Drug complex based on cyclic peptide with responsive self-assembly function and preparation method thereof

Abstract

The present invention belongs to the field of biotechnology, and discloses a drug complex based on a cyclic peptide with a responsive self-assembly function and a preparation method thereof, wherein the drug complex comprises a cyclic peptide formed by disulfide bond connection and a drug loaded on the cyclic peptide; positively charged lysine residues and arginine residues in a polypeptide sequence are combined with negatively charged groups in a drug molecule through electrostatic action, so that the cyclic peptide and the drug can be tightly combined, and when the drug complex enters a cell, under the action of a high concentration of glutathione in the cell, the disulfide bond of the cyclic peptide is broken, self-assembly is initiated, and the self-assembly behavior of the polypeptide itself will squeeze out the drug molecule, thereby completing its own self-assembly and realizing efficient release of the drug. At the same time, the present invention also provides a drug.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical technology, in particular to a pharmaceutical complex based on a cyclic peptide with a responsive self-assembly function and a preparation method thereof. Background Art

[0002] Nucleic acid drugs have become a research hotspot in the field of biomedicine. With the development of genomics, proteomics and other disciplines, nucleic acid drugs have shown great application potential. Although the clinical application of nucleic acid drugs has gradually increased, they still face some challenges. For example, nucleic acid molecules are large in size and have negative charges, making it difficult for them to pass through the cell membrane and enter the cytoplasm. At present, two relatively mature nucleic acid drug delivery systems have been developed, including liposome nanoparticle (LNP) technology and chemical coupling, adding a GalNAc technology to the small nucleic acid sequence to promote the effective entry of small nucleic acids into hepatocytes. However, the limitations of these two technologies are also very obvious, and they can only target the liver. Therefore, the development of delivery technology targeting other than the liver has become the key to the third generation of small nucleic acid delivery technology. Cell-penetrating peptides are a commonly used drug delivery carrier that can cross the cell membrane without causing significant cytotoxicity and deliver various macromolecules (such as nucleic acids, proteins, drugs, etc.) into the cell. However, the poor stability of peptides and the problem of how to efficiently release drugs after delivering them into the cell still need to be further solved.

[0003] Chinese patent CN 114099701 A provides a self-assembling composite polypeptide and its preparation method and medicine, the self-assembling composite polypeptide comprises a targeting recognition unit, a self-assembling unit and a functional unit connected in sequence. When the self-assembling composite polypeptide enters the tumor cell, due to the high glutathione environment in the tumor cell, the disulfide bond is broken under the action of glutathione, self-assembly is initiated, and nanomaterials are formed, so that the probe and / or drug connected to the self-assembling unit is deposited in the tumor cell, the action time is prolonged, and the targeted action is in the tumor cell. It can be seen from the patent that the self-assembling unit and the functional unit are connected by an amide bond. After self-assembly occurs, the functional unit (probe and / or drug) is still connected to the polypeptide and is not disconnected. Chinese patent CN 117720661 A discloses a self-assembling sulfur cyclic peptide with neuroprotective effect and its preparation method and application, the self-assembling sulfur cyclic peptide comprises: a transmembrane peptide, cysteine, an assembly peptide, a PSD-95 targeting peptide, and cysteine ​​connected in sequence, and two cysteines are connected by a disulfide bond to form a cyclic peptide structure. When the thiocyclic peptide enters the damaged nerve cells, the disulfide bonds in the thiocyclic peptide break under the action of the overexpressed reactive oxygen species in the damaged cells, thereby activating the self-assembly ability of the thiocyclic peptide and the PSD-95 targeting ability. The activated thiocyclic peptide self-assembles in situ while targeting the PDZ domain in the PSD-95 protein, further enhancing the binding effect, effectively inhibiting the binding of PSD-95 and nNOS, and achieving the effect of protecting nerve cells. The cyclic peptide mainly utilizes the effect of the PSD-95 targeting peptide and is not connected to a drug.

