Drug delivery carrier, preparation method and application thereof

Abstract

The invention discloses a drug delivery carrier as well as a preparation method and application thereof, and relates to the field of application of a protein recombinant expression technology and a pharmaceutical preparation technology. The composition comprises a recombinant protein of ferritin and targeting peptide and an anti-inflammatory drug, the targeting peptide is used for targeting P selectin; the targeting peptide is displayed on the surface of the ferritin, the anti-inflammatory drug is arranged in an inner cavity of a nanocage formed by the ferritin, the amino acid sequence of the ferritin is as shown in SEQ ID NO: 1, the amino acid sequence of the targeting peptide is as shown in SEQ ID NO: 2, and a connecting peptide is arranged between the ferritin and the targeting peptide. According to the protein nanocage obtained through recombinant expression, the medicine can be more efficiently delivered to an inflammation part, the curative effect is enhanced, meanwhile, the off-target effect is reduced, and the side effect is relieved. And an innovative and effective delivery platform is provided for various anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein recombinant expression technology and pharmaceutical preparation technology, in particular, a drug delivery carrier, a preparation method and application thereof. BACKGROUND

[0002] Ferritin is a protein cage that stores iron ions in cells under physiological conditions, with an outer cavity diameter of about 12 nanometers and an inner cavity diameter of about 8 nanometers, which can regulate the content of iron elements in cells and thus protect cells from cytotoxicity caused by excessive free iron ions. Ferritin is composed of two subunits under natural conditions, including ferritin heavy chain and ferritin light chain, both of which can be recombinantly expressed in bacteria to form a protein nanocage. However, it was found that the ferritin heavy chain has a function of binding to transferrin receptors in addition to the spatial structure of the protein cage, and the transferrin receptors are highly expressed on various tumor tissues, which endows the ferritin heavy chain with tumor targeting function. The ferritin nanocage mentioned later refers to the ferritin heavy chain nanocage. When loading drugs, the ferritin nanocage can utilize its response characteristics to acid, alkali, heat, and salt, such as pH adjustment (e.g., neutral-acid-neutral or neutral-alkaline-neutral), heat adjustment (e.g., 60 degrees Celsius for 4 hours), and salt adjustment (e.g., urea gradient co-renaturation) to encapsulate drugs in the inner cavity of the ferritin nanocage. Due to the recombinant expression method, the targeting polypeptide can be expressed at the amino terminus of ferritin through genetic engineering, thereby being displayed on the outer surface of ferritin, which endows ferritin with new targeting properties. The ferritin drug delivery system has good application advantages.

[0003] P-selectin, as a classic adhesion molecule, is expressed on the surface of activated platelets and vascular endothelium, and can interact with various P-selectin ligands on the surface of immune cells, such as PSGL-1, thereby guiding the infiltration of immune cells at the inflammation site. Using this principle, the process of immune cell infiltration can be simulated by connecting P-selectin ligands on various drug delivery systems, thereby achieving inflammation targeting. However, for example, cell membrane preparations, although they can effectively deliver drugs to the inflammation site and have P-selectin ligands on the surface, the complexity of the surface molecules and the difficulty of process scaling hinder their further application. Therefore, there is an urgent need to develop effective P-selectin-targeted inflammation-targeted drug delivery systems.

[0004] Dexamethasone is a strong synthetic glucocorticoid, which enters the nucleus after binding to intracellular glucocorticoid receptors, promotes the expression of anti-inflammatory and immunosuppressive related genes, and effectively controls various excessive inflammatory reactions. However, it also shows many side effects in clinical application, including but not limited to metabolic disorders such as central obesity, exacerbation of diabetes, insulin resistance, dyslipidemia, water and sodium retention; abnormal bone metabolism such as osteoporosis, femoral head necrosis; increased risk of infection caused by immunosuppression and the like. These side effects hinder the further application of dexamethasone in clinic.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide a drug delivery carrier, a preparation method and an application thereof to solve the technical bottleneck of low targeting efficiency and large off-target side effects of anti-inflammatory drugs.

