A brush-like peptide drug delivery system, its preparation method and application

Abstract

This invention discloses a brush-like peptide drug delivery system, its preparation method, and its applications, belonging to the fields of polymer chemistry and biomedicine. The method includes modifying the amino acid residues of a peptide drug with active functional groups through click or cycloaddition reactions; reacting the modified peptide drug with a polydisulfide backbone through click or cycloaddition reactions to obtain a brush-like peptide drug; and performing charge shielding and bioactivity modification on the lysine residues in the brush-like peptide drug to obtain the brush-like peptide drug delivery system. The brush-like peptide drug delivery system of this invention reduces peptide toxicity, enhances peptide targeting, improves peptide pharmacokinetics, and solves the problem of poor water solubility of some peptide drugs. It can be used in the treatment of tumors, cardiovascular diseases, neurological diseases, pathogenic bacterial or viral infections, or in the development of vaccines.

Description

Technical Field

[0001] This invention belongs to the fields of polymer chemistry and biomedicine, specifically relating to a brush-like structure polypeptide drug delivery system, its preparation method, and its application. Background Technology

[0002] Peptide drugs are short-chain or long-chain molecules composed of amino acids. Compared to traditional small-molecule drugs, peptide drugs exhibit significant advantages in target selectivity and safety. First, peptide drugs can precisely bind to specific biological targets (such as receptors and enzymes). Their high specificity allows them to exert their effects accurately, avoiding off-target side effects common with traditional drugs and reducing impacts on other non-target tissues and organs. Second, being composed of amino acids, peptide drugs can be excreted through normal metabolic pathways after degradation, effectively avoiding potentially toxic metabolites produced during the metabolism of traditional drugs, thus exhibiting high safety. Furthermore, since alterations to the amino acid sequence can modify the function of peptides, various peptide drugs with specific functions can be designed according to different disease targets and treatment needs.

[0003] However, peptide drugs still face significant challenges in their use, including poor stability (proteases), short in vivo circulation time, and some peptides exhibiting hemolytic toxicity. To address these technical problems, traditional methods typically involve amino acid chemical modification or alteration of the peptide structure. For example, Chinese patent document CN109400695A discloses a method for modifying peptides and its application. This method includes: (1) introducing X into the N-terminus of the peptide to obtain an X-peptide; (2) oxidizing the X to an aldehyde group; (3) adding a reducing agent and covalently coupling the oxidation product obtained in step (2) with PEG to obtain a PEG-modified peptide; wherein the X is threonine or serine. This modification method improves the stability and cycling half-life of the peptide. Chinese patent document CN119119207A screened and obtained a short-sequence peptide that can be separated in vitro. This short-sequence peptide, when applied to peptide drugs such as analogs or derivatives of GLP-1, can improve the stability of the peptide drug, prolong its duration of action, and enhance its biological activity.

[0004] However, the above methods are generally not universally applicable. While self-assembled drug delivery systems can address these issues, self-assembled nanoparticles are prone to disintegration and failure during in vivo circulation. Therefore, a universal drug delivery system is needed to solve these technical problems. Currently, there are no reports on peptide drug delivery systems that combine peptide chemical modification and structural design to simultaneously achieve no hemolysis or toxicity, good targeting, enzyme stability, and long in vivo circulation time. Summary of the Invention

[0005] To address the problems of poor stability, significant hemolysis and toxicity, lack of targeting, and poor pharmacokinetics of peptide drugs, this invention provides a brush-like peptide drug delivery system that is pH and reducing environment responsive and degradable, suitable for the delivery of various peptide drugs.

[0006] The specific technical solution adopted is as follows:

[0007] This invention provides a brush-shaped peptide drug delivery system, comprising: modifying the amino acid residues of the peptide drug with active functional groups by click reaction or cycloaddition reaction; reacting the modified peptide drug with the polydisulfide backbone by click reaction or cycloaddition reaction to obtain a brush-shaped peptide drug; and performing charge shielding and bioactivity modification on the lysine residues in the brush-shaped peptide drug to obtain the brush-shaped peptide drug delivery system.

[0008] The polypeptide drug is composed of 5-100 amino acids, with ≥1 lysine residue.

