Methotrexate-Catonic Polypeptide Conjugates, Their Preparation Methods and Applications

Abstract

This invention discloses a methotrexate-cationic polypeptide conjugate, its preparation method, and its applications, belonging to the field of medicinal chemistry. This conjugate chemically links methotrexate (MTX) to a cationic polypeptide with the amino acid sequence shown in SEQ ID NO:1, innovatively combining the membrane-binding properties of oligo-lysine with the transmembrane-penetrating ability of TAT peptides to construct a polypeptide carrier system with highly efficient cell penetration. Peptide modification imparts amphiphilicity and positive charge to MTX, enabling it to spontaneously form stable nanoparticles, effectively improving the problems of poor water solubility and acid instability of the active pharmaceutical ingredient. This conjugate significantly enhances the drug accumulation efficiency at the lesion site by utilizing the targeting properties of the polypeptide carrier, and prolongs the drug's duration of action through a precise delivery mechanism while reducing systemic toxicity. The preparation method employs mild coupling reaction conditions, resulting in a product with high purity and excellent stability; it is suitable for treating immune-mediated inflammatory diseases and certain malignant tumors, and also provides innovative solutions for the treatment of related diseases.

Description

Technical Field

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

[0002] Methotrexate (MTX), a widely used folic acid antagonist, interferes with DNA synthesis and cell proliferation by inhibiting dihydrofolate reductase, and is used to treat tumors, autoimmune diseases, and inflammatory diseases. However, its therapeutic window is narrow, and its toxicity is closely related to dosage and duration of treatment, leading to multi-systemic adverse reactions such as hematopoietic suppression (manifested as bone marrow suppression and pancytopenia), gastrointestinal mucosal damage (e.g., stomatitis and gastrointestinal ulcers), abnormal liver function (from elevated transaminases to cirrhosis), nephrotoxicity (typically manifested as acute tubular crystal deposition), and neurotoxicity (risk of intrathecal administration-related encephalopathy). Notably, patients with "third-space" effusion, renal dysfunction, or pregnancy exhibit higher risks associated with medication use. Current clinical management of MTX has developed a series of individualized strategies: reducing mucositis and hepatotoxicity through co-administration of folic acid, avoiding gastrointestinal irritation through subcutaneous injection, and preventing renal crystal formation through urine alkalinization. Based on the existing challenges, future research should focus on three main directions: 1) In precision medicine, promote pharmacogenomics testing to guide personalized drug delivery; 2) In structural optimization, develop low-toxicity drug conjugates; 3) In delivery technology, construct novel drug delivery platforms such as sustained-release systems and nano-formulations to achieve simultaneous improvement in efficacy and safety.

[0003] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by symmetrical polyarthritis, synovial hyperplasia, and progressive joint destruction. It 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. Metabolic acid sulfadiazine (MTX), a guideline-recommended core treatment for RA, exerts its anti-inflammatory effect by inhibiting immune cell proliferation and regulating the inflammatory cytokine network. Studies have shown that MTX can upregulate regulatory T cells (Tregs), correcting the Th17 / Treg imbalance 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 prostaglandin E2, which causes pain, thus relieving pain.

[0004] However, the main problems facing methotrexate (MTX) in clinical application are: its poor water solubility (<1 mg / mL) and instability in acidic environments, leading to easy degradation by gastric acid and gastrointestinal irritation during oral administration, resulting in significantly reduced bioavailability; to improve solubility, excipients such as sodium hydroxide need to be added to adjust the pH to above 8 for injectable formulations, but such strongly alkaline conditions are poorly compatible with the physiological environment. Furthermore, the toxicity of MTX cannot be ignored; the severity of toxic reactions is related to dosage, frequency of administration, and individual tolerance. Although local injection into joints can reduce systemic toxicity, the acidic microenvironment of rheumatoid arthritis (RA) joints (pH≈6.5) can lead to decreased drug solubility, increasing 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, resulting in inherently low delivery efficiency. Recent research indicates that conjugating small molecule drugs with transmembrane peptides (CPPs) or antimicrobial peptides can overcome three major physiological barriers: 1) significantly enhancing transmembrane transport capacity, enabling a shift from passive diffusion to active targeting; 2) effectively circumventing P-glycoprotein-mediated efflux effects; and 3) reversing multidrug resistance by regulating intracellular drug concentration. This technology can 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 conjugate of methotrexate and a peptide, with the peptide sequence RFLKRLDRNLW. This conjugate exhibits low cytotoxicity and significantly reduces the expression of intracellular inflammation-related genes. However, this type of conjugate does not solve the inherent problems of poor water solubility and acid instability of methotrexate (MTX), and it lacks verification of self-assembly properties. 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 structures remains a key research direction, mainly including three key strategies: first, improving the solubility of MTX through hydrophilic modification to solve the formulation development problem; second, introducing acid-stabilizing groups to enhance microenvironment tolerance; and third, 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] To address the aforementioned problems, this invention aims to provide a methotrexate-cationic peptide conjugate delivery system that improves the water solubility and stability of methotrexate (MTX). This system organically combines the membrane-binding properties of oligo-lysine with the transmembrane-penetrating ability of TAT peptides to construct a peptide carrier with highly efficient cell penetration. Peptide modification imparts amphiphilicity and positive charge to MTX, enabling it to spontaneously form stable nanoparticles, effectively overcoming the shortcomings of poor water solubility (<1 mg / mL) and instability in acidic environments of the active pharmaceutical ingredient. The targeting properties of the peptide carrier also significantly enhance drug accumulation in diseased joints. Furthermore, the precise delivery mechanism prolongs the drug's duration of action while significantly reducing systemic toxicity resulting from distribution in normal tissues. This formulation provides a novel MTX delivery regimen for the treatment of rheumatoid arthritis that combines high efficiency and safety.

