A nano-vaccine for thrombus prevention and a preparation method and application thereof

Abstract

The application specifically discloses a kind of nanovaccine for thrombosis prevention and its preparation method and application, it is related to thrombotic disease, especially related to the nanovaccine for treating thrombotic disease.The nanovaccine provided by the application has good immunogenicity without macromolecular heterologous protein carrier such as KLH;The process simplicity and repeatability are improved by reducing complex chemical coupling and purification steps;The dependence on traditional high-stimulating immune adjuvant is reduced, and the biological safety is improved;The stability of antigen in gastrointestinal environment is significantly improved;Active targeting delivery of M cells and dendritic cells is realized;Dendritic cell maturation and antigen presentation are promoted, and mucosal immunity and systemic immunity level are improved;The advantages such as effectively inhibiting thrombosis are realized, and a new type of oral antithrombotic vaccine system with high compliance suitable for long-term thrombosis prevention is constructed.

Description

Technical Field

[0001] This invention relates to thrombotic diseases, and more particularly to nanovaccines for the treatment of thrombotic diseases. Background Technology

[0002] Thrombotic diseases, including ischemic stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism, are major causes of death and disability worldwide. Among these, the S100A9 protein is considered a key risk molecule linking inflammation and thrombosis. S100A9 can be released by neutrophils, monocytes, and activated platelets, and promotes platelet activation, amplified inflammation, and thrombosis through receptors such as CD36. Studies have shown that blocking S100A9 can significantly inhibit thrombosis without significantly increasing the risk of bleeding, making it a highly promising target for antithrombotic vaccines. Although studies have validated the feasibility of S100A9 vaccines, current technologies still have many limitations.

[0003] First, existing peptide-based antithrombotic vaccines generally suffer from insufficient immunogenicity. Since S100A9 is an autoantigen, its natural immunogenicity is weak, and it is difficult to induce a sufficiently strong immune response when used alone. Therefore, current research typically requires conjugating the target peptide with a large molecular weight carrier protein to enhance immunogenicity. Commonly used carriers include keyhole lipocyanin (KLH), bovine serum albumin (BSA), and virus-like particles (VLPs). However, these carrier systems have significant drawbacks. Taking KLH as an example, it is a large heterologous protein derived from marine mollusks, with a complex composition, large molecular weight, and significant batch-to-batch variations, increasing the difficulty of vaccine preparation and quality control. Peptide-KLH conjugation usually requires chemical cross-linking systems such as EDC / NHS, involving multiple steps of conjugation, purification, and conjugation efficiency verification, resulting in a complex overall process that is not conducive to large-scale production and industrialization.

[0004] Furthermore, KLH itself possesses strong immunogenicity and may induce significant nonspecific immune responses. Existing studies have shown that KLH immunization may lead to local inflammation, granuloma formation, fever, and a strong Th2-type immune bias. During repeated immunizations, a "carrier epitope dominance" phenomenon may also occur, meaning the body primarily generates an immune response against the KLH carrier rather than the target antigen, thereby reducing the effective immunization efficiency against the target antigen.

[0005] Besides the issue of delivery vectors, existing antithrombotic vaccines typically rely heavily on traditional adjuvants to enhance the immune response. Examples include Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), alum, and CpG oligonucleotides. While CFA possesses strong immunomodulatory capabilities, its mycobacterial components result in significant inflammatory stimulation, potentially leading to substantial tissue damage, local necrosis, and granuloma formation, rendering it unsuitable for clinical human vaccines. Although IFA has lower toxicity than CFA, it can still induce persistent local inflammation. Alum-type adjuvants, while widely used clinically, primarily induce Th2-type immunity, with limited activation of mucosal and cellular immunity, and may cause local redness, pain, and delayed inflammatory responses. While novel adjuvants like CpG can enhance Th1-type immunity, excessive activation of innate immunity can still lead to increased release of inflammatory factors, systemic inflammatory responses, and potential autoimmune risks.

[0006] Therefore, how to reduce adjuvant-related toxicity while ensuring immunization efficacy remains a significant challenge in the current vaccine field.

[0007] On the other hand, most existing antithrombotic vaccines are administered via subcutaneous or intradermal injection, requiring multiple booster immunizations. This not only makes them highly invasive but also reduces patient compliance in long-term chronic disease prevention scenarios.

[0008] More importantly, current research on oral antithrombotic vaccines remains very limited. After oral administration, peptide antigens are highly susceptible to degradation by gastric acid and proteases. Furthermore, the intestinal mucus layer, epithelial barrier, and intestinal immune tolerance mechanisms further restrict antigen absorption and effective immune activation.

