Antigen and adjuvant co-delivery nanocapsule vaccine and preparation method thereof

Abstract

The invention discloses an antigen and adjuvant co-delivery nanocapsule vaccine and a preparation method thereof. The preparation method comprises the following steps: dissolving the polycaprolactone, the polycaprolactone-polyetherimide block copolymer, the adjuvant oil phase and the fat-soluble immune activator in ethanol to obtain a mixed solution; and carrying out solvent exchange on the obtained mixed solution and an isotonic solution in which the antigen is dissolved so as to obtain the antigen and adjuvant co-delivered nanocapsule vaccine. The nanocapsule vaccine is simple and efficient in preparation scheme, adopts a biocompatible material and a green solvent, is green and free of toxic and side effects, and can be suitable for large-scale vaccine production process popularization. The nanocapsule vaccine can realize co-delivery of the oil phase adjuvant, the water-soluble antigen and the water-soluble antigen, improves the immune response effect, and has a good prospect in the field of vaccine research and development.

Description

Technical Field

[0001] The present invention relates to the field of nano-vaccine research and development, and particularly to a nano-capsule vaccine for co-delivery of an antigen and an adjuvant and a preparation method thereof. Background Art

[0002] Current nano-vaccine technologies, especially in the field of cancer treatment, face multiple technical bottlenecks. Traditional nano-carriers, such as liposomes and polylactic acid nanoparticles, can improve the antigen delivery efficiency, but there are significant limitations in the accuracy and synergy of immune regulation. Existing delivery systems are difficult to simultaneously load hydrophilic / hydrophobic dual adjuvants, and the surface antigen display efficiency is low. Research shows that the antigen loading of most nano-vaccines is less than 5 wt%, and the surface antigen is prone to losing immunogenicity due to conformational changes, seriously restricting the intensity of cellular immune responses. Moreover, problems such as complex production processes of nano-carriers and poor carrier stability further limit their clinical transformation potential.

[0003] In recent years, compared with traditional nano-carriers, vaccine carriers based on nano-capsules have attracted attention due to their core-shell structure and controlled release characteristics. Their hydrophobic core can efficiently encapsulate lipophilic immunomodulators and achieve slow release of adjuvants through controlled degradation. Some studies have confirmed that nano-capsules constructed with polyester materials can improve the antigen uptake rate of dendritic cells and regulate the antigen release kinetics through core-shell interface engineering, such as pH-responsive release. In addition, the rigid structure of nano-capsules can resist serum protein adsorption, and their stability in the circulatory system is significantly better than that of liposomes.

[0004] Although nano-capsule vaccines show potential at the theoretical level, their practical applications still have significant technical bottlenecks. Existing nano-capsules can encapsulate hydrophobic adjuvants, but they cannot achieve co-delivery of antigens, and the spatio-temporal co-localization ability of antigens and adjuvants is insufficient, resulting in asynchronous immune activation signals. In addition, existing nano-capsule preparation processes rely on high-pressure homogenization or emulsion templating methods, with low productivity. Although it has been reported that block copolymers are used to improve interfacial compatibility, their self-assembly accuracy is insufficient, resulting in uneven charge distribution at the oil-water interface, unable to achieve efficient co-delivery of antigens and adjuvants, and difficult to achieve large-scale production, further hindering their clinical transformation. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention proposes a nano-capsule vaccine for co-delivery of an antigen and an adjuvant and a green preparation method thereof. The preparation scheme of the nano-capsule vaccine is simple and efficient, and uses biocompatible materials and green solvents, which are green, non-toxic and have no side effects, and can be applied to the popularization of large-scale vaccine production processes. The nano-capsule vaccine can achieve co-delivery of oil-phase adjuvants, water-soluble antigens and water-soluble antigens, improve the immune response effect, and has good prospects in the field of vaccine research and development.