[0007] The present application is realized as follows: In a first aspect, the present application provides a drug delivery carrier, comprising: a recombinant protein of ferritin and a targeting peptide and an anti-inflammatory drug; the targeting peptide is used for targeting P-selectin; the targeting peptide is displayed on the surface of the ferritin, and the anti-inflammatory drug is arranged in the inner cavity of the nanocage formed by the ferritin, the amino acid sequence of the ferritin is shown as SEQ ID NO: 1, the amino acid sequence of the targeting peptide is shown as SEQ ID NO: 2, and the linking peptide is between the ferritin and the targeting peptide.

[0008] In a second aspect, the present application provides a preparation method of the drug delivery carrier, wherein the anti-inflammatory drug is mixed with the recombinant protein, and then stirred and incubated at 60℃±5℃ for 2-6 hours, and the supernatant is obtained by centrifugation.

[0009] In a third aspect, the present application provides an application of the drug delivery carrier in preparing an anti-inflammatory drug.

[0010] The present application has the following beneficial effects: The present application uses genetic engineering technology to modify (i.e., display) the targeting peptide targeting P-selectin on the surface of ferritin, so as to obtain the ability to target the inflammatory site, increase the enrichment of the loaded drug at the inflammatory site, and reduce the off-target effect. Dexamethasone has strong anti-inflammatory effect, but its systemic side effects limit its use. By loading dexamethasone sodium phosphate with recombinant protein, the anti-inflammatory effect of dexamethasone is increased, and the side effects caused by free drugs in other organs or tissues are reduced. The present application verifies the treatment potential of the loaded anti-inflammatory drug in zebrafish tail amputation inflammation model and mouse acute lung injury model, and the drug delivery carrier provided by the present application has good delivery efficiency.

[0011] The drug delivery carrier provided by this invention can also be used to deliver other anti-inflammatory drugs besides dexamethasone, and can be extended to the targeted delivery of other anti-inflammatory drugs and the development of treatments for other inflammatory diseases.

[0012] This invention is the first to recombine the targeting peptide of P-selectin with the ferritin heavy chain for recombinant expression, achieving an integrated design of "anti-inflammatory targeted drug delivery" and overcoming the limitations of traditional dosage forms. The recombinant protein is prepared through microbial fermentation, the process is easily scaled up, and no complex purification steps are required, conforming to the principles of green chemistry. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 Sequencing results for ferritin modified with P-selectin-binding peptides; Figure 2 SDS-PAGE electrophoretic analysis of the supernatant after induction, the fragmented supernatant after induction, and the fragmented precipitate after induction; Figure 3 SDS-PAFE electrophoresis analysis of purified P-selectin-targeted modified ferritin; Figure 4 Particle size distribution of ferritin modified with P-selectin-binding peptide; Figure 5 Transmission electron microscopy image of ferritin modified with P-selectin-binding peptide; Figure 6 To detect the expression of P-selectin on the surface of 293T cells after transfecting them with a plasmid that overexpresses P-selectin, flow cytometry was used. Figure 7 To detect by flow cytometry the uptake of Cy5-modified P-selectin-targeted ferritin by 293T cells overexpressing P-selectin in normal 293T cells; Figure 8 To detect the uptake of Cy5-modified P-selectin-targeted ferritin by normal 293T cells overexpressing P-selectin in their nuclei using fluorescence microscopy; Figure 9 Enrichment of zebrafish tail amputation inflammatory sites by free Cy5 dyes and Cy5-modified P-selectin-targeted ferritin; Figure 10 High-performance liquid chromatography was performed to break down P-selectin-targeted ferritin loaded with dexamethasone sodium phosphate. Figure 11 P-selectin targeting modification of ferritin particle size distribution for loading dexamethasone sodium phosphate; Figure 12 Transmission electron microscopy image of P-selectin-targeted ferritin without dexamethasone sodium phosphate loading; Figure 13 Morphological images of macrophages induced by LPS under different drug treatment conditions; Figure 14 The content of nitric oxide in the cell supernatant of macrophages under different drug treatment conditions after LPS induction; Figure 15 The content of interleukin-1β in the cell supernatant of macrophages under different drug treatment conditions after LPS induction; Figure 16 After establishing a tail-severance model for transgenic zebrafish with neutrophil green fluorescent protein (Tg(MPO:EGFP)), the enrichment of neutrophils in the tail was photographed and statistically analyzed after different drug treatments. Figure 17 Images of the lungs and skin pathological tissue sections of a mouse model of acute lung injury after treatment with different drugs. Figure 18 The concentrations of interleukin-1β, interleukin-6, and tumor necrosis factor in bronchoalveolar lavage fluid of a mouse acute lung injury model after different drug treatments. Figure 19 Statistics and images of spleen weight in a mouse model of acute lung injury after treatment with different drugs; Figure 20 An EP tube containing curcumin-loaded ferritin modified with P-selectin targeting peptides and a graph showing the statistical results of NO secretion levels in LPS-induced cells treated with Cur@PSBP HFn. Detailed Implementation