[0009] To achieve the above objectives, 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, whose amino acid sequence is shown in SEQ ID NO:1, wherein the hexamethylenetetramine domain enhances 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) After immobilizing amino-protected arginine Fmoc-Arg(Pbf)-OH onto a resin support via solid-phase synthesis, the amino protecting group is removed.

[0012] 2) The polypeptide chain was extended by stepwise coupling of amino-protected amino acids to obtain the resin-loaded KKKKKKYGRKKRRQRRR sequence;

[0013] 3) MTX is activated and then coupled with resin-loaded peptides;

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

[0015] 5) After purification, the target conjugate is obtained by freeze-drying.

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

[0017] Further, in step 3), the equivalence ratio of MTX to peptide is 3:1, the activating agent is HATU or HBTU, preferably HATU, the reaction temperature is 25-40℃, and the reaction time is 1-3h.

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

[0019] The above-mentioned methotrexate-cationic polypeptide conjugate can be combined with a stabilizer at a mass-to-volume ratio of 0.02% to 0.5% to form a nanocomposite.

[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 complex 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 above self-assembled nanocomposite is as follows: after dispersing the methotrexate-cationic polypeptide conjugate and stabilizer in an aqueous phase, the high-pressure microfluidic homogenization treatment is carried out 1-5 times at a pressure of 5000-20000psi, preferably 2-3 times at a pressure of 8000-12000psi.

[0023] This 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, wherein the pharmaceutical composition is an injectable formulation or a sustained-release formulation.

[0024] The above-mentioned methotrexate-cationic polypeptide conjugate can be used to prepare drugs for treating immune-mediated inflammatory diseases, such as rheumatoid arthritis or psoriasis.

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

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

[0027] The beneficial effects of this invention are as follows:

[0028] 1. This application achieves a breakthrough innovation at the molecular level by modifying the structure of methotrexate (MTX) through peptide coupling technology. The introduction of hydrophilic peptides can significantly improve the water solubility of the compound, overcome the development obstacles of the original drug formulation, and create favorable conditions for the development of injectable dosage forms.

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

[0030] 3. The KTAT peptide module proposed in this application is an innovative structure integrating a lysine-rich region and a transmembrane peptide functional domain. The novel compound formed after coupling with methotrexate exhibits multiple technological advantages: This peptide module significantly enhances the transmembrane transport capacity of the drug through its transmembrane peptide component, enabling it to efficiently target abnormally activated immune cell populations, including key inflammatory mediators such as macrophages and T cells, thereby achieving targeted 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 delivery efficiency in target tissues but also effectively reduces common adverse reactions of traditional methotrexate treatment, such as hepatotoxicity and bone marrow suppression, by reducing the systemic circulating drug dose. Simultaneously, the immunomodulatory potential inherent in the peptide module can produce a synergistic therapeutic effect with methotrexate, enhancing local anti-inflammatory effects in joints while further optimizing the drug's safety profile by reducing its distribution in normal tissues. This application achieves a dual breakthrough of enhanced efficacy and reduced toxicity.

[0031] 4. This application constructs a novel drug form that combines 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 treatments, 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 realizing personalized precision medicine, which has important clinical translational significance.

[0032] 5. The method for preparing 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 a reliable technical guarantee for large-scale production.