[0004] The above-mentioned prior arts do not involve the problem of how to efficiently release the drug after delivering it into the cell. Therefore, the technical problem to be solved by this scheme is: how to efficiently release the drug after delivering it into the cell so that the drug can be separated quickly and effectively. Summary of the invention

[0005] The object of the present invention is to provide a drug complex based on a cyclic peptide with a responsive self-assembly function, which can achieve efficient drug release under the action of high concentration of glutathione (GSH) in cells.

[0006] Meanwhile, the invention also provides a medicine.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A drug complex based on a cyclic peptide with responsive self-assembly function, the drug complex comprising a cyclic peptide formed by disulfide bond connection and a drug loaded on the cyclic peptide; the drug is loaded on the cyclic peptide by charge adsorption, and after entering the cell by utilizing the membrane-penetrating function of the cyclic peptide, when the disulfide bond of the cyclic peptide is broken, a linear polypeptide is formed, and the polypeptide realizes efficient release of the drug by self-assembly.

[0009] In the present invention, a cyclic peptide formed based on a disulfide bond connection is used to load the drug, wherein the cyclic peptide is loaded on the macromolecular drug by non-covalent bonding and charge adsorption, and its charge distribution is as follows: the positively charged lysine residues and arginine residues in the cyclic peptide sequence are combined with the negatively charged groups in the drug molecules (such as the phosphate groups in the nucleic acid drug molecules) through electrostatic interaction, so that the cyclic peptide and the drug can be tightly bound. When the drug complex enters the cell, under the action of the high concentration of glutathione in the cell, the disulfide bonds of the cyclic peptide are broken, and self-assembly is initiated. The self-assembly behavior of the polypeptide itself will squeeze out the drug molecules, thereby completing its own self-assembly and achieving efficient release of the drug. This self-classification phenomenon is the theoretical basis for the separation of polypeptides and drugs. Self-classification here refers to the separation of polypeptides and drugs. The delivery process is like Figure 1 shown.

[0010] Preferably, the amino acid sequence of the cyclic peptide is as shown in SEQ ID NO 1 and / or SEQ ID NO 2, and its specific sequence is VKVWVKCYNGTKCVRVKV-NH 2 and / or VKVWVKCYNGTKVCRVKV-NH 2 .

[0011] More preferably, the structural formula of the cyclic peptide is as shown in Formula I or Formula II:

[0012] Formula I;

[0013] Formula II.

[0014] More preferably, the preparation method of the cyclic peptide is as follows: the amino acid is coupled to the resin on a fully automatic peptide synthesizer by a standard Fmoc solid phase synthesis method, the synthesized polypeptide is cut from the resin, the side chain protecting groups of the amino acid are removed, and the crude polypeptide product is obtained by precipitation, centrifugation, and washing, and then purified, and then freeze-dried to obtain a polypeptide freeze-dried powder, and finally cyclized to obtain a cyclic peptide freeze-dried powder.

[0015] Preferably, the drug is a negatively charged drug.

[0016] More preferably, the drug is one or more of a nucleic acid drug, a polypeptide drug, a protein drug, and an antibody drug.

[0017] More preferably, the nucleic acid drug is one or more of miRNA, siRNA, mRNA, ASO and aptamers;

[0018] The polypeptide drug is one or more of the drugs containing aspartic acid and / or glutamic acid residues that are negatively charged overall;

[0019] The protein drug is one or more of a negatively charged recombinant protein drug or a blood preparation-related protein;

[0020] The antibody drug is one or more monoclonal antibodies for treatment.

[0021] Finally, the present invention also discloses a medicine containing the above-mentioned drug complex.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] In the present invention, a cyclic peptide formed by disulfide bond connection is used to load the drug, wherein the cyclic peptide is loaded on the macromolecular drug by non-covalent bonding and charge adsorption, and its charge distribution is as follows: the positively charged lysine residues and arginine residues in the polypeptide sequence are combined with the negatively charged groups in the drug molecules by electrostatic action, so that the cyclic peptide and the drug can be tightly combined. When the drug complex enters the cell, under the action of the high concentration of glutathione in the cell, the disulfide bond of the cyclic peptide is broken, and self-assembly is initiated. The self-assembly behavior of the polypeptide itself will squeeze out the drug molecules, thereby completing its own self-assembly and achieving efficient release of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the drug delivery process;