[0009] The structural formula of the polydisulfide backbone is any one of the following:

[0010] ;

[0011] Where x is an integer from 1 to 500, y is an integer from 1 to 2000, and z is an integer from 1 to 1000; R2 is a quencher residue, and R3 is a click reaction or cycloaddition reaction active functional group that can react with the modified functional groups on the amino acid residues of the peptide drug via click reaction or cycloaddition reaction. R1 and R3 are the same or are methyl or methoxy (R1 is further preferably methyl or methoxy).

[0012] The method for charge shielding and bioactivity modification involves reacting at least one 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative with lysine residues in a brush-like polypeptide drug, thereby shielding the positive charge caused by lysine residues and modifying the bioactive functional groups. The structural formula of the 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative is shown below:

[0013] ;

[0014] R4 is a bioactive functional group.

[0015] Preferably, the cysteine ​​residues of the polypeptide drug are modified with active functional groups by click reaction or cycloaddition reaction.

[0016] Specifically, the peptide drug can be obtained by linking a cysteine ​​residue to the C-terminus or N-terminus of common peptide drugs (including but not limited to melittin, CALL, KLAK, LTX-315, etc.).

[0017] Furthermore, in the modified peptide drug, the click reaction or cycloaddition reaction active functional group is selected from any one of the following formulas:

[0018] .

[0019] Furthermore, in the polydisulfide backbone, R3 is selected from any one of the following formulas:

[0020] .

[0021] Furthermore, the molar ratio of the modified peptide drug to the polydisulfide backbone during click or cycloaddition reactions is 1:0.01-1; more preferably 1:0.01-0.5, and even more preferably 1:0.01-0.03; when a click reaction occurs, the reaction conditions are -20 to 100 °C for 2 to 48 h; when a cycloaddition reaction occurs, the reaction conditions are -20 to 100 °C for 1 to 72 h.

[0022] Furthermore, the bioactive functional group R4 is selected from any one of the following formulas:

[0023] ;

[0024] Where m, n, o, p, and q are each independently selected from integers from 1 to 1000.

[0025] Furthermore, during charge shielding and bioactivity modification, the molar ratio of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative to lysine residues in the brush-like peptide drug was 10-1000:1, and the reaction conditions were -20 to 100°C. ℃, 0.01-72 h.

[0026] Preferably, the lysine residues in the brush-like polypeptide drug are reacted with the polyethylene glycol conjugate of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid and the RGD cyclic peptide conjugate of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid; the molar ratio of the polyethylene glycol conjugate of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid and the RGD cyclic peptide conjugate of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid is 1:0.01-0.5.

[0027] In the above-mentioned preferred conditions, it is beneficial to improve the tissue penetration and targeting of the brush-structured peptide drug delivery system, or to achieve better disease treatment effects when combined with other drugs.

[0028] This invention designs a peptide drug delivery system by utilizing the reaction between the polydisulfide backbone and the peptide drug, the resulting brush-like structure, and the modification of lysine residues of the peptide by a 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative. This reduces peptide toxicity, enhances peptide targeting, improves peptide pharmacokinetics, and solves problems such as poor water solubility of certain peptide drugs. Depending on the type and quantity of the peptide drug carried and the function of the 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative, it can be used in the treatment of tumors, cardiovascular diseases, neurological diseases, pathogenic bacteria or viral infections, or in the development of vaccines.

[0029] The present invention also provides a method for preparing the brush-like structured peptide drug delivery system, comprising:

[0030] (1) In an organic solvent system, the peptide drug is reacted with maleimide with click reaction or cycloaddition reaction active functional groups to modify the amino acid residues of the peptide drug with click reaction or cycloaddition reaction active functional groups.

[0031] (2) In an organic solvent system or a mixed solvent system of organic solvent and water, the polydisulfide backbone and at least one modified polypeptide drug undergo a click reaction or cycloaddition reaction to obtain a brush-shaped polypeptide drug;

[0032] (3) The brush-structured polypeptide drug delivery system is obtained by reacting at least one 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative with a brush-shaped polypeptide drug.