[0033] 6. The methotrexate-cationic polypeptide conjugate developed in this application has broad application prospects. This technology is not only applicable to the treatment of rheumatoid arthritis, but can also be extended to multiple therapeutic areas such as anti-tumor, immunomodulation, and inflammatory diseases. The introduction of the cationic polypeptide significantly improves the drug's solubility and stability, laying an important foundation for developing diverse formulations. Based on this technology platform, various dosage forms, including injections and sustained-release formulations, can be developed to meet the personalized treatment needs of different disease characteristics, patient groups, and routes of administration. This potential for multi-indication and multi-dosage formulation development gives this technology a broad scope for clinical application. Attached Figure Description

[0034] Figure 1 The image shows the LC-MS chromatogram of the crude product prepared in step S4 of Example 1.

[0035] Figure 2 The image shows the LC-MS chromatogram of the purified sample prepared in step S5 of Example 1.

[0036] Figure 3 The image shows the LC-MS chromatogram of the crude product prepared in step S4 of Example 2.

[0037] Figure 4 The image shows the LC-MS chromatogram of the purified sample prepared in step S5 of Example 2.

[0038] Figure 5 The image shows the LC-MS chromatogram of the crude product prepared in step S4 of Example 3;

[0039] Figure 6 The image shows the LC-MS chromatogram of the purified sample prepared in step S5 of Example 3.

[0040] Figure 7 To enlarge the HPLC chromatogram of the synthesized target product MKTAT;

[0041] Figure 8 To magnify the LC-MS image of the synthesized target product MKTAT;

[0042] Figure 9 To amplify the NMR spectrum of the synthesized target product MKTAT;

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

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

[0045] Figure 12 The HPLC chromatogram of MTAT synthesized in Comparative Example 1 is shown.

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

[0047] Figure 14 The HPLC chromatogram of M19KTAT synthesized in Comparative Example 2 is shown.

[0048] Figure 15 LC-MS plot 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 To compare the stability of different drugs in PBS-simulated RA synovial fluid (pH=6.5);

[0051] Figure 18 The effects of different nanocomposites on the viability of Raw264.7 / MH7A cells;

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

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

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

[0055] Figure 22 Figure 1: Changes in the thickness of the right ankle joint of mice over time after immunization with different complexes.

[0056] Figure 23 The graph shows the change in mouse body weight over time after immunization with different complexes. Detailed Implementation

[0057] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0058] Example 1

[0059] This embodiment discloses a methotrexate-cationic polypeptide conjugate and its preparation method. The sequence of the conjugated cationic polypeptide is KKKKKKYGRKKRRQRRR (KTAT), which is a transmembrane peptide variant containing a lysine-rich region. YGRKKRRQRRR is a transmembrane peptide sequence that promotes polypeptide entry 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. The hexapeptide 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. There are currently no reports on its related functions.

[0061] The synthesized conjugate is designated MTX-KKKKKKYGRKKRRQRRR, or simply MKTAT. The six consecutive lysine (K) residues at the N-terminus provide strong hydrophilicity and positive charge, helping to improve the water solubility of MTX. The high-density positive charge of the lysine residues enhances molecular hydration, while the TAT membrane-penetrating peptide (YGRKKRRQRRR) itself is rich in arginine / lysine, further strengthening hydrophilicity, thus jointly addressing the problem of insufficient hydrophilicity in MTX. Furthermore, in acidic environments, the strong positive charge of the peptide sequence (high-density K / R) can reduce molecular aggregation through charge repulsion, lowering the risk of degradation.

[0062] The synthetic route for the coupling compound is as follows:

[0063]

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

[0065] The specific preparation process is as follows:

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

[0067] Weigh Wang resin (Wang-resin, degree of substitution 0.3 mmol / g) into a reactor, add dichloromethane (DCM) and soak for 2 hours, add N,N-dimethylformamide (DMF) at a ratio of 8 mL / g resin, wash the resin and dry it, repeat four times, and finally dry the resin for 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 the compound in DMF, add 3 eq of N,N-diisopropylcarbodiimide (DIC), shake until the solution is clear, and then add it to the reactor. React the solution in a shaker at 40°C for 4 h. Wash the solution with N,N-dimethylformamide (DMF) at a ratio of 8 mL / g resin and dry it. Repeat the process four times.

[0069] A certain amount of acetic anhydride / N,N-diisopropylethylamine (DIEA) / DMF mixture was used for a 0.5-hour end-capping reaction (the volume ratio of acetic anhydride, DIEA, and DMF was 1:1:2). The mixture was then washed with three times the resin volume of DMF and dried (repeated 4 times). A 20% piperidine / DMF solution (piperidine to DMF volume ratio of 1:4) was added, and the mixture was shaken on a decolorizing shaker for 20 minutes to remove the fluorenemethyloxycarbonyl (Fomc) group protecting the product at the amino terminus. The mixture was then washed with DMF and dried (repeated 6 times).