[0025] Figure 2 This is a diagram showing the effect of cP1 self-assembling to form a hydrogel under reducing conditions;

[0026] Figure 3 TEM image of P1 assembly hydrogel;

[0027] Figure 4 This is a diagram showing the effect of cP2 self-assembling to form a hydrogel under reducing conditions;

[0028] Figure 5 TEM image of P2 assembly hydrogel;

[0029] Figure 6 The cell viability results of SHED cells after 24 hours of treatment with different concentrations of cP1 and P1. The IC 50 The value is 136 μM, and the IC of P150 The value is 124 μM;

[0030] Figure 7 The cell viability results of SHED cells after 24 hours of treatment with different concentrations of cP2 and P2. The IC 50 The values ​​were all 118 μM;

[0031] Figure 8 This is the laser confocal image of the peptides entering the cells after cP1 and P1 were co-incubated with A549 cells for 4 hours;

[0032] Fig. 9 This is the laser confocal image of the peptides entering the cells after cP2 and P2 were co-incubated with A549 cells for 4 hours;

[0033] Fig.10 This is a biological transmission electron microscopy image of the cP1 polypeptide self-assembling into a nanofiber structure after entering the cell;

[0034] Fig.11 To simulate the intracellular reduction conditions in vitro, laser confocal microscopy and transmission electron microscopy were used to characterize the effective separation process of the peptide cP1 and the nucleic acid model drug DNA in the drug complex;

[0035] Fig.12 This is a confocal microscopy image after the drug complex was co-incubated with cells for 6 hours;

[0036] Fig.13 for Fig.12 The colocalization analysis results of the middle dashed rectangular area;

[0037] Fig.14 This is a laser confocal image of the drug entering the cell after cP1 and model nucleic acid drug DNA formed drug complexes at different charge ratios and co-incubated with A549 cells for 2 hours;

[0038] Fig.15 This is a WB experiment result diagram of the drug complex knocking down CD44 protein;

[0039] Fig.16 This is a laser confocal image of the drug entering the cells after cP1 and the model protein drug GFP formed a drug complex with a charge ratio of 10:1 and were co-incubated with A549 cells for 2 hours. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in 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.

[0041] Product information:

[0042] Fmoc-protected amino acids were purchased from Jier Biochemical (Shanghai) Co., Ltd.;

[0043] The activator HCTU was purchased from Jier Biochemical (Shanghai) Co., Ltd.;

[0044] Organic solvents such as DMF and anhydrous ether and piperidine were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0045] Fetal bovine serum and DMEM culture medium were purchased from Thermo Fisher (Suzhou) Instrument Co., Ltd.

[0046] Part I

[0047] Example 1: Preparation of cyclic peptide

[0048] Preparation method: The peptide was synthesized on a CSBio 316S fully automatic peptide synthesizer by a standard Fmoc solid phase synthesis method, with a synthesis amount of 0.2 mmol each time. The specific synthesis method is as follows: First, weigh the exact amount of resin according to the loading amount of the resin and put it into the reactor. The reactor was heated to 45°C in a water bath, and then the fully automatic synthesis program was started. Fmoc deprotection was completed in a DMF solution containing 20% ​​piperidine. The coupling reaction was carried out by adding a 4-fold excess of Fmoc-protected amino acid, 3.8-fold excess HCTU and 8-fold excess DIPEA. The coupling reaction lasted for 45 minutes. For amino acids with low coupling efficiency, the coupling reaction time was extended or the coupling was repeated once. The synthesized peptide was cut by adding 10 mL Regent B (TFA / phenol / water / triisopropylsilane = 88:5:5:2) to cut the peptide from the resin and remove the side chain protecting groups of the amino acid. After cutting at room temperature for 3 hours, cold ether was added for precipitation and centrifugation, and then washed twice with cold ether to obtain a crude peptide product. The crude product was purified by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and then lyophilized to obtain peptide lyophilized powder. The purified lyophilized peptide was analyzed and identified by MALDI-TOF mass spectrometry.