[0033] Specifically, an active lipid intermediate of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid can be obtained by condensing 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid with N-hydroxysuccinimide. This active lipid intermediate can then react with a compound containing an amino group and a bioactive functional group R4 to prepare a 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative.

[0034] The present invention also provides the application of the brush-like structure polypeptide drug delivery system in the preparation of disease treatment products.

[0035] The present invention also provides the application of the brush-like structure polypeptide drug delivery system in the preparation of disease prevention products.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) The brush-structured polypeptide drug delivery system provided by the present invention can solve the defects of hemolysis and non-specific toxic side effects of polypeptide drugs. The process route for synthesizing this drug delivery system has not been reported in the prior art.

[0038] (2) The brush-structured polypeptide drug delivery system provided by the present invention is universal, suitable for different polypeptide drugs, and can improve the water solubility and cellular uptake of polypeptide drugs.

[0039] (3) The brush-structured polypeptide drug delivery system provided by the present invention can improve the pharmacokinetics and in vivo distribution of polypeptide drugs, prolong the in vivo circulation time of polypeptides and target them to target organs and tissues.

[0040] (4) The brush-structured polypeptide drug delivery system provided by the present invention is based on a dual mechanism that can respond to acidic and reducing environments, realize safe and rapid release of polypeptide drugs without leaving traces and restore their function.

[0041] (5) The brush-structured polypeptide drug delivery system provided by the present invention can utilize 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivatives to achieve the binding of other bioactive molecules (drugs or functional molecules) with polypeptide drugs, thereby achieving combined treatment and medication to further improve the therapeutic effect. Attached Figure Description

[0042] Figure 1 Matrix-assisted laser desorption / ionization mass spectrometry analysis of compounds 9, 19 and 22.

[0043] Figure 2 This is the permeate gel chromatogram of compound 4.

[0044] Figure 3 The graph shows the hydrated particle size and potential analysis results for compounds 15, 23, and 26.

[0045] Figure 4 Transmission electron microscope images of compounds 15 and 23.

[0046] Figure 5 The graph shows the change in potential of compounds 18 and 24 as a function of pH.

[0047] Figure 6 The figure shows the experimental results of the degradation of compound 15 with dithiothreitol (DTT).

[0048] Figure 7The figure shows the experimental results of degradation of free melitoxin and compound 18 by chymotrypsin.

[0049] Figure 8 Hemolytic toxicity test of compounds 18 and 27 on mouse erythrocytes.

[0050] Figure 9 Confocal microscopy and flow cytometry results of uptake experiments of compounds 15, 16 and 18 in mouse oral squamous cell carcinoma (MOC1) cells.

[0051] Figure 10 The figure shows the results of cytotoxicity analysis of free melitoxin, compound 15, compound 16 and compound 18 on mouse breast cancer (4T1), mouse oral squamous cell carcinoma (MOC1) and human ovarian adenocarcinoma (SKOV3) cells.

[0052] Figure 11 This is a graph showing the pharmacokinetic results of compound 18 and free melivitin. **** indicates... p <0.0001.

[0053] Figure 12 Distribution map of compound 18 in tumors and major organs 24 hours after injection.

[0054] Figure 13 The graph shows the inhibitory effect of compound 18 on the growth of mouse tumor (4T1). Detailed Implementation

[0055] The present invention will be further illustrated below with reference to the embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0056] Example 1

[0057] (1) Synthesis of lipoic acid derivative monomers

[0058]

[0059] Weigh 30.0 g (compound 1, 0.145 mol) of lipoic acid into a 500 ml round-bottom flask, add 300 ml of anhydrous dichloromethane (DCM) to dissolve it completely, and add 25.9 g (0.160 mol) under ice-water bath conditions. N,N'- Carbonyl diimidazole (CDI) was stirred at room temperature for 30 minutes, followed by the addition of 13.2 g (0.131 mol) of 3-azidopropanol (compound 2), and then 1.8 g (14.5 mmol) of 4-dimethylaminopyridine (DMAP). The reaction was carried out overnight at room temperature. The organic phase was washed with 200 ml of water, collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residual sample was dissolved in 200 ml of diethyl ether, filtered to remove insoluble matter, and the mother liquor was washed twice with 200 ml of water. The organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The solution was then dried with an oil pump to obtain 34.4 g of yellow oily compound 3, with a yield of 82%.