[0009] Therefore, how to construct an antithrombotic vaccine system that does not require complex protein conjugation, can be delivered orally, and has both good immunogenicity and safety remains an important problem that existing technologies have not yet solved. Summary of the Invention

[0010] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a nanovaccine for thrombosis prevention, its preparation method, and its application. The nanovaccine provided by this invention achieves good immunogenicity without the need for large molecular weight heterologous protein carriers such as KLH; reduces complex chemical coupling and purification steps, improving process simplicity and reproducibility; reduces dependence on traditional highly irritating adjuvants, improving biosafety; significantly improves antigen stability in the gastrointestinal environment; achieves active targeted delivery to M cells and dendritic cells; promotes dendritic cell maturation and antigen presentation, improving mucosal and systemic immunity levels; and effectively inhibits thrombus formation. This invention constructs a novel oral antithrombotic vaccine system with high compliance and suitable for long-term thrombosis prevention.

[0011] (II) Technical Solution To achieve the above objectives, the present invention provides a nano-vaccine comprising S100A9 peptide, oxidized β-glucan, and quaternary ammonium cationic starch.

[0012] In one embodiment, the oxidized β-glucan is derived from yeast.

[0013] In another aspect, the present invention provides a method for preparing the above-mentioned nano-vaccine, comprising: S1: React β-glucan with an oxidizing agent to obtain oxidized β-glucan (OYG). S2: Oxidized β-glucan was reacted with S100A9 peptide to obtain a conjugate (OYG-pep). S3: The conjugate is reacted with quaternary ammonium cationic starch to obtain the nanovaccine (OYG-pep / QA).

[0014] In one embodiment, the β-glucan in S1 is derived from yeast.

[0015] In one embodiment, the oxidant in S1 includes sodium periodate, sodium hypochlorite, hydrogen peroxide, ozone, ammonium persulfate, sodium dichloroisocyanurate, sodium hypochlorite-sodium bromide system, and / or TEMPO-sodium hypochlorite system.

[0016] In one embodiment, the oxidant in S1 includes a sodium hypochlorite-sodium bromide system and a TEMPO-sodium hypochlorite system.

[0017] In one embodiment, the reaction conditions for S1 are: pH 10.0~12.0, reaction time 30~60 min.

[0018] In one embodiment, S2 specifically includes: T1: React the carboxyl activator with the oxidized β-glucan to obtain the oxidized β-glucan active ester; T2: NH2-PEG-Mal is reacted with oxidized β-glucan active ester to obtain amino-active ester conjugate (OYG-PEG-Mal). T3: The amino-active ester conjugate is reacted with the S100A9 peptide to obtain the conjugate (OYG-pep).

[0019] In one embodiment, the carboxyl activator includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), diisopropylcarbodiimide, N-hydroxysuccinimide (NHS), sulfon-N-hydroxysuccinimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, benzotriazole-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N'-carbonyldiimidazole, and / or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline.

[0020] In one embodiment, the carboxyl activator in T1 includes EDC and NHS.

[0021] In one embodiment, the reaction conditions for T1 are: pH 5.0~6.0, 10~20 min.

[0022] In one embodiment, the reaction conditions for T2 are: pH 6.0~7.0, 2~3h.

[0023] In one embodiment, the reaction conditions for T3 are: pH 6.0~7.0, 1~2h.

[0024] In one embodiment, the quaternary ammonium cationic starch in S3 is gelatinized and then reacts with the amino-active ester conjugate.

[0025] In one embodiment, the gelatinization conditions of the quaternary ammonium cationic starch in S3 are: 80~100℃, 0.5~1h.

[0026] In one embodiment, the reaction conditions for S3 are: 0.1~1h.

[0027] In another aspect, the present invention also provides a nano-vaccine obtained by the above preparation method.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective amount of the above-mentioned nano-vaccine; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0029] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0030] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0031] In one embodiment, the pharmaceutical product includes a vial or box.

[0032] In another aspect, the present invention also provides the use of the above-mentioned nanovaccines, pharmaceutical compositions, pharmaceutical preparations and / or pharmaceutical products in the preparation of drugs for the prevention and / or treatment of thrombosis.

[0033] In one embodiment, the thrombotic disease includes arterial thrombosis, venous thrombosis, and / or microcirculatory thrombosis.

