Pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a pharmaceutical composition as well as a preparation method and application thereof. The invention provides an anti-inflammatory pharmaceutical composition containing budesonide (derivative) and bortezomib, the pharmaceutical curative effect is remarkably improved by utilizing a synergistic effect, an accumulative effect and an enhancement effect caused by drug combination, the immune disorder microenvironment and the immune macroenvironment are improved at the same time, and when the anti-inflammatory pharmaceutical composition is used for anti-inflammatory treatment of various diseases, the anti-inflammatory effect is obviously improved. The traditional Chinese medicine composition has an excellent anti-inflammatory effect and can effectively delay relapse.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a pharmaceutical composition, its preparation method, and its application. Background Technology

[0002] Human and animal diseases (including infectious diseases, chronic non-infectious diseases, tumors, vascular diseases, and metabolic diseases) are often characterized by high morbidity, high disability rates, or high mortality rates, posing a serious threat to human health and socioeconomic development. Although existing treatments can achieve some efficacy, they remain limited at the level of fundamental treatment, suffering from slow disease control and frequent side effects, making it difficult to meet clinical needs. Therefore, finding new strategies is crucial. Currently, nano-drug delivery systems (NDDSs) such as liposomes, nanoemulsions, and lipid nanoparticles offer new opportunities for disease treatment due to their ability to improve drug distribution, enhance targeting, and reduce toxicity. To prolong the in vivo circulation time of NDDSs and increase targeted drug accumulation, researchers have focused on "polyethylene glycol (PEG) technology." Traditional PEGylated NDDSs, which avoid the clearance of nanoparticles by immune cells, have led to new challenges such as hindering target cell recognition and uptake, suppressing beneficial immune responses, inducing antibody production, and triggering allergic reactions. Therefore, when designing NDDSs for anti-inflammatory diseases (IDs), it is necessary to fully consider the interaction between formulation-related factors and the immune system. Only by establishing advanced NDDSs and gaining insight into the complex communication among members of the immune system can the progression of IDs be effectively curbed.

[0003] Research has found that the pathological processes of human diseases often involve immune dysregulation, including the uncontrolled aggregation and close cooperation of disease-associated immune cells (DAICs) (such as disease-associated dendritic cells, disease-associated T cells, disease-associated macrophages, and disease-associated neutrophils), and complex crosstalk between DAICs and disease-associated non-immune cells (DANICs) (such as disease-associated endothelial cells, disease-associated fibroblasts, and disease-associated stem cells). These cells do not exist independently but cooperate to build a tight "social network" in the bloodstream, lesion sites, and lymphoid tissues. This invention refers to these cells collectively as "disease-associated cell populations" (DACPs), including but not limited to "tumor-associated cell populations" (TACPs) and "inflammation-associated cell populations" (IACPs). DACPs embody the fundamental characteristics of a population: a relatively persistent and stable structure. It utilizes the interaction of multiple pathogenic elements to construct a complex compensation mechanism, maintain a pathogenic cascade reaction, and create a catastrophic disease environment.

[0004] Inflammation-associated cells (IACPs) possess a relatively persistent and stable structure. Through the interaction of various pro-inflammatory elements, they maintain a pro-inflammatory cascade response, becoming a major reason why many diseases are difficult to cure and frequently relapse. This mechanism makes inflammation play a crucial role in the pathogenesis of numerous diseases; almost all diseases are closely related to the inflammatory process. By deeply understanding the role of IACPs in different diseases, we can explore more effective treatment methods, providing new ideas and directions for disease prevention and treatment, thereby developing universal strategies for treating various diseases. Among the many individual members of IACPs, we selected key effectors of inflammation—inflammatory-associated dendritic cells, inflammatory-associated T cells, and inflammatory-associated macrophages—as typical representatives of IACPs. To design efficient and specific disease treatment strategies targeting IACPs, it is first necessary to identify the intrinsic commonalities among these individual members of inflammatory-associated cells.

[0005] Sialic acid (SA) is a nonacarbonic amino acid with an endogenous pyranose structure. It interacts with sialic acid-binding receptors (Siglecs / Selectins) on the cell surface to form the SA-Siglecs / Selectins axis. Studies have shown that inflammation-associated dendritic cells (DCs) and macrophages highly express Siglec-1; inflammation-associated T cells highly express Siglec-G; inflammation-associated neutrophils highly express L-selectin; and inflammation-associated endothelial cells highly express E-selectin. This highlights that the SA-Siglecs / Selectins axis may be a key target for intervention in intra-aqueous plasma coagulation (IACP) and comprehensive disease treatment. Therefore, based on the close relationship between disease, IACP, and the SA-Siglecs / Selectins axis, it is hoped that SA-mediated NDDSs (SA-NDDSs) can be used to target and intervene in IACP to treat diseases.

[0006] Glucocorticoids or their derivatives bind to glucocorticoid receptors to form the GC-GR complex, translocate into the nucleus, and directly bind to the p65 subunit of NF-κB, inhibiting NF-κB binding to DNA, blocking the NF-κB signaling pathway, and exerting anti-inflammatory and immunomodulatory effects. Boron peptide proteasome inhibitors bind to the 26S proteasome, inhibiting IκB ubiquitination and degradation, thereby inhibiting NF-κB signaling pathway activation and exerting anti-inflammatory and immunomodulatory effects. Furthermore, these drugs also inhibit the ubiquitin-proteasome pathway, causing the aggregation of unfolded or misfolded proteins, inducing endoplasmic reticulum stress, and leading to apoptosis. However, glucocorticoids (derived from) and boron peptide proteasome inhibitors are generally challenged in pharmaceutical applications by poor water solubility, low bioavailability, and low selectivity. In addition, clinical studies have confirmed many drugs that can exert anti-inflammatory and immunomodulatory effects; how to screen for anti-inflammatory drug combinations with synergistic effects has become one of the technical challenges that needs to be solved in this field. Summary of the Invention

[0007] To address the aforementioned technical challenges, this invention, through drug screening, proposes an anti-inflammatory drug composition with synergistic effects, and based on this, proposes the following technical solution.

[0008] In a first aspect, the present invention provides a pharmaceutical composition comprising: budesonide and bortezomib; or, a budesonide derivative and bortezomib.

[0009] This invention discovers that when budesonide or its derivatives are combined with bortezomib, the synergistic, additive, and enhancing effects of combined drug use can significantly improve drug efficacy. This drug composition exerts a synergistic anti-inflammatory effect by blocking the key pro-inflammatory NF-κB signaling pathway at two sites, reducing the dosage of single drugs and achieving a synergistic effect greater than the sum of its parts (1+1>2). Furthermore, the drug composition of this invention can simultaneously improve both the dysregulated immune microenvironment and the macro-immune environment.

[0010] Preferably, the budesonide derivative is esterified budesonide, more preferably budesonide palmitate.

[0011] Preferably, the molar ratio of budesonide (or its derivatives) to bortezomib is (0.25–4):1, more preferably (0.3–2):1, and even more preferably (0.4–0.6):1.

[0012] With the above ratio, the synergistic anti-inflammatory effect of the two is even better.

[0013] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients or carriers.

[0014] In some embodiments, the pharmaceutical composition is a solid dosage form, a semi-solid dosage form, or a liquid dosage form.

[0015] In some embodiments, the pharmaceutical composition is a nanoformulation; more preferably, the nanoformulation is a phospholipid complex nanoparticle, nanoliposome, nanosphere, nanocapsule, or polymer micelle.

[0016] Preferably, the pharmaceutical composition is a phospholipid complex nanoparticle, wherein the phospholipid is a natural, semi-synthetic, or fully synthetic phospholipid.

[0017] Phospholipid complex nanoparticles can improve the physicochemical properties of drugs, meet clinical needs, and can be administered via multiple routes such as intravenous, oral, and intranasal administration.

[0018] More preferably, the particle size of the phospholipid complex nanoparticles is 50-80 nm, and more preferably 50-70 nm, 50-60 nm, or 52-58 nm.

[0019] Preferably, the phospholipid is at least one of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and myocardial phospholipid; more preferably, it is at least one of egg yolk phosphatidylglycerol, hydrogenated egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, distearyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and dilauroyl phosphatidylglycerol; and more preferably, it is egg yolk phosphatidylglycerol (EPG).

[0020] The structural formula of egg yolk phosphatidylglycerol (EPG) is:

[0021] Preferably, the molar ratio of drug to phospholipid in the phospholipid complex nanoparticles is 1:(1-5); more preferably, it is 1:(1-3).

[0022] Preferably, the molar ratio of budesonide or its derivatives, bortezomib, and phospholipids is 1:(1.5-2.5):(3.5-5).

[0023] More preferably, the molar ratio of budesonide or its derivatives, bortezomib to phospholipids is 1:(1.5-2.5):(3.5-4.5).

[0024] When the above ratio is used, the phospholipid complex nanoparticles have a small particle size, narrow particle size distribution range, good dispersibility and high drug loading.

[0025] In some embodiments, the phospholipid complex nanoparticles further include sialic acid lipid derivatives; the sialic acid lipid derivatives are formed by coupling sialic acid with lipid fragments.

[0026] By using amphiphilic sialic acid lipid derivatives as modifying ligands, it is possible to target inflammation-associated dendritic cells, inflammation-associated T cells, and inflammation-associated macrophages. Compared with drug composition nanoformulations without sialic acid lipid derivatives, drug composition nanoformulations modified with sialic acid lipid derivatives showed a 2-3 fold increase in targeting efficiency against inflammation-associated cells.

[0027] Preferably, the sialic acid lipid derivative is a lipid fragment coupled to the carboxyl group at the 1-position or the hydroxyl group at the 9-position of sialic acid, wherein the lipid fragment is cholesterol, fatty acid, or a derivative thereof.

[0028] Preferably, the sialic acid and lipid fragments are linked by an amide bond or a carboxyl ester bond.

[0029] Preferably, the general formula of the sialic acid lipid derivative is shown in Formula I or Formula II: In Formula I, R1 is a H atom or a C1-C6 alkyl group; R2 is H or (CH2) m C2-C6 alkenyl, m = 1-17; R4 is -OH, -NHCOCH3, or -NHCOCH2OH; X is a H atom, an O atom, or a carbonyl group; When X is a H atom or an oxygen atom, R3 is (CH2)2 or a cholesterol group, n = 1 to 17; When X is a carbonyl group, R3 is a C12-C24 alkoxy group, a C12-C24 alkyl-substituted amino group, or a cholesterol group; In Equation II, R1 is -S-(CH2). n -or -SS-(CH2) n -, n = 1 to 6; R2 is -OH, -NHCOCH3, or -NHCOCH2OH; R3 is a C12–C24 alkoxy group, a C12–C24 alkyl-substituted amino group, or a cholesterol group.

[0030] Preferably, the molar amount of the sialic acid lipid derivative accounts for less than 30% of the total molar amount of the sialic acid lipid derivative and phospholipid, and more preferably 20% to 30%.

[0031] Preferably, the sialic acid lipid derivative is a sialic acid lipid derivative coupled with cholesterol or other derivatives; more preferably, the sialic acid lipid derivative is a sialic acid lipid derivative coupled with a double-chain fatty acid; even more preferably, the sialic acid lipid derivative is a sialic acid lipid derivative coupled with a single-chain fatty acid.

[0032] Preferably, the sialic acid lipid derivative is a sialic acid-cholesterol conjugate (SA-CH), with the following chemical formula:

[0033] The preparation method is described in Example 16 of CN 106188169 A.

[0034] Compared with other ligand modifications such as mannose and folic acid, sialic acid lipid derivatives have the following advantages: (i) Broad targeting effect: Based on the sialic acid-Siglecs / Selectin axis, the drug composition nanoformulation modified with sialic acid lipid derivatives can target a variety of inflammation-related cells, achieving comprehensive regulation of the "inflammation-related cell population"; (ii) Good biocompatibility and safety: Sialic acid is non-immunogenic. Modifying the surface of pharmaceutical nanoparticles with sialic acid at an appropriate density can improve the blood circulation behavior of the nanoparticles and reduce the body's clearance of the drug. Furthermore, it does not induce specific adverse immune responses or antibody production, demonstrating safety. (iii) By regulating inflammation-related cell populations, it has been preferentially verified that it can induce “strong memory immune tolerance” by assisting drug composition nano-formulations, and has excellent therapeutic effects and clinical application value for inflammation-related diseases.

[0035] In some embodiments, the phospholipid complex nanoparticles further include PEG lipid derivatives; the PEG lipid derivatives are formed by coupling PEG with lipid fragments.

[0036] Preferably, the PEG lipid derivative contains 1 to 2 PEG chains with a molecular weight of 200 to 20,000 Da.

[0037] Preferably, the PEG lipid derivative is a breakable PEG lipid derivative.

[0038] Preferably, the cleavable chemical bonds in the cleavable PEG lipid derivative are degradable or broken under any of the following conditions: light, heat, enzymes, acidity, redox, physiological conditions, or special pathological conditions.

[0039] Preferably, the cleavable chemical bonds of the cleavable PEG lipid derivative are at least one of the following: carboxylic acid ester bond, carbonate bond, phosphate ester bond, phosphonate bond, orthoester bond, thioester bond, thioester bond, dithioester bond, carbonate bond, thiocarbonate bond, dithiocarbonate bond, trithiocarbonate bond, carbamate bond, thiocarbamate bond, dithiocarbamate bond, sulfonate bond, sulfinate bond, urea carbamate bond, thiourea carbamate bond, hydrazine thiocarbamate bond, disulfide bond, vinyl ether bond, hydrazone bond, acylhydrazone bond, oxime bond, thiooxime ether bond, acetal, hemiacetal Aldehydes, cyclic acetals, azaacetals, heterocyclic acetals, dithioacetals, thiohemiacetals, ketals, heterocyclic ketals, imines, maleimides, benzoimides, hydrazines, acylhydrazines, thiocarbonylhydrazines, azocarbonylhydrazines, thioazocarbonylhydrazines, azo, guanidine, amidine, aminoguanidine, aminoamidinyl, imine, sulfonylhydrazines, sulfonylurea, phosphite, phosphonate, thioamide, sulfonamide, polyamide, phosphoramide, phosphorous acid, pyrophosphoramide, thiophosphoramide, polypeptide fragments, nucleotide (derivative) backbones, deoxynucleotide (derivative) backbones.

