IL-17A-targeting peptide-modified multifunctional drug-loaded nanoparticles, and preparation method and application thereof

Abstract

The application discloses an IL-17A-targeting peptide modified multifunctional drug-loaded nanoparticle and a preparation method and application thereof. The drug-loaded nanoparticle comprises a complex lipid carrier material and dexamethasone wrapped therein, the complex lipid carrier material is composed of S100 phospholipid, HS 15 and Pal-PEG-Mal, and the hydrophilic end of Pal-PEG-Mal is covalently coupled with a polypeptide molecule targeting IL-17A through maleimide-sulfhydryl. The nanoparticle has a particle size of 80-200 nm, a Zeta potential of-20 to-10 mV, an encapsulation efficiency of no less than 80%, a drug loading amount of no less than 4%, and a decrease in encapsulation efficiency of no more than 5% after storage at 4 DEG C for one month. The application realizes active targeting delivery of inflammatory lesions through the IL-17A-targeting peptide, has a double synergistic effect of IL-17A neutralization and dexamethasone anti-inflammation, significantly reduces systemic toxic side effects, and improves the drug concentration at lesions. The nanoparticle can be used for preparing a drug for treating IL-17A-mediated inflammatory diseases such as rheumatoid arthritis.

Description

Technical Field

[0001] This application relates to the field of compound preparation technology, specifically to a multifunctional drug-loaded nanoparticle modified with an IL-17A-targeting peptide, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of the synovium, destruction of articular cartilage, and erosion of periarticular bone, ultimately leading to severe physical disability. RA patients often experience synovitis, progressive bone destruction, and joint dysfunction. The pathogenesis of RA is not fully understood, but research suggests that its occurrence is related to environmental and genetic factors. This disease often severely impacts patients' lives, significantly shortening their life expectancy compared to the general population; therefore, early prevention and treatment are crucial.

[0003] Dexamethasone (DEX) is a classic glucocorticoid with potent anti-inflammatory and immunosuppressive effects, widely used clinically to treat rheumatoid arthritis, psoriasis, inflammatory bowel disease, and various autoimmune diseases. However, systemic administration of dexamethasone has several limitations: its non-specific distribution leads to significant systemic side effects, and long-term use can cause serious adverse reactions such as osteoporosis, glucose metabolism disorders, and hypothalamic-pituitary-adrenal axis suppression; furthermore, dexamethasone has poor water solubility and limited bioavailability, restricting its application in targeted therapy.

[0004] Liposomes, as a mature drug delivery carrier, can effectively improve the pharmacokinetic properties of drugs, prolong their circulation time in vivo, and accumulate in inflamed or tumor tissues through passive targeting (such as the EPR effect). In recent years, the rise of immunoliposomes (i.e., antibodies or fragments of antibodies conjugated to the surface of liposomes) has provided a new strategy for active targeted drug delivery. Immunoliposomes can achieve precise delivery to lesion sites by recognizing and binding to specific antigens or receptors on the surface of target cells through targeting ligands, thereby significantly improving therapeutic efficacy and reducing systemic toxicity.

[0005] Interleukin-17A (IL-17A) is a key pro-inflammatory cytokine secreted by Th17 cells, playing a central role in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis, psoriasis, and psoriatic arthritis. IL-17A binds to the IL-17 receptor on the surface of target cells, activating downstream signaling pathways such as NF-κB and MAPK, inducing the expression of various pro-inflammatory factors (such as TNF-α, IL-6, and IL-8) and chemokines, thus mediating tissue inflammation and destruction. Studies have shown that IL-17A neutralizing antibodies (such as secukinumab and ixekizumab) have demonstrated excellent clinical efficacy in the treatment of psoriasis and psoriatic arthritis. Therefore, IL-17A and its receptor are important targets for the development of targeted anti-inflammatory drug delivery systems.

[0006] Several patents targeting dexamethasone liposomes have been reported. For example, CN119499179A discloses a dexamethasone liposome targeting synovial fibroblasts, whose lipid bilayer contains DSPE-PEG2000-GPA-KLVFF-48. CN113827738A discloses a sialic acid-modified dexamethasone palmitate liposome. However, no reports have been found of immunoliposomes conjugated with IL-17A targeting peptides as targeting ligands to dexamethasone liposomes. Furthermore, existing liposome formulations still have room for improvement in terms of stability and targeting efficiency, and there is an urgent need to develop a dexamethasone immunoliposome with high stability, good biocompatibility, and excellent targeting properties.

[0007] Compared to therapeutic antibodies, de novo designed peptides or peptide chains offer advantages such as low immunogenicity, low cost, high purity, good tissue penetration, and ease of synthesis and modification. Furthermore, rational design and screening of peptide sequences based on the extracellular structural information of target proteins can yield peptide sequences with high affinity and selectivity, even targeting cryptic epitopes or non-classical binding sites that are difficult for traditional antibodies to recognize, potentially overcoming drug resistance issues caused by epitope mutations in existing antibody drugs. Simultaneously, peptide molecules have relatively simple structures, facilitating chemical modification (such as cyclization, polyethylene glycol conversion, and drug conjugation), and can be flexibly integrated into nanodelivery systems to achieve multifunctional synergistic therapy. Therefore, utilizing IL-17A-targeting peptides to replace traditional antibodies in the construction of active targeted nanodelivery systems holds significant clinical application potential.

[0008] However, achieving a highly stable, highly targeted, and low-immunogenic delivery system by conjugating IL-17A-targeting peptides to dexamethasone liposomes still faces numerous technical challenges. First, selecting suitable lipid carrier materials that ensure good biocompatibility while also possessing high encapsulation efficiency, high drug loading capacity, and excellent storage stability is a pressing technical problem. Second, stably and efficiently coupling the IL-17A-targeting peptide to the liposome surface, ensuring its targeting activity remains unaffected, and avoiding increased immunogenicity and shortened in vivo circulation time due to the introduction of the targeting ligand is another significant challenge for those skilled in the art. Furthermore, existing liposome technologies often use large molecules such as antibodies or hyaluronic acid as targeting ligands, which suffer from poor tissue penetration, non-specific distribution, or unstable coupling, failing to meet the needs of precise targeted therapy. Therefore, developing an IL-17A-targeting dexamethasone immunoliposome that simultaneously possesses high stability, high targeting, deep tissue penetration, and long-lasting circulation characteristics is of significant clinical importance and urgently needed for the precise treatment of inflammatory diseases such as rheumatoid arthritis. Summary of the Invention

