Phosphatidylserine modified gliadin nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a phosphatidylserine modified gliadin nanoparticle as well as a preparation method and application thereof. The phosphatidylserine modified gliadin nanoparticle is of a core-shell structure, gliadin is entrapped by polylactic acid-glycolic acid copolymer to serve as a core, and a lipid composite membrane formed by phosphatidylserine and DSPE-PEG2000 is coated outside the nanoparticle. The gliadin-loaded nanoparticles modified by phosphatidylserine are used for simulating membrane signal characteristics of apoptotic bodies, enhancing recognition and uptake of the nanoparticles by macrophages and inducing tolerance phenotypic transformation of the nanoparticles, so that immune tolerance of celiac disease patients to gliadin is recovered, and the gliadin-loaded nanoparticles can be used for treating celiac disease patients. On the basis of simplifying the preparation process, the problems of unstable tolerance effect and lack of metabolic regulation in the existing nano immunotherapy are further solved. The nano-particles prepared by the technical route have good particle size distribution and stability, can effectively deliver antigens and induce immune tolerance, and are a novel convertible celiac disease treatment nano-preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a phosphatidylserine-modified gliadin nanoparticle, its preparation method, and its application. Background Technology

[0002] Celiac disease (CeD) is an autoimmune enteropathy caused by the ingestion of gluten (primarily gliadin), characterized by villous atrophy, crypt hyperplasia, and intraepithelial lymphocyte (IEL) infiltration in the small intestine. The incidence of this disease can reach over 1% of the global population in individuals carrying susceptibility genes such as human leukocyte antigen (HLA) DQ2 or DQ8. Currently, the only effective treatment is a strict gluten-free diet (GFD), but due to food cross-contamination and poor patient adherence, the clinical remission rate is limited, and some patients still experience relapses or persistent inflammation.

[0003] To address this clinical challenge, recent research has focused on restoring the body's immune homeostasis to gliadin through antigen-specific immune tolerance. An important strategy is to utilize biodegradable nanoparticle carriers to deliver antigens, thereby inducing tolerance in the peripheral immune system rather than an inflammatory response.

[0004] Freitag et al. (2020) first proposed tolerogenic Immune Modifying Nanoparticles (TIMP-GLIA) loaded with gliadin in a study published in *Gastroenterology*. This nanosystem, administered intravenously, is taken up by antigen-presenting cells (APCs) in the spleen and liver, thereby downregulating the expression of co-stimulatory molecules (CD80 / CD86), upregulating the inhibitory ligand PD-L1, and promoting the activation of regulatory T cells (Tregs). Animal experiments showed that this system significantly reduced gliadin-specific T cell proliferation and the expression of inflammatory factors (IFN-γ, IL-17), and alleviated intestinal mucosal damage. This strategy provides important proof of concept for the immune tolerance therapy of celiac disease.

[0005] Building on this, Kelly et al. (2021) further completed a Phase I / IIa clinical trial of TAK-101 nanoparticles in *Gastroenterology*. TAK-101 consists of negatively charged PLGA nanoparticles encapsulating gliadin. Clinical results showed that it was safe and well-tolerated in subjects, and significantly reduced peripheral blood IFN-γ response and chorionic villus atrophy after oral gluten challenge. This study is the first to demonstrate the feasibility of inducing antigen-specific immune tolerance through nanoparticles in humans.

[0006] However, the above technologies still have certain limitations:

[0007] 1. The lack of biological recognition signals on the surface of nanoparticles results in limited efficiency in recognition and uptake by antigen-presenting cells such as macrophages;

[0008] 2. The regulatory role of macrophage metabolic state in the establishment of tolerance has not been fully considered, therefore the durability of the immunomodulatory effect is still limited;

[0009] 3. Systems such as TAK-101 rely solely on antigen delivery and lack a mechanism for actively inducing a tolerance microenvironment.