[0040] More preferably, the cleavable chemical bonds in the cleavable PEG lipid derivative are esterase-sensitive chemical bonds (e.g., chemical bonds sensitive to carboxylesterase or carbonate esterase).

[0041] In some embodiments, the breakable PEG lipid derivative has the following general structural formula:

[0042] In the formula, R is a C12-C24 alkoxy or cholesterol group, X1 is a cleavable chemical bond, and R2 is the functional end of PEG.

[0043] Preferably, the cleavable PEG lipid derivative is mPEGnCHEMS; Wherein, n represents the molecular weight of PEG, and n is 200 to 20000. mPEGnCHEMS is a compound with a molecular weight of PEG or a combination of multiple PEG molecular weight compounds.

[0044] Preferably, the breakable PEG lipid derivative is a polyethylene glycol monomethyl ether and cholesterol carbonate coupling compound (methoxy polyethylene glycol). 2000 -cholesterol hemisuccinate, mPEG 2000 CHEMS, mPEG 2000 CHE). mPEG 2000 CHEMS(mPEG 2000 The chemical structural formula of CHE is:

[0045] mPEG 2000 The preparation method of CHEMS can be found in mPEG in CN117159492A. 2000 CHE was prepared using a synthetic method.

[0046] This invention unexpectedly discovered that selecting mPEG n CHEMS(mPEG n When CHE, as a cleavable PEG lipid derivative, is combined with SA-CH co-modified phospholipid complex nanoparticles, it can prolong the blood circulation time of the phospholipid complex nanoparticles, even exceeding that of indestructible PEG lipid derivatives (such as mPEG). 2000 -DSPE) This reverses the previous understanding in the field that breakable PEG lipid derivatives can be broken by ester bonds in the bloodstream, resulting in a shorter blood circulation time than non-breakable PEG lipid derivatives.

[0047] Moreover, choosing mPEG n When CHEMS, as a cleavable PEG lipid derivative, is combined with SA-CH co-modified phospholipid complex nanoparticles, it helps reduce the excessive production of PEG-specific IgM. mPEG n When CHEMS and SA-CH are co-modified into phospholipid complex nanoparticles, mPEG 2000 -CHEMS can effectively reduce the formation of protein corona and promote the high accumulation of nanomedicines at the disease site, further avoiding damage to the healthy immune system caused by the drugs. Meanwhile, mPEG... 2000 -CHEMS can respond to CES at the disease site, effectively expose SA, and promote the targeted regulation of drugs on inflammation-associated immune cells (IAICs), thereby effectively inhibiting disease recurrence.

[0048] Preferably, the molecular weight of the PEG lipid derivative is 200 to 20000 Da.

[0049] Preferably, the molar amount of the breakable PEG lipid derivative accounts for 0.1% to 10% of the total molar amount of the breakable PEG lipid derivative and phospholipid; more preferably, it is 6% to 10%, and even more preferably, it is 6% to 8%.

[0050] The mPEG provided by this invention n CHEMS and SA-CH co-modified phospholipid complex nanoparticles can improve the blood circulation behavior of nanomedicines and deliver drugs to inflammation-related immune cells with high selectivity. They exert a triple therapeutic mechanism of immune depletion, immune regulation and anti-inflammation, promote the restoration of immune homeostasis, have excellent long-term efficacy in inflammation-related diseases and delay recurrence, and have versatility and clinical application value.

[0051] In some embodiments, the pharmaceutical composition is a liposome; the liposome has budesonide or a derivative thereof loaded in a phospholipid bilayer and a borate ester compound prepared from bortezomib and a polyol loaded in an aqueous phase.

[0052] Preferably, the polyol is a compound having cis-1,2-diol or 1,3-diol functionality.

[0053] Preferably, the polyol is at least one of the following: polyvinyl alcohol, catechin, monosaccharide (e.g., maltose, glucose, ribose, fructose or sorbitol), disaccharide, oligosaccharide, or polysaccharide.

[0054] Preferably, the polyol is glycerol, polyglycerol, or aminopolyol aminosorbitol.

[0055] In some embodiments, the liposomes have a high internal / low external ion gradient.

[0056] Preferably, the ion gradient is a hydrogen ion gradient (pH gradient), the pH inside the liposome is 7.5 to 8.5, and the pH outside the liposome is 6.0 to 7.0.

[0057] In a second aspect, the present invention provides a method for preparing the pharmaceutical composition in any of the above embodiments, comprising: The raw material is dissolved in an organic solvent and stirred at a temperature of 20°C to 70°C to remove the organic solvent, thereby obtaining the pharmaceutical composition.

[0058] Preferably, the preparation method includes: dissolving the raw material in an organic solvent, stirring at a temperature of 20°C to 70°C, removing the organic solvent, adding water, and filtering through a filter membrane to obtain the pharmaceutical composition.

[0059] Preferably, the material is filtered sequentially through filter membranes of 0.80μm, 0.45μm, and 0.22μm.

[0060] Preferably, the volume-to-mass ratio of water to drug is 1:1 to 5:1 in mL / mg.

[0061] Preferably, the preparation method includes: dissolving budesonide or its derivatives, bortezomib, phospholipids, sialic acid lipid derivatives and PEG lipid derivatives in an organic solvent, stirring at a temperature of 20°C to 70°C, refluxing in a closed system, removing the organic solvent, adding water, and filtering through a filter membrane to obtain the phospholipid complex nanoparticles; The concentration of budesonide or its derivative in the organic solvent is 3.5 mg·mL. -1 Hereinafter, the concentration of bortezomib in the organic solvent is 6 mg·mL. -1 the following.

[0062] In the above preparation method, the stirring temperature is preferably 20℃~65℃, 30℃~65℃, 40℃~65℃ or 50℃~65℃.

[0063] In the above preparation method, the stirring time is preferably 10-60 min, 20-60 min, 20-50 min, 20-40 min, 20-30 min, or 20-25 min.

[0064] Preferably, the preparation method includes: (1) Budesonide or its derivatives and liposome membrane material are dissolved in an organic solvent to obtain a membrane material solution. The organic solvent is removed to obtain a membrane material concentrate. The membrane material concentrate is mixed with a gradient substance solution to obtain a blank liposome initial product. (2) The blank liposome sample is extruded through a filter membrane to obtain a liposome suspension; (3) Remove the gradient-establishing substances from the aqueous phase outside the liposome suspension to obtain a gradient liposome suspension; (4) The drug composition (liposomes) is prepared by mixing the gradient liposome suspension with bortezomib.

[0065] More preferably, the preparation method includes: (1) Budesonide or its derivatives and liposome membrane material are dissolved in an organic solvent to obtain a membrane material solution. The organic solvent is removed to obtain a membrane material concentrate. The membrane material concentrate is mixed with a gradient substance solution to obtain a blank liposome initial product. (2) The blank liposome sample is extruded through a filter membrane to obtain a liposome suspension; (3) Remove gradient-building substances from the aqueous phase outside the liposome suspension using at least one technique, such as percolation, dialysis, centrifugation, size exclusion chromatography, or ion exchange, to obtain a gradient liposome suspension. Adjust the pH of the aqueous phase outside the gradient liposome suspension to below 7.0. (4) Mix the gradient liposome suspension with bortezomib and then perform the reaction at the principal phase transition temperature T of the liposome lipids. m The drug composition (liposomes) was prepared by incubation at the above temperature.

[0066] Preferably, the incubation temperature is 55℃~70℃, more preferably 60~70℃.

[0067] Preferably, the incubation time is 20 min, 30 min, 40 min, 50 min, or 60 min or more, and can also be up to 12 hours or more. The incubation time depends on the incubation temperature.

[0068] Preferably, after incubation, free (unencapsulated) bortezomib is removed by at least one technique, such as percolation, dialysis, centrifugation, size exclusion chromatography, or ion exchange.

[0069] Preferably, the gradient material comprises a polyol, and more preferably polyvinyl alcohol.

[0070] Thirdly, the present invention provides the use of the pharmaceutical composition or preparation method in any of the above embodiments in the preparation of anti-inflammatory drugs.

[0071] Preferably, the anti-inflammatory drug is used to treat at least one of the following diseases: Infectious or contagious diseases, autoimmune diseases, cancer, vascular diseases, and metabolic diseases.

[0072] The infectious or contagious diseases mentioned include, but are not limited to, acquired immunodeficiency syndrome (AIDS), viral pneumonia, novel coronavirus infection (SARS-CoV-2), severe acute respiratory syndrome (SARS), influenza, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes zoster, herpes simplex, herpes simplex type I, herpes simplex type II, smallpox, chickenpox, monkeypox, diphtheria, human papillomavirus (HPV) infection, Helicobacter pylori infection, plague, rabies, psittacosis, Ebola hemorrhagic fever, amoebiasis, Marburg virus, measles, tuberculosis, syphilis, tetanus, scarlet fever, leishmaniasis, typhoid fever, typhoid fever, rubella, and whooping cough. Cough), Pfeiffer fever, astrovirus infection, cholera, Acinetobacter homanis infection, anthrax, cytomegalovirus infection, dengue fever, aspergillosis, leprosy, malaria, mycoplasma pneumonia, ascariasis, babesiosis, giardiasis, schistosomiasis, shellfish ascariasis, human budema infection, black hair nodule disease, filariasis, dracunculiasis, liver fluke disease, ginger fascioliasis, cysticercosis, coccidioidomycosis, cutaneous larval migration (CLM), fox tapeworm, hookworm infection, paragonimiasis. Hymenolepis taeniasis, coccidiosis, toxoplasmosis, trichinosis, trichomoniasis, whipworm infection, Tripper, trypanosomiasis (sleeping sickness), scrub typhus, strongyloides infection, tapeworm infection, echinococcosis, pinworm infection, schistosomiasis, tinea capitis, tinea corporis, jock itch, tinea manuum and tinea pedis, tinea pedis, onychomycosis, pityriasis versicolor, tinea leukoplakia, rickets Hypoallergenic infection, Enriquezosis, Chlamydia trachomatis infection, Chlamydia pneumoniae infection, Botulinum toxin poisoning (and infantile botulism), Clostridium perfringens food poisoning, epidemic typhus, Epstein-Barr virus infectious mononucleosis, mumps, murine typhus (endemic typhus), sepsis, poliomyelitis, pertussis, yeast fungal spots, yellow fever, meningitis, Salmonella paratyphus, Salmonella typhus, salmonellosis, human bocavirus infection, hand-foot-mouth disease (HFMD). Hantavirus pulmonary syndrome (HPS), hemolytic uremic syndrome (HUS), leptospirosis, Listeria, Lyme spirochete infection, Lyme disease, lymphangiofilariasis (elephantiasis), lymphocytic choroid plexus meningitis, Marburg hemorrhagic fever (MHF), melioidosis (Whitmore disease), meningitis, conjunctivitis, keratitis, meningococcal infection, hemolytic bacillus infection, Staphylococcus aureus infection, Shigella infection (bacterial dysentery), condyloma acuminata, dermatophytosis, cutaneous leishmaniasis.

[0073] The autoimmune diseases (AIDs) mentioned include, but are not limited to, rheumatoid arthritis (RA), lupus nephritis, polymyositis rheumatoid arthritis, myasthenia gravis, autoimmune thyroid diseases (including thyroid autoimmune diseases and Graves' disease), autoimmune liver diseases (including autoimmune hepatitis and primary biliary cholangitis), rheumatoid pneumonia, autoimmune neuritis (Guillain-Barré syndrome), autoimmune ulcerative colitis (UC), rheumatoid lupus erythematosus, autoimmune gonaditis, thyroid nodular ophthalmopathy, autoimmune thrombocytopenic purpura, rheumatoid nephritis, and autoimmune lichen lupus. Rheumatic encephalomyelitis, autoimmune thyroiditis, ulcerative stomatitis, autoimmune pancreatitis, autoimmune encephalitis, rheumatoid pericarditis, systemic lupus erythematosus (SLE), autoimmune tubulointerstitial nephritis, autoimmune gonaditis, antiphospholipid antibody syndrome, dermatomyositis, autoimmune otitis media, Parkinson's disease, autoimmune myocarditis, rheumatoid cerebral arteritis, autoimmune myopathy, autoimmune eye disease, acute disseminated encephalomyelitis, autoimmune small vessel vasculitis, autoimmune diabetes mellitus, autoimmune insulin resistance, autoimmune thymic disease, autoimmune osteomyelitis, autoimmune skin disease, autoimmune nephritis.

[0074] The cancers mentioned include, but are not limited to, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, myelodysplastic syndrome, macroglobulinemia, chronic myelodysplastic disorder; lymphoma, AIDS-related lymphoma, central nervous system lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma; brain tumors, cerebellar astrocytoma, brainstem glioma, intracranial germ cell tumors, meningioma, acoustic neuroma, ependymoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, medulloblastoma, intracranial epidermoid cysts; ocular cancer, intraocular melanoma, retinoblastoma; nasal cavity and sinus cancer, nasopharyngeal carcinoma, sinus and nasal cavity cancer; oral cancer, lip cancer; laryngeal cancer; lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, basal cell lung cancer, small cell lung cancer, basal cell carcinoma. Cellular carcinoma, bronchial adenoma / carcinoid; sarcoma; Kaposi's sarcoma, osteosarcoma, leiomyosarcoma, lymphosarcoma, synovial sarcoma, Ewing's sarcoma, rhabdomyosarcoma, soft tissue sarcoma; bile duct carcinoma, gallbladder carcinoma; liver cancer, primary hepatocellular carcinoma; secondary liver metastases; esophageal cancer; bone cancer, malignant fibrous histiocytoma; breast cancer; gastric cancer; colorectal cancer; small bowel cancer; endometrial cancer; ovarian cancer, ovarian epithelial cancer; ovarian germ cell tumors, low-potential ovarian tumors; cervical cancer; neuroblastoma; skin cancer, Sezary syndrome, cutaneous melanoma, cutaneous Merkel cell carcinoma; pancreatic cancer, islet cells; parathyroid carcinoma; penile cancer; pheochromocytoma; bladder cancer; adrenal cortex; islet cells; germ cell tumors, gonadal extragerminal tumors, ovary; gestational trophoblastic tumors; kidney cancer; ureteral and renal pelvis transitional cell carcinoma; urethral cancer; vaginal cancer; testicular cancer; thymoma; thyroid cancer.