[0009] In view of this, the purpose of this application is to provide a multifunctional drug-loaded nanoparticle modified with IL-17A targeting peptide, its preparation method and application. This multifunctional drug-loaded nanoparticle uses amphiphilic phospholipid S100, polyethylene glycol 15-hydroxystearate HS15, and maleimide-modified amphiphilic polymer Pal-PEG-Mal as a composite lipid carrier material. It is co-assembled with dexamethasone to form liposome nanoparticles. A stable thioether bond is formed between the maleimide group at the end of Pal-PEG-Mal and the thiol group at the end of the IL-17A targeting peptide, covalently coupling the IL-17A targeting peptide to the liposome surface. This results in nanoparticles possessing high encapsulation efficiency, high drug loading capacity, excellent storage stability, and specific active targeting capability for IL-17A highly expressed at inflammatory lesions. Simultaneously, the nanoparticles achieve synergistic treatment through the anti-inflammatory effect of dexamethasone and the neutralizing and blocking effect of the IL-17A targeting peptide on pro-inflammatory factors. This significantly reduces the systemic toxicity of dexamethasone and increases drug concentration at the lesion site, providing a stable, efficient, and precise nanodelivery treatment solution for IL-17A-mediated inflammatory diseases such as rheumatoid arthritis.

[0010] To achieve the above objectives, this application provides the following technical solution:

[0011] In a first aspect, this application provides a multifunctional drug-loaded nanoparticle modified with an IL-17A-targeting peptide. The multifunctional drug-loaded nanoparticle comprises a composite lipid carrier material and a lipophilic drug encapsulated therein. The composite lipid carrier material is composed of S100 phospholipid, HS 15 (polyethylene glycol 15-hydroxystearate), and an amphiphilic polymer Pal-PEG-Mal (palmitic acid-polyethylene glycol-maleimide) modified with maleimide. The hydrophilic end of the Pal-PEG-Mal is modified with a polypeptide molecule targeting IL-17A. The polypeptide molecule targeting IL-17A reacts with the maleimide group at the hydrophilic end of the Pal-PEG-Mal through its terminal thiol group to form a thioether bond, thereby covalently coupling it to the surface of the nanoparticle.

[0012] The multifunctional drug-loaded nanoparticles described in this application, namely liposomes, refer to vesicle-like microparticles composed of lipids assembled in a membrane form and an internal aqueous phase (see D.D. Lasic, "Liposomes: From Basics to Applications," Elsevier Science Publishers, pp. 1-171, 1933). Liposomes typically contain an internal aqueous phase surrounded by a lipid membrane, but the liposomes described in this application also include lipid aggregates without an internal aqueous phase. This application does not impose any particular restrictions on the structure of the liposomes; they can be multilayered or monolayered liposomes, preferably monolayered liposomes with uniform particle size, which are more conducive to stable drug encapsulation and in vivo delivery.

[0013] Although there are no particular limitations on the size of the liposomes in this application, the average vesicle diameter is approximately 10 to 5000 nm, preferably approximately 50 to 500 nm, and more preferably 80-200 nm, and even more preferably 100-150 nm. The average vesicle diameter of the liposomes can be determined according to the principle of kinetic light scattering.

[0014] The S100 phospholipids described in this application can enhance lipid membrane stability and synergistically improve inflammatory tropism. As a typical amphiphilic phospholipid, its hydrophilic phosphocholine head and hydrophobic fatty acid chain structure can spontaneously assemble into a uniform and dense lipid bilayer in an aqueous phase, which is beneficial for obtaining liposomes with uniform particle size, high encapsulation efficiency, and good stability. Its composition is highly similar to that of biological membranes, and it has low immunogenicity.

[0015] HS 15 is pharmaceutical-grade polyethylene glycol 15-hydroxystearate, which improves membrane homogeneity, drug solubilization, and formulation stability. HS15 and S100 phospholipids have a synergistic membrane stabilizing effect. S100 provides the framework structure for forming the lipid bilayer, while HS15 embeds itself into the lipid bilayer through its hydrophobic hydroxystearic acid tail chain, interacting hydrophobically with phospholipid molecules to increase membrane order and rigidity. Simultaneously, the hydrophilic PEG chain in the HS 15 molecule extends to the liposome surface, forming a dense hydration layer that prevents aggregation and fusion between liposome particles through steric hindrance, thereby significantly improving the physical stability of liposomes during storage and in vivo circulation. The structure of HS 15 is shown below:

[0016] The average number of additions to ethylene oxide is n=15.

[0017] This application modifies the hydrophilic end of an amphiphilic polymer with a polypeptide molecule that targets arthritis lesions. After self-assembly, several polypeptide molecules are modified onto the surface of nanoparticles, endowing the nanoparticles with the ability to specifically target and bridge lesions, thereby enhancing the blocking of the pro-inflammatory factor IL 17A in lesions.

[0018] Preferably, the hydrophilic end of the amphiphilic polymer is modified with maleimide and then connected to the hydrophilic end of the amphiphilic polymer via a thiol group.

[0019] Preferably, the modified amphiphilic polymer is palmitic acid-polyethylene glycol-maleimide (Pal-PEG-Mal). Pal-PEG has a clear and controllable molecular structure, which can be functionally modified using chemical reactions. The palmitoyl group is inserted into the lipid bilayer, the PEG chain is extended for in vivo circulation, and the maleimide terminus is used for site-specific protein coupling, serving as a backbone for peptide display. Its structure is shown in the following formula:

[0020]

[0021] The essence of this reaction is that the sulfur atom of the free thiol group of the protein launches a nucleophilic attack on the electron-deficient double-bonded carbon in the maleimide ring, causing the maleimide ring to open and form a stable thioether bond (C–S), and the product is a thiosuccinimide derivative.

[0022] This application innovatively introduces HS 15 and Pal-PEG-Mal into a liposome system, forming a dual PEG-modified system. The HS 15 molecule itself carries a PEG chain (approximately 15 ethylene glycol units), while Pal-PEG-Mal carries a longer PEG2000 chain. The long and short PEG chains form a gradient hydration layer on the liposome surface, which can minimize serum protein adsorption and recognition and uptake by the mononuclear phagocyte system (MPS), significantly prolonging the half-life of liposomes in blood circulation. Simultaneously, the combination of long and short PEG chains provides a hydrophilic and interference-free reaction interface for the coupling of maleimide and the IL-17A targeting peptide, avoiding the reduction in coupling efficiency due to excessive steric hindrance of the long PEG chain.

[0023] In some embodiments, the mass ratio of S100 phospholipid, HS 15 and Pal-PEG-Mal in the composite lipid carrier material is 3~8:1~3:2~4.