[0010] In recent years, Jiang et al. (2025) reported in *ACS Nano* a rapamycin-gliadin composite nanoparticle (PLN-GR) that, by simultaneously delivering the immunomodulatory drugs rapamycin and gliadin, remodeled the energy metabolism of hepatic Kupffer cells (shifting from glycolysis to oxidative phosphorylation) and promoted the secretion of its metabolite itaconic acid, thereby driving the transformation of splenic dendritic cells to a PD-L1⁺-tolerant phenotype. This strategy significantly reduced intestinal inflammation and restored antigen-specific tolerance in animal models. However, the stability of the composite drug delivery system is poor, and rapamycin carries the potential immunosuppressive side effect.

[0011] In summary, existing technologies mainly focus on antigen-loaded PLGA nanoparticles to induce immune tolerance (such as TIMP-GLIA, TAK-101, and PLN-GR systems), but these technologies suffer from low target cell recognition efficiency, insufficient metabolic regulation, and unstable tolerance effects. Therefore, how to introduce natural signaling molecules with immune recognition functions (such as phosphatidylserine) to actively regulate phagocyte metabolism and epigenetic status while delivering antigens, in order to achieve stable and durable antigen-specific immune tolerance, has become a pressing technical challenge in this field. Summary of the Invention

[0012] The purpose of this invention is to address the shortcomings of existing technologies by providing phosphatidylserine-modified gliadin nanoparticles, their preparation method, and applications.

[0013] To achieve the above objectives, the present invention adopts the following technical solution: a phosphatidylserine-modified gliadin nanoparticle, wherein the nanoparticle has a core-shell structure, with gliadin encapsulated in polylactic acid-glycolic acid copolymer as the core, and is externally coated with a lipid composite membrane formed by phosphatidylserine and DSPE-PEG2000.

[0014] Furthermore, the nanoparticles have a particle size of 180–220 nm and a surface potential of –25–35 mV.

[0015] Furthermore, the mass ratio of phosphatidylserine to DSPE-PEG2000 is 1:(1-9), and the number average molecular weight of the polyethylene glycol segments in DSPE-PEG2000 is 2000 Da.

[0016] Furthermore, the polylactic acid-glycolic acid copolymer has a lactic acid to glycolic acid ratio of 50:50 and a weight-average molecular weight of 30-50 kDa.

[0017] Furthermore, the nanoparticles are encapsulated with a fluorescent marker, which is IR-780 iodide or Cousarin 6.

[0018] A method for preparing the above-mentioned phosphatidylserine-modified gliadin nanoparticles includes the following steps:

[0019] (1) Glycol protein and polylactic acid-glycolic acid copolymer are co-dissolved in dimethyl sulfoxide to form an organic phase;

[0020] (2) Dissolve DSPE-PEG2000 and phosphatidylserine in a 4% (v / v) ethanol aqueous solution to form a lipid-containing aqueous phase;

[0021] (3) Inject the organic phase into the lipid-containing aqueous phase under stirring conditions to allow the system to self-assemble into a nanoparticle suspension;

[0022] (4) After centrifuging and washing the nanoparticle suspension, purified phosphatidylserine-modified gliadin nanoparticles were obtained.

[0023] Furthermore, the mass ratio of gliadin to polylactic acid-glycolic acid copolymer is 1:(5-9); the ratio of phosphatidylserine to DSPE-PEG2000 is 1:(1-9).

[0024] Furthermore, step (2) specifically includes the following steps:

[0025] (a) Phosphatidylserine was dissolved in a chloroform-dimethyl sulfoxide mixed solvent in a volume ratio of 9:1;

[0026] (b) DSPE-PEG2000 was dissolved in a 95% (v / v) aqueous ethanol solution and preheated in a 65°C water bath for 10 min.

[0027] (c) The DSPE-PEG2000 dissolved in steps (a) and (b) and phosphatidylserine were added to a 4% ethanol aqueous solution, ultrasonically emulsified and uniformly dispersed, and then nanoprecipitated.

[0028] The above-mentioned phosphatidylserine-modified gliadin nanoparticles are used in the preparation of drugs for treating diseases caused by antigen-specific immune tolerance deficiency, including celiac disease, inflammatory bowel disease, and multiple sclerosis.

