Methotrexate-cationic polypeptide conjugate as well as preparation method and application thereof

Abstract

The invention discloses a methotrexate-cationic polypeptide conjugate as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. According to the conjugate, MTX and cationic polypeptide with the amino acid sequence shown as SEQ ID NO: 1 are connected through chemical bonding, the membrane binding characteristic of oligomeric lysine and the transmembrane capacity of TAT cell-penetrating peptide are creatively fused, and a polypeptide carrier system with efficient cell penetrating capacity is constructed; mTX obtains amphipathy and electropositivity through peptide fragment modification, stable nanoparticles can be spontaneously formed, and the problems of poor water solubility and instable acidity of raw material medicines are effectively improved; according to the conjugate, the accumulation efficiency of the medicine at a diseased region is remarkably improved by utilizing the targeting characteristic of the polypeptide carrier, the action time of the medicine is prolonged through a precise delivery mechanism, and meanwhile, the system toxicity is reduced. The preparation method adopts mild coupling reaction conditions, and the product is high in purity and excellent in stability; the compound is suitable for treating immune-mediated inflammatory diseases and specific malignant tumors, and provides an innovative solution thought for treating related diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and specifically discloses a methotrexate-cationic polypeptide conjugate, its preparation method, and its application. This conjugate, formed by covalently binding methotrexate to a cationic polypeptide, exhibits significant anti-inflammatory activity and can be widely used in the treatment of inflammatory diseases. Background Art

[0002] Methotrexate (MTX), a widely used folate antagonist, interferes with DNA synthesis and cell proliferation by inhibiting dihydrofolate reductase and is therefore used to treat tumors, autoimmune diseases, and inflammatory diseases. However, its therapeutic window is narrow, and its toxicity is closely related to dose and duration of treatment, leading to multisystem adverse reactions such as hematopoietic suppression (manifested as bone marrow suppression and pancytopenia), gastrointestinal mucosal damage (such as stomatitis and gastrointestinal ulcers), liver dysfunction (ranging from elevated transaminases to cirrhosis), nephrotoxicity (typically manifested as acute tubular crystal deposition), and neurotoxicity (risk of encephalopathy associated with intrathecal administration). Notably, patients with "third space" effusions, renal dysfunction, or pregnancy present with increased medication risks. Current clinical management of MTX has developed a series of personalized strategies: combining folic acid to alleviate mucositis and hepatotoxicity, using subcutaneous injection to avoid gastrointestinal irritation, and alkalinizing the urine to prevent renal crystal formation. Based on the existing challenges, future research should focus on three major directions: 1) In precision medicine, promoting pharmacogenomic testing to guide individualized drug delivery; 2) In structural optimization, developing low-toxic drug conjugates; and 3) In delivery technology, constructing new drug delivery platforms such as sustained-release systems and nanoformulations to achieve simultaneous improvements in efficacy and safety.

[0003] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by symmetrical polyarticular inflammation, synovial hyperplasia, and progressive joint destruction. The disease not only causes joint pain, swelling, and dysfunction, but can also lead to systemic complications such as cardiovascular disease, lung disease, and osteoporosis, severely impairing patients' quality of life and social function. MTX, a guideline-recommended core treatment for RA, exerts anti-inflammatory effects by inhibiting immune cell proliferation and regulating inflammatory factor networks. Studies have shown that MTX can upregulate regulatory T cells (Tregs) and correct Th17 / Treg imbalances to reduce the production of inflammatory mediators (such as IL-1, IL-6, and TNF-α), thereby alleviating the inflammatory response. Furthermore, it can inhibit the synthesis of pain-causing prostaglandin E2, achieving the purpose of pain relief.

[0004] However, the main challenges faced by methotrexate (MTX) in clinical application are: poor water solubility (<1 mg / mL) and instability in acidic environments, resulting in easy degradation by gastric acid and gastrointestinal irritation when administered orally, significantly reducing bioavailability. To improve solubility, injections require the addition of excipients such as sodium hydroxide to adjust the pH to above 8, but this strongly alkaline condition is poorly compatible with the physiological environment. Furthermore, the toxicity of MTX cannot be ignored, and the severity of its toxic reactions is related to dose, frequency of administration, and individual tolerance. Although local injection into joints can reduce systemic toxicity, the acidic microenvironment (pH ≈ 6.5) of rheumatoid arthritis (RA) joints can reduce drug solubility, leading to the risk of precipitation and degradation. These factors collectively restrict the clinical administration of MTX.

[0005] Furthermore, traditional small-molecule drugs (such as MTX) primarily enter cells via passive diffusion, inherently resulting in low delivery efficiency. Recent research indicates that by conjugating small-molecule drugs with peptides such as cell-penetrating peptides (CPPs) or antimicrobial peptides, three major physiological barriers can be overcome: 1) significantly enhancing transmembrane transport, enabling a shift from passive diffusion to active targeting; 2) effectively circumventing P-glycoprotein-mediated efflux; and 3) reversing multidrug resistance by regulating intracellular drug concentrations. This technology can also further enhance metabolic stability by optimizing conjugation methods and peptide sequence design, providing new insights for the precise delivery of MTX.

[0006] For example, Chinese patent CN 110621349 A discloses a methotrexate-peptide conjugate with the peptide sequence: RFLKRLDRNLW. This conjugate exhibits low cytotoxicity and significantly reduces the expression of genes associated with inflammatory responses in cells. However, this conjugate does not address the inherent poor water solubility and acid instability of MTX, and its self-assembly properties have not been verified. Its ability to form a nanoscale delivery system is unclear, and its targeted accumulation efficiency and drug delivery efficiency are unknown.

[0007] Therefore, the optimization design of peptide structure remains the focus of current research, which mainly includes three key strategies: improving MTX solubility through hydrophilic modification to solve the problem of formulation development; secondly, introducing acid-stable groups to enhance microenvironment tolerance; and thirdly, designing amphiphilic and positively charged structures to achieve molecular self-assembly to form a nanoscale delivery system, thereby improving tissue permeability. Summary of the Invention

[0008] In response to the above-mentioned problems, the present invention aims to provide a methotrexate-cationic polypeptide conjugate delivery system that improves the water solubility and stability of MTX. The system organically combines the membrane binding properties of oligolysine with the transmembrane ability of TAT transmembrane peptide to construct a polypeptide carrier with efficient cell penetration ability. MTX is modified to obtain amphiphilicity and positive charge through peptide modification, so that it can spontaneously form stable nanoparticles, effectively solving the defects of poor water solubility (<1mg / mL) and acid instability of the raw material drug; the targeting properties of the polypeptide carrier can also significantly improve the accumulation of drugs in diseased joints; in addition, the drug action time is extended through a precise delivery mechanism, while the systemic toxicity caused by normal tissue distribution is greatly reduced. This preparation provides a new MTX administration regimen with both high efficiency and safety for the treatment of rheumatoid arthritis.