[0060] The 1H NMR spectrum data of compound 3 are as follows: 1 H NMR (400 MHz, CDCl3) δ 4.16 (t, J = 6.0Hz, 2H), 3.62–3.52 (m, 1H), 3.39 (t, J = 6.8 Hz, 2H), 3.25–3.05 (m, 2H), 2.52–2.41 (m, 1H), 2.33 (t, J = 7.2 Hz, 2H), 1.98–1.85 (m, 3H), 1.77–1.61 (m, 4H), 1.56–1.39 (m, 2H).

[0061] (2) Synthesis of polydisulfide backbone

[0062]

[0063] Weigh out 30.4 mg (0.02 mmol) of polyethylene glycol thioglycolate (PEG). 2000 Compound 3 (-SH) was dissolved in 300 μL of anhydrous tetrahydrofuran (THF) in a 1.5 ml centrifuge tube under anaerobic conditions. Then, 1.9 μL (0.02 mmol) of tetramethylguanidine (Me4-guanidine) was added. The mixture was vortexed for 1 minute, and the solution became turbid. The turbid solution was aspirated in one go and quickly injected into a solution containing 800 μL (3.04 mmol) of compound 3 in 500 μL of tetrahydrofuran. The mixture was vortexed for 5 minutes and then stirred at room temperature for 2 hours. 39 μL (0.30 mmol) of trifluoroethyl acrylate was added to quench the reaction. After stirring at room temperature for 2 hours, the reaction solution was added dropwise to 30 ml of diethyl ether to precipitate the product. After vortexing and centrifugation, the supernatant was discarded. The remaining white solid was dissolved in a small amount of dichloromethane and then added dropwise to 30 ml of diethyl ether for precipitation. This precipitation was repeated 5 times and then dried to obtain 630 mg of the colorless, paste-like polydisulfide polymer backbone product compound 4 (Poly(LpN3)), with a yield of 72%.

[0064] The 1H NMR spectrum data of polydisulfide backbone 4 are as follows: 1 H NMR (400 MHz, CDCl3) δ 4.16 (t, J =6.4 Hz, 2H), 3.64 (s, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.90–2.66 (m, 3H), 2.34(t, J = 7.2 Hz, 2H), 2.10–1.83 (m, 4H), 1.72–1.59 (m, 4H), 1.55–1.39 (m, 2H).

[0065] The NMR fluorine spectrum data of polydisulfide backbone 4 are as follows: 19 F NMR (376 MHz, CDCl3) -73.68.

[0066] (3) Synthesis of cyclooctyne-modified polypeptide drugs

[0067] (3.1) Synthesis of cyclooctyne maleimide

[0068]

[0069] Bicyclo[6.1.0]non-4-yn-9-ylmethyl N - Succinimidyl carbonate (compound 5, 50 mg, 0.172 mmol), 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (compound 6, 36 mg, 0.2064 mmol) and N,N Diisopropylethylamine (DIPEA, 100 μL) was dissolved in anhydrous dichloromethane (DCM) in a round-bottom flask. The reaction mixture was stirred at room temperature for 1 hour, followed by washing with brine (3 × 50 mL). The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 20:1) to give compound 7 as a white solid (46.38 mg, yield: 85%).

[0070] The proton NMR spectrum data of the white solid compound 7 are as follows: 1 H NMR (400MHz, CDCl3) δ 6.72 (s, 2H), 4.89 (s, 1H), 3.95-3.93 (d, 2H), 3.70-3.67 (m, 2H), 3.40 (q, J = 5.8 Hz, 2H), 2.41-2.36 (m, 2H), 2.29 (t, J= 14.3 Hz, 2H), 2.15 (m, J) = 16.4, 3.0, 2.5 Hz, 2H), 1.40-1.31 (m, 2H), 1.27 (d, J = 12.4 Hz, 0H), 0.74 (s, 1H), 0.74-0.62 (m, 2H).