[0029] A pharmaceutical composition for treating celiac disease includes the above-mentioned phosphatidylserine-modified gliadin nanoparticles.

[0030] Phosphatidylserine (PS) modified gliadin-loaded nanoparticles (PS@PLGA-G) mimic the membrane signaling characteristics of apoptotic bodies, enhancing macrophage recognition and uptake of nanoparticles, inducing tolerance phenotype transformation, thereby restoring immune tolerance to gliadin in celiac disease patients and solving the problems of unstable tolerance effects and lack of metabolic regulation in existing nanoimmunotherapy.

[0031] The nanosystem provided by this invention significantly enhances macrophage recognition and phagocytic efficiency by mimicking the "eat-me" signal of apoptotic bodies through surface-exposed phosphatidylserine residues. After entering phagocytes, the nanoparticles promote increased glycolytic flux and lactate accumulation, activating lactylation of histone H3K9, thereby driving M2 macrophage polarization and transcriptional activation of anti-inflammatory genes (Arg1, IL-10, Mrc1). This chain of action constitutes a "metabolic-epigenetic dual-regulation" mechanism, ultimately rebuilding the intestinal mucosal tolerance microenvironment and effectively alleviating celiac disease-related inflammatory responses and tissue damage.

[0032] The PS@PLGA-G nanoparticles prepared by the above technical route have good particle size distribution and stability, and can effectively deliver antigens and induce immune tolerance. They are a novel and convertible nano-formulation for the treatment of celiac disease.

[0033] The beneficial effects of this invention: Compared with existing technologies (TIMP-GLIA, TAK-101, and other PLGA carrier systems), this invention has the following significant advantages:

[0034] (1) Efficient and stable preparation process: Nanoparticles are prepared by one-step precipitation self-assembly method, which greatly shortens the preparation time of nanosystems and significantly improves the uniformity of nanoparticle size and system stability compared with existing technologies.

[0035] (2) Enhanced biorecognition: By introducing phosphatidylserine on the surface of nanoparticles, the phagocytic efficiency of macrophages on apoptotic cells is significantly improved, achieving precise targeted delivery of immune cells.

[0036] (3) Innovation of immune regulation mechanism: This invention is the first to apply the concept of "lactic acid-driven epigenetic regulation of macrophage polarization" to the reconstruction of gluten tolerance in celiac disease, realizing the reconstruction of immune tolerance at both metabolic and epigenetic levels.

[0037] (4) Good safety and convertibility: The formulation components are all FDA-approved biodegradable materials (PLGA, PS), with no obvious liver and kidney toxicity, and are clinically feasible.

[0038] In summary, this invention provides an intelligent antigen nanocarrier that can mimic apoptosis signals and actively regulate metabolic and epigenetic states. It not only overcomes the shortcomings of traditional nanoimmunotherapy, such as poor targeting and unstable tolerance, but also provides a new technical route and theoretical basis for immune tolerance therapy of celiac disease and other autoimmune diseases. Attached Figure Description

[0039] Figure 1 Examples of preparation and characterization of phosphatidylserine-modified gliadin nanoparticles (PS@PLGA-G): (A) Transmission electron microscopy image and schematic diagram of nanostructure of phosphatidylserine-modified gliadin nanoparticles (PS@PLGA-G); (B) Nanoparticle size distribution; (C) Nanoparticle surface zeta potential; (D) IC50 value; (E) Cytotoxicity; (F) Liver biochemistry; (G) Hepatic chemiluminescence (HE) of important organs; (H) Fluorescence distribution of important organs; (I) Fluorescence pattern of macrophage uptake of nanoparticles.

[0040] Figure 2 Example PS@PLGA-G improves celiac disease: (A) Animal experiment flowchart; (B) Weight record line graph; (C) Foot swelling degree; (D) Small intestine length; (E) Intestinal villus-crypt ratio; (F) Duodenum HE.