[0009] In order to achieve the above-mentioned object, the technical solution adopted by the present invention is as follows: a methotrexate-cationic polypeptide conjugate, wherein the carboxyl group of the methotrexate molecule is covalently linked to the N-terminal amino group of the cationic membrane-penetrating polypeptide KKKKKKYGRKKRRQRRR with an amino acid sequence as shown in SEQ ID NO: 1, wherein the hexalysine domain enhances the membrane binding affinity through its positive charge characteristics, and the TAT membrane-penetrating peptide (YGRKKRRQRRR) domain not only has positive charge binding ability, but also has efficient cell penetration function.

[0010] The preparation method of the above-mentioned methotrexate-cationic polypeptide conjugate is as follows:

[0011] 1) The amino-protected arginine Fmoc-Arg(Pbf)-OH is immobilized on a resin support by solid-phase synthesis, and then the amino-protecting group is removed;

[0012] 2) Stepwise coupling of amino-protected amino acids to extend the polypeptide chain to obtain the resin-loaded KKKKKKYGRKKRRQRRR sequence;

[0013] 3) Activated MTX is coupled to the resin-loaded peptide;

[0014] 4) cracking the resin to obtain a crude product;

[0015] 5) After purification, freeze-drying is performed to obtain the target conjugate.

[0016] Furthermore, in step 1), DIC / HOBT is used as a condensing agent, the equivalent ratio of amino-protected arginine, HOBT and DIC is 1:1.5-3:1.5-3 (preferably 1:2:2), the reaction temperature is 35-45°C, and the reaction time is 3-5 hours; in step 2), when the polypeptide chain extension reaction is carried out, the condensing agent used is DIC / HOBT, the equivalent ratio of amino-protected amino acid, HOBT and DIC is 1:1.5-2.5:1.5-2.5 (preferably 1:2:2), the reaction temperature is 40-50°C, and the reaction time is 0.5-2 hours.

[0017] Furthermore, in step 3), the equivalent ratio of MTX to polypeptide is 3:1, the activation reagent is HATU or HBTU, preferably HATU, the reaction temperature is 25-40° C., and the reaction time is 1-3 h.

[0018] Furthermore, the resin is preferably Wang resin.

[0019] The methotrexate-cationic polypeptide conjugate is compounded with a stabilizer in a mass volume ratio of 0.02% to 0.5% to form a nanocomposite by self-assembly.

[0020] The stabilizer is selected from any one or more of SDS, CTAB, cationic cholesterol, polyethylene glycol, phospholipids, chitosan, povidone, and gelatin; the concentration of the methotrexate-cationic polypeptide conjugate is 500-1500 μg / mL (drug concentration is calculated as methotrexate);

[0021] The composite has an average particle size of ≤200 nm, a polydispersity index of <0.3, and a zeta potential of ≥30 mV.

[0022] The preparation method of the self-assembled nanocomplex is as follows: after dispersing the methotrexate-cationic polypeptide conjugate and the stabilizer in the aqueous phase, the mixture is homogenized by high-pressure microfluidization at a pressure of 5000-20000 psi for 1-5 times, preferably at a pressure of 8000-12000 psi for 2-3 times.

[0023] The present application also discloses a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned methotrexate-cationic polypeptide conjugate or nanocomplex and pharmaceutically acceptable excipients. The pharmaceutical composition is an injectable preparation or a sustained-release preparation.

[0024] The methotrexate-cationic polypeptide conjugate can be used to prepare a drug for treating immune-mediated inflammatory diseases, wherein the immune-mediated inflammatory disease is rheumatoid arthritis or psoriasis.

[0025] The methotrexate-cationic polypeptide conjugate can be used to prepare drugs for treating malignant tumors.

[0026] Furthermore, the drug is a nanocomplex preparation comprising the methotrexate-cationic polypeptide conjugate.

[0027] The beneficial effects of the present invention are:

[0028] 1. This application uses peptide coupling technology to structurally modify methotrexate (MTX), achieving breakthrough innovation at the molecular level. The introduction of a hydrophilic peptide can significantly improve the compound's water solubility, overcome the development barriers of the original drug formulation, and create favorable conditions for the development of injectable dosage forms.

[0029] 2. Coupling methotrexate with a peptide can change the spatial structure of MTX. The steric hindrance of the peptide can effectively protect the key active groups of MTX, reduce the risk of benzoyl hydrolysis to generate glutamic acid and pterinic acid, and essentially improve the chemical stability of the compound.

[0030] 3. The KTAT polypeptide module proposed in this application is an innovative structure that integrates a lysine-rich region and a membrane-penetrating peptide functional domain. The novel compound formed after coupling with methotrexate exhibits multiple technical advantages: the polypeptide module significantly enhances the transmembrane transport ability of the drug through the membrane-penetrating peptide component, enabling it to efficiently target abnormally activated immune cell populations, including key inflammatory mediators such as macrophages and T cells, thereby achieving directional accumulation of the drug at the lesion site; the introduction of the lysine-rich region further enhances the compound's biomembrane affinity, forming a dual-channel cellular uptake mechanism. This unique structural design not only significantly improves the drug's delivery efficiency in target tissues, but also effectively reduces common adverse reactions such as hepatotoxicity and bone marrow suppression associated with traditional methotrexate treatment by reducing the systemic circulating drug dose; at the same time, the immunomodulatory potential of the polypeptide module itself can produce a synergistic therapeutic effect with methotrexate, while enhancing the local anti-inflammatory effect of the joints, further optimizing the drug's safety profile by reducing normal tissue distribution; this application achieves a dual breakthrough in enhanced efficacy and reduced toxicity;

[0031] 4. This application constructs a novel drug form that combines both targeting and safety by molecularly coupling methotrexate with a specifically designed cationic peptide. This not only provides an alternative for patients with poor tolerance to traditional therapeutic drugs, but also achieves a balance between efficacy and safety through a precise delivery mechanism. This molecular design approach not only expands the treatment options for rheumatoid arthritis, but also provides a feasible technical path for achieving personalized precision medicine, with important clinical translational significance.

