A nano-micro bubble and a preparation method and application thereof

Abstract

The application provides a nanometer micro-bubble, a preparation method and application thereof. The nanometer micro-bubble comprises a gas phase, an aqueous phase and solid particles; wherein the gas phase is an internal dispersed phase, the aqueous phase is an external continuous phase, and the solid particles are attached to the gas-liquid interface between the gas phase and the aqueous phase. The nanometer micro-bubble can be used as a vaccine adjuvant, the bubble properties can be controlled and regulated, the obtained bubble is stable, the use of surfactants and oil phases is avoided, and the toxicity to human body and environmental pollution can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a nano-micro bubble as well as a preparation method and application thereof. BACKGROUND

[0002] A bubble is a two-phase system in which a gas phase is dispersed into a continuous phase such as water or oil. The gas phase in the bubble is dispersed in the continuous phase in the form of small air masses to form individual independent suspended air masses, and there is no mutual connection between the bubbles, which is different from the foam in which the boundary is connected to each other in an aggregated state. According to the difference of the continuous phase, the bubble can be divided into water-based or oil-based bubbles, and the water-based bubble is more common and has been applied to the fields of mineral flotation, foam fire extinguishing, aerated concrete manufacturing, cleaning and decontamination, and food.

[0003] Since the gas-liquid interface has high free energy, the bubble is in a thermodynamic unstable state and is easily broken and disappeared, and a surfactant needs to be added to reduce the gas-liquid interface tension so as to stabilize the bubble. However, the desorption energy of the surfactant on the gas-liquid interface is low, and the bubble stabilization effect is poor, and the biocompatibility of the surfactant is poor, which restricts its application in the field of biological medicine. In order to solve the above problems, a new dosage form, particle stabilized bubble, has attracted people's attention in recent years.

[0004] Particle stabilized bubble (PSB) refers to a bubble stabilized by a nano-micro particle with suitable hydrophilicity and hydrophobicity. Since the desorption energy of the particle on the gas-liquid interface is high, the PSB has higher stability than the surfactant stabilized bubble (SSB). At the same time, the PSB also has many advantages, such as being able to select different particles to control the properties of the bubble, not containing surfactant, reducing the toxic and side effects, etc. These advantages make the PSB have broad application prospects, especially in the field of biological medicine with high safety requirements.

[0005] At present, the application of PSB in the field of biological medicine mainly focuses on biological separation, ultrasonic contrast imaging, and drug delivery. Among them, the drug delivery system, also known as drug carrier, refers to a drug-loaded system that changes the way of drug entering the body and the distribution in the body, controls the release speed of the drug or transports the drug to the target tissue or organ, so as to achieve the purpose of improving the utilization rate, safety and effectiveness of the drug. Some studies mix nano-particles and micro-bubbles, and then inject them into the blood circulation through intravenous injection, and the application of ultrasound can enhance the targeted delivery of nano-particles.

[0006] There are also researchers who connect nanoparticles with microbubbles through physical or chemical methods to further enhance the delivery of nanoparticles after ultrasound microbubble destruction. For example, Bruke et al. connected bovine serum albumin (BSA)-stabilized microbubbles with lactic acid-glycolic acid copolymer (PLGA) drug-loaded nanoparticles through covalent cross-linking to prepare a microbubble-nanoparticle composite system, and the results showed that the targeting and uptake of the nanoparticles were significantly improved. However, this method requires the preparation of microbubbles first, and then the cross-linking of nanoparticles on the microbubbles through secondary operation, which is relatively cumbersome. To solve this problem, Morch et al. prepared polybutylcyanoacrylate (PEBCA) nanoparticles loaded with the anticancer drug gemcitabine, and used the nanoparticles to stabilize microbubbles PSBs, and used the PSBs for the treatment of prostate cancer model mice. However, in this system, the PEBCA nanoparticles alone are not enough to stabilize the bubbles, and surfactant sodium dodecyl sulfate (SDS) and surface-active proteins still need to be added to enhance the stability of the bubbles. Chen et al. prepared PSBs stabilized by pH-responsive gold nanoparticles (sAuNP) for the treatment of tumor model mice, but surfactants were also added to the system. The addition of surfactants or the cumbersome preparation steps can cause a decrease in the biocompatibility of the PSBs system, affecting its application in the field of biological medicine. Therefore, it is urgent to develop PSBs with better stability and biocompatibility.