[0049] Cyclization method: The lyophilized peptide powder was dissolved in a phosphate buffer solution (100 mM, pH 7.4) to obtain a solution with a concentration of 1 mM, and then a 2-fold excess of oxidized glutathione (GSSG) was added to the above peptide solution. After the obtained mixed solution was incubated at 37°C for 2 h, the reaction was monitored by high performance liquid chromatography to determine whether it was complete, and the molecular weight was identified by MALDI-TOF mass spectrometry. After the reaction was completed, it was purified by semi-preparative reversed-phase high performance liquid chromatography and lyophilized to obtain a lyophilized powder of the cyclic peptide.

[0050] The final amino acid sequence is VKVWVKCYNGTKCVRVKV-NH 2 Cyclic peptide cP1;

[0051] The amino acid sequence is VKVWVKCYNGTKVCRVKV-NH 2 Cyclic peptide cP2.

[0052] Example 2: Self-assembly verification of cyclic peptides cP1 and cP2

[0053] After adding 1.1 times the molar equivalent of tris(2-carboxyethyl)phosphine (TCEP, a reducing agent) to a 1.2 wt% cP1 peptide solution (peptide dissolved in a pH 7.4 BTP buffer solution), the disulfide bond can be broken to form P1, which then self-assembles and forms a hydrogel. The microstructure of the hydrogel was characterized by transmission electron microscopy (TEM). Related test results refer to Figure 2 and Figure 3 ;

[0054] Figure 2 This is a diagram showing the effect of cP1 self-assembling to form a hydrogel under reducing conditions;

[0055] Figure 3 TEM image of P1 assembly hydrogel.

[0056] 2. After adding 1.1 times the molar equivalent of TCEP to a 2 wt% cP2 peptide solution (peptide dissolved in a pH 7.4 BTP buffer solution), the disulfide bonds can be broken to form P2, which then self-assembles and forms a hydrogel. Figure 4 and Figure 5 ;

[0057] Figure 4 This is a diagram showing the effect of cP2 self-assembling to form a hydrogel under reducing conditions;

[0058] Figure 5 TEM image of P2 assembly hydrogel.

[0059] The above experiments show that cP1 and cP2 are reduction responsive and can self-assemble to form nanofiber structures.

[0060] Example 3: Cytotoxicity experiments of cP1 and P1 and cP2 and P2

[0061] Experimental method: Cytotoxicity was determined using the MTT method. The specific protocol was as follows: SHED cells were plated at a density of 5,000 cells / well in a 96-well plate, and 100 μL of DMEM complete medium containing 10% fetal bovine serum (FBS, v / v), 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well. The cells were incubated at 37°C, 5% CO 2 After 24 hours of incubation under the conditions, the medium was replaced with serum-free medium containing 0.01-500 µM peptide. Blank medium or medium containing 20% ​​DMSO was used as positive or negative control, respectively. After 24 hours of incubation, the medium was aspirated and DMEM (1:10, v / v) containing MTT (5 mg / mL) was added and incubated for another 4 hours. Then 100 µL of DMSO was added to each well and incubated in a shaker at 37°C for 0.5 hours to dissolve the methazolamide crystals. The absorbance at a wavelength of 490 nm was read using a microplate reader, and the percentage survival rate was calculated according to the following formula: (absorbance of peptide-treated cells / absorbance of untreated cells) × 100, the experimental results refer to Figure 6 and Figure 7 ;

[0062] Figure 6 The cell viability results of SHED cells after 24 hours of treatment with different concentrations of cP1 and P1. The IC 50 The value is 136 μM, and the IC of P1 50 The value is 124 μM.

[0063] Figure 7 The cell viability results of SHED cells after 24 hours of treatment with different concentrations of cP2 and P2. The IC 50 The values ​​were all 118 μM.

[0064] The experimental results showed that the IC 50 The values ​​were all over 100 μM, and the highest concentration used in subsequent experiments was 20 μM, so it would not cause damage to the cells.