[0024] In a preferred embodiment, the mass ratio of S100 phospholipid, HS 15 and Pal-PEG-Mal in the composite lipid carrier material is 4~6∶1~2∶2~3.

[0025] In a more preferred embodiment, the mass ratio of S100 phospholipid, HS 15 and Pal-PEG-Mal in the composite lipid carrier material is 5:1:2.5.

[0026] In some embodiments, the lipophilic drug is dexamethasone, and the mass ratio of dexamethasone to the composite lipid carrier material is 1:5 to 40.

[0027] In a preferred embodiment, the lipophilic drug is dexamethasone, and the mass ratio of dexamethasone to the composite lipid carrier material is 1:10~30.

[0028] In a more preferred embodiment, the lipophilic drug is dexamethasone, and the mass ratio of dexamethasone to the composite lipid carrier material is 1:20.

[0029] In some preferred embodiments, the amino acid sequence of the polypeptide molecule targeting IL-17A is selected from any one or more sequences shown in SEQ ID NO:1~14.

[0030] In some embodiments, the molecular weight of polyethylene glycol (PEG) in the Pal-PEG-Mal is 2000~6000 Da.

[0031] In a preferred embodiment, the molecular weight of polyethylene glycol (PEG) in the Pal-PEG-Mal is 2000 Da.

[0032] The aforementioned multifunctional drug-loaded nanoparticles are spherical nanovesicles with the following characteristics: the preferred average particle size is 80~200 nm; the zeta potential is -20~-10 mV; the encapsulation efficiency is ≥80% and the drug loading is ≥4%; after being stored at 4 ℃ in the dark for 1 month, the encapsulation efficiency decreases by ≤5% and the drug leakage rate is ≤3%.

[0033] Secondly, this application provides a method for preparing the IL-17A-targeting peptide-modified multifunctional drug-loaded nanoparticles described in the first aspect, comprising the following steps:

[0034] (1) S100 phospholipid, HS 15 and Pal-PEG-Mal and the lipophilic drug dexamethasone are dissolved together in an organic solvent, the organic solvent is removed to form a lipid film, a hydration medium is added to the lipid film for hydration treatment, ultrasonication and filtration are performed to obtain micelle nanoparticles loaded with dexamethasone.

[0035] (2) The peptide molecule targeting IL-17A is treated with a reducing agent to expose the thiol group. Then the reduced peptide molecule targeting IL-17A is mixed with the micellar nanoparticles described in step (1) so that the thiol group at the end of the peptide molecule targeting IL-17A is coupled with the maleimide group at the hydrophilic end of the Pal-PEG-Mal. After purification, the multifunctional drug-loaded nanoparticles modified with the IL-17A targeting peptide are obtained.

[0036] In some implementations, step (1) may further include one or more of the following conditions:

[0037] (a) The mass ratio of the S100 phospholipid, HS 15, Pal-PEG-Mal to dexamethasone is 1.5:0.6:3:0.25;

[0038] (b) The organic solvent is methanol, acetonitrile, or chloroform;

[0039] (c) The method for removing the organic solvent is rotary evaporation under reduced pressure at 30~50 °C until a uniform film is formed;

[0040] (d) The hydration medium is a 5% glucose solution, or it can be an aqueous solution of hydrochloric acid, an aqueous solution of sodium hydroxide, or a phosphate buffer solution, with a pH of 2 to 8;

[0041] (e) The hydration treatment time is 2~10 min;

[0042] (f) The ultrasound conditions are 20~30 ℃, 20~100 W, and 2~10 min.

[0043] In some implementations, one or more of the following conditions are met in step (2):

[0044] (a) The reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and the molar ratio of the reducing agent to the polypeptide molecule is 2 to 10:1;

[0045] (b) The reduction treatment is performed at a temperature of 4~25 °C for a time of 0.25~2 h;

[0046] (c) The terminal end of the polypeptide molecule targeting IL-17A is a cysteine ​​residue;

[0047] (d) The concentration of maleimide groups in the micelle nanoparticles is 0.07~4 mmol / L, and the molar ratio of the micelle nanoparticles to the polypeptide molecules is 1:5~5:1.

[0048] (e) The coupling reaction is carried out at a temperature of 4 to 25 °C for 2 to 24 h.

[0049] Thirdly, this application provides a pharmaceutical composition comprising the multifunctional drug-loaded nanoparticles described in the first aspect and a pharmaceutically acceptable carrier or excipient.

[0050] In some embodiments, the pharmaceutical composition is an injectable preparation that can be administered intravenously, subcutaneously, or intra-articularly.

[0051] Fourthly, this application provides the use of the multifunctional drug-loaded nanoparticles described in the first aspect or the multifunctional drug-loaded nanoparticles prepared by the preparation method described in the second aspect in the preparation of drugs for treating IL-17A-mediated inflammatory diseases.

[0052] Compared with the prior art, this application has at least the following advantages:

[0053] 1. This application employs S100 phospholipid, HS 15, and Pal-PEG-Mal synergistically as a composite lipid carrier material. S100 phospholipid provides the skeletal structure for forming a lipid bilayer, enhancing the stability of the liposome membrane. HS 15, through its hydrophobic tail chain embedding into the lipid bilayer, increases the membrane's order and rigidity, while its hydrophilic PEG chains extend to the liposome surface to form a dense hydration layer, preventing particle aggregation through steric hindrance. The PEG chains in Pal-PEG-Mal further prolong the in vivo circulation time. The synergistic effect of these three components results in liposomes that, after storage at 4 °C for one month, exhibit an encapsulation rate decrease of ≤5% and a drug leakage rate of ≤3%, significantly superior to existing dexamethasone liposome formulations. Furthermore, the encapsulation rate can reach over 80%, and the drug loading can reach over 4%, effectively solving the problems of poor stability, easy degradation, and low bioavailability of dexamethasone.

[0054] 2. This application utilizes a specific coupling reaction between the maleimide group at the hydrophilic end of the Pal-PEG-Mal peptide and the terminal thiol group of the IL-17A targeting peptide to form a stable thioether bond, firmly modifying the IL-17A targeting peptide onto the liposome surface with a coupling efficiency exceeding 41%. This IL-17A targeting peptide can specifically recognize and bind to IL-17A highly expressed at inflammatory lesions, achieving active and precise targeted delivery to lesions of inflammatory diseases such as rheumatoid arthritis. Simultaneously, the high expression of S100 phospholipids at inflammatory sites further enhances the inflammatory tropism of the liposomes, achieving a dual targeting effect. Compared to the HA-CD44 strategy, which suffers from non-specific uptake due to the widespread expression of CD44 receptors in normal tissues, this application targets IL-17A, a factor highly enriched at inflammatory sites but almost not expressed in normal tissues, resulting in higher target specificity. This significantly reduces drug distribution in normal tissues and lowers the systemic toxicity of dexamethasone.