[0006] Technical solution of the present invention: A nano-capsule vaccine for co-delivering an antigen and an adjuvant and a preparation method thereof, comprising the following steps:

[0007] S1: Dissolve polycaprolactone, polycaprolactone-polyetherimide block copolymer, adjuvant oil phase and lipophilic immune activator in ethanol to obtain a mixed solution;

[0008] S2: Then perform solvent exchange on the mixed solution obtained in step S1 and the isotonic solution containing the antigen. The adjuvant oil phase aggregates to form an oil core, the lipophilic immune activator is dissolved in the oil core, and polycaprolactone and polycaprolactone-polyetherimide self-assemble at the oil-water interface to form an oil-core nano-capsule. Then, through electrostatic interaction, the antigen is captured on the surface of the nano-capsule, thereby obtaining a nano-capsule vaccine co-loaded with the antigen and the adjuvant.

[0009] As a preferred embodiment of the present invention, in step S1, the molecular weight of the polycaprolactone is 1000-10000; the molecular weight of the polycaprolactone-polyetherimide block copolymer is 2000-15000; the polycaprolactone structure can be one or more of polycaprolactone or polycaprolactone polyol derivatives.

[0010] As a preferred embodiment of the present invention, the oil phase in step S1 is a pharmaceutical co-solvent that is immiscible with water and miscible with ethanol, including one or more of iodized oil, squalene oil, monoglyceride, diglyceride, polyethylene glycol glycerol caprylate / caprate, and propylene glycol monolaurate.

[0011] As a preferred embodiment of the present invention, the adjuvant oil phase in step S1 includes one or more mixtures of squalene oil, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, and mineral oil.

[0012] As a preferred embodiment of the present invention, the lipophilic immune activator in step S1 includes one or more of ginsenoside, lipophilic derivative of muramyl dipeptide, α-GalCer, PAM 3CSK4, and monophosphoryl lipid A.

[0013] As a preferred embodiment of the present invention, the antigen in step S2 includes one of antigens such as ovalbumin, influenza virus nucleoprotein, embryonic protein, glycoprotein, etc. that can cause corresponding immune responses; the concentration of the antigen in the isotonic solution is 10-100 μg / mL.

[0014] As a preferred embodiment of the present invention, the isotonic solution in step S2 is one of 0.9% normal saline or 5% glucose water. Further, the isotonic solution also includes CpG oligonucleotide with a concentration of 10-100 μg / mL; during the formation of the nano-capsule vaccine loaded with the antigen and the adjuvant, the CpG oligonucleotide is captured on the surface of the nano-capsule.

[0015] As a preferred embodiment of the present invention, in step S1, the concentration of polycaprolactone in ethanol is 20 mg / mL - 100 mg / mL, the concentration of polycaprolactone - polyetherimide block copolymer in ethanol is 2 mg / mL - 20 mg / mL, the concentration of adjuvant oil phase in ethanol is 25 - 50 μL / mL, and the concentration of lipophilic immune activator in ethanol is 0.2 - 1 mg / mL.

[0016] As a preferred embodiment of the present invention, for the preparation method according to claim 1, it is characterized in that: in step S2, the solvent exchange scheme includes the dropping method, the microchannel injection mixing method, and the microfluidic chip mixing method, and the ratio of ethanol to isotonic solution is 1:10 - 1:50.

[0017] The present invention also provides an antigen - and - adjuvant co - loaded nanocapsule vaccine prepared according to the above - mentioned preparation method.

[0018] The present invention also provides the application of the nanocapsule vaccine in the preparation of vaccine preparations.

[0019] For the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) The nanocapsule vaccine prepared by the scheme of the present invention has a size of 200 - 300 nm, can effectively encapsulate the adjuvant oil phase and the lipophilic immune adjuvant, and has an encapsulation efficiency of more than 95%, and has good dispersibility.

[0021] 2) The present technology proposes an antigen - adjuvant co - delivery system based on oil - core nanocapsules. This system can achieve the synergistic effect of hydrophobic and hydrophilic adjuvants, further enhancing the antigen immune response effect. In addition, by classically adsorbing antigens on the surface, the co - delivery of antigens and adjuvants can be achieved, realizing the activation of synchronous immune responses with spatiotemporal coordination, which has important clinical significance.

[0022] 3) The nanocapsules prepared by the present invention can effectively regulate the size and wall thickness of the nanocapsules according to needs by controlling the mixing solution, the isotonic solution formula, and the microfluidic shear process during the solvent exchange process, achieving the effect of drug slow release.