[0065] Example 4: Experiment on the cell membrane penetration ability of cP1 and cP2

[0066] Experimental method: A549 cells in the logarithmic growth phase were digested and plated in confocal dishes, with 150,000 cells per dish, and placed in a cell culture incubator for 24 hours. The culture medium was aspirated, and 5 μM fluorescently labeled (FITC-labeled) cP1 / P1 and cP2 / P2 serum-free culture medium was added and cultured for 4 hours. The culture medium was aspirated, and the cells were washed once with a culture medium containing 10% FBS (v / v) to remove non-specifically adsorbed peptides on the cell membrane surface, and then gently washed twice with PBS, and then the nuclear staining agent Hoechst 33342 was added for staining for 15 minutes. The stain was aspirated, and Opti-MEM culture medium was added. The entry of peptides into cells was observed under a confocal microscope. The experimental results refer to Figure 8 and Fig. 9 .

[0067] Figure 8 Laser confocal images of the peptides entering A549 cells after cP1 and P1 were co-incubated with the cells for 4 hours.

[0068] Fig. 9 Laser confocal images of cP2 and P2 peptides entering A549 cells after incubation with the cells for 4 hours.

[0069] The experimental results showed that cP1 and cP2 had good cell membrane penetration ability, while P1 and P2 without disulfide bond cyclization had no cell membrane penetration ability.

[0070] Example 5: Self-assembly experiment of cP1 in cells

[0071] Experimental method: A549 cells in the logarithmic growth phase were digested and plated in a 6-well plate with 500,000 cells per well, and placed in a cell culture incubator for 24 hours. The culture medium was aspirated, and serum-free culture medium containing 100 μM fluorescent-labeled peptides was added and cultured for another 8 hours. The culture medium was aspirated, and the cells were first washed once with a culture medium containing 10% FBS (v / v) to remove nonspecifically adsorbed peptides on the cell membrane surface, and then gently washed twice with PBS, and then trypsin was added to digest and collect the cells. The cells were scraped off with a cell scraper (do not scrape repeatedly, and scrape them off quickly at one time), centrifuged at 3000 rpm for 5 minutes, and after discarding the supernatant, 2.5% (v / v) glutaraldehyde fixative precooled at 4°C was slowly added along the tube wall, and then sent to the sample testing company for testing. Test result reference Fig.10 .

[0072] Fig.10 This is a biological transmission electron microscopy image of the cP1 polypeptide self-assembling into a nanofiber structure after entering the cell; Fig.10 A is a biological transmission electron microscopy image of cells in the blank control group. Fig.10B in the figure is a biological transmission electron microscopy image of cells treated with cP1 polypeptide; Fig.10 In A and B, from left to right they are gradually enlarged images, and the image on the right is the area shown in the box on the left.

[0073] The experimental results show that cP1 can self-assemble into a nanofiber structure after entering the cell, proving that after cP1 enters the cell, disulfide bond reduction occurs in the presence of a large amount of intracellular GSH to form P1, and P1 then self-assembles into a nanofiber structure.

[0074] Part 2

[0075] Example 6: In vitro simulation of nucleic acid drug delivery experiment

[0076] Experimental method: Take a certain amount of fluorescently labeled (Cy5 labeled) miRNA master solution (10 μM) and fluorescently labeled (FITC labeled) polypeptide master solution (1 mM) and pre-mix them at a charge ratio of 1:20 for 30 minutes. Then divide it into two equal parts, one of which is diluted 10 times and dropped on the confocal dish, and the other part is added with 2.5 times excess TCEP (the master solution is 5 mM), the pH is adjusted to 7.4, incubated at 37°C for 10 minutes, and also diluted 10 times, dropped on the confocal dish, and the distribution of miRNA and polypeptide is observed under a confocal microscope. At the same time, the sample is characterized by transmission electron microscopy (TEM). Experimental results reference Fig.11 .

[0077] Fig.11 In order to simulate intracellular reduction conditions in vitro, laser confocal microscopy and transmission electron microscopy were used to characterize the effective separation process of polypeptide cP1 and nucleic acid model drug miRNA in the drug complex.