[0070] Take a small amount of resin sample into the detection tube, add ninhydrin (Ninhydrin hydrate) detection reagent (2 drops each of test A and test B), react at 100℃ for 1 min and observe the color reaction. The obvious color change of the resin indicates that the C-terminal starting amino acid (Fmoc-Arg(Pbf)-OH) of the peptide has been successfully linked 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'-tetramethylurea hexafluorophosphate (HBTU) into a centrifuge tube, add an appropriate amount of DMF to dissolve, then add 3 eq of DIEA, shake well 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 dry it (repeat 4 times).

[0073] Take a small amount of resin for ninhydrin detection (two drops each of test A and test B, react at 100℃ for 1 min). If the resin is colorless, it indicates that the amino acid condensation is complete; if the resin is colored, 3 eq of the corresponding amino acid and condensing agent (such as HBTU / DIEA) need to be added and the reaction repeated. After confirming that the condensation is complete, add 8 mL of 20% piperidine / DMF solution (V / V = 1:4) per gram of resin, and shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group to obtain compound 4. Then wash the resin with DMF and dry it under vacuum (8 mL / g resin × 6 times), drying it each time.

[0074] Following the condensation process in step S2, the following Fmoc protected amino acids are sequentially coupled to the resin: 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, Fm The sequences 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 were used to obtain the final resin-loaded polypeptide sequence KKKKKKYGRKKRRQRRR, which was named KTAT-Wang-resin.

[0075] Step S3: Couple MTX

[0076] Add 3 eq of methotrexate (MTX, compound 5) and 3 eq of HATU to a centrifuge tube, dissolve in DMF, add 5 eq of pyridine, shake for 1 min until the solution is clear, transfer to a reactor containing resin (in KTAT-Wang-resin, the amount of KTAT is 1 eq), and react in a shaker at 35°C for 2 hours to complete MTX coupling. After the reaction, wash the resin sequentially with DMF (8 mL / g resin × 4 times, each time dried) and methanol (8 mL / g resin × 5 times, each time dried). The obtained resin product is named MKTAT-Wang-resin. Transfer the resin to a centrifuge tube for cutting.

[0077] Step S4: Cut the crude target coupling compound

[0078] Add an appropriate amount of resin (at a ratio of 1g resin to 10mL cutting solution) to a centrifuge tube, and inject 95% cutting solution (trifluoroacetic acid (TFA): 1,2-ethylenedithiol (EDT): triisopropylsilane (TIS): H2O = 95:2:2:1). Shake on a shaker at room temperature for 2.5h to complete the cutting. After filtering the reaction solution through a 0.45μm filter membrane, add pre-cooled diethyl ether (-20℃) at a volume ratio of 1:10. Vortex mix, centrifuge to precipitate the crude peptide, discard the supernatant, and repeat the ether precipitation and washing twice to finally obtain the crude product.

[0079] Step S5: Purify the target conjugate

[0080] The crude product was purified by preparative HPLC (C18 column, 5×30cm) using a dual-pump system: Pump A was 0.1% TFA aqueous solution, and Pump B was 0.1% TFA acetonitrile solution, at a flow rate of 60 mL / min, with gradient elution (Phase B 10%→40%, 60 min), monitored at 220 nm wavelength, with a target component retention time of 20-30 min. The target peak fraction was collected, and after removing acetonitrile by rotary evaporation, it was freeze-dried to obtain a high-purity target conjugate preparation solution, named MTX-KKKKKKYGRKKRRQRRR, abbreviated as MKTAT, and stored at -20℃ protected from light for later use.

[0081] Step S6: Freeze-drying

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

[0083] The crude product obtained in step S4 and the purified sample obtained in step S5 were analyzed by LC-MS, and the results are as follows: Figure 1-2 As shown in the mass spectrum, in addition to the main peak of the target conjugate, the crude product also contains a peak with a lower intensity (called a secondary peak). Even after purification, it still contains small secondary peaks.

[0084] Example 2-3

[0085] The difference between Examples 2-3 and Example 1 lies in the type and amount of condensing agent used in step S2, and the temperature at which the amide condensation reaction is carried out.