[0041] Figure 3Examples of PS@PLGA-G-induced histone lactation promoting M2 polarization: (A) Lactate content in the supernatant after co-incubation of Raw264.7 macrophages with nanoparticles; (B) Intracellular lactate content; (C) Western blotting verification of histone lactation; (D) Flow cytometry verification of Raw264.7 macrophage polarization; (E) Statistical graph of M1 macrophage proportion; (F) Statistical graph of M2 macrophage proportion; (G) Flow cytometry verification of mouse bone marrow-derived macrophage polarization; (H) Flow cytometry verification of mouse bone marrow-derived macrophage polarization after inhibiting lactate synthesis with the LDHA inhibitor Oxamate; (I) Lactate content in the supernatant and intracellular lactate content after inhibiting lactate synthesis with the LDHA inhibitor Oxamate; (J) Western blotting verification of histone lactation inhibition. Detailed Implementation

[0042] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0043] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product or device.

[0044] Experimental materials and equipment

[0045] The main raw materials and chemical reagents are shown in Table 1:

[0046] Table 1

[0047] name Specifications / Purity supplier CAS number Remark Polylactic acid-glycolic acid copolymer (PLGA, 50:50, MW 40 kDa) Analytical Pure Shanghai Shunna Biotechnology Co., Ltd. — Antigen carrier materials Gliadin ≥95% Macklin 9007-90-3 Celiac sensitizing antigen Phosphatidylserine (PS) ≥98% Macklin 51446-62-9 Surface-modified signal molecules DSPE-PEG2000 ≥98% Macklin 147867-65-0 Stability and surface coating materials Dimethyl sulfoxide (DMSO) Analytical Pure Sinopharm Group 67-68-5 organic solvents Anhydrous ethanol Analytical Pure Sinopharm Group 64-17-5 Aqueous solvent IR-780 ≥95% Shanghai McLean Biochemical Technology Co., Ltd. 207399-07-3 Far-infrared fluorescent markers Coumarin 6 ≥96% Shanghai TCI Chemical Industry Development Co., Ltd. 58336-35-9 fluorescent markers Oxamate ≥99% MCE 565-73-1 LDHA inhibitors

[0048] The main experimental equipment is shown in Table 2:

[0049] Table 2

[0050] name Model / Brand use Ultrasonic breaker JY92-IIDN (Ningbo Xinzhi) Emulsified phospholipid system Thermostatic magnetic stirrer IKA RCT Basic Nanoprecipitation stirring reaction Injection pump Harvard Apparatus 70-4500 Controlling the organic phase injection rate centrifuge Eppendorf 5810R Nanoparticle purification and washing Ultrafiltration centrifuge tubes Amicon Ultra-15 (MWCO 100 kDa, Millipore) Solvent removal and concentration Dynamic Light Scattering (DLS) Malvern Zetasizer Nano ZS90 Particle size and potential measurement Transmission electron microscopy (TEM) JEOL JEM-2100 Morphological observation Flow cytometer CytoFlex Cellular uptake and immunophenotyping analysis Small animal live imaging device Berto Company, USA Nanoscale organ distribution analysis

[0051] Example 1: Preparation of phosphatidylserine-modified gliadin nanoparticles (PS@PLGA-G)

[0052] 1. Organic phase preparation

[0053] Glycol protein (1 mg) and PLGA (5 mg) were dissolved together in 1 mL of dimethyl sulfoxide (DMSO) and vortexed for 1 min to obtain a homogeneous and transparent solution.

[0054] 2. Aqueous phase preparation

[0055] DSPE-PEG2000 and phosphatidylserine were added to a 4% (v / v) aqueous ethanol solution to prepare a phospholipid mixture at a mass ratio of 8:2.

[0056] • First, dissolve DSPE-PEG2000 in a 95% ethanol aqueous solution and preheat it in a 65°C water bath for 10 minutes to ensure complete dissolution;

[0057] • Dissolve phosphatidylserine in a chloroform:dimethyl sulfoxide mixture of 9:1 before slowly adding it to the aqueous phase described above;

[0058] • Emulsification was performed using an ultrasonic disruptor (400 W, 4 s / 4 s, 30 s total) to obtain a stable phospholipid emulsion.