[0023] 4) Compared with the traditional emulsion method, the preparation efficiency of the nanocapsule vaccine based on the solvent exchange scheme is higher, suitable for continuous production, conducive to the large - scale production and promotion of nanocapsule vaccines, and its stability is higher than that of conventional liposome vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the preparation flow chart of the nanocapsule vaccine of the method shown in the present invention;

[0025] Figure 2 is the charge change of the nanocapsule vaccine with different antigen loading amounts;

[0026] Figure 3 It is a schematic diagram of the core-shell structure of the nano-capsule vaccine.

[0027] Figure 4 It is a fluorescence image of the nano-capsule vaccine with fluorescent ovalbumin antigen loaded on the surface. Specific embodiments

[0028] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that for those of ordinary skill in the art in the technical field, without departing from the principle of the present invention, several improvements and retouches can also be made, and these improvements and retouches are also regarded as the protection scope of the present invention.

[0029] All raw materials used in the following examples are commercially available products.

[0030] Example 1: Nano-capsule vaccine for immune activation against H1N1 influenza virus

[0031] As Figure 1 shown, the specific steps for preparing the nano-microcapsules by the method of the present invention are as follows:

[0032] S1: Dissolve 30 mg of polycaprolactone (molecular weight 3000), 3 mg of polycaprolactone-polyetherimide (molecular weight 5000), 10 μL of squalene oil and 10 μL of α-tocopherol in 1 mL of ethanol;

[0033] S2: Pipette 100 μL of the mixed solution obtained in step S1, and then inject the mixed solution into 5 mL of an isotonic solution of 0.9 wt% normal saline containing 0.01 mg of H1N1 antigen and 0.05 mg of CpG oligonucleotide at a speed of 10 mL / min. During the rapid solvent exchange of ethanol and water, squalene oil and α-tocopherol precipitate to form an oil-phase core, and polycaprolactone and polycaprolactone-polyetherimide precipitate at the oil-water interface to form a positively charged surface nano-capsule. The negatively charged H1N1 antigen and CpG oligonucleotide are captured by the positively charged nano-capsule due to electrostatic interaction, thereby obtaining a nano-capsule vaccine loaded with H1N1 antigen and adjuvant;

[0034] With the adjustment of the content of polycaprolactone-polyetherimide in the formulation and the increase of the antigen amount, the surface charge of the formed nano-capsule changes accordingly, which proves the aggregation effect of polycaprolactone-polyetherimide at the nano-capsule interface and the successful loading of the antigen on the nano-capsule surface, as Figure 2 shown; the core-shell structure of the obtained nano-capsule is as Figure 3 shown, and the size of the prepared oil-core nano-capsule is about 250 nm.

[0035] Example 2: Nano-capsule vaccine for immune activation against influenza B

[0036] S1: Dissolve 50 mg of polycaprolactone (molecular weight 5000), 10 mg of polycaprolactone - polyetherimide (molecular weight 8000), 10 μL of squalene oil and 10 μL of mineral oil in 1 mL of ethanol;

[0037] S2: Drop 1 mL of the mixed solution obtained in step S1 into 50 mL of an isotonic solution of 0.9 wt% saline containing 0.1 mg of influenza B antigen at a rate of 0.1 mL / min, thereby obtaining a mineral oil / squalene oil core nanocapsule vaccine with influenza B antigen loaded on its surface.

[0038] Example 3: Nanocapsule vaccine for liver cancer immune activation

[0039] S1: Dissolve 30 mg of polycaprolactone, 10 mg of polycaprolactone - polyetherimide, 25 μL of squalene oil and 2 mg of monophosphoryl lipid A in 1 mL of ethanol;

[0040] S2: Mix 10 mL of the mixed solution obtained in step S1 with 100 mL of an isotonic solution of 5 wt% glucose containing 2 mg of liver cancer antigen through a microfluidic chip at a flow rate of 1:10, thereby obtaining a squalene oil core nanocapsule vaccine loaded with liver cancer cell surface antigen and monophosphoryl lipid A immune agonist.