[0071] The carbon NMR spectrum data of the white solid compound 7 are as follows: 13 C {1H} NMR (101 MHz, CDCl3) δ 170.8, 156.8, 134.2, 98.8, 69.3, 40.0, 37.8, 33.3, 23.7, 22.8, 21.4.

[0072] (3.2) Synthesis of cyclooctyne-modified Melittin-C

[0073]

[0074] In the preparation of cyclooctyne-modified Melittin-C, the polypeptide drug (Melittin-C, melittin N-terminus linked to cysteine ​​C, compound 8, 15 mg, 5 μmol, melittin amino acid sequence GIGAVLKVLTTGLPALISWIKRKRQQ) and compound 7 (6.43 mg, 20 μmol) were dissolved in anhydrous N,N-dimethylformamide (DMF). The reaction mixture was stirred at room temperature for 4 hours, and then precipitated with diethyl ether (3 × 12 mL). The resulting precipitate (i.e., cyclooctyne-modified Melittin-C, compound 9) was freeze-dried and stored at -20°C for subsequent use (yield: 14.44 mg, yield: 87%).

[0075] (4) Synthesis of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivatives

[0076] (4.1) Synthesis of active ester of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid

[0077]

[0078] 2-Propionic-3-methylmaleic anhydride (compound 10, 37 g), N-hydroxysuccinimide (compound 11, 11 g), and 4-dimethylaminopyridine (DMAP, 6 g) were dissolved in 5 L of anhydrous tetrahydrofuran (THF) in a round-bottom flask. Then, the mixture was added... N,N'-Dicyclohexylcarbodiimide (DCC, 24 g). The reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the mixture was filtered and precipitated with cold diethyl ether (3 × 5 L) to give compound 12 (11 g, yield: 40%), a brown solid.

[0079] The proton NMR spectrum data of compound 12 are as follows: 1 H NMR (400 MHz, CDCl3), δ 3.02 (m, 2H), 2.98-2.80 (m, 6H), 2.11 (s, 3H).

[0080] (4.2) Synthesis of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid-polyethylene glycol

[0081]

[0082] mPEG11-NH2 (compound 13, 2g) and N,N 100 mL of diisopropylethylamine was dissolved in 500 mL of anhydrous N,N-dimethylformamide (DMF) in a round-bottom flask, followed by the addition of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid active ester (compound 12, 20 g). The reaction was carried out at room temperature for 4 hours and monitored by thin-layer chromatography. After confirmation of the reaction completion, the synthesis of the product 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid-polyethylene glycol (compound 14) was confirmed by liquid chromatography-mass spectrometry.

[0083] (5) Construction of a brush-shaped polymer drug delivery system

[0084] (5.1) Brush-like Melittin-C

[0085]

[0086] The polydisulfide backbone (compound 4, 2.77 mg) and cyclooctyne-modified Melittin-C (compound 9, 25 mg) were weighed and dissolved in 655 μL of anhydrous dimethyl sulfoxide (DMSO) in a microcentrifuge tube (molar ratio of compound 4 to compound 9: 1:70). The reaction mixture was stirred overnight at room temperature, followed by dialyzing with pure water over a dialysis membrane for 8 hours (molecular weight cutoff: 70,000 Da). The dialyzed solution was lyophilized to give brush-like Melittin-C (compound 15), 26 mg, yield approximately 93%, and stored at -20 to 80 °C.

[0087] (5.2) Polyethylene glycol protected brush-like Melittin-C

[0088]

[0089] Brush-like Melittin-C (compound 15, 0.84 mg) and N,N-diisopropylethylamine (DIPEA, 5.4 μL, 0.031 mmol) were weighed and dissolved in water. Then, a dimethylformamide solution of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid-polyethylene glycol (compound 14) was added at 50 times the molar volume of lysine residues. The mixture was stirred vigorously at room temperature for 5 minutes. Impurities were removed by dialyzing against an aqueous solution for 2 days (molecular weight cutoff: 700,000 Da), yielding brush-like Melittin-C modified with 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid-polyethylene glycol, which is polyethylene glycol-protected brush-like Melittin-C (compound 16). This was then lyophilized and stored.