[0059] 3. Nanoprecipitation process

[0060] Under magnetic stirring (500 rpm), the organic phase was uniformly injected into the lipid-containing aqueous phase using a syringe pump at a rate of 0.2 mL / min. Stirring was continued for 10 min to complete solvent displacement and structural stabilization.

[0061] 4. Purification and Collection

[0062] The nanoparticle suspension was transferred to an ultrafiltration centrifuge tube (100 kDa cutoff diameter) and centrifuged at 4,000 × g for 15 min. The supernatant was discarded, and the suspension was washed three times with deionized water. After purification, the suspension was concentrated to 1.5 mL and stored at 4°C.

[0063] 5. Feature Detection

[0064] • Particle size: 200.6 ± 3.6 nm (measured by DLS);

[0065] • Zeta potential: –26.89 ± 13.68 mV;

[0066] • Morphology: TEM showed that the particles were spherical, had smooth surfaces, and exhibited a distinct shell-core structure;

[0067] • Stability: No significant aggregation or particle size increase was observed after 7 days of storage at 4°C.

[0068] See Figure 1 for schematic diagrams of the nanoparticle structure and morphology characterization results.

[0069] Example 2: Therapeutic effect in a celiac disease animal model

[0070] 1. Laboratory animals

[0071] Female C57BL6 / J mice (6–8 weeks old, weighing 18–22 g) were selected and housed in an SPF environment.

[0072] 2. Modeling and Grouping

[0073] 2.1 Preparation of sensitizing reagents for celiac disease mouse model

[0074] (1) Preparation of antigen sensitization emulsion: 5 mg / mL Gliadin solution (solvent: DMSO) was diluted 2.5 times with PBS solution and mixed with complete Freund's adjuvant (CFA) at a ratio of 1:1; 5 mg / mL Gliadin solution was diluted 5 times with PBS solution and mixed with incomplete Freund's adjuvant (ICFA) at a ratio of 1:1. After thorough emulsification using an antigen emulsification device, two sensitization emulsions were obtained.

[0075] (2) Prepare a 125 mg / mL Gliadin gavage solution using 0.1 mol / L acetic acid solution as solvent, and perform ultrasonic disruption in an ice-water bath (600 s, 4 s / 4 s) to ensure that Gliadin is fully dissolved.

[0076] 2.2 Therapeutic drug administration model of celiac disease in mice

[0077] (1) Healthy eight-week-old female C57BL / 6J mice were housed in an SPF-grade barrier facility, five mice per cage, at a temperature of 22°C, following a 12-hour light-12-hour dark cycle. After one week of acclimatization, the mice were sensitized to gluten. They were grouped as follows, with five mice per group:

[0078] 1) Control: Normal control group;

[0079] 2) CeD: Celiac model group + physiological saline;

[0080] 3) PLGA-G: Celiac model group + PLGA-G treatment;

[0081] 4) PS@PLGA-G: Celiac model group + PS@PLGA-G treatment.

[0082] (2) On day 0, mice in the model group were subcutaneously injected with 100 μL of a sensitizing emulsion of Gliadin and CFA on both sides of their flanks, while mice in the control group were subcutaneously injected with an equal volume of PBS solution. On day 7, mice in the model group were subcutaneously injected with 100 μL of a sensitizing emulsion of Gliadin and ICFA on both sides of their flanks, while mice in the control group were injected with an equal volume of PBS solution. The procedure of day 0 was repeated on day 14.

[0083] (3) On day 21, the successful establishment of the delayed gluten hypersensitivity model was assessed by a paw edema test. First, the initial paw pad thickness of the mice was measured. Then, 25 μL of Gliadin solution (concentration 2.5 mg / mL) was injected parallel to the sole of the mouse paw using an insulin syringe. An equal volume of DMSO solution was injected into the contralateral paw. The paw pad thickness was measured again 24 hours later, and the degree of paw pad swelling was calculated.