[0007] In addition to drug delivery systems, vaccine adjuvants are also an important research direction in the field of biological medicine. Particle adjuvants and oil emulsion adjuvants are the two most commonly used types of adjuvants. Particle adjuvants can enhance the uptake and presentation of antigens by antigen-presenting cells, and oil emulsion adjuvants can recruit antigen-presenting cells, thereby exerting immune-enhancing effects. However, the above systems have some problems. The particle material is a rigid system, which is difficult to deform under stress when it comes into contact with cells, which is not conducive to increasing the contact with cells and thus not conducive to the uptake of cells. In addition, the high solid content of microparticles brings a high degradation and metabolic burden to the body, and the small specific surface area is not conducive to the adsorption of antigens. Nanoparticles are not conducive to the loading of larger antigens such as bacterial antigens due to their small particle size. And the loading capacity of a single nanoparticle is limited, which is not conducive to the uptake of cells after entering the body. The emulsion system can deform under stress when it comes into contact with cells, but its surface is an oil-water interface, which is not conducive to the loading of antigens.

[0008] To solve the above problems, Xia et al. prepared a Pickering emulsion with PLGA nanoparticles as stabilizer and squalene as oil phase. This system has the following advantages: (1) a large number of particles are arranged on the surface of the emulsion droplets, providing a large specific surface area, which can increase the antigen load; (2) the emulsion droplets can deform flexibly on the surface of the cell membrane, increasing the contact area with the cells, thereby effectively enhancing the cellular and humoral immune effects. However, this system contains an oil phase, which is irritating, and the main source of squalene is still the liver oil of deep-sea sharks. From the perspectives of environmental protection and animal protection, the use of squalene should be avoided as much as possible. SUMMARY

[0009] In view of the deficiencies in the prior art, the purpose of the present application is to provide a nano-micro bubble and a preparation method and application thereof. In view of the current problems of vaccine adjuvants and the application of PSBs in drug delivery systems, the present application proposes to prepare PSBs for vaccine adjuvants, which have similar high specific surface area and flexible deformation characteristics as Pickering emulsion, and do not require the addition of surfactants and oil phases, have simple composition, obtain more safe and effective immune protection effect, and are more in line with the design concept of green environmental protection. There is no research on PSBs as vaccine adjuvants in the currently known literature or patents, and the known PSBs are not designed and optimized according to the needs of vaccine preparations, such as the PSBs used in drug preparations containing non-biodegradable particles or surfactants, which restricts their use in healthy populations for vaccine immunization. The nano-micro bubble is a particle-stabilized nano-micro bubble that can be used in the body of humans or other animals, and it is particularly important that the nano-micro bubble of the present application does not contain lipids or other soluble surfactants, but uses particles with hydrophilic-hydrophobic amphiphilic properties as bubble stabilizers, which can be used as vaccine adjuvants.

[0010] To achieve this purpose, the present application adopts the following technical solutions:

[0011] In a first aspect, the present application provides a nano-micro bubble, which comprises a gas phase, an aqueous phase and solid particles.

[0012] The gas phase is an internal dispersed phase, the aqueous phase is an external continuous phase, and the solid particles are attached to the gas-liquid interface between the gas phase and the aqueous phase.

[0013] In the present application, the nano-micro bubble is composed of a gas phase, an aqueous phase and at least one solid particle, wherein the gas phase is an internal dispersed phase, the aqueous phase is an external continuous phase, the solid particle is adsorbed on the gas-liquid interface and plays a role in stabilizing the bubble, the bubble is freely dispersed in the aqueous phase, and the bubbles are not connected to each other. The solid particles can be adsorbed on the gas-liquid interface in single or multiple layers to stabilize the bubble, and the solid particles can be cross-linked or not cross-linked.