[0086] The relevant reaction conditions for 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 and the purified sample obtained in step S5 of Example 2 were analyzed by LC-MS, and the results are as follows: Figure 3-4 As shown in the mass spectrum, the mass spectrometry analysis results indicate 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 and the purified sample obtained in step S5 of Example 3 were analyzed by LC-MS, and the results are as follows: Figure 5-6 As shown, the mass spectrometry analysis results indicate that the main peak position of the target conjugate in the crude product is accurate and there are no obvious impurity peaks. After purification, the peak area of ​​the target product increases significantly, indicating that the purification process is effective and the product yield is high.

[0092] The results above show that the type and amount of condensing agent and the reaction conditions have a significant impact on the purity and yield of the product during the preparation of peptides using amide condensation reaction.

[0093] Following the preparation process of Example 3, a 10-fold scale-up synthesis was performed, successfully obtaining the target conjugate MKTAT. Product purity was determined by HPLC, molecular weight and structure were verified by LC-MS, molecular structure was confirmed by 1H NMR, functional group characteristics were analyzed by Fourier transform infrared spectroscopy (FT-IR), and its crystal structure was characterized by X-ray diffraction (XRD).

[0094] Purity analysis and structural confirmation: HPLC analysis ( Figure 7 It was confirmed that the purity of the synthesized MKTAT was 95.04%, which is greater than the required 95% purity. Therefore, the synthesized MKTAT meets the purity requirements of the experiment. LC-MS analysis showed ( Figure 8 Its molecular weight is 2765.30, and its [M+6H] content is... 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 the [M+6H] of the synthesized MKTAT molecule. 6+ The measured value of the peak is 461.88, [M+5H] 5+ The measured value of the peak is 554.06, [M+4H] 4+ The measured value of the peak is 692.33, [M+3H] 3+The measured peak value was 922.88, which was consistent with the theoretical value, fully verifying the correctness of the target product's structure. Combined HPLC and LC-MS data confirmed the successful synthesis of high-purity MKTAT.

[0095] 1H NMR spectrum ( Figure 9 )show:

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

[0097] 4.0~4.4 (15H): 2H on amino acid G, 1H on amino acid Y (ortho-carboxyl hydrogen), 1H on amino acid R (ortho-carboxyl hydrogen), and 8H on amino acid K (ortho-carboxyl hydrogen).

[0098] 2.0~3.0 (34H): 2H on amino acid Y (benzylidene methylene pHCH2-), 16H on amino acid K (amino ortho-NH2CH2-), 12H on amino acid R (amino ortho-NH2CH2-), and 4H on amino acid Q (amide ortho-methylene -CH2-).

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

[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 3400–3200 cm⁻¹. -1 The broad peak at 2900–3000 cm⁻¹ is attributed to the stretching vibrations of the NH₂ group of the pteridine ring and the NH group of the amide bond; -1 The weak peaks within the range are due to the CH stretching vibrations of the pteridine and benzene rings; 1645 cm⁻¹ -1 The absorption peak at 1538 cm⁻¹ corresponds to the stretching vibrations of the amide and carboxyl C=O groups; -1 The characteristic peaks of the amide II band are visible at this location, originating from the coupling effect of the NH bending vibration of the amide and the peptide bond. These characteristic peaks collectively verify the functional group structure of the target molecule.

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

[0103] X-ray diffraction (XRD) analysis showed that MKTAT, formed by the conjugation of MTX and the peptide, exhibited an amorphous morphology, and the original crystalline characteristic peaks of MTX in its diffraction pattern completely disappeared, confirming the significant difference in crystal structure between the conjugated product and the active pharmaceutical ingredient. 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 membrane-penetrating peptide YGRKKRRQRRR (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, SEQ ID NO.2). The synthesis method of this membrane-penetrating peptide is the same as that in Example 3. After MTX is linked to this membrane-penetrating peptide through the same coupling reaction process, the resulting product MTX-YGRKKRRQRRR is named MTAT.

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

[0107] HPLC peak data ( Figure 12 The synthesized MTAT had a purity of 96.37%, exceeding the required 95% purity, thus meeting the experimental purity requirements. Subsequent LC-MS analysis confirmed the structure of MTAT, revealing a molecular weight of 1996.28 and a [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 the [M+5H] of the synthesized MKTAT molecule. 5+ The measured value of the peak is 400.25, [M+4H] 4+ The measured value of the peak is 500.07, [M+3H] 3+ The measured value of the peak was 666.55, and the measured value was consistent with the theoretical value. Figure 13 HPLC and LC-MS results 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 peptide coupled to methotrexate (MTX) is a transmembrane peptide variant with a higher lysine enrichment. The sequence of this peptide is KKKKKKKKKKKKKKKKYGRKKRRQRRR, and its amino acid sequence is Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Tyr-Gly-Arg-Lys-Lys-Arg-Gln-Arg-Arg-Arg, as shown in SEQ ID NO.3.