[0015] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0017] Definition of noun As used in this article, the term "prevention" means reducing the risk of disease in an individual or group, or mitigating the severity or slowing the progression of disease, through a series of proactive interventions. Disease prevention is a proactive health management concept that aims to avoid or mitigate the harm of disease through "intervention," and its value far exceeds that of treatment after the onset of disease.

[0018] As used in this article, the term "treatment" refers to a series of medical actions that involve interventions in an existing disease through medical means, techniques, or methods, with the aim of eliminating the cause, relieving symptoms, controlling disease progression, promoting tissue repair, restoring function, or alleviating pain.

[0019] In a first aspect, the present invention provides a drug delivery carrier comprising: a recombinant protein of ferritin and a targeting peptide and an anti-inflammatory drug; the targeting peptide is used to target P-selectin; the targeting peptide is displayed on the surface of ferritin, the anti-inflammatory drug is disposed in the cavity of a nanocage formed by ferritin, the amino acid sequence of ferritin is shown in SEQ ID NO:1, the amino acid sequence of the targeting peptide is shown in SEQ ID NO:2, and a linker peptide is present between ferritin and the targeting peptide.

[0020] This invention utilizes recombinant expression of ferritin and targeting peptides in *E. coli* to encapsulate anti-inflammatory drugs within the lumen of a nanocage formed by ferritin, thus combining drug delivery and inflammation targeting functions. This system aims to overcome the technical bottlenecks of low targeting efficiency and significant off-target side effects of anti-inflammatory drugs. Furthermore, its therapeutic potential after loading with anti-inflammatory drugs has been validated in zebrafish tail-cutting inflammation models and mouse acute lung injury models.

[0021] In a preferred embodiment of the present invention, the mass ratio of recombinant protein to anti-inflammatory drug is 20:1 – 5:1.

[0022] In a preferred embodiment of the present invention, the linking peptide is a flexible linking peptide or a rigid linking peptide. In a preferred embodiment of the present invention, the linker peptide is selected from (GGGGS). x (Gly)8, (Gly)6, (EAAAK) n ,A(EAAAK)4ALEA(EAAAK)4A,PAPAP,AEAAAKEAAAKA,(Ala-Pro) m (10 – 34 aa), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG or LE, x=1~4, n=1~3.

[0023] For example, the linker peptide is GGGGS.

[0024] In a preferred embodiment of the present invention, the anti-inflammatory drug is selected from dexamethasone, dexamethasone sodium phosphate, ibuprofen, aspirin, naproxen, prednisone, hydrocortisone, methotrexate, leflunomide, etanercept, adalimumab, or curcumin.

[0025] The preferred formulation is dexamethasone sodium phosphate, whose therapeutic potential after loading with the anti-inflammatory drug (dexamethasone sodium phosphate) has been verified in zebrafish tail-cutting inflammation model and mouse acute lung injury model.

