl ester, 4-nitrophenyl chloroformate and a base, and specifically comprises the following steps:

[0048] 4-(Hydroxymethyl)phenylboronic acid dihydropentadienyl ester and 4-nitrophenyl chloroformate are dissolved in solvents respectively, mixed, and then a base is added at 0-4°C, stirred and mixed, reacted at 20-25°C for 3-5h, the solvent is removed, diluted, washed, the organic layer is collected, concentrated, and purified to obtain NBC.

[0049] In this embodiment, specifically, the molar ratio of 4-(hydroxymethyl)phenylboronic acid dihydropentadienyl ester, 4-nitrophenyl chloroformate and the base is 1:1.1-1.2:2.

[0050] More preferably, the base is triethylamine (TEA).

[0051] More preferably, the solvent is tetrahydrofuran (THF).

[0052] In this embodiment, specifically, reacting the NBC with the polypeptide QE20 comprises the following steps:

[0053] The NBC and the polypeptide QE20 are dissolved separately, mixed and stirred, and reacted at 20-25° C. overnight to obtain a mixed solution, and the mixed solution is dialyzed to obtain the NBC-modified polypeptide QE20.

[0054] In this embodiment, specifically, the solvent used for dissolution is dimethyl sulfoxide (DMSO).

[0055] In this embodiment, specifically, the molar ratio of the NBC and the polypeptide QE20 after they are dissolved is 1.2-1.5:1; more preferably, the molar ratio is 1.5:1.

[0056] In this embodiment, specifically, the molecular weight cut-off of the dialysis is 1000Da.

[0057] More preferably, the dialysis is performed with DMSO and ultrapure water.

[0058] In this embodiment, specifically, the reaction of curcumin with the NBC-modified polypeptide QE20 comprises the following steps:

[0059] The curcumin and the NBC-modified polypeptide QE20 are mixed at 20-25° C., reacted for 20-30 minutes, added with PBS solution and stirred, reacted for 3-4 hours, and dialyzed to obtain the nanopeptide targeting Pannexin-1 channel protein.

[0060] In this embodiment, specifically, the volume ratio of the curcumin to the NBC-modified polypeptide is 1:3-4.

[0061] In this embodiment, specifically, the molecular weight cut-off of the dialysis is 3000-3500Da.

[0062] As a result, the hydrogen spectrum of the synthesized nanopeptide is as follows Figure 1 As shown, it can be seen Figure 1 The chemical shifts of each hydrogen atom in the QE20-N monomer molecular structure are annotated, indicating the successful construction of the monomer molecule.

[0063] Example 2

[0064] A nanopeptide targeting Pannexin-1 channel protein, wherein the nanopeptide targeting Pannexin-1 channel protein is formed by modifying polypeptide QE20 by NBC and curcumin, and the amino acid sequence of the polypeptide QE20 is shown in SEQ ID NO: 1.

[0065] FKHLWLPLNGSESQLSNKQE(SEQ ID NO:1);

[0066] In this embodiment, specifically, the method for synthesizing the nanopeptide targeting the Pannexin-1 channel protein comprises the following steps:

[0067] The polypeptide QE20 is synthesized by a solid phase method, stearic acid is conjugated to the N-terminus of the polypeptide QE20, and NBC is synthesized simultaneously;

[0068] reacting the NBC with the polypeptide QE20 to obtain the NBC-modified polypeptide QE20;

[0069] Curcumin is reacted with the NBC-modified polypeptide QE20 to obtain a nanopeptide targeting the Pannexin-1 channel protein.

[0070] In this embodiment, specifically, the synthesis method of NBC comprises the following steps:

[0071] 1.17 g of 4-(hydroxymethyl)phenylboronic acid dihydropentadienyl ester (5.0 mM) and 15 mL of anhydrous THF were added to a flask. After complete dissolution, TEA (1.40 mL, 10.0 mM) and 4-nitrophenyl chloroformate (1.12 g, 5.6 mM) dissolved in 15 mL of THF were added and added dropwise to the above solution at 0 ° C. The reaction was stirred at room temperature for 5 h. Most of the solvent was removed and diluted with ethyl acetate, washed with 1.0 M hydrochloric acid, saturated sodium carbonate solution and brine in turn. The organic layer was collected, dried, filtered and concentrated. Petroleum ether / ethyl acetate (volume ratio of 10 / 1) was used as an eluent, and silica gel column chromatography was performed to purify the crude product to obtain a white powder, which was NBC.

[0072] In this embodiment, specifically, reacting the NBC with the polypeptide QE20 comprises the following steps:

[0073] 5 mg of peptide QE20 was dissolved in 1.5 mL of anhydrous DMSO, and then 1.5 N NBC dissolved in DMSO (the molar ratio of NBC to the primary amine on the peptide was 150%) was added and stirred at room temperature overnight. The resulting solution was dialyzed against DMSO and ultrapure water (molecular weight cutoff of ∼1000 Da, 1 h each time) in order to remove unreacted NBC and dihydropentadienyl esters generated by hydrolysis.