[0090] (5.3) Brush-like structure peptide drug delivery system

[0091]

[0092] Weigh out brush-shaped Melittin-C (compound 15, 0.84 mg) and N,N-diisopropylethylamine (DIPEA, 5.4 μL, 0.031 mmol) and dissolve them in water. Then add 50 times the molar amount of lysine residues of a dimethylformamide mixture of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid-polyethylene glycol (compound 14) and 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid-RGD cyclic peptide (compound 17, commercially available) (molar ratio of compounds 14 and 17 is 100:1). Stir vigorously at room temperature for 5 minutes. Impurities were removed by dialyzing with an aqueous solution for 2 days (molecular weight cutoff: 700,000 Da), and a brush-structured peptide drug delivery system based on melitrix peptides (compound 18) was obtained and lyophilized. During the preparation of this brush-structured peptide drug delivery system, lysine residues (amino groups) in the peptide drug were randomly conjugated to the RGD cyclic peptide conjugate of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid-polyethylene glycol and 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid. The number of RGD cyclic peptides on the bottle-brush-shaped Melittin-C was 2.

[0093] Example 2

[0094] In this embodiment, the preparation method of the brush-structured peptide drug delivery system differs from that in Example 1 only in that the peptide drug used is CALL-C (a cell lysis peptide with a cysteine ​​C-terminus linked to its N-terminus, and the amino acid sequence of the cell lysis peptide is KWKLFKKIFKRIVQRIKDFLR). Under the same process parameters as in Example 1, cyclooctyne-modified CALL-C (compound 19) and brush-structured CALL-C (compound 20) are prepared, and finally, a brush-structured peptide drug delivery system based on cell lysis peptide (compound 21) is obtained.

[0095] Example 3

[0096] In this embodiment, the preparation method of the brush-structured peptide drug delivery system differs from that in Example 1 only in that the peptide drug used is KLAK-C (mitochondrial cleavage peptide with N-terminus linked to cysteine ​​C, and the amino acid sequence of the mitochondrial cleavage peptide is KLAKLAKKLAKLAK). Under the same process parameters as in Example 1, cyclooctyne-modified KLAK-C (compound 22) and brush-structured KLAK-C (compound 23) are prepared, and finally, a brush-structured peptide drug delivery system based on mitochondrial cleavage peptide (compound 24) is obtained. During the preparation of this brush-structured peptide drug delivery system...

[0097] Example 4

[0098] In this embodiment, the preparation method of the brush-structured peptide drug delivery system differs from that in Example 1 only in that the peptide drug used is LTX-315-C (the N-terminus of the anticancer peptide LTX-315 is linked to a cysteine ​​C, and the amino acid sequence of the anticancer peptide LTX-315 is KKWWKKWDipKNH2). Under the same process parameters as in Example 1, cyclooctyne-modified LTX-315-C (compound 25) and brush-structured LTX-315-C (compound 26) are prepared, and finally, a brush-structured peptide drug delivery system based on the anticancer peptide LTX-315 (compound 27) is obtained.

[0099] Sample Analysis

[0100] (1) Performance characterization

[0101] Figure 1 The matrix-assisted laser desorption / ionization mass spectrometry (MA / MS) spectra of cyclooctyne-modified Melittin-C (compound 9), cyclooctyne-modified CALL-C (compound 19), and cyclooctyne-modified KLAK-C (compound 22) are shown respectively. The results indicate that modifying peptide drugs with cyclooctyne maleimide is highly efficient and feasible.

[0102] Figure 2 The image shows a permeate gel chromatogram of the polydisulfide backbone (compound 4), indicating that the polydisulfide backbone obtained by this method has good dispersibility.

[0103] Figure 3 The figure shows the hydrated particle size and potential analysis results of brushed Melittin-C (compound 15), brushed KLAK-C (compound 23), and brushed LTX-315-C (compound 26). The particle size of brushed Melittin-C is 49 nm and the potential is 29 mV; the particle size of brushed KLAK-C is 43 nm and the potential is 27 mV; and the particle size of brushed LTX-315-C is 24 nm and the potential is 39 mV.