[0032] 5. The method for preparing the methotrexate-cationic polypeptide conjugate disclosed in this application has the characteristics of simple process, concise route and good reproducibility. Combined with environmentally friendly purification technology, it provides reliable technical support for large-scale production;

[0033] 6. The methotrexate-cationic peptide conjugate developed in this application has broad application prospects. This technology is not only suitable for the treatment of rheumatoid arthritis, but can also be expanded to multiple therapeutic areas such as anti-tumor, immunomodulation, and inflammatory diseases. The introduction of cationic peptides significantly improves the solubility and stability of the drug, laying an important foundation for the development of diversified formulations. Based on this technology platform, a variety of dosage forms, including injections and sustained-release preparations, can be developed to meet the personalized treatment needs of different disease characteristics, patient groups, and administration routes. This potential for development of multiple indications and dosage forms gives this technology broad clinical application space. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 LC-MS chart of the crude product prepared in step S4 of Example 1;

[0035] Figure 2 LC-MS image of the purified sample prepared in step S5 of Example 1;

[0036] Figure 3 LC-MS chart of the crude product prepared in step S4 of Example 2;

[0037] Figure 4 LC-MS image of the purified sample prepared in step S5 of Example 2;

[0038] Figure 5 LC-MS chart of the crude product prepared in step S4 of Example 3;

[0039] Figure 6 LC-MS image of the purified sample prepared in step S5 of Example 3;

[0040] Figure 7 This is the HPLC chart of the target product MKTAT obtained by amplification synthesis;

[0041] Figure 8 This is the LC-MS chart of the target product MKTAT obtained by amplification synthesis;

[0042] Figure 9 To amplify the NMR spectrum of the target product MKTAT obtained by synthesis;

[0043] Figure 10 The following are Fourier transform infrared (FTIR) spectra of MTX and the target product MKTAT obtained by amplified synthesis;

[0044] Figure 11 X-ray diffraction (XRD) patterns of MTX and target product MKTAT;

[0045] Figure 12 HPLC chart of MTAT synthesized in Comparative Example 1;

[0046] Figure 13 LC-MS chart of MTAT synthesized in Comparative Example 1;

[0047] Figure 14 HPLC chart of M19KTAT synthesized in Comparative Example 2;

[0048] Figure 15 LC-MS chart of M19KTAT synthesized in Comparative Example 2;

[0049] Figure 16 Solubility curves of MTX, MKTAT, MTAT and M19KTAT at different pH values;

[0050] Figure 17 Comparison of the stability of different drugs in PBS simulated RA synovial fluid (pH = 6.5);

[0051] Figure 18 Effects of different nanocomplexes on the viability of Raw264.7 / MH7A cells;

[0052] Figure 19 Effects of different nanocomplexes on LPS-induced TNF-α secretion in Raw264.7 cells;

[0053] Figure 20 Effects of different nanocomplexes on LPS-induced IL-6 secretion in Raw264.7 cells;

[0054] Figure 21 Effects of different nanocomplexes on LPS-induced IL-1β secretion in Raw264.7 cells;

[0055] Figure 22 The graph shows the changes in the thickness of the right ankle joint of mice over time after immunization with different complexes;

[0056] Figure 23 Figure 2 is a graph showing the changes in mouse body weight over time after immunization with different complexes. DETAILED DESCRIPTION

[0057] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0058] Example 1

[0059] This embodiment discloses a methotrexate-cationic polypeptide conjugate and a preparation method thereof. The sequence of the conjugated cationic polypeptide is KKKKKKYGRKKRRQRRR (KTAT), which is a membrane-penetrating peptide variant containing a lysine-rich region, wherein YGRKKRRQRRR is a membrane-penetrating peptide sequence that promotes the entry of the polypeptide into cells. It not only has positive charge binding ability but also has efficient cell penetration function; KKKKKK is the target sequence that mainly exerts its effect after entering the body, and the hexalysine domain provides positive charge binding ability, thereby improving membrane affinity.

[0060] The amino acid sequence of this polypeptide is: Lys-Lys-Lys-Lys-Lys-Lys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, as shown in SEQ ID NO. 1. Currently, there is no report on its related function.

[0061] The synthesized conjugate is designated MTX-KKKKKKYGRKKRRQRRR, abbreviated as MKTAT. The six consecutive lysine residues (K) at the N-terminus provide strong hydrophilicity and positive charge, helping to improve the water solubility of MTX. The high positive charge density of lysine residues enhances molecular hydration. The TAT transmembrane peptide (YGRKKRRQRRR) itself is rich in arginine / lysine residues, further enhancing hydrophilicity and jointly addressing the insufficient hydrophilicity of MTX. Furthermore, in an acidic environment, the strong positive charge of the peptide sequence (high K / R density) reduces molecular aggregation through charge repulsion, lowering the risk of degradation.

[0062] The synthetic route of the conjugate is as follows:

[0063]

[0064] Among them, R1, R2, R3, R5, R6, and R9 all represent variable side chains of arginine, R4 represents a variable side chain of glutamine, and R7, R8, R9 represent variable side chains of arginine, R1, R2, R3, R5, R6, and R9 represent variable side chains of arginine, R4 represents a variable side chain of glutamine, 12 、R 13 、R 14 、R 15 、R 16 、R 17 Both represent the variable side chain of lysine, R 10 represents the variable side chain of glycine, R 11 represents the variable side chain of tyrosine.

[0065] The specific preparation process is as follows:

[0066] Step S1: Connecting the C-terminal starting amino acid of the polypeptide to Wang resin

[0067] Wang resin (0.3 mmol / g substitution degree) was weighed into a reactor and soaked in dichloromethane (DCM) for 2 hours. N,N-dimethylformamide (DMF) was added at a ratio of 8 mL / g resin, and the resin was washed and dried. This was repeated four times, and the resin was finally dried for later use.