[0078] The experimental results show that the drug complex formed by nucleic acid drugs and cP1 can be separated under reducing conditions, and the formation of polypeptide assemblies can be observed.

[0079] Example 7: Colocalization analysis experiment

[0080] Experimental method: Fluorescently labeled cP1 and fluorescently labeled miRNA (non-functional nucleic acid drug model) first formed a complex with a charge ratio of 20:1. After the complex was co-incubated with A549 cells for 6 hours, it was imaged and observed by confocal microscopy, and co-localization analysis was performed. Fig.12 , Fig.13 .

[0081] Fig.12 This is a confocal microscopy image after the drug complex was co-incubated with cells for 6 hours;

[0082] Fig.13for Fig.12 The colocalization analysis results in the middle dashed rectangular area.

[0083] The experimental results show that the green fluorescent group of the labeled polypeptide and the red fluorescent group of the labeled miRNA hardly overlap, indicating that the polypeptide and miRNA are separated in the cell.

[0084] Example 8: cP1 intracellular delivery of nucleic acid drugs characterization experiment

[0085] Experimental method: This experiment selected single-stranded DNA (ssDNA) molecules as model nucleic acid drugs for research. The specific method is as follows: A549 cells in the logarithmic growth phase were digested and plated in confocal dishes, with 150,000 cells per dish, placed in a cell culture incubator for 24 hours, and the culture medium was aspirated. Different amounts of polypeptide master solution (1 mM) and a certain amount of fluorescently labeled DNA master solution (10 μM) were pre-mixed for 30 minutes at charge ratios of 2:1, 5:1 and 10:1, respectively. Serum-free culture medium was added to dilute to 200 μL, and the final concentration of DNA was 100 nM. The above solution was added to the cells and incubated for another 2 hours. The culture medium was aspirated, and the cells were washed once with a culture medium containing 10% FBS (v / v) to remove nonspecifically adsorbed complexes on the cell membrane surface, then gently washed twice with PBS, and then the nuclear stain hoechst 33342 was added for staining for 15 minutes. The stain was aspirated, and Opti-MEM culture medium was added to observe the entry of DNA into the cells under a confocal microscope. The experimental results are as follows Fig.14 shown.

[0086] Fig.14 These are laser confocal images of the drug entering A549 cells after cP1 and model nucleic acid drug DNA formed drug complexes at different charge ratios and were incubated with the cells for 2 hours.

[0087] Experimental results show that with the increase of charge ratio (cP1:ssDNA), the intracellular delivery efficiency of nucleic acid drugs increases significantly.

[0088] Example 9: WB experiment of cP1 delivering functional miRNA

[0089] Experimental method: Western blot experiment was used to verify the effect of cP1 delivering functional miRNA-34a to knock down CD44 protein expression. The specific method is as follows: cP1 and miRNA-34a were pre-mixed to form a complex, and then diluted with culture medium to a cP1 concentration of 10 μM (miRNA-34a concentration of 100 nM), and then added to T24 cells for incubation for 48 hours. The treated cells were collected, lysed, and protein samples were prepared. Protein quantification was performed using the BCA method. The amount of protein loaded was adjusted according to experimental requirements. After electrophoresis, transfer, and blocking, the primary antibody was added and incubated at 4 o Incubate overnight in refrigerator at C. After rinsing with 1×TBST, add secondary antibody and incubate at room temperature for 1 hour. Prepare substrate developer and drop the developer onto the PVDF membrane. Use protein luminescence imaging to perform exposure analysis to detect the effect of drug complex knocking down CD44 protein. The experimental results are shown in Fig.15 shown.

[0090] Fig.15 This is a graph showing the WB experimental results of knocking down CD44 protein using a drug complex.

[0091] The experimental results showed that the drug complex formed by using 10 μM cP1 and functional miRNA can effectively knock down the expression of the target protein, and its effect is better than the commercial transfection reagent Lipofectamine 2000.