[0055] 3. The liposomes of this application possess dual functions of IL-17A neutralization and dexamethasone delivery: on the one hand, they specifically neutralize and block the IL-17A-mediated pro-inflammatory signaling pathway through the IL-17A-targeting peptide, inhibiting the expression of downstream pro-inflammatory factors such as TNF-α, IL-6, and IL-8 at the source; on the other hand, they inhibit the inflammatory response downstream through the classic anti-inflammatory and immunosuppressive effects of dexamethasone. The synergistic intervention of both achieves a therapeutic effect of "1+1>2". Compared to dexamethasone-only formulations or ordinary liposomes, this application can significantly enhance the anti-inflammatory effect and reduce the dosage of dexamethasone, further reducing toxic side effects. Furthermore, the small molecular weight of the IL-17A-targeting peptide used in this application endows the liposomes with excellent deep tissue penetration ability, allowing them to penetrate deep into the synovial membrane and cartilage tissue of inflamed joints, overcoming the shortcomings of large molecular weight ligands such as HA, which mainly remain on the tissue surface and are difficult to deliver deeply.

[0056] 4. The preparation method of this application employs a conventional thin-film hydration method combined with thiol-maleimide site-directed coupling technology. This process is mature and simple to operate, involving only conventional unit operations such as rotary evaporation, ultrasonication, and ultrafiltration, requiring no special equipment and resulting in low production costs. Furthermore, the maleimide-thiol covalent coupling reaction conditions are mild, having minimal impact on peptide activity, and exhibiting high coupling efficiency. The resulting thioether bonds are irreversible, maintaining targeting function for a prolonged period in blood circulation, thus avoiding the defects of HA-modified liposomes, such as dependence on non-covalent anchoring or hydrophobic insertion, and susceptibility to degradation by hyaluronidase in vivo. This method has good reproducibility, is suitable for large-scale industrial production, and has significant industrial application prospects. Attached Figure Description

[0057] Figure 1 The particle size distribution of the nanoparticles prepared in Example 1 that were not modified with the IL 17A targeting peptide is shown.

[0058] Figure 2 Zeta potential diagrams for NPs and DEX-BNPs nanoparticles.

[0059] Figure 3 The particle size distribution of the multifunctional drug-loaded nanoparticles modified with the IL 17A targeting peptide prepared in Example 3 is shown in Figure 3.

[0060] Figure 4 The figure represents the protein linkage rate of IL 17A targeting peptide and Pal-PEG-Mal at different molar ratios in Example 3.

[0061] Figure 5 The results of the molecular docking simulation between the IL-17A targeting peptide and IL-17A in Example 4 are shown.

[0062] Figure 6 This is a flow cytometry image of different IL 17A targeting peptides displayed on the surface of yeast in Example 4.

[0063] Figure 7 The images show transmission electron microscopy (TEM) images of NPs and DEX-BNPs nanoparticles, with NPs on the left and DEX-BNPs on the right.

[0064] Figure 8 The graphs show the stability of nanoparticles measured in Example 5; the left graph shows the particle size variation, and the right graph shows the zeta potential variation.

[0065] Figure 9 The diagram shows the encapsulation efficiency and drug loading of nanoparticles in Example 6.

[0066] Figure 10 The MTT plots of RAW.254.7 cytotoxicity of DEX-Sol and DEX-NPs at different drug concentrations in Example 8 are shown; the left plot shows the drug cytotoxicity to normal RAW 264.7 cells, and the right plot shows the drug cytotoxicity to RAW 264.7 cells after LPS stimulation.

[0067] Figure 11 This is a cellular uptake diagram of DID-Sol, DID-NPs, and DID-BNPs in Example 9.

[0068] Figure 12 The results of the in vitro anti-inflammatory experiment of DEX-Sol, DEX-NPs, and DEX-BNPs in RAW 264.7 cells by ELISA are shown in Example 10; the left figure shows the TNF-α level and the right figure shows the IL-6 level.

[0069] Figure 13 This is a trend graph showing the distribution of DiD-Sol, DiD-NPs, and DiD-BNPs carrying fluorescent molecules in rats with rheumatoid arthritis over time, as described in Example 12.

[0070] Figure 14 This is a fluorescence intensity image of the heart, liver, spleen, lungs, kidneys, and soles of the feet taken at 48 hours in Example 12. Detailed Implementation

[0071] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0072] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0073] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0074] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following explanations of terminology are provided to better understand this application.

[0076] 1. S100 phospholipids: In this application, it refers to high-purity soybean lecithin with a phosphatidylcholine content of ≥94%. Its molecular structure contains a hydrophilic phosphocholine head and a hydrophobic fatty acid chain tail. In the aqueous phase, it can spontaneously assemble to form a lipid bilayer structure, which is the main component of the liposome membrane skeleton.

[0077] 2. HS 15: In this application, HS 15 is a pharmaceutical-grade nonionic surfactant with the chemical name polyethylene glycol 15-hydroxystearate (CAS No. 70142-34-6). The HS 15 molecule contains hydrophobic hydroxystearic acid chains and hydrophilic polyethylene glycol chains (approximately 15 ethylene glycol units), which can be embedded in the lipid bilayer to improve the stability and rigidity of the membrane. At the same time, its PEG chains form a hydration layer on the surface of liposomes, preventing particle aggregation through steric hindrance.

[0078] 3. Pal-PEG-Mal: Palmitic acid-polyethylene glycol-maleimide. This molecule consists of three parts: a hydrophobic palmitoyl group (intercalated into the lipid bilayer as an anchoring structure), a hydrophilic polyethylene glycol chain, and a terminal maleimide group (used for site-directed coupling reactions with thiol-containing peptides). Pal-PEG-Mal possesses three major functions: liposome membrane anchoring, prolonging in vivo circulation, and targeted ligand coupling.

[0079] 4. Composite Lipid Carrier Material: In this application, it refers to a liposome membrane material composition formed by mixing S100 phospholipid, HS15, and Pal-PEG-Mal in a certain proportion. The three components work synergistically: S100 provides the lipid bilayer framework, HS15 enhances membrane stability and forms a short PEG hydration layer, and Pal-PEG-Mal provides a long PEG hydration layer and targeting peptide coupling sites.