[0074] In this embodiment, specifically, the reaction of curcumin with the NBC-modified polypeptide QE20 comprises the following steps:

[0075] 5 μL of curcumin (10 mg / mL) was mixed with 20 μL of NBC-modified peptide QE20 (10 mg / mL) at room temperature for 30 min to form peptide-curcumin prodrug molecules. The molecule was then added to 2 mL of PBS solution and stirred at room temperature for 4 h. The resulting solution was dialyzed with ultrapure water (molecular weight cutoff 3000-3500 Da, 1 h each time) to remove unreacted curcumin, NBC-modified peptides and unassembled prodrug molecules.

[0076] result, Figure 2 This is the mass spectrometry result of the polypeptide (QE20-N) of this example, which clearly shows that the molecular weight of the QE20-N monomer molecule is 3145, further illustrating the successful construction of the monomer molecule.

[0077] Example 3

[0078] A nanopeptide targeting Pannexin-1 channel protein, which is different from Example 2 in that the curcumin is reacted with the NBC-modified polypeptide QE20, specifically comprising the following steps:

[0079] (1) The polypeptide QE20 prepared above was dissolved in 2.5% volume of DMSO (D2650, Sigma-Aldrich, the same below), and then 1×PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.2-7.4) buffer was added to a final concentration of 1 mM.

[0080] (2) The ROS-modified peptide QE20 (QE20-M) prepared above was dissolved in 2.5% DMSO, and then 1× PBS buffer was added to a final concentration of 1 mM.

[0081] (3) Curcumin (C805204, MACKLIN, Shanghai, China) was dissolved in 2.5% DMSO, and then 1× PBS buffer was added to a final concentration of 1 mM or 10 mg / mL or 10 mg / mL.

[0082] (4) The nanopeptide QE20 (QE20-N) prepared above was dissolved in 2.5% volume of DMSO, and then 1× PBS buffer was added to a final concentration of 1 mM.

[0083] Application Example 1

[0084] The application of the nanopeptide obtained in Example 3 in cytotoxicity specifically includes the following contents:

[0085] 1. Cell Viability Assay

[0086] Mouse normal hepatocytes AML12 were cultured in DMEM medium (Gibco, C11995500BT) (hereinafter referred to as complete medium) containing 10% FBS (Gibco, 10270-106) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37°C in a humidified incubator containing 5% CO2 (hereinafter referred to as normal incubator) until the logarithmic growth phase. The cell growth density reached more than 80%. After separation, 1×10 4 The cells were seeded into 24-well plates at a density of 1.5 cells / mL.

[0087] The cells were cultured for 24 to 48 hours until the cell growth density reached more than 80%. The culture medium was gently removed and the cells were washed once with 1×PBS preheated at 37°C. Complete culture medium was then added, followed by 1×PBS 50 μL without or with the following test samples, and the final concentrations of QE20, QE20-M, curcumin (i.e., Figure 3 Cur) and QE20-N.

[0088] The cells were cultured in a normal incubator for 24 h, and then the cell supernatant was aspirated. According to the instructions of the lactate dehydrogenase cytotoxicity assay kit (LDH Cytotoxicity Assay Kit, C0017, Bio-Tech), the LDH released by the cells was detected using a microplate reader (Thermo Fisher) to evaluate the cytotoxicity of the drug.

[0089] The results are as follows Figure 3 As shown, it can be seen that 25 μM QE20, 25 μM QE20-M, 25 μM curcumin, and 25 μM QE20-N are the optimal experimental concentrations for cells and do not cause obvious toxicity to cells.

[0090] 2. Cell Hypoxia and Reoxygenation Experiment

[0091] Mouse normal hepatocytes AML12 were cultured in DMEM medium (Gibco, C11995500BT) (hereinafter referred to as complete medium) containing 10% FBS (Gibco, 10270-106) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37°C in a humidified incubator containing 5% CO2 (hereinafter referred to as normal incubator) until the logarithmic growth phase. The cell growth density reached more than 80%. After separation, 5×10 5 The cells were seeded into 3.5 cm culture dishes at a density of 1.5 cells / mL.

[0092] The cells were cultured for 24 to 48 hours until the cell growth density reached more than 80%, and the culture medium was gently removed and the cells were washed once with 1×PBS preheated at 37°C. 1 mL of complete culture medium was added to each culture dish in the normal culture group (Normoxia), and 1 mL of sugar-free and serum-free DMEM culture medium (Gibco, 11966025) was added to each culture dish in the hypoxia / reoxygenation experiment group (H / R). Then, 50 μL of 1×PBS without or with the following test samples was added to the two groups, and the final concentrations were 25 μM QE20, 25 μM QE20-M, 25 μM curcumin, and 25 μM QE20-N, respectively.