[0068] Weigh 1.5 eq of the first C-terminal amino acid Fmoc-Arg(Pbf)-OH (i.e., compound 1), 3 eq of 1-hydroxy-benzotriazole (HOBT), and 0.5 eq of 4-dimethylaminopyridine (DMAP) into a centrifuge tube, dissolve them with DMF, add 3 eq of N,N-diisopropylcarbodiimide (DIC), shake until the solution is clear, and then add it to the reactor. Shake the reaction in a constant temperature shaker at 40°C for 4 h, wash with N,N-dimethylformamide (DMF) at a ratio of 8 mL / g resin, drain, and repeat four times.

[0069] A capping reaction was performed for 0.5 h using a mixture of acetic anhydride / N,N-diisopropylethylamine (DIEA) / DMF (the volume ratio of acetic anhydride, DIEA, and DMF was 1:1:2). The product was then washed with 3 times the volume of DMF and drained (repeated 4 times). A 20% piperidine / DMF solution (the volume ratio of piperidine to DMF was 1:4) was added and shaken on a decolorizing shaker for 20 min to remove the fluorenylmethyloxycarbonyl (Fomc) protecting the product at the amino terminus. The product was then washed with DMF and drained (repeated 6 times).

[0070] Take a small amount of resin sample in the test tube, add ninhydrin (Nine Wells Hydrated Ninhydrin) detection reagent (2 drops each of Test A and Test B) and react at 100°C for 1 minute. Observe the color response. The resin shows an obvious color change, indicating that the C-terminal starting amino acid of the polypeptide (Fmoc-Arg(Pbf)-OH) has been successfully connected to the resin to form compound 2, and the Fmoc protecting group has been completely removed.

[0071] Step S2: Elongation of the polypeptide chain

[0072] Weigh 3 eq of Fmoc-Arg(Pbf)-OH (i.e., compound 3) and 3 eq of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) into a centrifuge tube, add an appropriate amount of DMF to dissolve, add 3 eq of DIEA, shake for 1 min until the solution is clear, transfer to a reactor, and react on a shaker at room temperature for 1 h. After the reaction is complete, wash the resin with DMF and drain (repeat 4 times).

[0073] A small amount of resin was tested with ninhydrin (two drops each of Test A and Test B, 1 min at 100°C). If the resin remained colorless, the amino acid condensation was complete. If the resin developed color, 3 eq of the corresponding amino acid and a condensing agent (e.g., HBTU / DIEA) were added and the reaction was repeated. After confirming the condensation was complete, 8 mL of a 20% piperidine / DMF solution (V / V = 1:4) was added per gram of resin. The mixture was shaken on a decolorizing shaker for 20 min to remove the Fmoc protecting group, yielding compound 4. The resin was then washed with DMF and drained (8 mL / g resin x 6 times), draining each time.

[0074] According to the condensation process of step S2, the following Fmoc-protected amino acids were coupled to the resin in sequence: Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc- oc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Lys(Boc)-OH, and finally the resin-loaded polypeptide sequence KKKKKKYGRKKRRQRRR was obtained, which was named KTAT-Wang-resin.

[0075] Step S3: Coupling with MTX

[0076] 3eq methotrexate (MTX, compound 5) and 3eq HATU were added to a centrifuge tube, dissolved in DMF, and then 5eq pyridine was added. The mixture was shaken for 1 min until the solution was clear and transferred to a reactor containing resin (in KTAT-Wang-resin, the amount of KTAT was 1eq). The reaction was shaken at 35°C for 2 hours to complete the MTX coupling. After the reaction, the resin was washed with DMF (8 mL / g resin × 4 times, each time drained) and methanol (8 mL / g resin × 5 times, each time drained). The obtained resin product was recorded as MKTAT-Wang-resin, and the resin was transferred to a centrifuge tube for cutting.

[0077] Step S4: Cleavage of crude target conjugate

[0078] An appropriate amount of resin (1 g of resin corresponds to 10 mL of cutting solution) was added to a centrifuge tube, and 95% cutting solution (trifluoroacetic acid (TFA): 1,2-ethanedithiol (EDT): triisopropylsilane (TIS): H2O = 95:2:2:1) was injected. The reaction mixture was shaken on a shaker at room temperature for 2.5 h to complete the cutting. After filtering the reaction solution through a 0.45 μm filter membrane, pre-cooled ether (-20°C) was added at a volume ratio of 1:10. After vortex mixing, the crude peptide was centrifuged and the supernatant was discarded. The ether precipitation and washing were repeated twice to finally obtain the crude product.

[0079] Step S5: Purification of target conjugate

[0080] The crude product was purified by preparative HPLC (C18 column, 5×30 cm) using a dual-pump system: Pump A was 0.1% TFA in water, Pump B was 0.1% TFA in acetonitrile, flow rate 60 mL / min, gradient elution (phase B 10% → 40%, 60 min), monitoring at 220 nm, and retention time of the target component was 20-30 min. The target peak fractions were collected, rotary evaporated to remove acetonitrile, and then freeze-dried to obtain a high-purity target conjugate preparation solution, named MTX-KKKKKKYGRKKRRQRRR, abbreviated as MKTAT, and stored at -20°C in the dark until use.

[0081] Step S6: Lyophilization

[0082] The target conjugate preparation solution with qualified purity was freeze-dried using a freeze dryer for 48 hours to obtain the target conjugate dry product MKTAT.

[0083] The crude product obtained in step S4 and the purified sample obtained in step S5 were tested by LC-MS. Figure 1-2 As shown, the mass spectrum shows that in addition to the main peak of the target conjugate, a peak with lower intensity (called a secondary peak) also appears in the crude product. After purification, the small secondary peak is still contained.

[0084] Example 2-3

[0085] The difference between Example 2-3 and Example 1 lies in the different types and amounts of the condensing agent used in step S2, and the different temperatures during the amide condensation reaction.

[0086] The relevant reaction conditions of step S2 in Examples 1-3 are shown in Table 1:

[0087] Table 1. Reaction conditions for amide condensation reaction in step S2 of different embodiments

[0088]

[0089]

[0090] The crude product obtained in step S4 of Example 2 and the purified sample obtained in step S5 were detected by LC-MS. Figure 3-4 As shown, the mass spectrum shows that the mass spectrometry analysis results show that the main peak position of the target conjugate in the crude product is correct, but there are many impurity peaks; after purification, although the impurities are significantly reduced, the peak area of the target product is significantly reduced, and the overall yield is not ideal.