[0080] The main raw materials used in the embodiments of this application are described as follows:

[0081] S100 phospholipids: High-purity soybean lecithin (Lipoid S100, phosphatidylcholine content ≥94%, CAS No. 97281-47-5), purchased from Aladdin Company.

[0082] HS 15: Polyethylene glycol 15-hydroxystearate (CAS No. 70142-34-6-10), pharmaceutical grade nonionic surfactant, purchased from Meilun Biotechnology Co., Ltd.

[0083] Pal-PEG-Mal: Palmitic acid-polyethylene glycol-maleimide (palmitic acid-PEG-maleimide), wherein the polyethylene glycol has a molecular weight of 2000 Da and was purchased from Aladdin Company.

[0084] Dexamethasone: CAS No. 50-02-2, purchased from Yuanye Company.

[0085] Tris(2-carboxyethyl)phosphonic hydrochloride (TCEP): CAS No. 51805-45-9, purchased from Aladdin Company.

[0086] IL-17A targeting peptide: The amino acid sequence is shown in Table 1.

[0087] Table 1. List of IL-17A target peptide sequences

[0088]

[0089] Unless otherwise specified, all reagents used in the following examples are pharmaceutical grade or analytical grade and were purchased from conventional commercial channels.

[0090] Example 1: Preparation of blank nanoparticles (NPs)

[0091] This embodiment provides a method for preparing functional nanoparticles (NPs). The specific steps are as follows:

[0092] S100 phospholipid, HS 15, and Pal-PEG-Mal were weighed and dissolved in methanol to obtain S100 phospholipid solution, HS 15 solution, and Pal-PEG-Mal solution with a final concentration of 5 mg / mL. 600 μL of S100 phospholipid solution, 300 μL of Pal-PEG-Mal solution, and 120 μL of HS 15 solution were added to a 10 mL round-bottom flask and rotary evaporated under reduced pressure at 37 °C for 3 min to form a uniform polymer film on the inner wall of the flask. 2 mL of 5% glucose solution was added, and the film was detached by shaking at room temperature. The mixture was then sonicated at 28 °C for 4 min at a power of 100 W. The solution was filtered through a 0.45 μm filter to obtain a homogeneous NPs dispersion.

[0093] In the above embodiments, the mass ratio of S100, HS 15 and Pal-PEG-Mal can also be other ratios within the range of 3~8:1~3:2~4.

[0094] Results: The nanoparticles prepared in this embodiment were analyzed using a Malvern particle size analyzer, such as... Figure 1 As shown, the nanoparticles have a particle size of 80–160 nm and a particle size distribution (PDI) of less than 0.3; Figure 2 As shown, the nanoparticles have a negatively charged surface (Zeta potential of approximately -15±2 mV), resulting in satisfactory performance.

[0095] Example 2: Preparation of Dexamethasone-loaded Nanoparticles (DEX-NPs)

[0096] S100 phospholipid solution (5 mg / mL), HS 15 solution (5 mg / mL) and Pal-PEG-Mal solution (5 mg / mL) were prepared according to the method in Example 1. A 2 mg / mL dexamethasone (DEX) solution was also prepared.

[0097] 600 μL of S100 phospholipid solution, 300 μL of Pal-PEG-Mal solution, 120 μL of HS 15 solution, and 125 μL of DEX solution (2 mg / mL) were added to a 10 mL round-bottom flask (S100:HS 15:Pal-PEG-Mal:DEX mass ratio of 1.5:0.6:3:0.25). The mixture was rotary evaporated under reduced pressure at 37 °C for 3 min to form a uniform polymer film on the inner wall of the flask. 2 mL of 5% glucose solution was added, and the film was detached by shaking at room temperature. The mixture was then sonicated at 28 °C for 4 min at a power of 100 W. The solution was filtered through a 0.45 μm filter to obtain a homogeneous DEX-NPs dispersion.

[0098] In the above embodiments, the mass ratio of DEX to the composite lipid material (S100+HS 15+Pal-PEG-Mal) can also be other ratios in the range of 1:5 to 40, which will not have a significant impact on the structure of the nanoparticles, but may affect the encapsulation efficiency and drug loading.

[0099] Example 3 Preparation of drug-loaded nanoparticles modified with IL-17A targeting peptide (DEX-BNPs)

[0100] This embodiment provides a method for preparing multifunctional drug-loaded nanoparticles modified with DEX-loaded IL-17A targeting peptides (DEX-BNPs). The specific steps are as follows:

[0101] (1) Peptide reduction treatment

[0102] Tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP), a reducing agent, was added to a solution of IL-17A targeting peptide (sequence selected from Table 1, terminally cysteine). The solution was incubated at room temperature (4–25 °C) in the dark for 0.25–2 h to reduce the disulfide bonds in the IL-17A targeting peptide and expose the thiol groups. The molar ratio of reducing agent to peptide molecules was 2–10:1.

[0103] (2) Coupling reaction

[0104] The reduced target peptide solution was slowly added dropwise to the DEX-NPs dispersion prepared in Example 2 (the concentration of maleimide groups of Pal-PEG-Mal in DEX-NPs was 0.07~4 mmol / L). The molar ratio of DEX-NPs to peptide molecules was 1:5~5:1. The mixture was incubated at 4~25℃ in the dark for 2~24 h to allow the maleimide groups of Pal-PEG-Mal to specifically react with the thiol groups at the end of the target peptide to form stable thioether bonds.

[0105] (3) Purification

[0106] After incubation, uncoupled free IL-17A targeting peptides and reducing agents were removed by ultrafiltration. Ultrafiltration centrifuge tubes (molecular weight cutoff 100 kDa) were used at 3000–5000 rpm for 10–15 min, repeated 2–3 times to obtain a DEX-BNPs dispersion.

[0107] like Figure 3 As shown, the particle size of the drug-loaded nanoparticles modified with IL-17A targeting peptide (DEX-BNPs) is 120±15 nm, and the particle size distribution PDI is less than 0.3, which is basically consistent with the particle size of NPs.

[0108] In the coupling reaction, the amount of peptide added directly affects its coupling density on the nanoparticle surface, and thus affects the targeting efficiency of the nanoparticles. In this embodiment, the molar ratio of DEX-NPs to peptide molecules is 1:5 to 5:1, which can be adjusted within the above range according to the actual required targeting ligand density.

[0109] Connectivity determination: Under the condition of fixed DEX-NPs concentration, IL-17A targeting peptides with different molar ratios were coupled with Pal-PEG-Mal. After the reaction, the content of uncoupled free peptides was determined by BCA method, and the protein connectivity was calculated.