[0026] Secondly, this invention provides a method for preparing a drug delivery carrier, which involves mixing an anti-inflammatory drug with a recombinant protein, stirring and incubating at 60℃±5℃ for 2-6 hours, and then centrifuging to collect the supernatant. Temperature-controlled drug loading does not disrupt the assembly structure of ferritin, thus significantly improving its loading efficiency and stability.

[0027] Thirdly, the present invention provides the application of drug delivery carriers in the preparation of anti-inflammatory drugs.

[0028] In a preferred embodiment of the present invention, the drug is administered by injection or orally.

[0029] In a preferred embodiment of the present invention, anti-inflammatory drugs are used to treat acute lung injury or chronic inflammatory diseases.

[0030] In a preferred embodiment of the present invention, the chronic inflammatory disease is selected from rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, gout, inflammatory bowel disease, nervous system diseases, or cardiovascular diseases.

[0031] In a preferred embodiment of the present invention, the drug also includes pharmaceutically acceptable excipients.

[0032] In a preferred embodiment of the present invention, the excipients include, but are not limited to, conventional pharmaceutical excipients, carriers, or diluents.

[0033] Drugs also include pharmaceutically acceptable excipients, such as water, saline, sugars, polysaccharides, buffers, excipients, biodegradable polymers, liposomes, stabilizers, etc.

[0034] Pharmaceutically acceptable excipients include, but are not limited to, fillers, lubricants, disintegrants, binders, and flow aids.

[0035] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, hydroxypropyl methylcellulose, starch and its derivatives, polyethylene glycol and its derivatives, lactose, lactose-starch complex, lactose-cellulose complex, sucrose, mannitol, mannitol-starch complex, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, povidone, copovidone, calcium hydrogen phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, low-substituted hydroxypropyl cellulose, croscarmellose sodium cellulose, croscarmellose, magnesium stearate, sodium stearate fumarate, talc, and stearic acid, or a combination thereof.

[0036] In an alternative embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, and gelling agents (e.g., methylcellulose), as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the reagent selected.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] Example 1 This embodiment provides a drug delivery carrier comprising a recombinant protein of ferritin and a targeting peptide and an anti-inflammatory drug; the targeting peptide is used to target P-selectin; the targeting peptide is displayed on the surface of ferritin, and the anti-inflammatory drug is disposed in the lumen of a nanocage formed by ferritin; the amino acid sequence of ferritin is shown in SEQ ID NO:1, the amino acid sequence of the targeting peptide is shown in SEQ ID NO:2, and a linker peptide GGGGS is present between ferritin and the targeting peptide.

[0039] SEQ ID NO:1: MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES.

[0040] SEQ ID NO:2: DAEWVDVS.

[0041] Its preparation method is as follows: 1. Preparation of recombinant proteins containing ferritin and targeting peptides: (1) Plasmid construction and protein expression Oligonucleotides with NdeI and EcoRI sticky ends on both sides, which simultaneously encode P-selectin-binding polypeptides and GGGGS linker polypeptides, are annealed to form double strands. The nucleotide sequence is as follows: Forward sequence: 5'-TATGGACGCAGAATGGGTGGATGTGAGCGGCGGTGGTGGTTCTG-3' Reverse sequence: 5'-AATTCAGAACCACCACCGCCGCTCACATCCACCCATTCTGCGTCCA-3' The annealing product was digested with NdeI and EcoRI, purified, and incubated with ligase at 16°C for 12 hours. It was then transformed into E. coli (Rosetta) competent cells via heat shock. Positive clones were selected the following day and confirmed by sequencing. The positive clones were inoculated into LB medium and incubated at 160 rpm at 37°C for 4 hours. Then, IPTG at a final concentration of 5 mM was added to induce expression for 5 hours.

[0042] (2) Key process parameters Molar ratio: The molar ratio of annealed product to plasmid is 3:1; Purification process: After expression, the bacterial cells were collected, the supernatant was collected and lysed, and the supernatant was collected after treatment at 70 degrees Celsius for 10 minutes. The supernatant was then precipitated with 50% ammonium sulfate, redissolved in phosphate-buffered saline, and dialyzed overnight. The final protein obtained had a purity of over 95%.