[0091] The crude product obtained in step S4 of Example 3 and the purified sample obtained in step S5 were detected by LC-MS. The results were as follows: Figure 5-6 As shown, the mass spectrometry analysis results showed that the main peak position of the target conjugate in the crude product was accurate and there was no obvious impurity peak. After purification, the peak area of the target product increased significantly, indicating that the purification process was effective and the product yield was high.

[0092] From the above results, it can be seen that in the process of preparing polypeptides using amide condensation reaction, differences in the type, dosage and reaction conditions of the condensing agent have a significant impact on the purity and yield of the product.

[0093] The target conjugate, MKTAT, was successfully obtained by a 10-fold scale-up synthesis according to the preparation process of Example 3. The product purity was determined by HPLC, the molecular weight and structure were verified by LC-MS, the molecular structure was confirmed by proton nuclear magnetic resonance spectroscopy (1H NMR), the functional group characteristics were analyzed by Fourier transform infrared spectroscopy (FT-IR), and the crystalline structure was characterized by X-ray diffraction (XRD).

[0094] Purity analysis and structure confirmation: HPLC analysis ( Figure 7 ) confirmed that the purity of the synthesized MKTAT was 95.04%, which was greater than the quality requirement of 95%, so the synthesized MKTAT met the purity requirement of the experiment. LC-MS analysis showed ( Figure 8 ), its molecular mass is 2765.30, its [M+6H] 6+ The theoretical value of the peak is 461.72, [M+5H] 5+ The theoretical value of the peak is 554.07, [M+4H] 4+ The theoretical value of the peak is 692.08, [M+3H] 3+ The theoretical value of the peak is 922.11, and the mass spectrum shows that the [M+6H] 6+ The peak was found to be 461.88, [M+5H] 5+ The peak was found to be 554.06, [M+4H] 4+ The peak was found to be 692.33, [M+3H] 3+The measured peak value was 922.88, which was consistent with the theoretical value, fully verifying the structural correctness of the target product. Combined HPLC and LC-MS data confirmed the successful synthesis of high-purity MKTAT.

[0095] H NMR spectroscopy ( Figure 9 )show:

[0096] Chemical shift σ: 8.68 1H (1H on the aromatic ring of methotrexate), 4.87 (2H on the methylene group of methotrexate);

[0097] 4.0~4.4 total 15H: 2H on amino acid G, 1H on amino acid Y (adjacent hydrogen of carboxyl group), 1H on amino acid R (adjacent hydrogen of carboxyl group), 8H on amino acid K (adjacent hydrogen of carboxyl group);

[0098] 2.0 to 3.0, a total of 34H: 2H on amino acid Y (benzyl methylene pHCH2-), 16H on amino acid K (NH2CH2- at the amino acid adjacent position), 12H on amino acid R (NH2CH2- at the amino acid adjacent position), and 4H on amino acid Q (-CH2- at the amide adjacent position).

[0099] 1.25~1.78, a total of 72H: 48H (methylene-CH2-CH2-) on amino acid K, and 24H (methylene-CH2-CH2-) on amino acid R.

[0100] NMR results showed that MTX was successfully linked to the peptide.

[0101] Fourier transform infrared spectroscopy (FTIR) analysis showed that ( Figure 10 ), the characteristic absorption peaks of MKTAT correspond to: 3400~3200cm -1 The broad peak at 2900-3000 cm is attributed to the stretching vibration of the pteridine ring NH2 and the amide bond NH; -1 The weak peaks in the range are the CH stretching vibrations of the pteridine ring and the benzene ring; 1645 cm -1 The absorption peak at 1538 cm corresponds to the stretching vibration of amide and carboxyl C=O; -1 The characteristic peaks of the amide II band are shown at the bottom, which are derived from the coupling of the amide NH bending vibration and the peptide bond. These characteristic peaks together verify the functional group structure of the target molecule.

[0102] The results of NMR and IR jointly confirmed the successful synthesis of MKTAT.

[0103] X-ray diffraction (XRD) analysis showed that MKTAT formed after MTX and peptide coupling was amorphous, and the original crystal characteristic peak of MTX in its diffraction pattern completely disappeared, confirming the significant difference in crystal structure between the coupling product and the raw material drug ( Figure 11 ).

[0104] Comparative Example 1

[0105] The difference between this comparative example and Example 3 is that the polypeptide coupled to methotrexate (MTX) is only the cell-penetrating peptide YGRKKRRQRRR (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, SEQ ID NO. 2), and the synthesis method of the cell-penetrating peptide is the same as that in Example 3; after MTX is connected to the cell-penetrating peptide through the same coupling reaction process, the resulting product MTX-YGRKKRRQRRR is named MTAT.

[0106] The purity of MTAT was analyzed by HPLC, and the molecular weight and structure were confirmed by LC-MS.

[0107] HPLC peak data showed ( Figure 12 ), the purity of the synthesized MTAT was 96.37%, which was greater than the quality requirement of 95%, so the synthesized MTAT met the purity requirement of the experiment. The molecular weight of MTAT was then analyzed by LC-MS for structural confirmation, and its molecular mass was 1996.28, and its [M+5H] 5+ The theoretical value of the peak is 400.26, [M+4H] 4+ The theoretical value of the peak is 500.07, [M+3H] 3+ The theoretical value of the peak is 666.40, and the mass spectrum shows that the [M+5H] 5+ The peak was found to be 400.25, [M+4H] 4+ The peak was found to be 500.07, [M+3H] 3+ The measured value of the peak is 666.55, which is consistent with the theoretical value ( Figure 13 ). The results of HPLC and LC-MS confirmed the successful synthesis of high-purity MTAT.

[0108] Comparative Example 2

[0109] The difference between this comparative example and Example 3 is that the polypeptide coupled to methotrexate (MTX) is a cell-penetrating peptide variant with higher lysine enrichment, and the sequence of the polypeptide is KKKKKKKKKKKKKKKKKKKYGRKKRRQRRR, and its amino acid sequence is Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, as shown in SEQ ID NO.3.