[0084] (4) On days 22 and 29, the nano-concentrated solution was diluted with physiological saline to a final volume of 3 mL. 200 μL of nano solution was injected into the tail vein of mice in each nano-treatment group, and an equal volume of physiological saline was injected into the tail vein of the CeD group.

[0085] (5) Starting from day 35, mice in the model group and nanotherapy group were administered 0.2 mL of Gliadin solution (containing 25 mg of Gliadin) by gavage for two consecutive weeks, and their weight and mental status were recorded daily.

[0086] (6) The foot swelling test was repeated on day 49. The thickness of the foot pad was measured 24 hours later and analyzed and compared.

[0087] (7) On day 51, tissue samples were collected from all mice. Mice were first anesthetized with pentobarbital, and then blood was collected from the eyeballs. The collected blood samples were centrifuged at 3000 g for 15 min at 4°C, and the supernatant serum was collected. The entire intestinal segment was removed, and the length of the small intestine was measured. The duodenal segment was cut and frozen in liquid nitrogen, while the remaining portion was fixed with 4% paraformaldehyde. The histological morphology of the duodenum was observed using hematoxylin and eosin (H&E) staining, and the degree of villus atrophy was assessed.

[0088] 3. Results

[0089] • Body weight: The PS@PLGA-G group mice maintained a high body weight during gluten-enhanced gavage, similar to the normal control group, and significantly improved compared to the CeD model group;

[0090] • Foot swelling: Similar to the group with antigen-loaded nanoparticles (PLGA-G), the PS@PLGA-G group showed good gluten tolerance;

[0091] • Small intestine length: The small intestine length in the PS@PLGA-G group was significantly longer than that in the CeD model group;

[0092] • Histology: HE staining showed an increased villus / crypt ratio (V / C);

[0093] See Figure 2, which shows the improvement in celiac disease.

[0094] Example 3: Verification of the role and mechanism of nanoparticles in macrophages

[0095] 1. Cell materials and processing

[0096] Mouse macrophage cell line (Raw264.7) was cultured in DMEM medium containing 10% FBS, with PLGA-G and PS@PLGA-G (final concentration 10 μg / mL) added respectively, and incubated for 48 h, followed by LPS stimulation for 24 h to simulate an inflammatory environment. To inhibit intracellular lactate synthesis, after nano-co-culture and before LPS treatment, cells were co-cultured with 50 mmol / L Oxamate for 24 h.

[0097] 2. Cell phenotype detection

[0098] • Western blot results showed that the histone lactation signal was significantly enhanced in the PS@PLGA-G treatment group, and the histone lactation signal was weakened after lactate synthesis was inhibited.

[0099] • Flow cytometry showed an increased proportion of CD206⁺ M2 macrophages, which, after inhibition of lactate synthesis, showed an increase in CD206… + There was no significant difference in the proportion of M2 macrophages among the groups.

[0100] See Figure 3 for a schematic diagram of the mechanism of lactate-driven M2 polarization.

[0101] Example 4: Evaluation of biosafety and targeting

[0102] 1. Biological distribution

[0103] PS@PLGA-G was prepared by intravenous injection (0.2 mg / 20 g) into mice via the tail vein. Serum and vital organs (heart, liver, spleen, lung, and kidney) were collected 24 hours later and stained with hematoxylin and eosin (HE). ALT, AST, BUN, and Cr levels were all within the normal range, and no obvious pathological abnormalities were observed in the major organs (heart, liver, spleen, lung, and kidney).

[0104] 2. Biological Targeting

[0105] PS@PLGA-G containing the far-infrared fluorescent marker IR-780 was prepared and injected into mice via the tail vein (0.2 mg / 20 g). After 24 hours, vital organs (heart, liver, spleen, lung, and kidney) were collected and imaged using a small animal in vivo visible light imaging system (Ex / Em=745 / 840 nm). It was observed that PS@PLGA-G had a stronger fluorescence signal accumulation in the liver than PLGA-G, demonstrating the liver-targeting ability of PS@PLGA-G.