[0043] (3) Gene sequencing and structural characterization Sequencing results showed that the DNA sequence encoding the P-selectin-binding polypeptide (i.e., the targeting peptide that targets P-selectin) and the DNA sequence encoding the GGGGS flexible fragment were inserted between the NdeI restriction site and the EcoRI restriction site. Figure 1 The gene encoding human ferritin heavy chain lies between its 3' EcoRI restriction site and XhoI restriction site.

[0044] Protein expression and purification: Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis: SDS-PAGE electrophoresis showed that P-selectin-targeting peptide-modified ferritin (PSBP HFn) was expressed in E. coli after IPTG induction. Figure 2 After being disrupted by ultrasound, the protein is mainly distributed in the supernatant. Following a series of purification strategies, its purity can reach over 95%. Figure 3 ).

[0045] DLS particle size distribution and transmission electron microscopy morphology Dynamic light scattering (DLS) results showed that the ferritin modified with P-selectin-targeting peptides had a uniform particle size distribution, with a diameter of approximately 15.63 nanometers. Figure 4 After negative staining, a uniformly distributed protein cage structure was also observed under a transmission electron microscope, with a size of approximately 10 nanometers. Figure 5 ).

[0046] (4) P-selectin targeting and in vivo inflammation targeting characterization Construction and Targeting of Cells with High P-selectin Expression A cell model with high P-selectin expression was obtained by transfecting the P-selectin eukaryotic expression vector into 293T cells. Figure 6 Cy5-conjugated P-selectin-binding peptide-modified ferritin was incubated in 293T cells before and after transformation. Flow cytometry results showed that 293T cells with high P-selectin expression significantly increased their uptake of P-selectin-binding peptide-modified ferritin. Figures 7-8 This demonstrates that the ferritin cage specifically targets P-selectin.

[0047] Zebrafish tail amputation inflammation model targeted A model of localized inflammation was created by cutting the tail of zebrafish using a needle. Two hours later, free Cy5 and Cy5-conjugated P-selectin-binding peptide-modified ferritin were injected intravenously into the zebrafish. Three hours later, photographs of the zebrafish tails were taken for comparison. The results showed that, compared to free Cy5, Cy5-conjugated P-selectin-binding peptide-modified ferritin was significantly enriched at the tail amputation site. Figure 9 ).

[0048] 2. Loading of dexamethasone sodium phosphate Drug loading strategy: Dexamethasone sodium phosphate was mixed with the recombinant protein from step 1 at a mass ratio of 3:2, stirred and incubated at 60 degrees Celsius for 4 hours, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was dialyzed overnight in phosphate-buffered saline to obtain the P-selectin-targeted ferritin drug complex (i.e., drug delivery carrier).

[0049] (1) Drug loading and characterization High performance liquid chromatography analysis Dexamethasone sodium phosphate was loaded onto ferritin modified with P-selectin-targeting peptides. After the ferritin was cleaved with 0.1 M hydrochloric acid, the drug loading concentration was analyzed by high-performance liquid chromatography. The results showed that the drug loading rate was close to 14.3%. Figure 10 Instrument model: Agilent 120; HPLC conditions: C18 column; column temperature: 25 degrees Celsius; flow rate: 1 ml / min; mobile phase ratio: acetonitrile:water 75:25; detector: UV detector; wavelength: 254 nm; injection volume: 10 μL.

[0050] DLS particle size distribution and transmission electron microscopy morphology analysis: Dynamic light scattering (DLS) results showed that the ferritin modified with P-selectin-targeting peptides loaded with dexamethasone sodium phosphate had a uniform particle size distribution, with a diameter of approximately 17.49 nm. Figure 11 After negative staining, a uniformly distributed protein cage structure was also observed under a transmission electron microscope, with a size of approximately 10 nanometers. Figure 12 ).

[0051] (2) In vitro and in vivo anti-inflammatory characterization of dexamethasone sodium phosphate loaded in ferritin nanocages targeting inflammation.