[0110] Result: As Figure 4 As shown, the connection rate between the IL-17A targeting peptide and Pal-PEG-Mal can reach over 41%, indicating that the maleimide-thiol covalent coupling reaction is efficient and reliable.

[0111] Multifunctional drug-loaded nanoparticle dispersions modified with DEX-loaded IL-17A targeting peptides 1, 2, 3, 4, and 5 (Table 1) were prepared according to the preparation method of multifunctional drug-loaded nanoparticles modified with DEX-loaded IL-17A targeting peptide 1.

[0112] Example 4: Verification of the affinity between IL-17A targeting peptide and IL-17A

[0113] The molecular docking simulation results of IL-17A targeting peptide and IL-17A are as follows: Figure 5 As shown, the targeting peptides can form a stable binding conformation with the extracellular region of IL-17A. This embodiment further verifies the binding ability of different IL-17A targeting peptides to IL-17A protein through yeast surface display experiments.

[0114] The purified His-tagged IL-17A protein was expressed using E. coli. Yeast vector plasmids displaying the target peptides IL-17A1, 2, 3, 4, and 5 were constructed and transformed into competent yeast cells using lithium acetate conversion. Protein expression was induced using induction medium.

[0115] Yeast cells displaying the target peptides IL-17A1, 2, 3, 4, and 5 on their cell surface were incubated with 1 μM IL-17A for 20 min at room temperature. After washing away unbound receptors, the cells were labeled with Anti-His and Anti-Flag dual fluorescent antibodies, and the fluorescence intensity was detected by flow cytometry.

[0116] Result: As Figure 6 As shown, compared with the blank control yeast cells, significant fluorescence signals were detected in each group of yeast cells displaying target peptide sequences 1-5, indicating that these 5 target peptides have good binding ability with IL-17A.

[0117] Example 5: Characterization of the overall performance of liposomes

[0118] (1) Morphological observation

[0119] The morphology of NPs, DEX-NPs and DEX-BNPs was observed using transmission electron microscopy.

[0120] (2) Particle size and zeta potential detection

[0121] The foregoing examples have shown the results of using dynamic light scattering (Malvin particle size analyzer) to detect particle size and zeta potential.

[0122] (3) Encapsulation efficiency and drug loading detection

[0123] Encapsulation efficiency and drug loading were determined using high performance liquid chromatography (HPLC).

[0124] (4) Storage stability test

[0125] Liposomes were stored at 4°C in the dark for one month, and the encapsulation efficiency and drug leakage rate were tested.

[0126] The results show:

[0127] Morphology: Transmission electron microscopy results of NPs and DEX-BNPs are as follows Figure 7 As shown, the liposomes are spherical, uniform in shape, and show no obvious aggregation.

[0128] Particle size: The average particle size of DEX-BNPs was 120±15 nm, which was basically consistent with that of NPs, indicating that drug loading and targeting peptide conjugation did not cause significant particle aggregation.

[0129] Zeta potential: Both NPs and DEX-BNPs are negatively charged, and the zeta potential is approximately -15 ± 2 mV (e.g., Figure 2 (as shown)

[0130] Encapsulation efficiency: 82.3%, drug loading: 4.1%;

[0131] Stability: such as Figure 8 As shown, after storage at 4 ℃ in the dark for 1 month, the encapsulation rate was 78.1%, a decrease of 4.2%, and the drug leakage rate was 2.5%, demonstrating excellent stability.

[0132] Example 6: Determination of Encapsulation Efficiency and Drug Loading

[0133] A DEX-loaded multifunctional drug-loaded nanoparticle solution with a DEX concentration of 0.125 mg / mL was prepared. 2 mL of this solution was precisely transferred, and the free drug was separated by ultrafiltration centrifugation (molecular weight cutoff 100 kDa, 5000–8000 r / min, centrifugation 10–15 min).

[0134] The content of dexamethasone was determined by high performance liquid chromatography (HPLC). Chromatographic conditions: reversed-phase C18 column (250 mm × 4.6 mm × 5 μm), mobile phase: water-acetonitrile (60:40), detection wavelength: 242 nm, flow rate: 1 mL / min, injection volume: 20 μL.

[0135] Based on the standard curve plotted from the dexamethasone standard and the peak area detected in the sample, the concentration of dexamethasone in the sample was calculated, and the encapsulation efficiency (encapsulation efficiency = drug loading / total drug dosage × 100%) and drug loading (drug loading = drug loading / total mass of liposomes × 100%) were calculated.

[0136] Result: As Figure 9 As shown, the encapsulation efficiency reached 84.27% ± 2.76%, and the drug loading reached 4.24% ± 0.14% or higher. The conjugation of the IL-17A targeting peptide had no significant effect on the encapsulation efficiency, indicating that the conjugation reaction occurred on the liposome surface and did not affect the integrity of the drug encapsulation within the liposomes.

[0137] Example 7 Cell Culture

[0138] The macrophage RAW 264.7 was derived from the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0139] RAW 264.7 cells were cultured in high-glucose DMEM medium containing 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM D-glutamine, and 10% fetal bovine serum, and maintained at 37 °C and 5% CO2. Cells were passaged by pipetting after reaching 80% confluence.

[0140] Example 8 Cytotoxicity Detection (MTT Assay)

[0141] RAW 264.7 cells in logarithmic growth phase were seeded at an appropriate density into 96-well plates and incubated at 37 °C in a 5% CO2 incubator for 24 hours. After 24 hours, the old culture medium in the 96-well plates was carefully aspirated, and the plates were washed once with PBS. The following experimental groups were set up (each group had 3 replicates):

[0142] Blank control group: only 100 μL of serum-free DMEM medium containing 0.1% DMSO was added (solvent control);

[0143] Free dexamethasone group (DEX-Sol): 100 μL of serum-free DMEM medium containing different concentrations of free dexamethasone was added, with the dexamethasone concentration gradient set at 0.2, 0.4, 1, 2, 3, 4, and 5 μg / mL;

[0144] Dexamethasone liposome group (DEX-NPs): 100 μL of dexamethasone liposome suspension containing different concentrations of dexamethasone was added, with the dexamethasone concentration gradient being consistent with that of the free group (0.2, 0.4, 1, 2, 3, 4, 5 μg / mL).