[0075] The vascular-related diseases mentioned include, but are not limited to, coronary heart disease, hypertension, atherosclerosis, stroke, myocardial infarction, aneurysm, varicose veins, thrombosis, vasculitis, vascular malformations, hypercholesterolemia, venous thromboembolism, rheumatic fever, cardiomyopathy, congenital cardiovascular malformations, hyperlipidemia, arterial stenosis, arrhythmia, vascular dementia, thrombotic thrombocytopenic purpura, thrombotic microangiopathy, vascular headache, hypertensive encephalopathy, aneurysm, angioedema, vascular deafness, vascular retinopathy, vascular infarction, angioedema, vascular anaphylactic shock, vasospasm, vascular skin lesions, vascular nephropathy, vascular pain, vascular dyspnea, vascular anemia, vascular brain injury, vascular cardiomyopathy, vascular liver disease, and vascular bowel disease.

[0076] The metabolic-related diseases mentioned include, but are not limited to, diabetes, hyperlipidemia, obesity, metabolic syndrome, insulin resistance syndrome, hepatic steatosis, hyperuricemia, hypothyroidism, hyperthyroidism, insulin-resistant polycystic ovary syndrome, hyperinsulinemia, osteoporosis, hyperuricemia, gout, malnutrition, hyperinsulinemia, malignant hypercalcemia, primary hyperparathyroidism, primary aldosteronism, fatty liver, hyperuricemia, hyperinsulinemia, metabolic acidosis, metabolic alkalosis, metabolic bone disease, metabolic headache, metabolic fatigue syndrome, metabolic sexual dysfunction, and metabolic skin diseases.

[0077] More preferably, the anti-inflammatory drug is used to treat arthritis or ulcerative colitis.

[0078] This invention summarizes the interactions of key autoimmune elements and proposes for the first time the concept of the autoimmune cycle (AIC). In the AIC process, the information transmission and interaction among members of the body's immune system are crucial. For example... Figure 1 As shown, firstly, an imbalance of cytokines and / or the failure of dendritic cells (DCs) to respond correctly to signals leads to DC dysfunction①. This causes them to present self-antigens to Th precursor cells (Th0), transmitting autoimmune signals②. Subsequently, effector T cells (Teff, such as Th1 / Th17, etc.) are activated, causing the balance between Teff and regulatory T cells (Treg) to tilt towards Teff. Teff-mediated B cell activation③ is accompanied by the production of autoantibodies④ and the formation of immune complexes⑤, which in turn activates complement⑥ and effector cells (such as macrophages and NEs, etc.)⑦, promoting DC dysfunction①, thereby forming a positive immune chain reaction and initiating AIC. Simultaneously, the development of Teff is accompanied by the release of pro-inflammatory cytokines (such as TNFα, IL-1β, IL-17A, and IFN-γ) and chemokines, as well as subsequent DC dysfunction① and the recruitment and activation of effector cells (such as monocytes / macrophages and NEs, etc.)⑦. The latter further promotes DC dysfunction①. In summary, this series of processes constitutes a positive feedback loop, forming an AIC, which leads the immune system to launch a continuous attack on its own tissues, forming an abnormal immune response and driving the continuous progression of AIDs. The proposal of AIC provides important clues and scientific basis for developing more effective treatment strategies and interventions.

[0079] This invention, based on the heterogeneity and holistic nature of the immune system, proposes for the first time the concept of a memory immune tolerance cycle (MITC). In contrast to AIC, MITC is a key regulatory mechanism in the immune system, characterized by its ability to avoid attacking normal tissues through the formation, stability, and renewal of peripheral immune tolerance and regulatory immune memory. Figure 2 As shown, the MITC process involves effectively induced tolerogenic dendritic cells (tolDCs)① which, by transmitting immune tolerance information, induce the production of regulatory immune cells, such as Tregs and regulatory B cells (Bregs), while memory regulatory T cells (mTregs) differentiate under the mediation of tolDCs②. Further, tolDC-induced regulatory immune cells stimulate tolDC production① by producing tolerance cytokines, such as IL-10 and TGF-β, forming a tolerance-inducing microcirculation. Simultaneously, regulatory immune cells inhibit Teff differentiation by producing tolerance cytokines③. Furthermore, the activation of autoreactive B cells and the production of self-antigen-specific IgG antibodies④, along with a series of downstream immune responses (such as the formation of immune complexes⑤ and complement activation⑥), are all suppressed. This suppression of the series of responses is closely linked to the tilt of macrophages towards the M2 phenotype⑦. Furthermore, Teff differentiation inhibition③ also contributes to the tilt of macrophages towards the M2 phenotype⑦, accompanied by a tilt of cytokines towards tolerance cytokines, which also helps induce the production of tolDCs①. Thus, the above steps collectively construct a complex and coordinated MITIC, a regulatory mechanism crucial for maintaining the homeostasis of the immune system and helping to prevent inflammatory damage caused by excessive immune activity. Upon re-exposure to the same self-antigen, the memory characteristics of regulatory immune cells (such as mTregs) (including specificity, persistence, rapid responsiveness, and enhancing effects) cause them to specifically proliferate, producing a more rapid and intense response, suppressing secondary inflammatory responses, and delaying / inhibiting the recurrence of AIDs. In summary, MTIC, by regulating the immune system's response and maintaining immune homeostasis, helps to prevent tissue damage caused by excessive immune activity in the long term, demonstrating the diversity of the immune system.

[0080] Furthermore, this invention proposes that immune tolerance can be divided into "strong memory immune tolerance", "medium memory immune tolerance" and "weak memory immune tolerance".

[0081] "Strong memory immune tolerance" is a stable and potent regulatory acquired immune response, reflecting memory-based immune tolerance established through appropriate treatment. It manifests in long-term efficacy against a disease, excellent prognosis, delayed / suppressed relapse, and even disease cure. "Medium memory immune tolerance" represents a variable type of memory, falling between "weak memory immune tolerance" and "strong memory immune tolerance." "Weak memory immune tolerance" represents a harmful type of memory, where the immune system may not exhibit memory function or even show unfriendly effects in complex environments.

[0082] This method of classifying memory-type immune tolerance into strong, medium, and weak categories represents a significant breakthrough in researchers' understanding of the immune system's response mechanisms and the relationship between nanomedicine delivery systems and immunity. It expands the boundaries of knowledge in the field of immunology and holds promise for playing a key role in designing more effective treatment strategies.

[0083] The phospholipid complex nanoparticles provided by this invention exhibit excellent targeting of inflammation-related immune cells (especially inflammation-related dendritic cells, inflammation-related T cells, and inflammation-related macrophages) in the inflammatory microenvironment mediated by the sialic acid-Siglecs / Selectins axis. Furthermore, through a triple therapeutic mechanism of immune exhaustion, immunomodulation, and anti-inflammation, they block the autoimmune cycle (AIC), initiate a memory-based immune tolerance cycle (MITC), and establish "strong memory-based immune tolerance," demonstrating excellent long-term efficacy in treating inflammation-related diseases and delaying relapse. This confirms their versatility and clinical application value in the treatment of autoimmune diseases.

[0084] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides an anti-inflammatory drug composition comprising budesonide (derived from) and bortezomib, which significantly improves drug efficacy by utilizing the synergistic, additive, and enhancing effects of combined drug administration. Simultaneously, budesonide-bortezomib phospholipid complex nanoparticles co-modified with sialic acid and a breakable PEG lipid derivative were prepared. These nanoparticles improve the blood circulation behavior of the nanomedicine and selectively deliver the drug to inflammation-associated immune cells, exerting a triple therapeutic mechanism of immune depletion, immunomodulation, and anti-inflammation. This blocks the autoimmune cycle, initiates a memory-type immune tolerance cycle, and functions as a "non-antigen-loaded vaccine," stimulating a stable and potent regulatory acquired immune response similar to a vaccine, inducing "strong memory-type immune tolerance," and demonstrating excellent long-term efficacy in inflammation-related diseases and delaying recurrence. It possesses versatility and clinical application value, providing a new approach for immunotherapy of inflammatory diseases. This invention compares the therapeutic effects of budesonide-bortezomib phospholipid complex nanoparticles co-modified with sialic acid and fractured PEG lipid derivatives on inflammatory diseases such as rheumatoid arthritis with the clinical drug adalimumab (anti-TNF-α antibody). Unlike the rapid relapse after discontinuation of anti-TNF-α drugs, BUD@BTZ PcSNPs exhibit better long-term efficacy against rheumatoid arthritis, potentially improving the current situation of "treating the symptoms but not the root cause" in the clinical treatment of AIDs. This invention proposes the concepts of autoimmune cycle (AIC) and memory immune tolerance cycle (MITC), providing a new perspective for elucidating the pathogenesis of AIDs and optimizing treatment. Furthermore, by proposing a method to classify memory immune tolerance into strong, moderate, and weak categories, this invention represents a significant breakthrough in researchers' understanding of the immune system response mechanisms and the relationship between nanomedicine delivery systems and immunity, expanding the boundaries of knowledge in the field of immunology. This concept is expected to play a key role in designing more effective treatment strategies. Attached Figure Description

[0085] Figure 1 This is a schematic diagram of the autoimmune cycle.

[0086] Figure 2 This is a schematic diagram of memory-type immune tolerance.

[0087] Figure 3 To screen the optimal synergistic dose ratio (n / n) of BUD and BTZ using enzyme-linked immunosorbent assay (ELISA); A, B, and C represent the BMDCs (A), CD4+, and CD4+ of different formulations after 12 hours of treatment, respectively. + Levels of TNF-α released by T cells (B) and BMDMs (C).

[0088] Figure 4 Incubation of different DiI-labeled phospholipid complex nanoparticles to activate BMDCs(A) and CD4 + Flow cytometry results of T cells (B) and BMDMs (C).

[0089] Figure 5 To activate BMDCs(A), CD4 + T cells (B) and BMDMs (C) exposed to BUD@BTZ NPs DiI BUD@BTZSNPs DiI BUD@BTZ PdSNPs DiI and BUD@BTZ PcSNPs DiI (with or without 40 U·L) -1 Confocal laser scanning microscopy image of carboxylesterase (CES) after 2 hours, scale bar = 20 μm.

[0090] Figure 6 For BUD@BTZ NPs DiR BUD@BTZ SNPs DiR BUD@BTZ PdSNPs DiR and BUD@BTZ PcSNPs DiR The concentration of DiR in plasma after injection into Wistar rats.

[0091] Figure 7 To detect the serum anti-PEG IgM titer of each group using the DAC-ELISA method; A is the fitting curve of serum dilution ratio Log10 value and optical density value; B is the summary data of serum antibody logEC50 of each group.

[0092] Figure 8 To evaluate the therapeutic effects of different formulations based on joint scores; A is the joint score-time curve; B is the experimental endpoint mouse joint score.

[0093] Figure 9 To evaluate the therapeutic effects of different formulations based on foot volume; A is the foot volume-time curve; B is the foot volume of mice at the experimental endpoint.

[0094] Figure 10 The body weight (A) and swelling inhibition index (SI) of CIA mice were compared with those of different formulations. index (B) curve and pharmacodynamic endpoint (SI) index (C is a statistical chart, and D is a statistical table).

[0095] Figure 11 CD11c in the inflamed ankle joint + DCs(A), CD3 + CD4 + T cells (B) and CD11b + F4 / 80 + The percentage of macrophages (C).

[0096] Figure 12 CD11c in inflammatory joints + CD86 + DCs(A) and CD11c + CD80 + The percentage of DCs(B).

[0097] Figure 13 The percentages of Th1(A) and Th17(B) in inflammatory joints.

[0098] Figure 14 To detect the mRNA levels of CD86, TNF-α, iNOS, CD206, Arg-1, and CD163 in inflamed joint tissues of CIA mice using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR).

[0099] Figure 15 The percentage of Tregs in the draining lymph nodes of CIA mice treated with different phospholipid complex nanoparticles.

[0100] Figure 16 The titers of serum anti-CII IgG and the subtype IgG2a level in mice treated with different phospholipid complex nanoparticles are shown in Figure 1; A and C are the fitting curves of serum dilution ratio Log10 value and optical density value; B and D are the summary data of serum antibody logEC50 for each group.

[0101] Figure 17 Response of disease-remission mice to type II collagen rechallenge: CIA incidence statistics.

[0102] Figure 18 The response of disease-remission mice to type II collagen rechallenge: changes in joint score (A); changes in paw volume (B).

[0103] Figure 19 For each treatment group, Treg(CD4) was re-exposed to the antigen. + FoxP3 + The proportion of ).

[0104] Figure 20 To evaluate the therapeutic effects of anti-TNF-α and BUD@BTZ PcSNPs based on joint scores; (A) Joint score-time curve; (B) Joint score at the experimental endpoint.

[0105] Figure 21 To evaluate the therapeutic effects of anti-TNF-α and BUD@BTZ PcSNPs based on foot volume; (A) Foot volume-time curve; (B) Foot volume as the experimental endpoint.

[0106] Figure 22The relative body weights of mice in the healthy group, model group, anti-TNF-α group, and BUD@BTZ PcSNPs group are shown in (A) and (B) are the relative body weight curves. The area under the curve of the relative body weight curves is shown in (AOC; baseline = 1.1).

[0107] Figure 23 The Disease Activity Index (DAI) score is used.

[0108] Figure 24 Macroscopic images of the colon (A) and a statistical chart of colon length (B) for each group.