[0052] Cellular-level anti-inflammatory effects: At the cellular level, after RAW264.7 cells were activated by LPS, they were treated with ferritin modified with P-selectin-targeting peptides loaded with dexamethasone sodium phosphate (DexSPG) at a final drug concentration of 2 μmol / L. Macrophage polarization was then observed, showing that the drug-treated group significantly inhibited macrophage polarization and deformation. Figure 13 The concentrations of nitric oxide and IL-1β in cell supernatant were measured. The results showed that ferritin modified with P-selectin targeting peptide loaded with dexamethasone effectively reduced LPS-induced nitric oxide and IL-1β production at the cellular level compared to the model group. Figures 14-15 ).

[0053] Anti-inflammatory effects of zebrafish tail amputation inflammation model: Free dexamethasone sodium phosphate and ferritin modified with a P-selectin-targeting peptide loaded with dexamethasone sodium phosphate were injected intravenously into zebrafish. Four hours later, the tails of transgenic zebrafish expressing green fluorescent protein (Tg(MPO:EGFP)) were dissected using a needle to create a local inflammation model. Twenty-four hours later, the zebrafish tails were photographed for comparison, and the number of neutrophils enriched in the tails was counted. The results showed that, compared to the model group and the free group, ferritin modified with a P-selectin-targeting peptide loaded with dexamethasone sodium phosphate significantly reduced neutrophil infiltration at the tail dissection site. Figure 16 ).

[0054] Example 2 This embodiment characterizes the anti-inflammatory effects and side effects of a mouse model of acute lung injury.

[0055] Model establishment: An acute lung injury model in mice was induced by intratracheal administration of LPS at a dose of 5 mg / kg.

[0056] Treatment regimen: Experimental group: Ferritin modified with P-selectin-targeting peptide loaded with dexamethasone sodium phosphate; Control group: Free dexamethasone sodium phosphate; Treatment duration: 2 days.

[0057] Therapeutic indicators and results: The images of mouse lungs and the results of skin pathological tissue sections are shown below. Figure 17 The lungs of mice treated with ferritin modified with P-selectin-targeting peptides loaded with dexamethasone sodium phosphate showed the most similar appearance to the normal group, with less hemorrhage and edema compared to the model group and the free drug group. Histological sections showed less pulmonary hemorrhage and less immune cell infiltration. The concentrations and levels of interleukin-6, tumor necrosis factor, and interleukin-1β in bronchoalveolar lavage fluid were also measured. Compared to the model group and the free dexamethasone sodium phosphate group, the concentrations of interleukin-1β, interleukin-6, and tumor necrosis factor in the bronchoalveolar lavage fluid of the ferritin treatment group loaded with P-selectin-targeting peptides loaded with dexamethasone sodium phosphate were significantly reduced. Figure 18 This demonstrates its promising application prospects in the treatment of acute inflammatory diseases. Simultaneously, the spleen weight of mice in each group was characterized. Typically, LPS-induced acute inflammation leads to immune cell depletion, resulting in a decrease in spleen weight, while the toxicity of dexamethasone sodium phosphate to immune cells further suppresses systemic immunity. The ferritin treatment group loaded with dexamethasone sodium phosphate and modified with P-selectin-targeting peptides significantly reduced the immunosuppressive effect of free dexamethasone sodium phosphate, and the spleen weight of mice was significantly higher than that of the model group and the free drug group. Figure 19 ).

[0058] Example 3 This embodiment studies the drug loading and cellular-level anti-inflammatory effects of another anti-inflammatory drug, curcumin.

[0059] Drug delivery method: Cur@PSBP HFn nanoparticles (NPs) were prepared via a disassembly / reassembly process. The pH of the PSBP HFn solution (0.5 mg / mL, 0.15 M NaCl, 3 mL) was adjusted to 12.5 by adding an appropriate volume of 1 M NaOH solution. After stirring for 15 minutes, freshly dissolved Cur in 0.1 M sodium hydroxide solution was added. The pH was then gradually lowered to 7.4 using 1 M HCl solution. To protect Cur from oxidation, nitric oxide was charged into a brown glass vial and stirred at room temperature for 1 hour to promote protein assembly. The solution was then centrifuged at 10,000 rpm for 10 minutes to remove undissolved Cur. The supernatant was dialyzed (MWCO = 3500 Da) overnight in PBS to remove unencapsulated drug. The product was stored at -80°C for further use.