[0041] Example 4: Chinese patent medicine - loaded nanocapsule vaccine for breast cancer immune activation

[0042] S1: Dissolve 20 mg of polycaprolactone, 5 mg of polycaprolactone - polyetherimide, 10 μL of β - tocopherol and 1 mg of ginsenoside in 1 mL of ethanol;

[0043] S2: Pipette 100 μL of the mixed solution obtained in step S1, and then inject the mixed solution into 5 mL of an isotonic solution of 0.9 wt% saline containing 0.01 mg of breast cancer antigen and 0.05 mg of CpG oligonucleotide at a rate of 10 mL / min, thereby obtaining a β - tocopherol core nanocapsule vaccine loaded with breast cancer antigen, ginsenoside immune agonist and CpG oligonucleotide adjuvant.

[0044] Example 5: Nanocapsule vaccine for cancer immune activation with fluorescent ovalbumin loaded on its surface

[0045] S1: Dissolve 100 mg of polycaprolactone, 10 mg of polycaprolactone - polyetherimide, 50 μL of squalene oil in 1 mL of ethanol;

[0046] S2: Pipette 500 μL of the mixed solution obtained in step S1, and then inject the mixed solution into 3 mL of an isotonic solution of 0.9 wt% physiological saline containing 0.1 mg of FITC-ovalbumin at a rate of 10 mL / min, thereby obtaining a nano-capsule vaccine loaded with fluorescent protein antigen on the surface. The results are as Figure 4 shown, and it can be seen that the protein antigen is attracted to the surface of the nano-capsule through electrostatic interaction.

[0047] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for preparing a nano-capsule vaccine for co-delivery of an antigen and an adjuvant, characterized in that: It includes the following steps: S1: Dissolve polycaprolactone, polycaprolactone-polyetherimide block copolymer, adjuvant oil phase and liposoluble immune activator in ethanol to obtain a mixed solution; S2: Then perform solvent exchange on the mixed solution obtained in step S1 and the isotonic solution dissolved with antigen. The adjuvant oil phase aggregates to form an oil core, the liposoluble immune activator is dissolved in the oil core, and polycaprolactone and polycaprolactone-polyetherimide self-assemble at the oil-water interface to form an oil-core nanocapsule. Then, through electrostatic interaction, the antigen is captured on the surface of the nanocapsule, thereby obtaining an antigen and adjuvant-loaded nanocapsule vaccine.

2. The preparation method according to claim 1, wherein: In step S1, the molecular weight of the polycaprolactone is 1000 - 10000; the molecular weight of the polycaprolactone-polyetherimide block copolymer is 2000 - 15000; the polycaprolactone structure is one or more of polycaprolactone or polycaprolactone polyol derivatives.

3. The preparation method according to claim 1, wherein: The adjuvant oil phase described in step S1 includes one or more mixtures of squalene oil, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol or mineral oil.

4. The preparation method according to claim 1, characterized in that: The liposoluble immune activator described in step S1 includes one or more of ginsenoside, lipophilic derivative of muramyl dipeptide, α-GalCer, PAM 3CSK4 or monophosphoryl lipid A.

5. The preparation method according to claim 1, characterized in that: The antigen in step S2 is one of ovalbumin, influenza virus nucleoprotein, embryonic protein, glycoprotein; the concentration of the antigen in the isotonic solution is 10 - 100 μg / mL; the isotonic solution is one of 0.9wt% normal saline or 5wt% glucose water.

6. The preparation method according to claim 1, characterized in that: The isotonic solution also includes CpG oligonucleotide with a concentration of 10 - 100 μg / mL.

7. The preparation method according to claim 1, characterized in that: In step S1, the concentration of polycaprolactone in ethanol is 20 mg / mL - 100 mg / mL, the concentration of polycaprolactone-polyetherimide block copolymer in ethanol is 2 mg / mL - 20 mg / mL, the concentration of adjuvant oil phase in ethanol is 25 - 50 μL / mL, and the concentration of liposoluble immune activator in ethanol is 0.2 - 1 mg / mL.

8. The preparation method according to claim 1, characterized in that: In step S2, the solvent exchange scheme includes the dropwise addition method, the microchannel injection mixing method or the microfluidic chip mixing method, and the volume ratio of ethanol to the isotonic solution is 1:10 - 1:

50.

9. An antigen and adjuvant co-loaded nanocapsule vaccine prepared by the preparation method according to any one of claims 1 - 7.

10. Use of the nanocapsule vaccine according to claim 8 in the preparation of a vaccine preparation.

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