[0109] Figure 25 Appearance images of BUD@BTZ NPs prepared with different phospholipid molar ratios. Detailed Implementation

[0110] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0111] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0112] In the following examples, budesonide (BUD) was purchased from Shanghai Maclean Biotech Co., Ltd.; bortezomib (BTZ) was purchased from Shanghai Aladdin Biotech Co., Ltd.; and budesonide palmitate was purchased from a CRO company.

[0113] Example 1: Screening for the optimal dose ratio of budesonide (BUD) to bortezomib (BTZ) using bone marrow-derived dendritic cells (BMDCs) and CD4+. + T cells (CD4) + T cells and bone marrow-derived macrophages (BMDMs) were seeded into 6-well plates. After stabilization for 12 hours, the BMDMs were then incubated in a solution containing 2.0 μg / mL of [unspecified substance]. -1Mature dendritic cells (mDCs) were obtained by induction in LPS medium for 4 hours. CD4 cells were then induced using anti-CD3 mAb and anti-CD28 mAb co-stimulatory molecules. + T cell proliferation and activation. This was achieved using 1 μg / mL... -1 LPS stimulation of BMDMs for 12 hours activated macrophages. The activated cells were then incubated for 12 hours in culture media containing pre-set concentrations of BUD solution (BUD), BTZ solution (BTZ), or a mixture of BUD and BTZ at different molar ratios (BUD+BTZ), with consistent total drug concentration in all groups. Following the instructions for use, BMDMs and CD4 counts were detected using an ELISA kit. + The levels of TNF-α in the culture supernatant of T cells and BMDMs. The results are as follows: Figure 3 As shown in Table 1, the area under the curve (AUC) of each experimental group was calculated using Graphpad Prism 9 software to more intuitively and comprehensively compare the level of TNF-α expression in cells of each experimental group.

[0114] Table 1. Area under the curve of TNF-α concentration in different experimental groups Group BMDCs <![CDATA[CD4 + T cells]]> BMDMs BUD 942.70±21.29 3280.33±62.39 1570.33±4.62 BTZ 1015.67±10.41 3607.00±80.29 1667.00±11.23 4：1 836.07±25.59 2990.00±98.97 1422.33±16.26 3：1 801.47±24.06 3049.67±54.59 1408.67±8.08 2：1 581.03±23.69 2358.67±138.90 1219.33±21.22 1：1 693.23±18.08 2487.33±67.88 1339.33±36.83 1：2 446.20±30.78 1834.67±101.69 1101.33±9.71 1：3 584.53±7.99 2189.67±110.63 1215.33±6.66 1：4 809.10±32.72 2857.67±66.86 1396.67±23.29 According to the results, both BUD / BTZ single-drug solutions reduced the activation of BMDCs and CD4. + The levels of TNF-α released by T cells and BMDMs were observed. The anti-inflammatory effect gradually increased with increasing drug concentration, exhibiting a dose-dependent relationship, indicating that BUD / BTZ has an inhibitory effect on the inflammatory response of IAICs. Notably, the anti-inflammatory effect of the combination drug group was enhanced to some extent compared to the single-drug solution group at the same dose. Specifically, BUD+BTZ (1:2, n / n) downregulated BMDCs and CD4+. + T cells and BMDMs showed the strongest effect in releasing TNF-α levels, indicating that they can effectively inhibit the release of pro-inflammatory factors in IAICs and achieve the best synergistic anti-inflammatory effect.

[0115] Example 2: Preparation of different budesonide and bortezomib complex nanoparticles This embodiment provides a pharmaceutical composition, which is a BUD and BTZ phospholipid complex nanoparticle, and the preparation method is as follows: Unmodified BUD@BTZ phospholipid complex nanoparticles (BUD@BTZ NPs) were prepared by setting the feed ratio of BUD, BTZ, and EPG to 1:2:4 (n / n). The drugs and excipients were weighed according to the formulation in Table 2, and anhydrous ethanol was added to adjust the reaction concentrations of BUD and BTZ to 3.4 and 6.0 mg·mL, respectively. -1 The mixture was then dissolved in a 50°C water bath by stirring. After reflux for 20 minutes in a closed system, stirring was continued to evaporate most of the anhydrous ethanol (approximately 2% of the final volume of the formulation remained, v / v). Sterile water for injection preheated to the same temperature was then added and stirred for 20 minutes. The mixture was then passed sequentially through 0.80, 0.45, and 0.22 μm polyvinylidene fluoride microporous membranes to remove uncomposite drug particles, yielding unmodified BUD and BTZ phospholipid complex nanoparticles (BUD@BTZ NPs). Following the same method, sialic acid-cholesterol conjugate (SA-CH) and non-breakable methoxylated polyethylene glycol were additionally added during the initial feeding. 2000 - Distearatelphosphatidylethanolamine (mPEG) 2000 -DSPE) or breakable methoxy polyethylene glycol 2000 -cholesterol hemisuccinate (mPEG) 2000 -CHEMS), respectively, to prepare BUD@BTZ phospholipid complex nanoparticles modified with sialic acid-cholesterol conjugate (SA-CH) (BUD@BTZSNPs), and SA-CH and indestructible methoxy polyethylene glycol. 2000 - Distearatelphosphatidylethanolamine (mPEG) 2000 -DSPE co-modified BUD and BTZ phospholipid complex nanoparticles (BUD@BTZ PdSNPs) and SA-CH with breakable methoxy polyethylene glycol 2000 -cholesterol hemisuccinate (mPEG) 2000 -CHEMS co-modified BUD and BTZ phospholipid complex nanoparticles (BUD@BTZPcSNPs).

[0116] Table 2 Formulations of different BUD and BTZ phospholipid complex nanoparticles Group Ingredients(w / w) BUD@BTZ NPs BUD / BTZ / EPG (0.57 / 1 / 4) BUD@BTZ SNPs BUD / BTZ / EPG / SA-CH(0.57 / 1 / 4 / 1) BUD@BTZ PdSNPs <![CDATA[BUD / BTZ / EPG / SA-CH / mPEG 2000 -DSPE(0.57 / 1 / 4 / 1 / 0.87)]]> BUD@BTZ PcSNPs <![CDATA[BUD / BTZ / EPG / SA-CH / mPEG 2000 -CHEMS(0.57 / 1 / 4 / 1 / 0.77)]]> In this embodiment, a sialic acid-cholesterol derivative (SA-CH) is used to modify the surface of the nanoparticles, which endows the nanoparticles with the potential to target inflammation-related immune cells. Furthermore, a breakable mPEG is employed. 2000 -CHEMS, together with SA-CH, is modified on the surface of nanoparticles to achieve long-term nanoparticle circulation while ensuring effective recognition and uptake by target cells.

[0117] Example 3 Characterization of different budesonide and bortezomib complex nanoparticles The particle size and zeta potential of nanoparticles were determined using a Nicomp-380 laser particle size and zeta potential analyzer. The method for detecting the recombination rate (CE%) is as follows: High-performance liquid chromatography (HPLC) conditions: Column: ODS C18 column (4.6 × 200 mm, 5 μm, Kromasil); Detection wavelength: BUD 240 nm, BTZ 270 nm; Column temperature: 25℃; Elution flow rate: 1.0 mL / min -1 Mobile phase: methanol-water (containing 0.4% triethylamine) (70 / 30, v / v); Injection volume: 20 μL.

[0118] It is known that the solubility of both BUD and BTZ in water is less than 0.1 mg·mL. -1 According to the solubility standards in the 2020 edition of the Chinese Pharmacopoeia, BUD and BTZ are practically insoluble or insoluble drugs. Therefore, taking advantage of the water insolubility of BUD and BTZ, the CE% of BUD and BTZ in nanoparticles was measured using a membrane filtration method. Samples were sequentially passed through 0.80, 0.45, and 0.22 μm polyvinylidene fluoride microporous membranes to remove uncomposite drug particles. 0.1 mL of the filtrate was accurately transferred to a 5 mL volumetric flask, dissolved thoroughly in methanol, diluted to the mark, and mixed well. 20 μL of the solution was injected, and the peak area A2 of BUD or BTZ was recorded. A separate sample that had not passed through a membrane was taken, and the peak area A1 of BUD or BTZ was measured using the same method. The CE% of the nanoparticles was calculated using the following formula.

[0119] CE%=A2 / A1×100%.

[0120] The results are shown in Table 3.

[0121] Table 3 Example 4 Cell Uptake Experiment Using flow cytometry and laser scanning confocal microscopy imaging techniques, with DiI as the fluorescent probe, the activation of BMDCs and CD4 was investigated. + The uptake of various nanoparticles by T cells and BMDMs.

[0122] Flow cytometry detection: BMDCs, CD4 + T cells and BMDMs were dispersed in complete culture medium to prepare 1×10⁻⁶ cells / mL. 6 cells·mL -1 Cell suspensions of high density were seeded into 6-well plates and stabilized for 12 hours in a 37°C, 5% CO2 incubator. After inducing cell activation and maturation, the culture plates were removed, and the original culture medium was replaced with one containing BUD@BTZ NPs.DiI BUD@BTZSNPs DiI BUD@BTZ PdSNPs DiI Or BUD@BTZ PcSNPs DiI (With or without carboxylesterase (CES); concentration: 40 U·L) -1 Serum-free culture medium (DiI final concentration 0.3 μg·mL⁻¹) -1 The cells were incubated at 37°C for 2 hours. The reagent solution was discarded, and the cells were washed three times with PBS before collection. Finally, the levels of activated DCs and CD4+ were measured by flow cytometry (FCM). + The uptake of nanoparticles by T cells and macrophages is shown in the following results. Figure 4 .

[0123] Results analysis: Activation of BMDCs, CD4 + T cells and BMDMs, BUD@BTZ SNPs DiI The average fluorescence intensity of DiI in the group was slightly higher than that of BUD@BTZ NPs. DiI The results showed that BUD@BTZ SNPs could more selectively target inflammation-associated immune cells. In contrast, BUD@BTZ PdSNPs... DiI The group exhibited the lowest DiI fluorescence intensity, which may be due to the presence of a hydration layer on the surface of BUD@BTZPdSNPs, hindering the recognition and uptake of IAICs. This invention is the first to term this as "immune indifference." Notably, in the absence of carboxylesterase CES, the fluorescence intensity of BUD@BTZ SNPs... DiI Compared to the previous group, BUD@BTZPcSNPs DiI Cellular uptake was significantly reduced, but this effect was gradually reversed in the presence of CES, comparable to the BUD@BTZSNPs group (p>0.05). This indicates that CES can effectively cleave the mPEG on the surface of BUD@BTZ PcSNPs. 2000 -Ester bonds in CHEMS expose SA, thereby selectively targeting inflammation-associated immune cells.

[0124] Laser scanning confocal microscopy imaging: BMDCs, CD4 + T cells and BMDMs were dispersed in complete culture medium to prepare 1×10⁻⁶ cells / mL. 5 cells·mL -1 Cell suspensions of high density were seeded into 24-well plates containing cell spreaders and stabilized for 12 hours in a 37°C, 5% CO2 incubator. After inducing cell activation and maturation, the culture plates were removed, and the original culture medium was replaced with one containing BUD@BTZ NPs. DiIBUD@BTZ SNPs DiI BUD@BTZ PdSNPs DiI and BUD@BTZ PcSNPs DiI (with or without 40 U·L) - 1 Serum-free culture medium (DiI final concentration 0.3 μg / mL) for CES -1 The cells were incubated at 37°C for 2 hours. The formulation solution was discarded, and the cells were washed three times with PBS. Cells were then fixed with 4% paraformaldehyde, incubated with Lysotracker Green working solution in the dark for 1 hour to label lysosomes, and the nuclei were counterstained with DAPI in the dark for 20 minutes. Cell slides were removed from the 24-well plate and placed on slides containing anti-fluorescence quenching mounting medium. The slides were then imaged using a laser scanning confocal microscope (CLSM) to observe the effects of the formulation on DCs and CD4+. + Distribution of T cells and macrophages, results are shown in Figure 5 .

[0125] Results analysis: Each nanoparticle co-localized with lysosomes in the cytoplasm, exhibiting a dotted distribution (overlapping green and red fluorescence resulted in yellow fluorescence). (Compared with BUD@BTZ NPs) DiI In comparison, BUD@BTZ SNPs DiI To activate BMDCs, CD4 + The red fluorescence signal transmitted in T cells and BMDMs was significantly increased, and the yellow fluorescence was stronger. In the absence of CES, BUD@BTZ PcSNPs DiI The fluorescence intensity of the formulation in the cells was weak, and cellular uptake was significantly reduced. However, this phenomenon was reversed in the presence of CES, as observed in BUD@BTZ SNPs. DiI The fluorescence intensities of the groups were comparable. Furthermore, BUD@BTZ PdSNPs DiI The lowest fluorescence intensity of the formulation was observed in the group of cells, indicating that inflammation-associated immune cells had the weakest uptake capacity for BUD@BTZ PdSNPs. In summary, FCM and CLSM imaging results showed the same trend.

[0126] Example 5: Pharmacokinetic Study of Different BUD and BTZ Phospholipid Complex Nanoparticles. DiR was used as a tracer to prepare DiR-labeled phospholipid complex nanoparticles, and the plasma pharmacokinetic behavior of different BUD and BTZ phospholipid complex nanoparticles in Wistar rats was investigated.

[0127] Establishment of the in vivo DiR assay method: 100 μL of rat blank plasma was precisely transferred to a 1.5 mL EP tube. 100 μL of a series of concentrations of DiR anhydrous ethanol solution were added, followed by 800 μL of anhydrous ethanol. The mixture was vortexed for 5 min to mix. The resulting suspension was centrifuged at 10,000 rpm for 10 min. 600 μL of the supernatant was transferred to another 1.5 mL EP tube and centrifuged at 10,000 rpm for 10 min. 200 μL of the supernatant was loaded into a 96-well plate and analyzed using a microplate reader (λ). ex =750nm, λ em The fluorescence intensity F was measured at 790 nm. Another 100 μL of blank plasma was added to 900 μL of anhydrous ethanol, and the fluorescence intensity F0 was measured using the same method. The sample F value was subtracted from the blank plasma F0 value to obtain the ΔF value. The ΔF value was then compared with the DiR concentration C (ng·mL⁻¹). -1 Linear regression was performed to obtain the regression curve. 100 μL of drug-containing rat plasma was precisely transferred to a 1.5 mL EP tube, and 900 μL of anhydrous ethanol was added. The remaining steps were the same as described above. The fluorescence intensity F was measured using a microplate reader to obtain the ΔF value. This ΔF value was then substituted into the standard curve to calculate the DiR concentration.