[0106] 3. Macrophage targeting

[0107] PS@PLGA-G and PLGA-G containing the fluorescent marker coumarin 6 were prepared. Mouse macrophages (Raw264.7) were cultured in DMEM medium containing 10% FBS. PLGA-G and PS@PLGA-G (final concentration 10 μg / mL) containing coumarin 6 were added and incubated for 24 h. The fluorescence intensity of each group was detected using a fluorescence microscope. It was observed that the fluorescence intensity of the PS@PLGA-G group was stronger than that of the PLGA-G group, indicating that macrophages had a stronger phagocytic efficiency for PS@PLGA-G.

[0108] See Figure 1 for the results on the biosafety and biotargeting of the nanoparticles.

[0109] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A phosphatidylserine-modified gliadin nanoparticle, characterized in that: The nanoparticles have a core-shell structure, with polylactic acid-glycolic acid copolymer encapsulating gliadin as the core, and are coated with a lipid composite membrane formed by phosphatidylserine and DSPE-PEG2000.

2. The phosphatidylserine-modified gliadin nanoparticles according to claim 1, characterized in that: The nanoparticles have a particle size of 180–220 nm and a surface potential of –25–35 mV.

3. The phosphatidylserine-modified gliadin nanoparticles according to claim 1, characterized in that: The mass ratio of phosphatidylserine to DSPE-PEG2000 is 1:(1-9), and the number average molecular weight of the polyethylene glycol segments in DSPE-PEG2000 is 2000 Da.

4. The phosphatidylserine-modified gliadin nanoparticles according to claim 1, characterized in that: The polylactic acid-glycolic acid copolymer has a lactic acid to glycolic acid ratio of 50:50 and a weight-average molecular weight of 30-50 kDa.

5. The phosphatidylserine-modified gliadin nanoparticles according to claim 1, characterized in that: The nanoparticles are encapsulated with a fluorescent marker, which is either IR-780 iodide or Courmarin 6.

6. A method for preparing phosphatidylserine-modified gliadin nanoparticles according to any one of claims 1 to 5, characterized in that... Includes the following steps: (1) Glycol protein and polylactic acid-glycolic acid copolymer are co-dissolved in dimethyl sulfoxide to form an organic phase; (2) Dissolve DSPE-PEG2000 and phosphatidylserine in a 4% (v / v) ethanol aqueous solution to form a lipid-containing aqueous phase; (3) Inject the organic phase into the lipid-containing aqueous phase under stirring conditions to allow the system to self-assemble into a nanoparticle suspension; (4) After centrifuging and washing the nanoparticle suspension, purified phosphatidylserine-modified gliadin nanoparticles were obtained.

7. The preparation method according to claim 6, characterized in that: The mass ratio of gliadin to polylactic acid-glycolic acid copolymer is 1:(5-9); the ratio of phosphatidylserine to DSPE-PEG2000 is 1:(1-9).

8. The preparation method according to claim 6, characterized in that: Step (2) specifically includes the following steps: (a) Phosphatidylserine was dissolved in a chloroform-dimethyl sulfoxide mixed solvent in a volume ratio of 9:1; (b) DSPE-PEG2000 was dissolved in a 95% (v / v) aqueous ethanol solution and preheated in a 65°C water bath for 10 min. (c) The DSPE-PEG2000 dissolved in steps (a) and (b) and phosphatidylserine were added to a 4% ethanol aqueous solution, ultrasonically emulsified and uniformly dispersed, and then nanoprecipitated.

9. The use of phosphatidylserine-modified gliadin nanoparticles according to any one of claims 1 to 5 in the preparation of medicaments for treating diseases caused by antigen-specific immune tolerance deficiency, characterized in that: Diseases caused by antigen-specific immune tolerance deficiency include celiac disease, inflammatory bowel disease, and multiple sclerosis.

10. A pharmaceutical composition for treating celiac disease, characterized in that: Including the phosphatidylserine-modified gliadin nanoparticles according to any one of claims 1 to 5.

Images