[0110] The synthesis method of this polypeptide is consistent with that of Example 3; after connecting MTX to this polypeptide through the same coupling reaction process, the resulting product MTX-KKKKKKKKKKKKKKKKKKKKYGRKKRRQRRR is named M19KTAT.

[0111] The purity of MTAT was analyzed by HPLC, and the molecular weight and structure were confirmed by LC-MS.

[0112] HPLC peak data showed ( Figure 14 ), the purity of the synthesized M19KTAT was 95.94%, which was greater than the quality requirement of 95%, so the synthesized M19KTAT met the purity requirement of the experiment. The molecular weight of M19KTAT was then analyzed by LC-MS for structural confirmation, and its molecular mass was 4431.58, and its [M+6H] 6+ The theoretical value of the peak is 739.60, [M+5H] 5+ The theoretical value of the peak is 887.32, [M+4H] 4+ The theoretical value of the peak is 1109.90, [M+3H] 3+ The theoretical value of the peak is 922.11, and the mass spectrum shows that the [M+6H] 6+ The peak was found to be 739.60, [M+5H] 5+ The peak was found to be 887.32, [M+4H] 4+ The measured value of the peak is 1108.90, which is consistent with the theoretical value ( Figure 15 ). The results of HPLC and LC-MS confirmed the successful synthesis of high-purity M19KTAT.

[0113] Related performance tests

[0114] 1. Equilibrium solubility test

[0115] Aqueous solutions with a series of pH values (pH = 1.2, 2.0, 3.0, 4.0, 5.0, 6.8, 7.4) were prepared using 0.1 M HCl and NaOH. MTX, MKTAT, MTAT, and M19KTAT were added, respectively. The solutions were shaken at 37°C for 30 min to form supersaturated solutions. The solutions were then centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The drug absorbance was measured at 302 nm, and the equilibrium solubilities of MTX, MKTAT, MTAT, and M19KTAT under different pH conditions were calculated.

[0116] Results see Figure 16As can be seen from the figure, the solubility of the MTX-peptide conjugate (MKTAT / MTAT / M19KTAT) in different pH environments (pH = 1.2 to 7.4) is significantly better than that of free MTX; in the acidic microenvironment (pH ≈ 6.5) characteristic of rheumatoid arthritis (RA), the solubility of the conjugate is significantly improved compared with MTX. This pH-responsive solubility characteristic is highly compatible with the microenvironment of diseased joints.

[0117] 2. Chemical stability test

[0118] In view of the acidic characteristics of the synovial microenvironment of rheumatoid arthritis (RA) (pH ≈ 6.5), this study used pH 6.5 PBS to simulate RA synovial fluid and dynamically monitored the chemical stability of MTX and its peptide conjugates (MKTAT / MTAT / M19KTAT). The experimental results showed that from 0 hours to 240 hours, the relative percentage of MTX decreased from 100% to about 80%, indicating that its stability in the simulated RA synovial fluid was poor and free MTX was significantly degraded in the acidic environment; the curves of MKTAT, MTAT and M19KTAT showed that within 240 hours, their relative percentages remained basically at around 100%, indicating that these three drugs had good stability in the simulated RA synovial fluid, stable chemical properties, and were not easily degraded, confirming that peptide modification can significantly enhance the stability of drugs in the pathological microenvironment ( Figure 17 ).

[0119] 3. Self-assembly performance evaluation

[0120] The stabilizer, MTX, and MTX-peptide conjugate (MKTAT prepared in Example 3, MTAT prepared in Comparative Example 1, and M19KTAT prepared in Comparative Example 2) were accurately weighed and dispersed in pure water. The mixture was homogenized using a high-pressure microfluidizer to prepare a self-assembled nanocomposite. Particle size and PDI were analyzed using a particle size analyzer, and zeta potential was measured using a potentiometer to characterize system stability.

[0121] Wherein, calculated as MTX, the concentration of the MTX-polypeptide conjugate is 625 μg / mL (specifically, taking MKTAT as an example, the molar molecular weight of MKTAT is 2765.30, the molar molecular weight of MTX is 454.44, the molar ratio of the two is about 6, and the actual concentration of MKTAT during feeding is 3.8 mg / mL), the stabilizer is preferably a phospholipid (such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), other optional polymers (chitosan, povidone, gelatin, etc.) or other surfactants (sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), cholesterol, polyethylene glycol, etc.), the dosage range is 0.02-0.5% (w / v), preferably 0.025%-0.05%; the homogenization pressure is 5000-20000 psi (preferably 8000-12000 psi), and the number of homogenizations is 1-5 times (preferably 2-3 times).

[0122] See Table 2 for specific parameters.

[0123] Table 2. Comparison of key parameters for self-assembly of MTX-peptide conjugates

[0124]

[0125]

[0126] The results showed that the average particle size of the unconjugated MTX and M19KTAT self-assemblies was above 300 nm, and the PDI of the MTX group was greater than 0.3. The particle size of MKTAT and MTAT was about 150-170 nm, and the PDI was less than 0.3, indicating that the particle uniformity of the conjugates was improved and the physical stability was enhanced. Zeta potential analysis showed that the potential of the MTX group was about 10 mV, and the zeta potential of the MTX-peptide conjugated group was above 30 mV, indicating that after MTX was conjugated with the peptide, the surface potential of the complex was significantly increased to a strong positive charge, confirming that the introduction of the peptide effectively reversed the electrical characteristics of MTX, endowed MTX with cationic properties, and could significantly improve the colloidal stability.

[0127] 4. Cell viability test

[0128] Test the effect of different concentrations of MTX-peptide conjugates on RAW264.7 / MH7A cell viability

[0129] The nanocomplexes prepared during the self-assembly performance evaluation test (respectively designated as MTX complex, MKTAT complex, MTAT complex, and M19KTAT complex) were used as test objects. The samples were diluted to a drug concentration of 360 ng / mL (in terms of MTX) using Opti-MEM medium at pH 6.5 as a solvent and set aside.

[0130] The experimental groups of Raw 264.7 cells were as follows: (1) Control, (2) LPS (1 μg / mL), (3) MTX complex + LPS (1 μg / mL), (4) MKTAT complex + LPS (1 μg / mL), (5) MTAT complex + LPS (1 μg / mL), and (6) M19KTAT complex + LPS (1 μg / mL).