[0145] All experimental groups were prepared with serum-free DMEM medium to eliminate interference from serum proteins on liposome-cell interactions. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0146] MTT testing:

[0147] After 24 hours of drug administration, add 20 μL of MTT reagent (5 mg / mL, prepared with PBS) to each well and incubate for another 4 hours in a 37 ℃, 5% CO2 incubator in the dark. After 4 hours, carefully aspirate the supernatant from the wells (avoiding the purple formazan crystals at the bottom of the wells), add 150 μL of DMSO to each well, and shake on a shaker at low speed for 15 minutes to completely dissolve the formazan crystals. After the crystals have fully dissolved, measure the absorbance (OD value) of each well at 570 nm using a full-wavelength microplate reader, using 650 nm as the reference wavelength, and record the final detection results.

[0148] Result: As Figure 10As shown, within the concentration range of 0.2–5 μg / mL, the survival rate of normal RAW264.7 cells in both the DEX-Sol and DEX-NPs groups decreased in a concentration-dependent manner. At the same drug concentration, the cell survival rate in the DEX-NPs group was slightly higher than that in the DEX-Sol group, indicating that liposome encapsulation can reduce the direct cytotoxicity of dexamethasone to macrophages and has good biocompatibility. However, for LPS-stimulated RAW 264.7 cells, there was no significant difference in cell survival rate with increasing drug concentration, suggesting that for RAW 264.7 cells in an inflammatory state, the inhibitory effect of the drug on cell proliferation is transformed into a therapeutic effect on inflammation.

[0149] Example 9 Cell Uptake Experiment

[0150] Principle: RAW 264.7 cells have IL-17A receptors on their surface. Adding 500 ng / mL of IL-17A to the culture medium simulates the IL-17A-rich environment of lesions. Liposomes with IL-17A targeting peptides first bind to IL-17A, and then are targeted to the cell surface by IL-17A.

[0151] Preparation of fluorescently labeled liposomes: Following the liposome preparation method in Example 3, fluorescent substance DiD was added during dissolution in an organic solvent to prepare fluorescently labeled DiD-NPs and DiD-BNPs respectively.

[0152] Experimental procedure: When the RAW 264.7 cells were in good condition and the density of growth reached about 80%, the cell suspension was collected by centrifugation (900 rpm, 3 min). The cells were then centrifuged at 2.5 × 10⁻⁶ cells / min. 5 Cells were seeded at a density of 500 μL of cell suspension per well in 24-well plates. After 24 h of cell adhesion, an inflammation model was established by stimulating the cells with LPS. After successful stimulation, 500 μL of DiD-Sol, DiD-NPs, and DiD-BNPs at a concentration of 0.4 μg / mL were added, respectively.

[0153] After drug administration, the cell culture plate was placed in an incubator and incubated for 4 hours. After the incubation period, the cells were washed three times with PBS solution to remove residual DiD on the cell surface. 300 μL of PBS solution was added to each well, and the cells were gently dispersed by blowing on the cell culture plate wall. The cells were collected in centrifuge tubes, filtered through a 40 μm cell sieve, and then added to sample loading tubes. Finally, the fluorescence intensity of cell uptake was obtained using flow cytometry.

[0154] Result: As Figure 11As shown, the mean fluorescence intensity of cells in the DiD-BNPs group was significantly higher than that in the DiD-NPs and DiD-Sol groups (p<0.01), indicating that the modification with the IL-17A targeting peptide significantly enhanced the uptake of liposomes by macrophages. The cellular uptake efficiency of the DiD-NPs group was also higher than that of the DiD-Sol group, which may be related to the passive endocytosis mechanism of the nanoparticles. These results confirm that the modification with the IL-17A targeting peptide endows liposomes with the ability to actively target macrophages.

[0155] Example 10: In vitro anti-inflammatory activity assay (ELISA method)

[0156] RAW 264.7 cells in logarithmic growth phase were seeded in 24-well plates and incubated overnight at 37 °C in a 5% CO2 incubator. The next day, the culture medium in the wells was aspirated, and fresh medium containing LPS was added. The cells were incubated for 4 h to establish an inflammation model. The culture medium in the wells was aspirated again, and fresh medium solutions containing DEX-Sol, DEX-NPs, and DEX-BNPs were added according to the groups, respectively. The cells were incubated together for 24 h. The culture supernatant was collected from the wells.

[0157] The concentrations of IL-6 and TNF-α in cell supernatant were determined using an ELISA kit, strictly following the manufacturer's instructions. A brief summary of the steps is as follows:

[0158] (1) Sample addition: After washing, add 100 μL of standard or appropriately diluted cell supernatant to each well, seal the plate and incubate at room temperature for 2 hours.

[0159] (2) Add detection antibody: After washing, add 100 μL of biotin-labeled detection antibody working solution to each well and incubate at room temperature for 1 hour.

[0160] (3) Add enzyme conjugate: After washing, add 100 μL of horseradish peroxidase-labeled streptavidin working solution to each well and incubate at room temperature in the dark for 30 minutes.

[0161] (4) Color development: After washing, add 100 μL of TMB color development substrate solution to each well and incubate at room temperature in the dark for 15-20 minutes. After a clear blue gradient appears in the standard wells, add 50 μL of stop solution (2M H2SO4) to each well to terminate the reaction.

[0162] (5) Detection: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a full-wavelength microplate reader, and 570 nm was used as the reference wavelength for calibration.

[0163] (6) Quantitative calculation: Plot a standard curve with the standard concentration as the abscissa and the OD value as the ordinate, and calculate the concentrations (pg / mL) of IL-6 and TNF-α based on the OD value of the sample.

[0164] Result: As Figure 12 As shown, LPS stimulation significantly increased the levels of IL-6 and TNF-α in the cell supernatant of the model group. Compared with the model group, the DEX-Sol, DEX-NPs, and DEX-BNPs treatment groups all significantly reduced the levels of IL-6 and TNF-α (p<0.05). Among them, the DEX-BNPs group showed the most significant inhibitory effect, with IL-6 and TNF-α levels significantly lower than those in the DEX-Sol and DEX-NPs groups (p<0.01). This indicates that liposomes modified with IL-17A targeting peptides actively target and deliver dexamethasone to inflammatory cells, while the IL-17A targeting peptides neutralize IL-17A-mediated pro-inflammatory signals, achieving a synergistic anti-inflammatory effect.

[0165] Example 11: Establishment of an Adjuvant-Induced Arthritis (AIA) Mouse Model

[0166] The pathogenesis of AIA in mice was induced using complete Freund's adjuvant (CFA).

[0167] Healthy male C57 mice, weighing 20±2 g and aged 6-8 weeks, were injected subcutaneously into the left hind paw on day 0. The CFA suspension was shaken well before injection. After modeling, the disease progression was monitored daily, ensuring the animals had sufficient food and water. The disease typically peaked about two weeks after modeling.