[0104] Figure 4 Transmission electron microscope images of brush-shaped Melittin-C (compound 15) and brush-shaped KLAK-C (compound 23). The dry particle size of the nanoparticles is about 30-40 nm, which is consistent with the hydrated particle size test results, indicating that the brush-shaped structure was successfully synthesized.

[0105] Figure 5 The graph shows the potential changes with pH for the brush-shaped peptide drug delivery system based on melitoxin (compound 18) and the brush-shaped peptide drug delivery system based on KLAK peptide (compound 24). The results indicate that the targeted charge-shielded brush obtained by this method is pH-responsive, and the charge-shielding molecule (containing the RGD moiety) can automatically detach in the pH environment of the lysosome, restoring its surface charge. This demonstrates the successful acid-responsive design of the brush-shaped peptide drug delivery system.

[0106] (2) Degradation experiment

[0107] Figure 6 The image shows the dithiothreitol (DTT) degradation results of the brush-shaped Melittin-C (compound 15) prepared in Example 1. The results indicate that the brush-shaped peptide drug delivery system based on melitrix prepared in this invention can achieve 90% degradation within 15 minutes and complete degradation within 1 hour under the action of DTT, demonstrating the peptide release capability of the brush-shaped peptide drug delivery system prepared in this invention under the reducing environment of tumors.

[0108] (3) Enzyme stability experiment

[0109] Figure 7 The image shows the effects of chymotrypsin degradation experiments on free meliofactin and a brush-structured peptide drug delivery system based on meliofactin (compound 18). The results indicate that this brush-structured peptide drug delivery system can significantly increase the enzymatic stability of peptides, demonstrating their stability in vivo circulation.

[0110] (4) Experiment to overcome hemolysis defects

[0111] Figure 8Figure 18 shows the results of a brush-structured peptide drug delivery system based on meliofemort peptide (compound 18), a brush-structured peptide drug delivery system based on the anticancer peptide LTX-315 (compound 27), and the hemolytic toxicity test results of the corresponding free meliofemort peptide and anticancer peptide LTX-315 on mouse erythrocytes. The results indicate that the brush-structured peptide drug delivery system can completely eliminate the hemolytic toxicity of the peptides and has good safety in vivo.

[0112] (5) Cell uptake experiment

[0113] Figure 9 Confocal microscopy and flow cytometry images show the uptake results of mouse oral squamous cell carcinoma (MOC1) cells using brush-shaped Melittin-C (compound 15), polyethylene glycol-protected brush-shaped Melittin-C (compound 16), and a melioficinale-based brush-shaped peptide drug delivery system (compound 18). The results indicate that the brush-shaped peptide drug delivery system can effectively target and enter tumor cells and regulate their uptake by incorporating the targeting molecule RGD cyclic peptide.

[0114] (6) Cytotoxicity test

[0115] Figure 10 Cytotoxicity analysis was performed on mouse breast cancer (4T1), mouse oral squamous cell carcinoma (MOC1), and human ovarian adenocarcinoma (SKOV3) cells using free melittin, brush-shaped melittin-C (compound 15), polyethylene glycol-protected brush-shaped melittin-C (compound 16), and a melittin-based brush-shaped peptide drug delivery system (compound 18). The results showed that the toxicity of the melittin-based brush-shaped peptide drug delivery system was comparable to that of free melittin. These results fully demonstrate that the brush-shaped peptide drug delivery system prepared in this invention can not only regulate cellular uptake but also specifically regulate its toxicity against tumor cells.

[0116] (7) Pharmacokinetic studies

[0117] Figure 11 Using cyanogenamide dye Cy5 (1564286-24-3) as a fluorescent label, the plasma concentrations of Cy5 at different time points following tail vein injection of a brush-shaped peptide drug delivery system (compound 18) based on melivesiculosine and free melivesiculosine were analyzed. The results showed that this brush-shaped peptide drug delivery system significantly prolonged the in vivo circulation time of the peptide, extending the half-life by four times and increasing the area under the curve (AUC) by 3.5 times.

[0118] (8) In vivo distribution experiment

[0119] Figure 12The distribution of a brush-like peptide-based drug delivery system (compound 18) using cyanogen dye Cy7 (1564286-24-3) as a fluorescent label in tumors and major organs 24 hours after tail vein injection is shown. The results indicate that the brush-like peptide-based drug delivery system is mainly distributed in tumors, demonstrating that RGD cyclic peptide modification can achieve tumor-targeted delivery of peptide drugs.