[0131] The experimental groups of MH7A cells were as follows: (1) Control, (2) TNF-α (40 ng / mL), (3) MTX complex + TNF-α (40 ng / mL), (4) MKTAT complex + TNF-α (40 ng / mL), (5) MTAT complex + TNF-α (40 ng / mL), and (6) M19KTAT complex + TNF-α (40 ng / mL).

[0132] Cell culture (RAW 264.7): Remove the frozen cells and place them in a 37°C constant temperature water bath for thawing. After disinfection with 75% ethanol, place them in a clean bench for use. Remove the sterilized 15mL centrifuge tube, add 5mL of DMEM medium (containing 10% fetal bovine serum, 100U / mL streptomycin and 100U / mL penicillin), then add 1mL of the thawed cell freezing solution, mix well, centrifuge at 1500rpm for 5 minutes, and discard the supernatant. Add 5mL of DMEM medium to resuspend the cells and transfer them to a T25 culture flask. Add 5mL of culture medium and gently shake the culture flask in a cross shape to evenly distribute the cell resuspension. Then place the culture flask in a 5% CO2 incubator with a relative humidity of 95% and a temperature of 37°C for culture. Regularly replace with fresh DMEM medium for culture and use.

[0133] Culture of arthritis synovial fibroblasts (MH7A cells): Remove frozen cells and thaw in a 37°C constant temperature water bath. Disinfect with 75% ethanol and place in a clean bench for later use. Remove a sterilized 15mL centrifuge tube and add 5mL of DMEM medium (containing 15% fetal bovine serum, 100U / mL streptomycin, and 100U / mL penicillin). Add 1mL of the thawed cell suspension and mix thoroughly. Centrifuge at 1500rpm for 5 minutes and discard the supernatant. Resuspend the cells in 5mL of DMEM medium and transfer to a T25 culture flask. Add 5mL of medium and gently shake the culture flask in a cross shape to evenly distribute the cell suspension. Then, place the culture flask in a 5% CO2 incubator at 95% relative humidity and 37°C. Regularly replace with fresh DMEM medium for further culture and use.

[0134] Raw 264.7 and MH7A cells were seeded in 96-well plates, with 3 replicates per group and 5×10 cells per well. 3Cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. Once the cells were fully adhered and stable, the original culture medium was discarded and the corresponding culture medium was added for co-incubation. The Raw 264.7 group was incubated for 24 hours. At the 22nd hour, 10 μL of CCK-8 reagent was added to each well and incubated in the cell culture incubator for another 2 hours. The MH7A group was incubated for 48 hours. At the 44th hour, 10 μL of CCK-8 reagent was added to each well and incubated in the cell culture incubator for another 4 hours. The OD value of each well was measured at a wavelength of 450 nm using a microplate reader, and the cell viability was calculated according to the following formula:

[0135]

[0136] LPS and TNF-α were used to induce the inflammatory model of Raw264.7 and MH7A cells, respectively. After co-incubation, Raw264.7 and MH7A showed significant compensatory cell proliferation, and the relative cell viability increased to 129% and 115%, respectively. This is because TNF-α activated the NF-κB and MAPK-p38 pathways, promoted cell proliferation and enhanced anti-apoptosis ability; as a tetrahydrofolate reductase inhibitor and NF-κB inhibitor, MTX effectively reduced cell viability to a level slightly lower than that of the control group, completely offsetting the cell proliferation induced by LPS / TNF-α, and the MTX-peptide conjugate complex group could further inhibit inflammation-related abnormal proliferation. From the experimental results, the cell viability of the MTX, MKTAT, MTAT and M19KTAT complex groups was significantly lower than that of the LPS / TNF-α group, and the cell viability of the MKTAT, MTAT and M19KTAT complex groups was significantly lower than that of the control group, verifying its inhibitory effect ( Figure 18 The results of in vitro cell viability experiments showed that MTX and its conjugates effectively blocked pathological compensatory proliferation by synergistically inhibiting inflammatory signals and cell cycle progression, providing molecular-level experimental evidence for the treatment of RA.

[0137] 5. Evaluation of anti-inflammatory activity

[0138] Based on enzyme-linked immunosorbent assay (ELISA) technology, this study evaluated the immunomodulatory effects of different nanocomplexes on macrophage Raw264.7 cells by detecting the secretion levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). The experimental design co-incubated cells with MTX complex, MKTAT complex, MTAT complex, and M19KTAT complex (the samples were also diluted to a drug concentration of 360 ng / mL (in terms of MTX) according to the above method), and quantitatively analyzed the changes in cytokine concentrations in the culture supernatant to clarify the differential regulatory effects of each nanocomplex on the expression of inflammatory factors. This approach effectively combines the core role of macrophages in the RA pathological model with the specific detection advantages of ELISA technology.

[0139] The experimental groups were: (1) Control, (2) LPS (1 μg / mL), (3) MTX complex + LPS (1 μg / mL), (4) MKTAT complex + LPS (1 μg / mL), (5) MTAT complex + LPS (1 μg / mL), and (6) M19KTAT complex + LPS (1 μg / mL).

[0140] The incubation time of Raw264.7 cells and each group of samples was 24 hours. After the incubation, the culture medium was aspirated and centrifuged at 3000 rpm for 10 minutes. The supernatant was aspirated for ELISA experiment to detect the concentration levels of TNF-α, IL-1β and IL-6.

[0141] ELISA test results showed that after Raw264.7 cells were co-incubated with different nanocomplexes, the levels of inflammatory factors in all experimental groups (TNF-α, IL-1β and IL-6) were significantly lower than those in the LPS stimulation group (p<0.0001). Although the inflammatory response was not completely eliminated, it showed significant anti-inflammatory efficacy. Among them, the MKTAT and MTAT complex groups showed very excellent anti-inflammatory effects, with the MKTAT complex group having the best effect. Its inflammatory factor inhibition rate was significantly better than that of the MTX complex group, indicating that the difference in polypeptide sequence is the key factor affecting the efficacy ( Figures 19-21 ). These results confirm that the peptide coupling strategy can significantly enhance the anti-inflammatory efficacy of the nanocomplex.