[0034] In one embodiment, the arterial thrombosis includes coronary atherosclerotic heart disease: when thrombosis forms in the branches or main trunk of the coronary arteries, causing myocardial ischemia, angina pectoris or myocardial infarction may occur; cerebral artery thrombosis: which can lead to transient ischemic attacks, cerebral infarction causing hemiplegia, altered consciousness, or central retinal artery occlusion causing blindness; peripheral arterial occlusive diseases: such as mesenteric artery thrombosis and limb arterial thrombosis, causing limb pain and limb ischemic necrosis, etc.

[0035] In one embodiment, the venous thrombosis includes thrombophlebitis; deep vein thrombosis; and pulmonary embolism.

[0036] In one embodiment, the microcirculatory thrombotic disease includes thrombotic thrombocytopenic purpura; hemolytic uremic syndrome; extracorporeal circulation thrombosis; and others such as fulminant purpura and disseminated intravascular coagulation.

[0037] (III) Beneficial Effects This invention provides a nano-vaccine for thrombosis prevention, its preparation method, and its application. Compared with existing technologies, it has the following advantages: 1. Flow cytometry analysis of BMDC maturity and antigen presentation capacity demonstrates that the nanovaccine provided by this invention can significantly stimulate BMDC maturity and antigen presentation.

[0038] 2. Inoculation with the nano-vaccine provided by this invention can significantly inhibit the formation of inferior vena cava thrombosis in mice. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This is the Zeta potential diagram of the product; Figure 2 This is a transmission electron microscope image of a nanovaccine; Figure 3 This is a diagram showing the uptake of nano-vaccines by dendritic cells and the maturity analysis of dendritic cells; Figure 4 This is a chart assessing the quality and area of ​​a thrombus. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Terms and Definitions As used herein, the term "pharmaceutical composition" refers to a composition comprising a nanovaccine formulated together with one or more pharmaceutically acceptable carriers.

[0043] The formulation of the pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: liquid suspensions (mainstream for injectable and nasal nanovaccines); lyophilized nanopowder injections (to improve the room-temperature stability of LNP and PLGA nanoparticles); in-situ nanosphere gels (gelling after subcutaneous injection for long-lasting sustained release); and oral nanocapsules.

[0044] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0045] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0046] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0047] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0048] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0049] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to accomplishing one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0050] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0051] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0053] Example 1: Preparation of OYG: 2 g of yeast-derived β-glucan was dissolved in 200 mL of ultrapure water, and the pH of the solution was adjusted to 10.0–12.0 using sodium hydroxide (NaOH). The mixture was stirred at 4°C in an ice bath for 1 hour. Subsequently, 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO, 17.32 mg), sodium bromide (NaBr, 0.384 g), and 20 mL of sodium hypochlorite (NaClO) solution were added to initiate the oxidation reaction. During the reaction, the pH was maintained at 10.0–12.0 by adding NaOH, and the reaction time was 45 min.

[0054] Add 10 mL of ethanol and adjust the pH to 5.0-6.0 using 1 M hydrochloric acid (HCl). Then add the reaction solution to 800 mL of ethanol and let it stand overnight.

[0055] The resulting product was collected by vacuum filtration, washed multiple times with 95% ethanol, and subjected to multiple dialysis cycles to ensure purification before freeze-drying.

[0056] Example 2: Preparation of OYG-pep: Dissolve 100 mg of OYG obtained in Example 1 in 10 mL of MES (pH 5.0~6.0) buffer, stir magnetically at room temperature, add 50 mg of EDC and 29 mg of NHS, and activate for 15 min.

[0057] Dissolve 10.0 mg of NH2-PEG-Mal in approximately 100 µL of deionized water. Slowly add the NH2-PEG-Mal solution to the activated OYG solution, while simultaneously adjusting the pH to 6.0–7.0 with a small amount of NaOH. Stir gently at room temperature for 2–3 hours. Transfer the reacted solution to a 3.5 kDa MWCO dialysis bag and dialyze with a large volume of PBS (pH 6.8, containing 1 mM EDTA).

[0058] Dissolve 5.0–10.0 mg of S100A9 peptide (CGSGRGHGHSHGKG) in 1 mL of PBS (pH 6.8, containing 1 mM EDTA). Add 100–200 µL of the peptide solution dropwise to the OYG-PEG-Mal solution. Maintain the pH at 6.0–7.0 and react at room temperature for 1–2 h. After the reaction is complete, place the reaction solution in a MWCO 3.5 kDa dialysis bag and dialyze against a large volume of deionized water at 4 °C or room temperature for 24–48 h, changing the water at least 4–6 times. Finally, freeze-dry the product (OYG-pep) and store it at -20 °C.