[0120] (9) Mouse model tumor growth inhibition experiment

[0121] Figure 13 The figure shows the results of a mouse tumor (4T1) growth inhibition experiment using a brush-like peptide-based peptide drug delivery system (compound 18). The results demonstrate that this brush-like peptide drug delivery system significantly inhibited tumor growth rate by targeting and delivering peptides to the tumor, validating the peptide delivery efficacy of this invention.

[0122] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A brush-like structure peptide drug delivery system, characterized in that, This includes modifying the amino acid residues of a peptide drug with active functional groups through click or cycloaddition reactions, reacting the modified peptide drug with the polydisulfide backbone through click or cycloaddition reactions to obtain a brush-shaped peptide drug, and performing charge shielding and bioactivity modification on the lysine residues in the brush-shaped peptide drug to obtain the brush-shaped peptide drug delivery system. Polypeptide drugs are composed of 5-100 amino acids, with ≥1 lysine residue; The structural formula of the polydisulfide backbone is any one of the following: ； Where x is an integer from 1 to 500, y is an integer from 1 to 2000, and z is an integer from 1 to 1000; R2 is a trifluoroethyl acrylate group, and R3 is a click reaction or cycloaddition reaction active functional group that can react with the modified functional groups on the amino acid residues of the peptide drug via click reaction or cycloaddition reaction. R1 and R3 are the same or are methyl or methoxy. The method for charge shielding and bioactivity modification involves reacting at least one 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative with lysine residues in a brush-like polypeptide drug. The structural formula of the 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative is shown below. ; Wherein, R4 is a bioactive functional group, and the bioactive functional group R4 is selected from any one of the following formulas: ； Where m, n, o, p, and q are each independently selected from integers from 1 to 1000; In the modified peptide drug, the click reaction or cycloaddition reaction active functional group is selected from any one of the following formulas: ； In the polydisulfide backbone, R3 is selected from any of the following formulas: 。 2. The brush-like structure polypeptide drug delivery system according to claim 1, characterized in that, The molar ratio of the modified peptide drug to the polydisulfide backbone during click or cycloaddition reactions was 1:0.01-1. When a click reaction occurred, the reaction conditions were -20 to 100 °C for 2 to 48 h; when a cycloaddition reaction occurred, the reaction conditions were -20 to 100 °C for 1 to 72 h.

3. The brush-like structure polypeptide drug delivery system according to claim 1, characterized in that, When performing charge shielding and bioactivity modification, the molar ratio of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative to lysine residues in brush peptide drugs is 10-1000:1, and the reaction conditions are -20-100℃, 0.01-72 h.

4. The brush-like structure polypeptide drug delivery system according to claim 1, characterized in that, The reaction was carried out with lysine residues in brush-like polypeptide drugs using polyethylene glycol conjugates of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid and RGD cyclic peptide conjugates of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid; the molar ratio of polyethylene glycol conjugates of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid and RGD cyclic peptide conjugates of 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid was 1:0.01-0.

5.

5. The brush-like structure polypeptide according to any one of claims 1-4 A method for preparing a drug delivery system, characterized in that it includes: (1) In an organic solvent system, the peptide drug is reacted with maleimide with click reaction or cycloaddition reaction active functional groups to modify the amino acid residues of the peptide drug with click reaction or cycloaddition reaction active functional groups. (2) In an organic solvent system or a mixed solvent system of organic solvent and water, the polydisulfide backbone and at least one modified polypeptide drug undergo a click reaction or cycloaddition reaction to obtain a brush-shaped polypeptide drug; (3) The brush-structured polypeptide drug delivery system is obtained by reacting at least one 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid derivative with a brush-shaped polypeptide drug.

6. The use of the brush-like structure polypeptide drug delivery system according to any one of claims 1-4 in the preparation of disease treatment products.

7. The use of the brush-like peptide drug delivery system according to any one of claims 1-4 in the preparation of disease prevention products.

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