[0128] Pharmacokinetic studies: Fifteen Wistar rats were randomly divided into 5 groups of 3 rats each, and administered 0.3 mg / kg of the drug. -1 DiR dosage, tail vein injection of BUD@BTZ NPs DiR BUD@BTZ SNPs DiR BUD@BTZ PdSNPs DiR and BUD@BTZ PcSNPs DiR Blood samples were collected from the orbital venous plexus at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after drug administration and placed in heparinized tubes. Plasma was separated by centrifugation at 4000 rpm for 10 min. Plasma samples were processed according to the in vivo DiR analysis method, and the DiR concentration in plasma was calculated. The drug-time curve is shown in [Figure number missing]. Figure 6 Pharmacokinetic parameters were analyzed using DAS 2.0 pharmacokinetic software, and the results are shown in Table 4.

[0129] Table 4 Group <![CDATA[AUC 0-t ]]> <![CDATA[T 1 / 2 ]]> <![CDATA[BUD@BTZ NPs DiR ]]> 6692.006±291.998 6.770±0.191 <![CDATA[BUD@BTZ SNPs DiR ]]> 8389.094±140.646 8.694±0.708 <![CDATA[BUD@BTZ PdSNPs DiR ]]> 14407.403±238.063 12.188±0.977 <![CDATA[BUD@BTZ PcSNPs DiR ]]> 14637.847±162.391 12.476±0.817 Results Analysis: Compared to BUD@BTZ NPs, BUD@BTZ SNPs exhibited a significantly prolonged blood circulation time, indicating that SA-CH modification can improve the blood circulation of nanoparticles. This may be due to the low immunogenicity of SA; high-density SA-CH modification can effectively reduce the immunogenicity of nanoparticles, thus facilitating their evasion by the body. Conversely, once BUD@BTZ NPs enter the body, they are recognized as foreign substances and cleared by the body through multiple pattern recognition receptors. Furthermore, modification with (non)breakable PEG lipid derivatives further improved the blood circulation characteristics of nanoparticles. This may be because PEG lipid derivative modification can form a hydration layer on the nanoparticle surface, enhancing hydrophilicity and thus reducing the formation of protein crowns, thereby prolonging the blood circulation time of nanoparticles. Notably, BUD@BTZ PdSNPs and BUD@BTZ PcSNPs exhibited similar long blood circulation characteristics, which is consistent with previous findings on mPEG. 2000- CHEMS can be broken by ester bonds in the bloodstream, resulting in a weaker ability to prolong the in vivo circulation time of nano-formulations compared to mPEG. 2000 The results of the DSPE study differ. Possible reasons include: (1) SA-CH and mPEG. 2000 There may be intermolecular interactions between -CHEMS, which weaken the effect of esterase on mPEG. 2000 - The degree of attack of CHEMS; (2) The level of esterase in peripheral blood is relatively low, which may not reach the level of SA-CH and mPEG. 2000 -CHEMS co-modification conditions for mPEG 2000 -The level required for CHEMS fracture. This SA-CH with mPEG 2000 -CHEMS co-modification-induced mPEG 2000 -CHEMS fracture passivation phenomenon helps to improve the problem of unstable PEG fracture response rate caused by large individual patient differences in the clinical application of fractureable PEG materials.

[0130] Example 6 Determination of Anti-PEG IgM Content Further investigation was conducted at the molecular level (antibody content) to preliminarily examine the potential impact of (non)-fragile PEG lipid derivative modification on the ABC phenomenon in nanoparticles. Based on existing mechanisms, the amount of anti-PEG IgM induced by the initial injection of (non)-fragile PEG lipid derivative modified nanoparticles is a crucial factor influencing the ABC phenomenon. Therefore, the serum anti-PEG IgM level was measured on day 7 after the initial injection of PEG lipid derivative modified nanoparticles. The experimental method is as follows: Blood was collected from Wistar rats on day 7 after the first injection of the formulation in unheparinized EP tubes. After incubation at room temperature for 2 hours, serum was separated by centrifugation at 4000 rpm for 10 min and stored at -20℃ for later use. Anti-PEG IgM in the serum was measured using ELISA. 50 μL of mPEG was added to 96-well transparent polystyrene plates at room temperature. 2000 -DSPE solution (0.56 mg·mL) -1 (dissolved in anhydrous ethanol) or mPEG 2000 -CHEMS solution (0.50 mg·mL) -1 The solution was dissolved in anhydrous ethanol and coated overnight to allow it to air dry completely. Then, 100 μL of PBS (pH = 7.4, PBSB) containing 1% bovine serum albumin (BSA) was added to each well. 1％ Block at room temperature for 1 hour. Use PBS (pH = 7.4, PBSB) containing 0.1% BSA. 0.1％ After washing 5 times, the serum sample was placed in PBSB. 1％ Dilute to medium (1:10–1:1325000, v / v) and incubate at room temperature for 1 h. PBSB 0.1％ After washing five times, add 100 μL of HRP-conjugated goat anti-rat IgM antibody (1:500 dilution, v / v) to each well and incubate at room temperature for 1 h. After washing, add 100 μL of freshly prepared o-phenylenediamine solution (1.0 mg / mL) to each well. -1 The antibody was prepared in a pH 5.0 phosphate-citrate buffer (containing 9.7 mM hydrogen peroxide) and incubated at room temperature for 15 min. Then, 100 μL of 2 M H₂SO₄ was added to each well to terminate the reaction. The absorbance was measured using a microplate reader at 450 nm within 15 min after reaction termination. Data analysis was performed using a GraphPad Prism 9. A four-parameter sigmoid logistic curve was used for fitting analysis, with the X-axis representing the log₁₀ value of the dilution factor and the Y-axis representing the OD value. The half-maximum value (EC50) of the antibody in each sample was then calculated using software. The results are shown in [Figure number missing]. Figure 7 .

[0131] Results Analysis: The initial injection of (non)fractureable PEG lipid derivative-modified nanoparticles resulted in differences in PEG-specific IgM titers in rat serum. Compared to BUD@BTZ NPs, initial injection of BUD@BTZ PdNPs or BUD@BTZ PdSNPs significantly increased IgM titers (***p<0.001). Conversely, initial injection of BUD@BTZ PcSNPs reduced the upregulation of PEG-specific IgM titers in rat serum (*p<0.05). Compared to the BUD@BTZ PdNPs and BUD@BTZ PdSNPs groups, the logEC50 values ​​of the BUD@BTZ PcSNPs group decreased by 30.8% and 32.9%, respectively, indicating that mPEG... 2000 -CHEMS modification helps reduce the excessive production of PEG-specific IgM.

[0132] Example 7: Pharmacodynamic evaluation of anti-collagen-induced arthritis Establishment of a mouse model of collagen-induced arthritis: Immunograde bovine type II collagen (CII) and incomplete Freund's adjuvant (IFA) were emulsified in a 1:1 ratio in an ice-water bath to obtain an inducer. The inducer was injected subcutaneously at multiple sites on the tail root and back of DBA / 1 mice for the primary immunization of a collagen-induced arthritis (CIA) model. A second booster immunization was performed 21 days after the primary immunization using the same procedure. Throughout the modeling process, changes in paw swelling were continuously observed. One week after the second immunization, rapid swelling of the paws occurred and gradually stabilized, with no spontaneous remission of inflammation observed, indicating that the CIA mouse model was successfully established and suitable for subsequent research.

[0133] Dosage regimen: Collagen-induced arthritis (CIA) mice were randomly divided into 8 groups (n=5): Model (saline), BUD solution (BUD), BTZ solution (BTZ), BUD and BTZ mixture (BUD+BTZ), BUD@BTZ NPs, BUD@BTZ SNPs, BUD@BTZ PdSNPs, and BUD@BTZ PcSNPs groups. Healthy mice served as the negative control group. The corresponding formulations were administered via tail vein injection on days 28, 30, 32, 34, and 36 after the initial immunization, and observation continued until day 57 after discontinuation of administration. The dosages of BUD and BTZ were 0.17 mg / kg and 0.30 mg / kg, respectively. -1 The Model group was administered at a dose of 5.0 mL / kg. -1The mice were administered saline via tail vein injection at a dose specified in the drug. To assess the therapeutic effects of each treatment group on CIA, the hind paw volume of mice was measured every other day after the start of treatment using a toe volume analyzer. Simultaneously, the severity of joint pathology in mice was graded from 0 to 4 based on the degree of joint edema, erythema, and deformity (0 points: no signs of erythema or swelling; 1 point: erythema and mild swelling only in the tarsal or ankle joints; 2 points: erythema and mild swelling from the ankle to the tarsal joints; 3 points: erythema and moderate swelling from the ankle to the metatarsal joints; 4 points: erythema and severe swelling in the ankle, metatarsal joints, and toes, or joint stiffness). The severity of arthritis in each CIA mouse was monitored, and the scores of the four paws were summed to obtain a total score (0–16 points). To assess the inhibition of joint swelling in each treatment group, the hind paws of mice were photographed on the last day of the efficacy experiment (day 57). Mice were euthanized, and tissues such as the hindfoot and ankle joints were collected for subsequent studies. Joint scoring results are shown below. Figure 8 The results of the volume test are shown below. Figure 9 .

[0134] Results Analysis: Pharmacodynamic studies showed that, compared with single NF-κB small molecule inhibitor treatment alone, the combination therapy of BUD and BTZ (BUD+BTZ) significantly delayed the progression of CIA, thanks to the dual-site blocking effect of the two therapeutic agents on the core pro-inflammatory NF-κB signaling pathway. These results suggest that combination therapy with multiple NF-κB small molecule inhibitors targeting different sites of the NF-κB signaling pathway has potential in the treatment of IDs. Unlike previous combination therapies that blocked multiple inflammatory signaling pathways to treat IDs, this invention focuses on different sites of key pro-inflammatory pathways and has demonstrated the synergistic efficacy of this strategy, providing a new approach to the treatment of IDs.

[0135] Unlike the drug solution group, all nanoparticle groups showed a trend of blocking CIA progression. During administration, the joint inflammatory response in all groups gradually decreased, and no recurrence occurred within 21 days after discontinuation of administration, highlighting the significant advantages of nanomedicine in improving drug targeting and efficacy. The nanoparticle design enabled the co-delivery of BUD and BTZ, maintaining a superior synergistic effect. Among them, the anti-CIA efficacy of the BUD@BTZ SNPs group was superior to that of the BUD@BTZ NPs group, possibly because SA-CH modification can improve the pharmacokinetics and targeting of nanoparticles, which is conducive to drug accumulation at the site of inflammation and enhances the targeted immunomodulatory ability of BUD@BTZ SNPs. SA-CH modification can selectively target IAICs (including inflammation-associated DCs, inflammation-associated T cells, and inflammation-associated macrophages), which is beneficial for BUD@BTZ SNPs to block the autoimmune circulation (IAC) at multiple sites, thereby effectively regulating CIA progression. The anti-CIA effect of the BUD@BTZ PdSNPs group was significantly worse than that of the BUD@BTZ SNPs group, which may be due to mPEG. 2000 -DSPE is difficult to break, and PEG is difficult to detach, causing the stable hydration layer on the surface of BUD@BTZ PdSNPs to become a barrier for IAICs to recognize and take up nanoparticles at the disease site, resulting in an "immune indifference" phenomenon. This problem was also reflected in the early stages of administration of the BUD@BTZPdSNPs group, where it failed to inhibit the development of CIA in a timely and effective manner, even comparable to the BUD@BTZ NPs group, with poor timeliness and delayed drug efficacy. Conversely, the anti-CIA effect of the BUD@BTZ PcSNPs group was significantly better than that of the BUD@BTZSNPs group, which is attributed to mPEG. 2000 -CHEMS not only improves the in vivo biodistribution behavior of nanomedicines, but also effectively responds to specific pathological conditions at the disease site, inducing the detachment of PEG chains at the target site, exposing SA ligands, and further promoting the recognition and uptake of formulations by IAICs.

[0136] Example 8: Body weight and anti-tumor index During the pharmacodynamic studies, the body weight of mice in each group was measured every other day. To more comprehensively compare the effectiveness of the different formulations, the "tumor inhibition index" (SI) for each treatment group was calculated using the following formula. index ,g·mL -1 The results are shown below. Figure 10 .

[0137] SI index =Body weight / Mean paw volume Based on experimental results and daily observations, the Healthy group mice were more active and gained weight steadily during the experiment, with the fastest rate of weight gain. Conversely, the Model group CIA mice were lethargic and had reduced appetite, resulting in slower weight gain, directly reflecting the significant impact of CIA on the mice's quality of life. Furthermore, after drug intervention, the daily activity levels of CIA mice in all treatment groups significantly increased, and at the experimental endpoint, the weight of the nanoparticle group CIA mice was similar to that of the Healthy group. Figure 10 A) This may be related to the effective inhibition of CIA progression by nanoparticles. The above results highlight that, in CIA treatment, in addition to classic indicators such as joint score and paw volume, the body weight of CIA mice is also an important indicator for evaluating the efficacy of anti-CIA treatment, a point that is often overlooked.