[0092] Example 10: cP1 intracellular delivery protein drug characterization experiment

[0093] Experimental method: In this experiment, green fluorescent protein (GFP) was selected as a model protein drug for research. The specific method is as follows: A549 cells in the logarithmic growth phase were digested and plated in confocal dishes, with 150,000 cells per dish, placed in a cell culture incubator for 24 hours, and the culture medium was aspirated. Different amounts of polypeptide master solution (1 mM) and a certain amount of GFP master solution (10 μM) were pre-mixed at a charge ratio of 10:1 for 30 minutes. Serum-free culture medium was added to dilute to 200 μL, and the final concentration of GFP was 0.25 μM. The above solution was added to the cells and incubated for another 2 hours. The culture medium was aspirated, and the cells were washed once with a culture medium containing 10% FBS (v / v) to remove nonspecifically adsorbed complexes on the cell membrane surface, then gently washed twice with PBS, and then the nuclear stain hoechst 33342 was added for staining for 15 minutes. The stain was aspirated, and Opti-MEM culture medium was added to observe the entry of GFP into the cells under a confocal microscope. The experimental results are as follows Fig.16 shown.

[0094] Fig.16This is a laser confocal image of the drug entering the cells after cP1 and the model protein drug GFP formed a drug complex with a charge ratio of 10:1 and were co-incubated with A549 cells for 2 hours.

[0095] Experimental results show that cP1 can efficiently deliver the model protein drug GFP into cells.

[0096] Summary: In summary, the drug complex based on the cyclic peptide with responsive self-assembly function provided by the present invention includes a cyclic peptide formed by a disulfide bond connection and a drug loaded on the cyclic peptide; wherein the cyclic peptide has a disulfide bond, which can be broken under reducing conditions and self-assembled to form a hydrogel; after verification by cytotoxicity experiments, the cyclic peptide and the polypeptide formed after the disulfide bond is broken will not cause damage to cells; moreover, the cyclic peptide has good cell membrane penetration ability, which provides a good basis for drug delivery.

[0097] In addition, the drug is combined with the cyclic peptide by charge adsorption, and when the disulfide bond of the cyclic peptide is broken, the cyclic peptide releases the drug by self-assembly. Relevant experiments have verified that the drug can be released quickly and efficiently, with almost no residue on the polypeptide, and as the charge ratio (cyclic peptide: drug) increases, the intracellular delivery efficiency of the drug increases significantly.

[0098] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A drug complex based on a cyclic peptide with responsive self-assembly function, characterized in that: The drug complex comprises a cyclic peptide formed by disulfide bond connection and a drug loaded on the cyclic peptide; the drug is loaded on the cyclic peptide by charge adsorption, and after entering the cell by utilizing the membrane-penetrating function of the cyclic peptide, when the disulfide bond of the cyclic peptide is broken, the polypeptide releases the drug by self-assembly; the structural formula of the cyclic peptide is shown in Formula I or Formula II: Formula I; Formula II.

2. The drug complex according to claim 1, characterized in that The preparation method of the cyclic peptide is as follows: amino acids are coupled to a resin on a fully automatic peptide synthesizer by a standard Fmoc solid phase synthesis method, the synthesized peptide is cut from the resin, the side chain protecting groups of the amino acids are removed, a crude polypeptide product is obtained by precipitation, centrifugation and washing, the crude polypeptide product is purified, and then freeze-dried to obtain a polypeptide freeze-dried powder, and finally the cyclic peptide freeze-dried powder is obtained by cyclization.

3. The drug complex according to claim 1, characterized in that The drug is a drug with a negative charge.

4. The drug complex according to claim 3, characterized in that The drug is one or more of nucleic acid drugs, polypeptide drugs, protein drugs, and antibody drugs.

5. The drug complex according to claim 4, characterized in that The nucleic acid drug is one or more of miRNA, siRNA, mRNA, ASO and aptamers; The polypeptide drug is one or more of the drugs containing aspartic acid and / or glutamic acid and having an overall negative charge; The protein drug is one or more of a negatively charged recombinant protein drug or a blood preparation-related protein; The antibody drug is one or more monoclonal antibodies for treatment.

6. A drug, characterized in that The drug contains the drug complex according to any one of claims 1 to 5.

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