[0168] Example 12: In vivo targeted inflammatory joint experiment

[0169] On the 5th day after AIA modeling, AIA mice were randomly divided into 3 groups (3 mice / group) and injected via tail vein with 100 μL of free DiD solution (0.5% DMSO, DiD 4 μg / 100 μL), DiD-labeled DEX-NPs (DiD 4 μg / 100 μL), and DiD-labeled DEX-BNPs (DiD 4 μg / 100 μL), respectively. This was recorded as h0.

[0170] Subsequently, at different time points (1 h, 6 h, 48 h), in vivo fluorescence distribution was collected using a small animal in vivo imaging system (IVIS). Mice were sacrificed at 48 h, and major organs (heart, liver, spleen, lung, kidney) and paws were harvested. The fluorescence distribution of the ex vivo tissues was then collected using the IVIS system.

[0171] Result: As Figure 13As shown, the free DiD solution group rapidly distributed throughout the body 1 h after injection, the fluorescence signal significantly weakened after 6 h, and was basically cleared by 48 h. The DiD-NPs group showed some fluorescence enrichment in the joint region at 6 h and 48 h after injection, but the signal intensity was relatively limited. The DiD-BNPs group showed the strongest fluorescence signal in the joint region at 6 h and 48 h after injection, and the signal maintained a high intensity until 48 h, indicating that IL-17A targeting peptide modification significantly enhanced the targeting enrichment and retention ability of nanoparticles in inflamed joints.

[0172] like Figure 14 As shown, 48 h after drug administration, in vitro organ fluorescence imaging revealed that the fluorescence intensity of the DiD-BNPs group in the ankle joint (inflammatory joint) was significantly higher than that of the DiD-NPs group and the free DiD group (p<0.01), while the distribution in organs with the mononuclear phagocyte system, such as the liver and spleen, was not significantly different from that of the DiD-NPs group. These results indicate that DEX-BNPs can achieve specific enrichment in inflamed joints through an IL-17A-mediated active targeting mechanism, without significantly increasing non-specific uptake by the reticuloendothelial system.

[0173] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A multifunctional drug-loaded nanoparticle modified with an IL-17A-targeting peptide, wherein the multifunctional drug-loaded nanoparticle comprises a composite lipid carrier material and a lipophilic drug encapsulated therein, the composite lipid carrier material being composed of S100 phospholipid, HS 15 and a maleimide-modified amphiphilic polymer Pal-PEG-Mal, wherein the hydrophilic end of the Pal-PEG-Mal is modified with a polypeptide molecule targeting IL-17A, and the IL-17A-targeting polypeptide molecule is covalently coupled to the surface of the nanoparticle by reacting its terminal thiol group with the maleimide group at the hydrophilic end of the Pal-PEG-Mal to form a thioether bond.

2. The multifunctional drug-loaded nanoparticles according to claim 1, wherein the mass ratio of S100 phospholipid, HS 15 and Pal-PEG-Mal in the composite lipid carrier material is 3~8∶1~3∶2~4.

3. The multifunctional drug-loaded nanoparticles according to claim 1, wherein the lipophilic drug is dexamethasone, and the mass ratio of dexamethasone to the composite lipid carrier material is 1:5~40.

4. The multifunctional drug-loaded nanoparticles according to claim 1, wherein the amino acid sequence of the polypeptide molecule targeting IL-17A is selected from any one or more sequences shown in SEQ ID NO:1~14.

5. The multifunctional drug-loaded nanoparticles according to claim 1, wherein the molecular weight of polyethylene glycol (PEG) in the Pal-PEG-Mal is 2000~6000 Da.

6. The method for preparing the IL-17A-targeting peptide-modified multifunctional drug-loaded nanoparticles according to claim 1, comprising the following steps: (1) S100 phospholipid, HS 15 and Pal-PEG-Mal and the lipophilic drug dexamethasone are dissolved together in an organic solvent, the organic solvent is removed to form a lipid film, a hydration medium is added to the lipid film for hydration treatment, ultrasonication and filtration are performed to obtain micelle nanoparticles loaded with dexamethasone. (2) The peptide molecule targeting IL-17A is treated with a reducing agent to expose the thiol group. Then the reduced peptide molecule targeting IL-17A is mixed with the micellar nanoparticles described in step (1) so that the thiol group at the end of the peptide molecule targeting IL-17A is coupled with the maleimide group at the hydrophilic end of the Pal-PEG-Mal. After purification, the multifunctional drug-loaded nanoparticles modified with the IL-17A targeting peptide are obtained.

7. The preparation method according to claim 6, wherein step (1) further comprises one or more of the following conditions: (a) The mass ratio of the S100 phospholipid, HS 15, Pal-PEG-Mal to dexamethasone is 1.5:0.6:3:0.25; (b) The organic solvent is methanol, acetonitrile, or chloroform; (c) The method for removing the organic solvent is rotary evaporation under reduced pressure at 30~50 °C until a uniform film is formed; (d) The hydration medium is a 5% glucose solution, or it can be an aqueous solution of hydrochloric acid, an aqueous solution of sodium hydroxide, or a phosphate buffer solution, with a pH of 2 to 8; (e) The hydration treatment time is 2~10 min; (f) The ultrasound conditions are 20~30 ℃, 20~100 W, and 2~10 min.

8. The preparation method according to claim 6, wherein step (2) satisfies one or more of the following conditions: (a) The reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and the molar ratio of the reducing agent to the polypeptide molecule is 2 to 10:1; (b) The reduction treatment is performed at a temperature of 4~25 °C for a time of 0.25~2 h; (c) The terminal end of the polypeptide molecule targeting IL-17A is a cysteine ​​residue; (d) The concentration of maleimide groups in the micelle nanoparticles is 0.07~4 mmol / L, and the molar ratio of the micelle nanoparticles to the polypeptide molecules is 1:5~5:

1. (e) The coupling reaction is carried out at a temperature of 4 to 25 °C for 2 to 24 h.

9. A pharmaceutical composition comprising the multifunctional drug-loaded nanoparticles of any one of claims 1-5 and a pharmaceutically acceptable carrier or excipient; said pharmaceutical composition being an injectable preparation for intravenous, subcutaneous, or intra-articular injection.

10. The use of the multifunctional drug-loaded nanoparticles according to any one of claims 1-5 or the multifunctional drug-loaded nanoparticles prepared by any one of claims 6-8 in the preparation of medicaments for treating IL-17A-mediated inflammatory diseases.

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