[0110] The synthesis method of this polypeptide is the same as that in Example 3; after MTX is linked to this polypeptide through the same coupling reaction process, the resulting product MTX-KKKKKKKKKKKKKKKKYGRKKRRQRRR is named M19KTAT.

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

[0112] HPLC peak data ( Figure 14 The synthesized M19KTAT had a purity of 95.94%, exceeding the required 95% purity, thus meeting the experimental purity requirements. Subsequent LC-MS analysis confirmed the structure of M19KTAT, revealing a molecular weight of 4431.58 and a [M+6H] [molecule name missing]. 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 the [M+6H] of the synthesized M19KTAT molecule. 6+ The measured value of the peak is 739.60, [M+5H] 5+ The measured value of the peak is 887.32, [M+4H] 4+ The measured value of the peak was 1108.90, and the measured values ​​were consistent with the theoretical values. Figure 15 HPLC and LC-MS results confirmed the successful synthesis of high-purity M19KTAT.

[0113] Related performance tests

[0114] 1. Equilibrium solubility test

[0115] A series of aqueous solutions with different pH values ​​(pH = 1.2, 2.0, 3.0, 4.0, 5.0, 6.8, 7.4) were prepared using 0.1M HCl and NaOH. MTX, MKTAT, MTAT, and M19KTAT were added to each solution, and 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 absorbance of the drugs was measured at 302 nm. The equilibrium solubility of MTX, MKTAT, MTAT, and M19KTAT under different pH conditions was calculated.

[0116] See results Figure 16As can be seen from the figure, the solubility of MTX-peptide conjugates (MKTAT / MTAT / M19KTAT) is significantly better than that of free MTX in different pH environments (pH=1.2~7.4); in the characteristic acidic microenvironment of rheumatoid arthritis (pH≈6.5), the solubility of the conjugates is significantly improved compared with MTX, and this pH-responsive solubility characteristic is highly matched with the microenvironment of the diseased joints.

[0117] 2. Chemical stability test

[0118] To address the acidic microenvironment (pH≈6.5) of the synovial microenvironment in rheumatoid arthritis (RA), this study used pH 6.5 PBS to simulate RA synovial fluid and dynamically monitored the chemical stability of metronidazole (MTX) and its peptide conjugates (MKTAT / MTAT / M19KTAT). The results showed that from 0 to 240 hours, the relative percentage of MTX decreased from 100% to approximately 80%, indicating poor stability in the simulated RA synovial fluid and significant degradation of free MTX in the acidic environment. The curves for MKTAT, MTAT, and M19KTAT, however, showed that their relative percentages remained relatively stable at around 100% over 240 hours, indicating good stability, chemical stability, and resistance to degradation in the simulated RA synovial fluid. This confirms that peptide modification can significantly enhance drug stability in the pathological microenvironment. Figure 17 ).

[0119] 3. Self-assembly performance evaluation

[0120] The stabilizer, MTX, and MTX-peptide conjugates (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 microfluidic homogenizer 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 the system's stability.

[0121] The concentration of MTX-peptide 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). Phospholipids (such as 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, DOPE) are preferred as stabilizers. Other optional polymers (chitosan, povidone, gelatin, etc.) or other surfactants (sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), cholesterol, polyethylene glycol, etc.) are used in a range of 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 homogenization cycles is 1-5 (preferably 2-3).

[0122] See Table 2 for specific parameters.

[0123] Table 2. Key Parameters for Self-Assembly of MTX-Peptide Conjugates

[0124]

[0125]

[0126] The results showed that the average particle size of the uncoupled MTX and M19KTAT self-assembled particles 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 of both 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 conjugate group was above 30 mV. This indicates that after MTX was conjugated with the peptide, the surface potential of the complex increased significantly 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 significantly improved colloidal stability.

[0127] 4. Cell viability test

[0128] The effect of different concentrations of MTX-peptide conjugate on the viability of RAW264.7 / MH7A cells was tested.

[0129] The nanocomposites prepared during the self-assembly performance evaluation test (denoted as MTX complex, MKTAT complex, MTAT complex and M19KTAT complex, respectively) were used as test objects. Opti-MEM medium at pH 6.5 was used as the solvent to dilute the samples to a drug concentration of 360 ng / mL (calculated as MTX) for later use.

[0130] Raw 264.7 cells were grouped 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), (6) M19KTAT complex + LPS (1 μg / mL).