[0138] This invention combines foot volume and body weight, introducing a new evaluation index, the "Inhibition Swelling Index" (SI). index SI index A larger paw print indicates less paw swelling, higher quality of life for the mouse, stronger overall anti-arthritis ability of the formulation, and better therapeutic effect. Figure 10 BD shows that SI for each experimental group index The order from largest to smallest is: BUD@BTZ PcSNPs > BUD@BTZ SNPs > BUD@BTZPdSNPs > BUD@BTZ NPs > BUD+BTZ > BTZ > BUD > Model. Compared to the solution group, each nanoparticle group successively showed SI after day 33. index The gradually increasing levels indicate that they can effectively block the progression of CIA, inhibit paw edema, and improve the quality of life in mice. The BUD@BTZ SNPs group showed a better overall anti-CIA effect compared to the BUD@BTZ NPs group. This may be because SA-CH modification enhances the targeting ability of nanoparticles, thereby improving the anti-CIA effect and the body's quality of life. Compared to the BUD@BTZ SNPs and BUD@BTZ PdSNPs groups, the BUD@BTZ PcSNPs group showed the best overall effect in treating CIA. This may be because BUD@BTZ PcSNPs possess both long circulation and effective exposure to SA ligands, achieving excellent arthritis targeting ability and facilitating the recognition and uptake of IAICs in the inflammatory microenvironment, thus maximizing the drug's therapeutic effect.

[0139] Body weight is an important indicator of the quality of life in laboratory animals. Disease progression is often accompanied by negative emotions and reduced spontaneous activity, leading to decreased appetite, reduced food intake, and consequently, weight loss. Therefore, during disease treatment, changes in body weight can scientifically reflect the restoration of homeostasis or the animal's overall health level. However, in pharmacodynamic studies primarily focused on respiratory tract infections (RA), body weight measurement is often overlooked. In SIindex In the calculation, when the mouse weight values ​​are similar, SI index The larger the foot volume, the smaller the foot volume, and the better the anti-swelling effect of this treatment group; conversely, when the foot volumes of CIA mice in different groups are similar, SI index A higher value indicates a larger body weight in the CIA mice, and a better quality of life in the treatment group. It is noteworthy that the foot volume was similar in the BUD and BTZ groups. Figure 9 ), but the SI of the BUD group index Significantly lower than the BTZ group ( Figure 10 Clearly, BUD is less effective than BTZ, reflecting SI's... index The value of SI. In summary, SI index The introduction of this method can amplify the pharmacodynamic differences between different groups, and more accurately help to screen the best treatment plan, which has important clinical guiding significance.

[0140] Example 9: Effects of Different Phospholipid Complex Nanoparticles on the Infiltration of Inflammation-Related Immune Cells in Joints. The day after drug administration, mice were sacrificed, ankle joints were dissected, and fur was removed to obtain single-cell suspensions of inflamed joint tissue. After washing the cell samples twice with pre-cooled PBS, DCs (FITC-CD11c mAb) and CD4+ were analyzed at 4°C using the corresponding fluorescently labeled mAbs according to the instructions. + Cell surface molecular staining of T cells (APC-CD3 mAb and FITC-CD4 mAb) and macrophages (APC-CD11b mAb and FITC-F4 / 80 mAb) was performed for 20 min, and CD11c was detected using FCM. + DCs, CD3 + CD4 + T cells and CD11b + F4 / 80 + The percentage of macrophages in all extracted cells was analyzed to assess the infiltration of intra-aqueous intravascular coagulation (IAICs) in the ankle joint tissues of mice in each treatment group. All FCM data were analyzed on FlowJo 10, and the results are shown below. Figure 11 .

[0141] Results analysis: After CIA was established, DCs (CD11c) in the inflamed joints + CD4 + T cells (CD3) + CD4 + ) and macrophages (CD11b + F4 / 80 +The proportions of [unspecified cells] were significantly increased in all groups compared to healthy mice, indicating that rheumatoid arthritis (RA) is a complex immune-related disease involving multiple immune cells. Compared to the Model group, treatment with each nanoparticle resulted in the secretion of large amounts of cytokines in the inflamed joints and the induction of DCs and CD4+ in severe inflammation and tissue damage characteristic of RA. + The proportions of T cells and macrophages were significantly reduced. Specifically, compared with the Model group, the proportions of inflammatory infiltrating DCs in the BUD@BTZ NPs, BUD@BTZ SNPs, BUD@BTZ PdSNPs, and BUD@BTZPcSNPs groups decreased by 39.1%, 60.0%, 48.4%, and 68.6%, respectively; inflammatory infiltrating CD4+... + The proportion of T cells decreased by 31.6%, 45.3%, 36.0%, and 56.2%, respectively; the proportion of inflammatory infiltrating macrophages decreased by 27.5%, 45.6%, 37.6%, and 55.3%, respectively. Clearly, BUD@BTZ PcSNPs treatment had the strongest effect in downregulating IAIC abundance, demonstrating excellent immunomodulatory capabilities. This may be attributed to the high accumulation of BUD@BTZ PcSNPs at inflammatory sites and their active recognition and reduction of IAICs mediated by SA.

[0142] Example 10: Effects of different phospholipid complex nanoparticles on dendritic cell phenotype in inflamed joints. This example uses FCM technology to quantify the phenotype of CD11c in inflamed joints. + CD80 + and CD11c + CD86 + The percentage of dendritic cells (DCs) was used as an indicator of tolDCs to further investigate the in vivo regulatory function of BUD@BTZ PcSNPs on tolDCs. On day 38 post-modeling, mice were sacrificed, and their ankles were dissected to obtain single-cell suspensions. Cell samples were washed twice with pre-cooled PBS and stained with anti-mouse FITC-CD11c mAb, PE-CD80 mAb, and APC-CD86 mAb at 4°C according to the manufacturer's instructions. DC phenotype was detected using FCM. All data were analyzed on FlowJo 10, and the results are shown below. Figure 12 .

[0143] Results analysis: Compared with the Model group, mice treated with nanoparticles showed significantly reduced inflammatory infiltration of CD11c. + CD86 + and CD11c + CD80 +The expression levels of DCs were significantly reduced. Specifically, compared to the Model group, the expression levels of CD11c in the BUD@BTZ NPs, BUD@BTZ SNPs, BUD@BTZPdSNPs, and BUD@BTZ PcSNPs treatment group were significantly lower. + CD86 + The levels of DCs decreased by 18.4%, 32.0%, 22.6%, and 40.5%, respectively; CD11c + CD80 + The levels of dendritic cells (DCs) decreased by 23.9%, 41.3%, 35.8%, and 52.2%, respectively. Clearly, BUD@BTZ PcSNPs showed the strongest ability to downregulate the expression of co-stimulatory molecules (CD80 and CD86), indicating its ability to reverse the dysregulation of DC activation in the inflammatory microenvironment. In conclusion, BUD@BTZ PcSNPs can effectively regulate the tolDC phenotype, which helps to transmit immune tolerance information to the body via antigen-presenting cells (APCs), achieving indirect regulation of antigen-specific regulatory cells and the establishment of immune tolerance in vivo.

[0144] Example 11 Effects of different phospholipid complex nanoparticles on Th1 and Th17 differentiation in inflammatory joints. In RA, CD4 + T cells differentiate into Th1 and Th17 cells, playing a crucial role in bone loss and joint destruction. Therefore, FCM detection of Th1 (CD4) cells in inflamed joints is important. + IFN-γ + ) and Th17 (CD4) + IL-17A + Subpopulation proportions, evaluating CD4 content of BUD@BTZ PcSNPs in CIA mice. + Inhibition of T cell differentiation. On day 38 after modeling, mice were sacrificed, and their ankles were separated and processed to obtain single-cell suspensions. Following the manufacturer's instructions, mouse tissue leukocyte separation kits were used to separate leukocyte suspensions via density gradient centrifugation. Cell samples were washed twice with pre-cooled PBS, and then stained with fluorescently labeled mAbs. Following the manufacturer's instructions, cell surface molecular staining with FITC-CD4 mAb at 4°C was performed, followed by fixation and permeabilization treatment to fix and permeabilize the cells. Cells were then stained with APC-IFN-γ mAb or PE-IL-17A mAb, washed twice, and resuspended in PBS for FCM analysis. All data were processed on FlowJo 10, and the results are shown below. Figure 13 .

[0145] Results analysis: Compared with the Healthy group, the Model group mice showed increased inflammatory infiltration of CD4+. +The proportion of T cells secreting IFN-γ and IL-17A was significantly increased, indicating that CD4+ cells... + T cell differentiation into autoimmune Th1 and Th17 cells was enhanced. Furthermore, all nanoparticle therapies were observed to downregulate Th1 / Th17 levels to some extent. Specifically, BUD@BTZ PcSNPs inhibited CD4... + The T cells exhibited the strongest differentiation into Th1 / Th17 cells, downregulating the proportions of Th1 and Th17 cells by 64.0% and 47.1%, respectively, compared to the Model group, demonstrating the strongest immunomodulatory effect. These results further support the view that BUD@BTZ PcSNPs effectively improve RA by selectively regulating inflammation-related T cell differentiation.

[0146] Example 12: Effects of Different Phospholipid Complex Nanoparticles on Macrophage Phenotype in Inflamed Joints. RT-qPCR was used to detect the mRNA levels of M1 markers (CD86, TNF-α, and iNOS) and M2 markers (CD206, Arg-1, and CD163) in inflamed joints of CIA mice, verifying the in vivo regulatory effect of BUD@BTZ PcSNPs on macrophage phenotype. On day 38 after modeling, hindfoot ankle joint tissues from mice in each group were collected and placed in conical tubes without RNase / DNase. The obtained tissues were minced with a scalpel blade and homogenized in the presence of TRIzol. Total RNA was then isolated from the tissues using TRIzol according to the manufacturer's instructions, and purified and quantified using the NanoDrop method. cDNA was synthesized using the GoScript reverse transcription system, and RT-qPCR was performed using SYBERgreen to detect the expression of CD86, TNF-α, iNOS, CD206, Arg-1, and CD163 mRNA. The expression levels of M1 / M2 marker mRNA relative to GAPDH mRNA in each group were quantified using the ΔΔCT method. Results Figure 14 .

[0147] Results Analysis: After CIA induction, M1-specific biomarkers such as CD86, TNF-α, and iNOS mRNA levels increased, while nanoparticle therapy significantly reduced their levels. Due to macrophage infiltration, CIA also stimulated the expression of M2 marker mRNAs (CD206, Arg-1, and CD163), and each nanoparticle therapy promoted the expression of immunosuppressive M2 markers to some extent. Among them, the BUD@BTZ PcSNPs group showed the lowest M1 marker mRNA levels and the highest upregulation of M2 marker mRNA levels, indicating an increase in M2 content and a decrease in M1 content, exhibiting the most significant M1-to-M2 phenotype transition. These results suggest that BUD@BTZ PcSNPs can effectively downregulate the M1-like characteristics of macrophages, converting them into anti-inflammatory M2, reducing the secretion of inflammatory factors, providing a non-inflammatory environment for inducing tolDCs, and contributing to the establishment of immune tolerance.

[0148] In summary, after systemic treatment, BUD@BTZ PcSNPs inhibit the autoimmune response of inflammation-associated immune cells, leading to significant changes in the "inflammation-associated cell population" (IACP) at the disease site, which will facilitate the successful establishment of immune tolerance.

[0149] Example 13 Effects of different phospholipid complex nanoparticles on Treg differentiation in collagen-induced arthritis mice. On day 57 after modeling, CD4+ in draining lymph nodes (dLNs) was assessed by FCM. + Differentiation of Tregs in T cells. Inguinal and popliteal lymph nodes were collected, tissues were cut into small pieces, and treated with digestive solution in a 37°C water bath shaker for 1 hour. All samples were then filtered through a 70μm nylon cell filter and resuspended in erythrocyte lysis buffer to remove erythrocytes. Cells were washed twice with pre-chilled PBS to obtain a single-cell suspension. Target cells were further stained with fluorescently labeled mAbs. Following the instructions, cell surface molecular staining was performed with FITC-CD4 mAb and APC-CD25 mAb at 4°C. Cells were then fixed and permeabilized with Foxp3 / transcription factor staining buffer, and further stained with PE-Foxp3 mAb at 4°C for 1 hour to label Tregs (CD4+). + CD25 + FoxP3 + After washing twice, the samples were resuspended in PBS for FCM analysis. All data were analyzed on FlowJo 10, and the results are shown below. Figure 15 .

[0150] Results analysis: All nanoparticle treatments increased Treg levels to some extent. In the BUD@BTZ PcSNPs treatment group, Treg(CD4+) levels were significantly increased.+ CD25 in + FoxP3 + The proportion of Tregs in dLN increased to about 30.3%, which was 3.46, 1.55, 1.30 and 1.42 times that of the Model (8.8%), BUD@BTZ NPs (19.5%), BUD@BTZ SNPs (23.2%) and BUD@BTZ PdSNPs (21.3%) groups, respectively, showing the best Treg induction ability.

[0151] In summary, BUD@BTZ PcSNPs mediate the production of tolDCs, further promoting T cell differentiation towards immunomodulatory phenotypes and mediating the induction of immune tolerance, which will be beneficial to the restoration of immune homeostasis.

[0152] Example 14 Serum anti-CII IgG and subtype levels Serum was separated on day 57 after CIA modeling was performed according to the steps in Example 7. A 96-well transparent polystyrene plate was taken and incubated at 4°C with 100 μL of bovine CII (10.0 μg / mL). -1 The sample was coated overnight with 0.1M bicarbonate buffer (pH 9.6) and washed with PBS (pH 7.4, PBST) containing 0.05% Tween 20. Then, it was coated with PBS (pH 7.4, PBSB) containing 2% BSA. 2％ Block at room temperature for 2 hours. After washing 5 times with PBST, add diluted serum sample (1:10–1:1325000, v / v) and incubate at room temperature for 1 hour. After washing 5 times with PBST, add 100 μL of HRP-conjugated goat anti-mouse IgG or rabbit anti-mouse IgG2a antibody (1:10000 dilution, v / v) to each well and incubate at room temperature for 1 hour. After washing, add 100 μL of freshly prepared tetramethylbenzidine solution (1.0 mg / mL) to each well. -1 Color development was performed, and the mixture was incubated at room temperature for 15 min. Then, 100 μL of 1M H₂SO₄ was added to each well to terminate the reaction. The OD value was measured using a microplate reader at 450 nm within 15 min after reaction termination. Data analysis was performed using GraphPad Prism 9. A four-parameter sigmoid logistic curve was used for fitting analysis, with the X-axis representing the log₁₀ value of the dilution factor and the Y-axis representing the OD value. The half-maximum (EC50) of the antibody in each sample was then calculated using software to obtain the EC50 value of the antibody in each sample. The results are shown in [Figure number missing]. Figure 16 .