[0142] 5. In vivo animal evaluation

[0143] 5.1 Assessment of rat joint swelling

[0144] To construct an animal model of rheumatoid arthritis (RA), we used a collagen-induced arthritis (CIA) rat model induced by complete Freund's adjuvant (CFA) combined with bovine type II collagen (CII). This model is highly similar to human RA characteristics in terms of clinical symptoms, pathological characteristics, and immune response, and can simulate typical RA lesions.

[0145] Before modeling, all male Wistar rats were acclimated for 7 days in an SPF-grade environment at 25°C ± 1°C and 50%-60% humidity. They had free access to food and water to minimize environmental influences on the experimental results. Subsequently, the rats were given a primary immunization with a subcutaneous injection of 250 μL of adjuvant emulsion into the tail, designated as day 0. Seven days later, the rats were given another injection of adjuvant emulsion for immune boosting. Continuous observation after modeling revealed that starting on day 10 after the initial immunization, the rats developed a significant inflammatory response, manifested by redness and swelling of the hind limb joints, elevated skin temperature, lameness, and limited mobility, indicating that the CIA model was successfully established.

[0146] Wistar rats weighing approximately 220 ± 20 g were randomly divided into 6 groups, each with 5 rats. Five rats without CIA modeling served as the blank group, and 20 rats with successful CIA modeling served as the drug-treated group. The groups were as follows: (1) Control group, (2) CIA model group, (3) MTX complex group, (4) MKTAT complex group, (5) MTAT complex group, and (6) M19KTAT complex group.

[0147] The drug was administered once a day from the 10th to the 14th day after the first immunization, with a dose of 100 μL (calculated as MTX, drug concentration 625 μg / mL). Three-point injection intervention was performed through the intra-articular and lateral synovial tissues. The weight changes of the rats were monitored daily from the 14th to the 19th day of the experiment, and the thickness of the right hind limb ankle joint was measured with a vernier caliper.

[0148] Experimental results reference Figure 22-23 The ankle thickness of the mice in the control group increased by less than 0.5 mm, which was caused by the normal weight growth of the rats. On the 10th day after CIA modeling, the rats began to show obvious inflammatory response. On the 15th day, the ankle thickness increased to 9-10 mm and the body weight decreased significantly. In the following days, the ankle thickness began to decrease slightly and the body weight began to increase. The growth rate of the ankle thickness of the mice in the complex group was significantly slower than that of the CIA group within 14-19 days, and the symptoms began to ease. This was particularly evident in the MKTAT complex group. On the 18th day, the average ankle thickness dropped to below 7 mm and the body weight increased further. It had basically recovered on the 19th day, with no significant difference from the control group. The other experimental groups failed to fully recover and showed significant differences from the control group. MTX showed the best anti-inflammatory effect after being coupled with the polypeptide prepared in Example 3.

[0149] The above shows and describes the basic principles, main features, and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any person skilled in the art without departing from the technical solution of the present invention are intended to be within the scope of the present invention.

Claims

1. A methotrexate-cationic polypeptide conjugate, characterized in that: The carboxyl group of the methotrexate molecule is covalently linked to the N-terminal amino group of the cationic membrane-penetrating polypeptide KKKKKKYGRKKRRQRRR with an amino acid sequence as shown in SEQ ID NO:

1.

2. The method for preparing the methotrexate-cationic polypeptide conjugate according to claim 1, wherein: The steps include: 1) After the amino-protected arginine is immobilized on a resin support by solid-phase synthesis, the amino-protecting group is removed; 2) Stepwise coupling of amino-protected amino acids to extend the polypeptide chain to obtain the resin-loaded KKKKKKYGRKKRRQRRR sequence; 3) Activated MTX is coupled to the resin-loaded peptide; 4) cracking the resin to obtain a crude product; 5) After purification, freeze-drying is performed to obtain the target conjugate.

3. The method for preparing the methotrexate-cationic polypeptide conjugate according to claim 2, wherein: In step 1), DIC / HOBT is used as a condensing agent, the equivalent ratio of amino-protected arginine, HOBT and DIC is 1:1.5-3:1.5-3, the reaction temperature is 35-45°C, and the reaction time is 3-5 hours; in step 2), when the polypeptide chain extension reaction is carried out, the condensing agent used is DIC / HOBT, the equivalent ratio of amino-protected amino acid, HOBT and DIC is 1:1.5-2.5:1.5-2.5, the reaction temperature is 40-50°C, and the reaction time is 0.5-2 hours.

4. The method for preparing the methotrexate-cationic polypeptide conjugate according to claim 2, wherein: In step 3), the equivalent ratio of MTX to polypeptide is 3:1, the activation reagent is HATU or HBTU, the reaction temperature is 25-40° C., and the reaction time is 1-3 h.

5. A self-assembled nanocomposite, characterized in that It comprises the methotrexate-cationic polypeptide conjugate according to claim 1 and a stabilizer in a mass volume ratio of 0.02% to 0.5%; The stabilizer is selected from any one or more of SDS, CTAB, cationic cholesterol, polyethylene glycol, phospholipids, chitosan, povidone, and gelatin. The drug concentration is calculated as methotrexate, and the concentration of the methotrexate-cationic polypeptide conjugate is 500-1500 μg / mL; The composite has an average particle size of ≤200 nm, a polydispersity index of <0.3, and a zeta potential of ≥30 mV.

6. The method for preparing the self-assembled nanocomposite according to claim 5, wherein: After the methotrexate-cationic polypeptide conjugate and the stabilizer are dispersed in the aqueous phase, a high-pressure microfluidization homogenization treatment is performed 1-5 times at a pressure of 5000-20000 psi.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of the methotrexate-cationic polypeptide conjugate according to claim 1 or the nanocomplex according to claim 5, and pharmaceutically acceptable excipients, wherein the pharmaceutical composition is an injectable preparation or a sustained-release preparation.

8. Use of the methotrexate-cationic polypeptide conjugate according to claim 1 in the preparation of a drug for treating immune-mediated inflammatory diseases, characterized in that: The immune-mediated inflammatory disease is rheumatoid arthritis or psoriasis.

9. Use of the methotrexate-cationic polypeptide conjugate according to claim 1 in the preparation of a drug for treating malignant tumors.

10. The use according to any one of claims 8 to 9, characterized in that The drug is a nanocomplex preparation comprising the methotrexate-cationic polypeptide conjugate.

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