[0060] Experimental Method: To detect the anti-inflammatory effect of curcumin loaded onto PSBP HFn, 1*10^5 Raw 264.7 cells were seeded in 24-well plates and cultured for 24 hours to allow cell adhesion. Then, 100 ng / mL LPS was added to each well to induce polarization of RAW246.7 cells. Simultaneously, the treatment group received Cur@PSBP HFn with a final curcumin concentration of 8 μg / mL. After 24 hours of conditioning, the nitric oxide content in the cell supernatant was measured.

[0061] Experimental results: Curcumin was successfully loaded using ferritin modified with P-selectin targeting peptide. Figure 20 Furthermore, after Raw264.7 cells were induced by LPS and then treated with Cur@PSBP HFn, NO secretion was significantly reduced, demonstrating the anti-inflammatory effect of Cur@PSBP HFn at the cellular level. Figure 20 ).

[0062] In summary, this invention is the first to recombine and express P-selectin-binding peptides with ferritin heavy chains, achieving an integrated design of "inflammation-targeted drug delivery" and breaking through the limitations of traditional dosage forms. Process advantages: microbial fermentation, easy process scale-up, no need for complex purification steps, in line with green chemistry principles; Delivery efficiency and therapeutic effects in animal inflammation models support its potential as a novel treatment option for acute lung injury, and it can be extended to the targeted delivery of other anti-inflammatory drugs and the development of treatments for other inflammatory diseases.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A drug delivery carrier, characterized in that, It comprises: a recombinant protein of ferritin and a targeting peptide and an anti-inflammatory drug; the targeting peptide is used for targeting P-selectin; the targeting peptide is displayed on the surface of the ferritin, the anti-inflammatory drug is arranged in the inner cavity of the nanocage formed by the ferritin, the amino acid sequence of the ferritin is shown as SEQ ID NO: 1, the amino acid sequence of the targeting peptide is shown as SEQ ID NO: 2, and the ferritin and the targeting peptide have a connecting peptide therebetween.

2. The drug delivery vehicle of claim 1, wherein, The mass ratio of the recombinant protein and the anti-inflammatory drug is 20:1-5:

1.

3. The drug delivery vehicle of claim 1, wherein, The connecting peptide is a flexible connecting peptide or a rigid connecting peptide. Preferably, the connecting peptide is selected from (GGGGS) x , (Gly)8, (Gly)6, (EAAAK) n , A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro) m (10 - 34 aa), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, E DVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG or LE, x = 1-4, n = 1-3.

4. The drug delivery vehicle of claim 1, wherein, The anti-inflammatory drug is selected from dexamethasone, dexamethasone sodium phosphate, ibuprofen, aspirin, naproxen, prednisone, hydrocortisone, methotrexate, leflunomide, etanercept, adalimumab or curcumin.

5. A method of preparing a drug delivery vehicle according to any one of claims 1 to 4, wherein, The anti-inflammatory drug is mixed with the recombinant protein, and the mixture is incubated at a temperature of 60℃±5℃ with stirring for 2-6 hours, and then centrifuged to obtain the supernatant.

6. Use of the drug delivery carrier according to any one of claims 1-4 in the preparation of an anti-inflammatory drug.

7. Use according to claim 6, characterized in that, The drug is administered by injection or orally.

8. Use according to claim 6, characterized in that, The anti-inflammatory drug is used for preventing or treating acute lung injury or chronic inflammatory disease.

9. Use according to claim 8, characterized in that, The chronic inflammatory disease is selected from rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, gout, inflammatory bowel disease, nervous system disease or cardiovascular disease.

10. Use according to claim 6, characterized in that, The drug further comprises a pharmaceutically acceptable excipient.

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