[0153] Results Analysis: Compared with the Model group, all nanoparticle treatments reduced CII-specific IgG titers to some extent. Among them, the BUD@BTZ PcSNPs group had the lowest logEC50 value for CII-specific IgG, which was 38.3%, 21.2%, and 38.8% lower than the BUD@BTZ NPs, BUD@BTZSNPs, and BUD@BTZ PdSNPs groups, respectively, indicating that the BUD@BTZ PcSNPs group had the lowest CII-specific IgG titer. Since overexpressed autoimmune IgG2a plays a decisive role in mediating autoimmunity and is generally associated with Th1 / Th17-driven B-cell immune responses, the IgG2a titer was measured to further evaluate the impact of various treatments on the Th1 / Th17 response. Compared with other groups, the BUD@BTZ PcSNPs group showed the lowest logEC50 value for IgG2a, indicating that this treatment group had the lowest CII-specific IgG2a titer, further suggesting that BUD@BTZ PcSNPs has the ability to inhibit Th1 / Th17-specific immune responses.

[0154] Example 15: Response of mice with collagen-induced arthritis treated with BUD@BTZ PcSNPs to antigen rechallenge. Mice in remission (n=5) from the nanoparticle treatment group were selected according to the following criteria: (1) the mice had arthritis; (2) the joint score was stable for more than one week; (3) the mice had one or more paws with a score of 0 or 1. On day 52 after immunization induction, mice were induced with type II collagen (CII), and rechallenge mice were continuously observed.

[0155] Incidence of collagen-induced arthritis: After the second challenge, the number of claws recurring with CIA was recorded every 3 days, and the incidence of CIA was calculated. Only the number of claws scoring 0 or 1 before the second challenge was recorded, and claws recurring with CIA were defined as those progressing from 0 or 1 to 2–4. Results are shown in […]. Figure 17 .

[0156] Joint scoring and foot volume: After the second challenge, mice underwent joint scoring and hind foot volume measurement every two days. The results are shown in [Table missing]. Figure 18 .

[0157] Results Analysis: Typically, CIA mice in all treatment groups reached a stable state by day 45 after the initial induction. Therefore, in this experiment, mice that had achieved disease remission were selected on day 52 after the initial induction and subjected to a secondary immune challenge with IFA-emulsified CIA. The results showed that nanoparticle therapy delayed CIA recurrence to varying degrees. In contrast to BUD@BTZ NPs (where two out of 12 paws relapsed on day 12 after secondary induction, with a recurrence rate of 100.0% (12 / 12) by day 48 after secondary induction), BUD@BTZ SNPs significantly delayed CIA recurrence (where only one paw relapsed on day 20 after secondary induction, with a recurrence rate of only 31.6% (6 / 19) by day 48 after secondary induction), and the rate of increase in foot swelling and joint pathology scores in the relapsed paws was significantly slowed. This may be because BUD@BTZ NPs possess the advantage of passive targeting and accumulation of nanoparticles at the target site, which helps establish immune tolerance. However, due to its lack of selective targeting ability for IAICs, it performs poorly in targeted immune regulation and causes damage to normal immune cells, which is detrimental to the regulation of immune tolerance and the restoration of immune homeostasis. BUD@BTZ... The active accumulation of SNPs mediated by SA-CH and their effective targeted regulation of IAICs circumvent interference with normal immune cells, ultimately maintaining the healthy foundation of the immune system while selectively regulating IAICs, thus achieving effective regulation of immune tolerance. Importantly, BUD@BTZ PcSNPs exhibit the strongest inhibitory effect on CIA relapse, due to mPEG... 2000 -CHEMS effectively reduces the formation of protein corona and promotes the high accumulation of nanomedicines at the disease site, further avoiding damage to the healthy immune system caused by the drugs. Meanwhile, mPEG... 2000-CHEMS responses to CES at the disease site effectively expose SA, promoting the targeted regulation of IAICs by the drug. These two advantages ultimately achieved the optimal immune tolerance induction effect of BUD@BTZ PcSNPs. On day 48 after the second antigen challenge, only 3 paws (out of 20 mice) in the BUD@BTZ PcSNPs group showed mild relapse, and CIA inflammatory symptoms were the mildest. Notably, compared to the BUD@BTZSNPs group, the inhibitory ability of BUD@BTZ PdSNPs on CIA relapse was significantly reduced, even slightly inferior to the BUD@BTZ NPs group (the BUD@BTZ PdSNPs group showed the earliest paw relapse in mice). Paradoxically, in pharmacodynamic studies, the inflammation relief in CIA mice was significantly better in the BUD@BTZPdSNPs group than in the BUD@BTZ NPs group. In response, this invention attempts to analyze the issue from the perspectives of "treating the symptoms" and "treating the root cause": As the mechanism investigation results above show, the BUD@BTZ PdSNPs group was not significantly superior to the BUD@BTZ NPs group in terms of immunomodulatory capacity, which may be due to the indestructible mPEG. 2000 The application of DSPE shields immune cells from recognizing and capturing nanomedicines, significantly reducing the targeting efficiency of IAICs and failing to fundamentally address CIA caused by immune dysregulation, demonstrating a certain deficiency in immune tolerance regulation. The BUD@BTZ PdSNPs group showed a certain advantage in alleviating CIA symptoms compared to the BUD@BTZ NPs group. This may be attributed to the fact that BUD@BTZ PdSNPs facilitates the large-scale non-selective accumulation of drugs at the target site, resulting in a non-specific therapeutic effect during drug release, manifested as relief of surface symptoms. Overall, the above experimental results preliminarily demonstrate that BUD@BTZ PdSNPs treatment does not achieve a comprehensive cure.

[0158] Example 16 Investigation of antigen-specific reaction To assess antigen-specific responses, dLNs were collected from mice in each group on day 7 after CII antigen rechallenge to obtain single-cell suspensions, and Treg(CD4+) were analyzed. + FoxP3 + FCM analysis was performed to examine the proportion of Tregs after re-exposure to the antigen. Mice in the corresponding control group that did not undergo secondary challenge served as controls. All data were analyzed on FlowJo 10, and the results are shown below. Figure 19 .

[0159] Results Analysis: mTreg cells exhibited a durable and specific response to specific antigens. Upon re-exposure to the same antigen, these cells showed a tendency to proliferate specifically, providing a self-protective mechanism to prevent relapse of autoantigens (AIDs), suggesting the spontaneous initiation of a memory-based immune tolerance cycle (MITC). A comprehensive analysis of the CIA re-challenge study results showed that different nanoparticle groups demonstrated varying degrees of effectiveness in slowing CIA relapse. Combined with pharmacodynamic results, BUD@BTZ PcSNPs treatment effectively blocked the development of CIA and maintained disease activity at a low level, exhibiting persistence, stability, and a degree of conservation. Upon re-exposure to autoantigens, the vast majority of mice in the BUD@BTZ PcSNPs group remained stable for a prolonged period, effectively delaying or inhibiting approximately 85% of CIA paw relapses within 48 days after re-challenge, indicating the establishment of a "strong memory-based immune tolerance" mechanism. Conversely, BUD@BTZ PdSNPs and BUD@BTZ NPs treatments failed to effectively resist the re-challenge of autoantigens, causing the immune balance to shift towards an autoimmune response, rapidly inducing CIA relapse, exhibiting characteristics of "weak memory immune tolerance." However, BUD@BTZ SNPs treatment could maintain a relatively stable state in some organisms for a certain period, delaying or inhibiting approximately 68.4% of CIA paw relapses within 48 days after re-challenge, exhibiting characteristics of "moderate memory immune tolerance," falling between "weak memory immune tolerance" and "strong memory immune tolerance." Based on this, by examining the changes in antigen-specific Treg levels before and after secondary stimulation, we preliminarily explored the potential mechanism by which BUD@BTZ PcSNPs induce "strong memory immune tolerance." The results showed that mice in the BUD@BTZ PcSNPs group exhibited the strongest antigen-specific Treg response amplification capacity after a second CIA antigen challenge. The above results suggest that BUD@BTZ PcSNPs treatment can effectively promote the production of mTregs. When the same antigen is encountered again, they will undergo specific amplification, producing a more rapid and intense response. This is conducive to the spontaneous initiation of MITCs and the construction of "strong memory immune tolerance", thereby inhibiting secondary inflammatory responses and delaying / inhibiting the recurrence of AIDs.

[0160] Example 17: Comparison of the efficacy of BUD@BTZ PcSNPs and anti-TNF-α antibodies in treating collagen-induced arthritis. Anti-TNF-α antibodies (Anti-TNF-α) are used as one of the benchmarks for treating arthritis-related diseases (AIDs). In this example, foot volume and joint score were used as indicators to evaluate their pharmacodynamic efficacy against BUD@BTZ PcSNPs. The results are shown in […]. Figure 20 and Figure 21 .

[0161] Results analysis: During the administration period, anti-TNF-α treatment was less effective in blocking CIA progression compared to BUD@BTZ PcSNPs. After discontinuation of administration, during the observation period, mice in the BUD@BTZ PcSNPs group experienced long-term remission of arthritis, while mice in the anti-TNF-α group showed significant relapse of arthritis. This may be because anti-TNF-α therapy targets only a single target, neglecting the highly complex and overall coordinated nature of the "inflammation-associated cell population" (IACP).

[0162] Specifically, the complexity of IACP is mainly manifested in the following aspects: (1) Synergistic effect of multiple cell types: Multiple types of inflammation-associated (non-)immune cells work together during the inflammatory process. They transmit information through rich cytokines, receptor-ligand interactions, cell contact, etc., forming a highly complex cell population, and jointly participate in the immune response to pathogens and damaged tissues; (2) Functional diversity: A single cell type may include multiple cell subtypes with different functions, which play a role in different stages of the immune response; (3) Spatiotemporal dynamics: The number and state of immune cells will change dynamically during the inflammatory process and are regulated by a variety of factors, including pathogen infection, tissue damage, and the role of regulatory cells. This spatiotemporal dynamics increases the complexity of the immune response.

[0163] The experimental results above indicate that anti-TNF-α therapy primarily targets TNF-α pro-inflammatory factors, while its regulatory capacity on other cytokines is relatively weak. Therefore, IACP may not be fully and effectively regulated, leading to arthritis recurrence upon discontinuation of treatment. In contrast, BUD@BTZ PcSNPs, due to their multi-target and multi-functional regulation at the cellular and molecular levels, more comprehensively influence IACP function, demonstrating superior efficacy in CIA mouse models. This further showcases its clinical application potential, potentially providing patients with more durable therapeutic effects and offering new insights for developing more effective immunomodulatory therapies.

[0164] Example 18: Pharmacodynamic study of BUD@BTZ PcSNPs against chronic ulcerative colitis (UC) Experimental protocol: Fifteen mice were randomly divided into three groups (n=5): Model (saline), anti-TNF-α, and BUD@BTZ PcSNPs groups. Healthy mice (not receiving DSS stimulation or treatment) served as the control group. Starting from the second DSS cycle (day 10), the drugs were administered every other day. The BUD@BTZ PcSNPs group received intravenous injection of BUD@BTZ PcSNPs (0.17 mg BUD·kg). -1 0.30 mg BTZ·kg -1The anti-TNF-α group received intraperitoneal injection of anti-TNF-α (3.0 mg / kg). -1 On day 30, administration was discontinued. The Model group received an equal volume of physiological saline intravenously, and mouse weight was monitored throughout the experiment. On the second day after administration (day 30 post-modeling), DAI scores were assessed for each group of mice, and colon and serum samples were collected from each group for subsequent studies.

[0165] Weight loss: Weight loss is one of the main symptoms in chronic UC mice. To investigate whether BUD@BTZ PcSNPs have a protective effect on chronic UC mice, the weight of mice in each group was monitored every two days during treatment, and the weight was continuously recorded after drug administration was stopped. The spontaneous recovery of mouse weight was observed, and a curve of relative weight change over time was plotted. The results are shown in [Figure 1]. Figure 22 The area on curve (AOC; baseline = 1.1) of each experimental group was statistically analyzed using Graphpad Prism 9 software to more intuitively and comprehensively compare the fluctuations in mouse weight caused by chronic ulcerative colitis (UC). Furthermore, the weight loss rate (WLR) and weight recovery rate (WRR) of each group were calculated using the following formulas to further compare the differences in weight changes among the experimental groups. The results are shown in Table 5.

[0166] WLR = (W Initial -W Min ) / W Initial ×100% WRR = (W Max -W Min ) / W Min ×100% Among them, W Initial W represents the initial weight before weight loss. Min W represents the lowest weight reached during the current phase. Max This indicates the weight at its highest point during the current phase.

[0167] Table 5 Group Model anti-TNF-α BUD@BTZ PeSNPs WLR% (18th day) 25.40±1.80 23.51±4.44 18.51±2.87 WRR% (24th day) 14.82±3.09 15.54±4.85 16.65±5.31 WLR% (26th day) - 6.78±2.75 2.74±1.30 WLR% (28th day) 8.21±2.52 - - WRR% (30th day) 5.62±2.41 7.05±2.04 7.70±1.97 Disease Activity Index (DAI): To investigate the effects of BUD@BTZ PcSNPs on clinical symptoms in chronic UC mice, we used the DAI score to represent the severity of clinical symptoms. On the second day after administration (day 30 post-modeling), the sum of three scores—percentage of body weight loss, stool characteristics, and occult blood test results (o-toluidine method)—was used as the Disease Activity Index (DAI). Results are shown below. Figure 23 .

[0168] Representative images of the colon and colon length in different treatment groups: The classic pathological feature of chronic UC mice is a significantly shortened colon. On day 30 after modeling, mice were sacrificed, and the entire colonic tissue from the anus to the terminal cecum was removed. Colon length was measured and photographed. The results are shown below. Figure 24 .