[0059] Figure 1 The results of the Zeta potential of the products obtained in each step of the synthesis process indicate that each step of the synthesis was successful and the peptide was successfully modified into OYG.

[0060] Example 3: Preparation of OYG-pep / QA: Add 100 mg of quaternary ammonium cationic starch (QA) to 10 mL of deionized water and gelatinize at 90 °C for 0.5–1 h. Cool to room temperature for later use.

[0061] Add 2 mL of 0.5 mg / mL OYG-pep solution (solvent: PBS buffer) to 1 mL of 5 mg / mL QA solution, and stir continuously at 400-600 rpm for 0.5 h. After synthesis, let stand for 0.5 h to obtain the final reaction product OYG-pep / QA.

[0062] Figure 2 These are transmission electron microscope images of the synthesized OYG-pep / QA, indicating that the nanoparticle vaccine was successfully synthesized and has a spherical morphology.

[0063] Example 4: In vitro cell experiment verification: Fluorescently labeled nanovaccines were co-incubated with DC2.4 cells and observed using a confocal microscope. Figure 3 As shown in A, the nano-vaccine can be efficiently taken up by DC2.4 cells.

[0064] Furthermore, dendritic cells (BMDCs) derived from primary bone marrow of C57BL / 6 mice were extracted, induced and cultured in vitro, and then treated with nano-vaccines. The maturity and antigen-presenting capacity of the BMDCs were analyzed by flow cytometry. Figure 3 As shown in B, nanovaccines can significantly stimulate BMDC maturation and antigen presentation.

[0065] Example 5: In vivo model experimental verification: C57BL / 6 mice were randomly divided into three groups. Vaccination was administered on days 0, 3, 6, 9, and 12. Different treatment groups were administered PBS, free S100A9 peptide, or OYG-pep / QA nanovaccine via gavage, respectively. The vaccinated mice were then used for subsequent in vivo model experiments to validate the vaccine.

[0066] After anesthetizing the mice, the inferior vena cava was exposed and the surrounding tissues were gently dissected. Collateral veins between the renal vein and the iliac vein were ligated. A 5-0 polypropylene suture was placed parallel to the inferior vena cava as a pad, followed by partial ligation of the inferior vena cava using a 4-0 polypropylene suture. The pad was then removed, restoring some blood flow and thus establishing a stable venous thrombosis model.

[0067] Forty-eight hours after thrombus formation, the thrombus is removed, weighed, and the effectiveness of the vaccine is assessed. Figure 4 The results showed that vaccination with nano-vaccines could significantly inhibit the formation of inferior vena cava thrombosis in mice.

[0068] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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. Such 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 nano-vaccine, characterized in that, It includes S100A9 peptide, oxidized β-glucan, and quaternary ammonium cationic starch.

2. The nano-vaccine according to claim 1, characterized in that, The oxidized β-glucan is derived from yeast.

3. The method for preparing the nano-vaccine according to claim 1 or 2, characterized in that, include: S1: React β-glucan with an oxidizing agent to obtain oxidized β-glucan; S2: The conjugate is obtained by reacting oxidized β-glucan with S100A9 peptide; S3: The conjugate is reacted with quaternary ammonium cationic starch to obtain the nano-vaccine.

4. The preparation method according to claim 3, characterized in that, S2 specifically includes: T1: React the carboxyl activator with the oxidized β-glucan to obtain the oxidized β-glucan active ester; T2: NH2-PEG-Mal was reacted with oxidized β-glucan active ester to obtain an amino-active ester conjugate; T3: The amino-active ester conjugate is reacted with the S100A9 peptide to obtain the conjugate.

5. The preparation method according to claim 4, characterized in that, The carboxyl activator includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, diisopropylcarbodiimide, N-hydroxysuccinimide, sulfon-N-hydroxysuccinimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, benzotriazole-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, N,N'-carbonyldiimidazole and / or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline.

6. A nano-vaccine, characterized in that, It is obtained by the preparation method according to any one of claims 3 to 5.

7. A pharmaceutical composition, characterized in that, include: (1) A therapeutically effective amount of the nanovaccine as described in claim 1, 2 or 6; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

8. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition of claim 7.

9. A pharmaceutical product, characterized in that, Includes the pharmaceutical preparation described in claim 8.

10. The use of the nanovaccine of claim 1, 2, or 6, the pharmaceutical composition of claim 7, the pharmaceutical formulation of claim 8, and / or the pharmaceutical product of claim 9 in the preparation of a medicament for the prevention and / or treatment of thrombosis.

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