[0093] The cells in the H / R group were first incubated in a normal incubator for 30 min, and then transferred to a hypoxic incubator at 37°C, containing 1.0% O2, 5% CO2, and 94% N2 for 12 h (this is the hypoxia stage). After 12 h of incubation, the cell culture medium of the Normoxia group and the H / R group was removed, and then washed once with 1×PBS incubated at 37°C, replaced with 1 mL of fresh complete culture medium incubated at 37°C, and 1×PBS, 25 μM QE20, 25 μM QE20-M, 25 μM curcumin, and 25 μM QE20-N were added to the corresponding wells, and then continued to be cultured in a normal incubator for 6 h (this is the reoxygenation stage). The cells in the Normoxia group were cultured in a normal incubator throughout the process.

[0094] After the cell culture was completed, 100 μL of the culture medium was aspirated and transferred to a 96-well black plate. The relative ATP content was determined using an enhanced ATP detection kit using a chemiluminometer (BioTek, SYNERGY LX) according to the instructions (enhanced ATP detection kit, S0027, Beyotime Biotechnology, Shanghai, China).

[0095] The results are as follows Figure 4 As shown, it can be seen that 25 μM QE20-N has the most significant effect in inhibiting cellular ATP release.

[0096] The culture medium was discarded, and the cells were washed once with 1×PBS. Then, 0.25% trypsin solution without EDTA (Beyotime, C0205) was added and incubated at 37°C for 2 min. Then, complete culture medium was added to neutralize the reaction. After collecting the cells, centrifugation (1000 rpm) was performed for 3 min. The supernatant was discarded, and the cells were washed twice with 1×PBS pre-cooled at 4°C. Then, antibody staining was performed according to the steps of the cell apoptosis detection kit (BD PharmingenTM FITC Annexin V Apoptosis Detection Kit I, 556547). After staining, the cells were detected by flow cytometry (BD LSRFortessaTM).

[0097] The results are as follows Figure 5 As shown, 25 μM QE20-N had the most significant effect in alleviating cell apoptosis.

[0098] Application Example 2

[0099] The application of the nanopeptide obtained in Example 3 to liver ischemia-reperfusion comprises the following steps:

[0100] Material preparation: 8-week-old male C57BL / 6J mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and fed with SPF rat growth and breeding feed (Beijing Keao Xieli Feed Co., Ltd.) under pathogen-free conditions and fasted for 12 h before surgery.

[0101] 1. Establishment of the liver blood flow occlusion model

[0102] The mice in the experimental group were anesthetized by intraperitoneal injection of 1.25% avertin, and then the limbs of the mice were fixed, the abdomen was disinfected with alcohol, and the abdomen was opened layer by layer along the midline of the abdomen to expose the hilum of the liver. The hilum of the mouse was ligated with 4-0 non-absorbable surgical sutures to construct a 70% liver (left lobe and middle lobe) blood flow occlusion model (the liver color turned grayish white).

[0103] Then, the incision was covered with wet gauze and placed under a warm lamp. After 1 hour, the ligature was loosened and the ligature was gently rubbed with a cotton swab. After the blood flow to the liver was restored, the abdominal cavity was closed and the mice were placed under a warm lamp to wake up. The sham operation group (Sham, 5 mice) underwent the same surgical procedure, but the liver hilum was not ligated.

[0104] Blood was collected from the eye sockets at 3 h (5 mice), 6 h (5 mice), 12 h (5 mice), and 24 h (5 mice) after surgery. The blood was placed in a 1.5 mL EP tube and allowed to stand at room temperature for 30 min. The blood was then centrifuged at 4°C, 5000 rpm, and the serum was transferred to another 1.5 mL EP tube. Alanine aminotransferase (ALT) (Jiangsu Kangshang Biomedical Technology Co., Ltd.) and aspartate aminotransferase (AST) (Jiangsu Kangshang Biomedical Technology Co., Ltd.) were detected according to the procedures of the kit.

[0105] The results are as follows Figure 6 As shown, the liver function (ALT, AST) test of mice showed that the liver damage was most serious after 1h of ischemia and 6h of reperfusion in mice.

[0106] 2. Mouse liver ischemia-reperfusion injury experiment (intravenous injection)

[0107] The average weight of 8-week-old mice was 22 g. The mouse liver ischemia-reperfusion model was the same as described in step 1, that is, the mouse liver was ischemic for 1 h and then reperfused for 6 h. The experimental groups were divided into NC (Normal Control) group (4 mice) and QE20 group. 1 h before surgery, the NC group was intravenously injected with 100 μL sterile 1×PBS, and the QE20 group was intravenously injected with the same volume of polypeptide (the final concentration of polypeptide in blood was about 10 uM, 25 uM and 50 uM, 4 mice each). The following experimental steps were the same as step 1.