[0169] Results Analysis: Anti-TNF-α biologics are widely used in the treatment of IBDs. This example established a chronic UC mouse model and compared the efficacy of BUD@BTZ PcSNPs with anti-TNF-α. The results showed that, compared to anti-TNF-α, BUD@BTZ PcSNPs significantly inhibited weight loss, DAI elevation, and colon length shortening in mice, and reduced tissue damage in colitis, indicating its good efficacy in the treatment of chronic UC.

[0170] BUD@BTZ PcSNPs may have the following advantages in the treatment of UC: (1) Multiple anti-inflammatory pathways: UC is a group of multifactorial diseases, and its pathogenesis involves multiple cell types, inflammatory mediators and signaling pathways. Anti-TNF-a mainly treats by inhibiting TNF-α, but cannot fully cover other pro-inflammatory mediators, and the therapeutic effect may be limited. BUD@BTZPcSNPs, on the other hand, blocks the key pro-inflammatory NF-κB signaling pathway at two sites and regulates multiple key inflammatory mediators at the same time, thereby achieving the regulation of multiple anti-inflammatory pathways and is expected to more comprehensively reduce intestinal inflammatory response. (2) Multiple immune regulation: UC involves abnormal activities of multiple cell types, including inflammation-associated DCs, T cells and macrophages. Anti-TNF-a indirectly affects the activity of immune cells by neutralizing TNF-α, while BUD@BTZ PcSNPs can directly target these inflammation-associated immune cells, regulate their activity and immune response, and help restore immune homeostasis.

[0171] In summary, compared with anti-TNF-α, BUD@BTZ PcSNPs have a more comprehensive regulatory effect in the treatment of UC, providing a more flexible option for the treatment of IBDs. This invention preliminarily verifies the universality of the strategy of targeting inflammation-related immune cells in the inflammatory microenvironment.

[0172] Example 19: Comparison of preparation processes for budesonide and bortezomib complex nanoparticles 1. Investigate the effect of different phospholipid molar ratios. BUD@BTZ NPs were prepared according to the preparation method of Example 2, the only difference being that the feed ratio of BUD, BTZ, and EPG was set to 1:2:1, 1:2:2, 1:2:3, 1:2:4, and 1:2:5 (n / n), respectively. The appearance was as shown. Figure 25As shown in Table 6, the test results for particle size, PDI, and CE% were obtained by repeating the experiment three times. The data are expressed as mean ± standard deviation.

[0173] Table 6 like Figure 25 As shown in Table 6, when BUD:BTZ:EPG = 1:2:1, 1:2:2, and 1:2:3 (n / n), white particles were observed to precipitate in the formulation. The number of white particles decreased significantly with increasing EPG ratio, while the CE% of BUD and BTZ gradually increased. When BUD:BTZ:EPG = 1:2:1, 1:2:2, and 1:2:3 (n / n), the CE% of both BUD and BTZ was below 95%, and after removing free drug, the PDI of the nanoparticles was relatively large. When BUD:BTZ:EPG = 1:2:4 and 1:2:5 (n / n), the formulation was clear and transparent, the nanoparticle size range was 50–60 nm, the particle size distribution was narrow, and the CE% of both BUD and BTZ was greater than 98%.

[0174] 2. Investigate the effect of BTZ concentration on the reaction between BTZ and BUD. BUD@BTZ NPs were prepared according to the preparation method in Example 2, except that the reaction concentration of BTZ was set to 3.0, 6.0, and 12.0 mg·mL, respectively. -1 (The corresponding BUD reaction concentrations were 1.7, 3.4, and 6.8 mg·mL, respectively) -1 The test results for particle size, PDI and CE% are shown in Table 7. The experiment was repeated 3 times, and the data are expressed as mean ± standard deviation.

[0175] Table 7 The results show that when the BTZ concentration is 3.0 and 6.0 mg·mL, the reaction is effective. -1 (The corresponding BUD reaction concentrations were 1.7 and 3.4 mg·mL, respectively) -1 At this concentration, the main characterization changes of BUD@BTZ NPs were relatively small, with the particle size remaining around 55 nm, a narrow particle size distribution, and a CE% greater than 95%. When the BTZ reaction concentration reached 12.0 mg·mL⁻¹, the changes were minimal. -1 At that time (the corresponding BUD reaction concentration was 6.8 mg·mL) -1 As the nanoparticles become larger, their particle size uniformity decreases, leading to a decline in CE%. Therefore, the reaction concentrations of BUD and BTZ in anhydrous ethanol should be controlled at 3.4 and 6.0 mg / mL, respectively. -1 and below.

[0176] 3. Investigate the effect of reaction temperature BUD@BTZ NPs were prepared according to the method in Example 2, except that the reaction temperatures were set to 50°C, 55°C, 60°C, and 65°C, respectively. The test results for particle size, PDI, and CE% are shown in Table 8. The experiment was repeated three times, and the data are expressed as mean ± standard deviation.

[0177] Table 8 Temperature (°C) Particle size (nm) polydispersion coefficient CE%(BUD) CE%(BTZ) 50 55.4±2.1 0.211±0.031 98.55±1.79 99.71±1.64 55 56.7±2.5 0.221±0.016 97.53±2.05 98.42±1.95 60 55.8±3.4 0.211±0.019 98.91±2.38 99.07±1.96 65 56.9±1.9 0.206±0.021 98.73±1.42 98.84±2.03 The results show that within the investigated reaction temperature range, the reaction temperature between the drug and phospholipid has little effect on the main characterization of BUD@BTZ NPs. The particle size of the nanoparticles remains at around 55 nm, the particle size distribution is narrow, and the CE% is greater than 95%.

[0178] Example 20: Budesonide palmitate and bortezomib co-loaded liposomes This embodiment provides a pharmaceutical composition, which is a co-loaded liposome of budesonide palmitate and bortezomib, and the preparation method is as follows: Weigh out budesonide palmitate and liposome membrane material (distearylphosphatidylcholine (DSPC), distearylphosphatidylglycerol (DSPG), and cholesterol (CH) in a molar ratio of 7:2:1), add 10% (v / v) of anhydrous ethanol to the final volume of the formulation, and stir to dissolve in a 65°C water bath. After the membrane material is completely dissolved, continue stirring to evaporate most of the anhydrous ethanol (approximately 2% of the final volume of the formulation remains, v / v). Then, inject a preheated polyvinyl alcohol aqueous solution (pH = 7.5) to the same temperature into the membrane material, and stir in a 65°C water bath for 20 min to obtain the initial budesonide palmitate liposome product.

[0179] The liposome pre-form was granulated using a liposome extruder under pressure from a polycarbonate membrane and nitrogen in a 65°C water bath. The liposome pre-form was then sequentially passed through polycarbonate membranes at 800 nm, 400 nm, 200 nm, 100 nm, and 50 nm to obtain liposome suspensions (phospholipid concentration 43.0 mg / mL). -1 ).

[0180] After extrusion, 0.2 mL of the above liposome suspension was loaded onto the top of a dextran gel column (provided by Shanghai Maclean Reagent Co., Ltd.) that had been pretreated by centrifugation. After standing for 10 min, the column was centrifuged at 2000 r / min for 4 min. 0.1 mL of deionized water was added, and the column was centrifuged at 2000 r / min for 4 min. This process was repeated three times. The filtrate was collected to obtain gradient budesonide palmitate liposomes with a high concentration of polyols in the internal aqueous phase and very little or no polyols in the external phase. After the liposomes were formed, the pH of the external aqueous phase of the gradient budesonide palmitate liposomes was adjusted to 6.0, and the pH inside the liposomes was 7.5.

[0181] Bortezomib was then added to a gradient of budesonide palmitate liposomes and incubated in a water bath at 60°C for 20 min with stirring. The mixture was then immediately cooled in an ice bath to obtain budesonide palmitate and bortezomib co-loaded liposomes.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, include: Budesonide and bortezomib; Alternatively, budesonide derivatives and bortezomib.

2. The pharmaceutical composition according to claim 1, characterized in that, The budesonide derivative is esterified budesonide, preferably budesonide palmitate.

3. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of budesonide (or its derivatives) to bortezomib is (0.25–4):1, preferably (0.3–2):1, and more preferably (0.4–0.6):

1.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The pharmaceutical composition may also include pharmaceutically acceptable excipients or carriers.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is a solid dosage form, a semi-solid dosage form, or a liquid dosage form.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is a nano-formulation; preferably, the nano-formulation is a phospholipid complex nanoparticle, nanoliposome, nanosphere, nanocapsule, or polymer micelle.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is a phospholipid complex nanoparticle, wherein the phospholipid is natural, semi-synthetic or fully synthetic.

8. The pharmaceutical composition according to claim 7, characterized in that, The phospholipid is at least one of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and myocardial phospholipid; preferably at least one of egg yolk phosphatidylglycerol, hydrogenated egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, distearyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, and dilauroyl phosphatidylglycerol, more preferably egg yolk phosphatidylglycerol.

9. The pharmaceutical composition according to claim 7, characterized in that, In the phospholipid complex nanoparticles, the molar ratio of drug to phospholipid is 1:(1-5); Preferably, the molar ratio of budesonide or its derivatives, bortezomib, and phospholipids is 1:(1.5-2.5):(3.5-5); More preferably, the molar ratio of budesonide or its derivatives, bortezomib to phospholipids is 1:(1.5-2.5):(3.5-4.5).

10. The pharmaceutical composition according to claim 7, characterized in that, The phospholipid complex nanoparticles also include sialic acid lipid derivatives; the sialic acid lipid derivatives are formed by coupling sialic acid with lipid fragments.

11. The pharmaceutical composition according to claim 10, characterized in that, The sialic acid lipid derivative is a lipid fragment coupled to the carboxyl group at the 1-position or the hydroxyl group at the 9-position of sialic acid, wherein the lipid fragment is cholesterol, fatty acid or a derivative thereof. Preferably, the sialic acid and the lipid fragment are linked by an amide bond or a carboxyl ester bond; Preferably, the general formula of the sialic acid lipid derivative is shown in Formula I or Formula II: In Formula I, R1 is a H atom or a C1-C6 alkyl group; R2 is H or (CH2) m C2-C6 alkenyl, m = 1-17; R4 is -OH, -NHCOCH3, or -NHCOCH2OH; X is a H atom, an O atom, or a carbonyl group; When X is a H atom or an oxygen atom, R3 is (CH2)2 or a cholesterol group, n = 1 to 17; When X is a carbonyl group, R3 is a C12-C24 alkoxy group, a C12-C24 alkyl-substituted amino group, or a cholesterol group; In Equation II, R1 is -S-(CH2). n -or -SS-(CH2) n -, n = 1 to 6; R2 is -OH, -NHCOCH3, or -NHCOCH2OH; R3 is a C12–C24 alkoxy group, a C12–C24 alkyl-substituted amino group, or a cholesterol group.

12. The pharmaceutical composition according to claim 10, characterized in that, The molar number of the sialic acid lipid derivative accounts for less than 30% of the total molar number of the sialic acid lipid derivative and phospholipid.

13. The pharmaceutical composition according to claim 10, characterized in that, The phospholipid complex nanoparticles also include PEG lipid derivatives; the PEG lipid derivatives are formed by coupling PEG with lipid fragments.

14. The pharmaceutical composition according to claim 13, characterized in that, The PEG lipid derivative is a breakable PEG lipid derivative; Preferably, the cleavable chemical bonds in the cleavable PEG lipid derivative are esterase-sensitive chemical bonds; More preferably, the breakable PEG lipid derivative is mPEG. n CHEMS; Where n represents the molecular weight of PEG, and n ranges from 200 to 20000.

15. The pharmaceutical composition according to claim 14, characterized in that, The molar number of the breakable PEG lipid derivative accounts for 0.1% to 10% of the total molar number of the breakable PEG lipid derivative and phospholipid.

16. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is a liposome; the phospholipid bilayer of the liposome encapsulates budesonide or its derivatives, and the aqueous phase encapsulates a borate ester compound prepared from bortezomib and a polyol.

17. A method for preparing the pharmaceutical composition according to any one of claims 1 to 16, characterized in that, include: The raw material is dissolved in an organic solvent and stirred at a temperature of 20°C to 70°C to remove the organic solvent, thereby obtaining the pharmaceutical composition.

18. The preparation method according to claim 17, characterized in that, The process includes: dissolving the raw material in an organic solvent, stirring at a temperature of 20°C to 70°C, removing the organic solvent, adding water, and filtering through a filter membrane to obtain the pharmaceutical composition. Preferably, the material is filtered sequentially through filter membranes of 0.80μm, 0.45μm, and 0.22μm.

19. The preparation method according to claim 17, characterized in that, include: Budesonide or its derivatives, bortezomib, phospholipids, sialic acid lipid derivatives and PEG lipid derivatives were dissolved in an organic solvent, stirred at 20℃~70℃, and water was added after removing the organic solvent. The phospholipid complex nanoparticles were then obtained by filtering through a filter membrane. The concentration of budesonide or its derivative in the organic solvent is 3.5 mg·mL. -1 Hereinafter, the concentration of bortezomib in the organic solvent is 6 mg·mL. -1 the following.

20. The preparation method according to claim 17, characterized in that, include: (1) Budesonide or its derivatives and liposome membrane material are dissolved in an organic solvent to obtain a membrane material solution. The organic solvent is removed to obtain a membrane material concentrate. The membrane material concentrate is mixed with a gradient substance solution to obtain a blank liposome initial product. (2) The blank liposome sample is extruded through a filter membrane to obtain a liposome suspension; (3) Remove the gradient-establishing substances from the aqueous phase outside the liposome suspension to obtain a gradient liposome suspension; (4) The drug composition is prepared by mixing the gradient liposome suspension with bortezomib.

21. The use of the pharmaceutical composition of any one of claims 1 to 16 or the preparation method of any one of claims 17 to 20 in the preparation of an anti-inflammatory drug.

22. The application according to claim 21, characterized in that, The anti-inflammatory drug is used to treat at least one of the following diseases: Infectious or contagious diseases, autoimmune diseases, cancer, vascular diseases, metabolic diseases; More preferably, the anti-inflammatory drug is used to treat arthritis or ulcerative colitis.

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