[0131] The MH7A cell experimental groups were: (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 (RAW 264.7) culture: Frozen cells were thawed in a 37°C water bath, sterilized with 75% ethanol, and placed in a clean bench for later use. A sterilized 15mL centrifuge tube was taken, and 5mL of DMEM medium (containing 10% fetal bovine serum, 100U / mL streptomycin, and 100U / mL penicillin) was added. Then, 1mL of the thawed cell cryopreservation solution was added, mixed thoroughly, and centrifuged at 1500rpm for 5min, discarding the supernatant. The cells were resuspended in 5mL of DMEM medium and transferred to a T25 culture flask. 5mL of medium was added, and the flask was gently agitated in a cross-shaped motion to distribute the cell resuspension evenly. The flask was then placed in a 5% CO2 incubator at 95% relative humidity and 37°C, with the DMEM medium changed periodically for future use.

[0133] Culture of synovial fibroblasts (MH7A cells) for arthritis: Frozen cells were thawed in a 37°C water bath, sterilized with 75% ethanol, and placed in a clean bench for later use. A sterilized 15mL centrifuge tube was taken, and 5mL of DMEM medium (containing 15% fetal bovine serum, 100U / mL streptomycin, and 100U / mL penicillin) was added. Then, 1mL of the thawed cell cryopreservation solution was added and mixed thoroughly. The mixture was centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 5mL of DMEM medium and transferred to a T25 culture flask. 5mL of medium was added, and the flask was gently agitated in a cross-shaped motion to distribute the cell resuspension evenly. The culture flask was then placed in a 5% CO2 incubator at 95% relative humidity and 37°C, with the DMEM medium changed periodically.

[0134] Raw 264.7 and MH7A cells were seeded into 96-well plates, with 3 replicates per group and 5 × 10⁶ cells per well. 3Cells were cultured at 37°C in a 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. For the Raw 264.7 group, after 24 hours of incubation, 10 μL of CCK-8 reagent was added to each well at the 22nd hour, and incubation continued for 2 hours. For the MH7A group, after 48 hours of incubation, 10 μL of CCK-8 reagent was added to each well at the 44th hour, and incubation continued for 4 hours. The OD value of each well was measured using a microplate reader at a wavelength of 450 nm, and cell viability was calculated using the following formula:

[0135]

[0136] Inflammation models of Raw264.7 and MH7A cells were induced by LPS and TNF-α, respectively. After co-incubation, both Raw264.7 and MH7A cells showed significant compensatory cell proliferation, with relative cell viability increasing to 129% and 115%, respectively. This is because TNF-α activated the NF-κB and MAPK-p38 pathways, promoting cell proliferation and enhancing anti-apoptotic ability. As a tetrahydrofolate reductase inhibitor and NF-κB inhibitor, MTX effectively reduced cell viability to a level slightly lower than the control group, completely offsetting the LPS / TNF-α-induced cell proliferation. The MTX-peptide conjugate complex group further inhibited inflammation-related abnormal proliferation. The experimental results showed that 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 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 RA treatment.

[0137] 5. Evaluation of anti-inflammatory activity

[0138] Based on enzyme-linked immunosorbent assay (ELISA), this study evaluated the immunomodulatory effects of different nanocomplexes on Raw264.7 macrophages by detecting the secretion levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). The experimental design involved co-incubating cells with MTX, MKTAT, MTAT, and M19KTAT complexes (samples were diluted to a drug concentration of 360 ng / mL (based on MTX) as described above), and quantitatively analyzing changes in cytokine concentrations in the culture supernatant to elucidate the differential regulatory effects of each nanocomplex on inflammatory cytokine expression. 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] Raw264.7 cells were incubated with each group of samples for 24 hours. After incubation, the culture medium was aspirated, centrifuged at 3000 rpm for 10 min, and the supernatant was aspirated for ELISA experiments to detect the concentration levels of TNF-α, IL-1β and IL-6.

[0141] ELISA 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-stimulated group (p<0.0001). Although the inflammatory response could not be completely eliminated, significant anti-inflammatory efficacy was demonstrated. Among them, the MKTAT and MTAT complex groups showed excellent anti-inflammatory effects, with the MKTAT complex group showing the best effect. Its inflammatory factor inhibition rate was significantly better than that of the MTX complex group, indicating that the difference in peptide sequence is a key factor affecting the efficacy. Figures 19-21 This result confirms that the peptide conjugation strategy can significantly enhance the anti-inflammatory efficacy of nanocomposites.