[0108] The results are as follows Figure 7As shown, the effect of polypeptide QE20 in preventing and inhibiting ischemia-reperfusion injury in mouse liver depends on the concentration of polypeptide in the blood, and the liver protective effect increases with increasing concentration.

[0109] 3. Mouse liver ischemia-reperfusion injury experiment (intravenous injection)

[0110] The average weight of 8-week-old mice was 22 g. The mouse liver ischemia-reperfusion model was established as described above, that is, the mouse liver was ischemic for 1 h and then reperfused for 6 h. The experimental groups were divided into NC (Normal Control) group (5 mice), QE20 group (5 mice), QE20-M group (5 mice), curcumin group (Cur group, 5 mice) and QE20-N group (5 mice).

[0111] 1h before surgery, the NC group was intravenously injected with 100 μL sterile 1×PBS, the QE20 group was intravenously injected with the same volume of peptide (final blood concentration of about 50uM), the QE20-M group was intravenously injected with the same volume of peptide (final blood concentration of about 50uM), the QE20-N group was intravenously injected with the same volume of peptide (final blood concentration of about 50uM), and the Cur group was intravenously injected with the same volume of 10mg / ml Cur. The following experimental steps were the same as step 1.

[0112] The results are as follows Figure 8 As shown, QE20-N was the best in preventing and inhibiting liver ischemia-reperfusion injury in mice.

[0113] It can be seen from the above embodiments that the nanopeptide targeting Pannexin-1 channel protein provided by the present invention is formed by modifying the polypeptide QE20 with NBC and curcumin. The nanoparticle encapsulation makes the small molecule polypeptide more stable in the acidic environment of oxidative stress in vivo; the nanoparticle encapsulation makes the small molecule polypeptide liver-targeted in vivo; at the same time, the polypeptide modification makes the small molecule polypeptide have ROS response function, and the curcumin modification makes the small molecule polypeptide have stronger antioxidant effect.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanopeptide targeting Pannexin-1 channel protein, characterized in that: The nanopeptide targeting Pannexin-1 channel protein is formed by modifying polypeptide QE20 by NBC and curcumin, and the amino acid sequence of the polypeptide QE20 is shown in SEQ ID NO:

1.

2. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 1, characterized in that: The following steps are involved: The polypeptide QE20 is synthesized by a solid phase method, stearic acid is conjugated to the N-terminus of the polypeptide QE20, and NBC is synthesized simultaneously; reacting the NBC with the polypeptide QE20 to obtain the NBC-modified polypeptide QE20; Curcumin is reacted with the NBC-modified polypeptide QE20 to obtain a nanopeptide targeting the Pannexin-1 channel protein.

3. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 2, characterized in that: The NBC is prepared by reacting 4-(hydroxymethyl)phenylboronic acid dihydropentadienyl ester, 4-nitrophenyl chloroformate and a base, and specifically comprises the following steps: 4-(Hydroxymethyl)phenylboronic acid dihydropentadienyl ester and 4-nitrophenyl chloroformate are dissolved in solvents respectively, mixed, and then a base is added at 0-4°C, stirred and mixed, reacted at 20-25°C for 3-5h, the solvent is removed, diluted, washed, the organic layer is collected, concentrated, and purified to obtain NBC.

4. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 2, characterized in that: The NBC is reacted with the polypeptide QE20, comprising the following steps: The NBC and the polypeptide QE20 are dissolved separately, mixed and stirred, and reacted at 20-25° C. overnight to obtain a mixed solution, and the mixed solution is dialyzed to obtain the NBC-modified polypeptide QE20.

5. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 4, characterized in that: The molar ratio of the NBC and the polypeptide QE20 after being dissolved is 1.2-1.5:

1.

6. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 5, characterized in that: The molecular weight cut-off of the dialysis was 1000 Da.

7. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 2, characterized in that: The step of reacting curcumin with the NBC-modified polypeptide QE20 comprises the following steps: The curcumin and the NBC-modified polypeptide QE20 are mixed at 20-25° C., reacted for 20-30 minutes, added with PBS solution and stirred, reacted for 3-4 hours, and dialyzed to obtain the nanopeptide targeting Pannexin-1 channel protein.

8. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 7, characterized in that: The volume ratio of the curcumin to the NBC-modified polypeptide is 1:3-4.

9. The method for synthesizing a nanopeptide targeting Pannexin-1 channel protein according to claim 7, characterized in that: The molecular weight cut-off of the dialysis is 3000-3500Da.

10. Use of the nanopeptide targeting Pannexin-1 channel protein according to claim 1 in liver ischemia-reperfusion injury and other liver injuries.

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