[0142] 5. Animal in vivo evaluation

[0143] 5.1 Assessment of the degree of joint swelling in rats

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

[0145] Before modeling, all male Wistar rats were acclimatized for 7 days in an SPF-grade environment with a temperature of 25℃±1℃ and a humidity of 50%-60%, with free access to food and water to minimize the interference of environmental factors on the experimental results. Subsequently, 250μL of adjuvant emulsion was injected subcutaneously into the tail of the rats to complete the primary immunization, which was recorded as day 0. Seven days later, the adjuvant emulsion was injected again for immunization enhancement. Continuous observation after modeling showed that from day 10 of the primary immunization, the rats exhibited obvious inflammatory responses, manifested as redness and swelling of the hind limb joints, increased skin temperature, accompanied by lameness and limited mobility, indicating that the CIA model was successfully established.

[0146] Wistar rats weighing approximately 220±20g were randomly divided into 6 groups of 5 rats each. Five rats that did not develop CIA were used as the control group, and 20 rats that successfully developed CIA were used as the drug treatment group. The groupings 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 rats were given the drug once daily from day 10 to day 14 after the first immunization. The dosage was 100 μL (625 μg / mL as MTX). The drug was injected at three points in the synovial tissue inside and outside the joint cavity. The rats' body weight was monitored daily from day 14 to day 19 of the experiment, and the thickness of the right hind limb ankle joint was measured with calipers.

[0148] Experimental results reference Figure 22-23 In the control group, the ankle joint thickness of mice increased by less than 0.5 mm, which was due to the normal weight gain of the rats. On the 10th day after the CIA model was established, the rats began to show obvious inflammatory response. On the 15th day, the ankle joint thickness increased to 9-10 mm and the weight decreased significantly. In the following days, the ankle joint thickness began to decrease slightly and the weight began to increase. In the complex group, the increase in ankle joint thickness was significantly slower than that in the CIA group from 14 to 19 days, and the symptoms began to be relieved. This was especially evident in the MKTAT complex group. On the 18th day, the average ankle joint thickness dropped to below 7 mm and the weight increased further. By the 19th day, the mice had basically recovered and there was no significant difference from the control group. Other experimental groups failed to fully recover and showed significant differences from the control group. MTX showed the best anti-inflammatory effect after being conjugated with the peptide prepared in Example 3.

[0149] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered 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 transmembrane polypeptide KKKKKKYGRKKRRQRRR, whose amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing the methotrexate-cationic polypeptide conjugate as described in claim 1, characterized in that, Includes the following steps: 1) After immobilizing amino-protected arginine onto a resin support via solid-phase synthesis, the amino protecting group is removed. 2) The polypeptide chain was extended by stepwise coupling of amino-protected amino acids to obtain the resin-loaded KKKKKKYGRKKRRQRRR sequence; 3) MTX is activated and then coupled with resin-loaded peptides; 4) Crack the resin to obtain the crude product; 5) After purification, the target conjugate is obtained by freeze-drying.

3. The method for preparing the methotrexate-cationic polypeptide conjugate as described in claim 2, characterized in that, In step 1), DIC / HOBT is used as the condensing agent, and the equivalent ratio of amino-protected arginine, HOBT and DIC is 1:1.5-3:1.5-3. The reaction temperature is 35-45℃ and the reaction time is 3-5h. In step 2), when the polypeptide chain elongation reaction is carried out, DIC / HOBT is used as the condensing agent, and 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℃ and the reaction time is 0.5-2h.

4. The method for preparing the methotrexate-cationic polypeptide conjugate as described in claim 2, characterized in that, In step 3), the equivalence ratio of MTX to peptide is 3:1, the activating agent is HATU or HBTU, the reaction temperature is 25-40℃, and the reaction time is 1-3h.

5. A self-assembled nanocomposite, characterized in that, It comprises the methotrexate-cationic polypeptide conjugate as described in claim 1 and a stabilizer at a mass-volume ratio of 0.02% to 0.5%. The stabilizers are cationic cholesterol and DOPE, and the drug concentration is calculated as methotrexate, with the concentration of the methotrexate-cationic polypeptide conjugate being 500-1500 μg / mL. In preparation, the methotrexate-cationic polypeptide conjugate and stabilizer are dispersed in an aqueous phase and then subjected to high-pressure microfluidic homogenization treatment 1-5 times at a pressure of 5000-20000psi to obtain the complex. The complex has an average particle size of ≤200 nm, a polydispersity index of <0.3, and a zeta potential of ≥30 mV.

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

7. The use of the methotrexate-cationic polypeptide conjugate as described in claim 1 in the preparation of drugs for treating immune-mediated inflammatory diseases, characterized in that, The immune-mediated inflammatory disease mentioned is rheumatoid arthritis.

8. The application as described in claim 7, characterized in that, The drug is a nanocomposite formulation containing the methotrexate-cationic polypeptide conjugate.

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