[0014] In the present application, for the first time, solid particles are combined with bubble preparations to prepare particle-stabilized nanomicrobubbles without surfactants and oil phases, which are applied to the development field of vaccine adjuvants. The addition of solid particles not only improves the biocompatibility of the preparation, avoids the adverse effects of surfactants on the human body, animals or vaccines, but also more effectively stimulates immune cells and triggers immune regulation functions. At the same time, the properties of solid particles are easy to control, which can be surface modified or coated, or solid particles with different properties (such as composition, morphology, structure, particle size, etc.) can be selected to exert different immune enhancement mechanisms, and antigens can be embedded, adsorbed or coupled, serving as antigen delivery carriers, using their antigen release control ability to regulate immune responses, and can be applied to various immunization methods. The use of nanomicrobubble preparations not only avoids the metabolic burden of organic solvents or excessive polymer materials, but also has a light mass and a fast in vivo circulation speed, which is conducive to its uptake by antigen-presenting cells and homing to lymph nodes, thereby improving the immune response effect.

[0015] Preferably, the particle size of the nanomicrobubbles is 10 nm-20 μm, for example, it can be 10 nm, 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, 200 nm, 400 nm, 600 nm, 800 nm, 1 μm, 2 μm, 4 μm, 6 μm, 8 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, 20 μm, etc., preferably 100 nm-10 μm.

[0016] In the present application, the nanomicrobubbles can be stably stored at room temperature for 10 min-1 month without separation, and can be stably stored at low temperature (for example, at 4℃) for 1 month-6 months without separation.

[0017] Preferably, the gas phase is a clinically used gas, preferably any one or a combination of at least two of air, nitrogen, sulfur hexafluoride, octafluoropropane, decafluorobutane, oxygen or nitric oxide.

[0018] In the present application, the gas phase of the particle-stabilized nanomicrobubbles is a clinically used gas such as air, nitrogen, sulfur hexafluoride, octafluoropropane, decafluorobutane, oxygen, nitric oxide, etc., preferably a gas with low water phase solubility and relatively large molecular weight, including but not limited to oxygen, carbon tetrafluoride, sulfur hexafluoride, octafluoropropane, decafluorobutane, etc.

[0019] Preferably, the water phase is a clinically available aqueous liquid, preferably any one or a combination of at least two of purified water, water for injection, glycerol aqueous solution or buffer saline solution, further preferably any one or a combination of at least two of water for injection, phosphate buffer, citric acid buffer or Tris buffer.

[0020] Preferably, the pH of the aqueous buffer solution is 5.0-8.1, for example, it can be 5.0, 5.5, 6, 6.5, 7, 7.5, 8, 8.1, etc.

[0021] In the present application, the aqueous phase can optionally contain pharmaceutical auxiliary substances, such as pH adjusters, buffers, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, human serum albumin, essential amino acids, non-essential amino acids, L-arginine hydrochloride, sucrose, anhydrous D-trehalose, mannitol, mannose, starch, gelatin.

[0022] Preferably, the solid particles have hydrophilic-hydrophobic amphiphilicity.

[0023] In the present application, the particle-stabilized nanomicro-bubbles contain at least one solid particle, which is adsorbed onto the liquid-gas interface between the aqueous phase and the gas phase, playing a role in stabilizing the bubbles, and the average particle size of the solid particle is in the nanometer to micrometer range.

[0024] Preferably, the solid particles include any one or a combination of at least two of high molecular polymers, polysaccharides or polysaccharide derivatives, preferably any one or a combination of at least two of polylactic acid, lactic acid-glycolic acid copolymer, polyethylene glycol-lactic acid copolymer, chitosan, chitosan derivatives, alginate, alginate derivatives, cellulose or cellulose derivatives.

[0025] In the present application, the polysaccharides or polysaccharide derivatives include, but are not limited to, chitosan, alginate, gelatin, dextran, konjac glucomannan, heparin, pectic polysaccharide, hyaluronic acid, chondroitin sulfate, etc., and their related salts, as well as polysaccharide derivatives obtained by quaternization, carboxymethylation, hydroxylation, alkylation, acylation, sulfonation, nitration, halogenation, etc. of the above polysaccharides. Preferred polysaccharides or polysaccharide derivatives are chitosan, alginate, gelatin and dextran.

[0026] The skilled person can determine the appropriate molecular weight according to the size of the antigen used, the desired release rate, etc. For chitosan, for example, a suitable molecular weight is about 500,000-9 million Daltons, for example, it can be 500,000 Daltons, 600,000 Daltons, 700,000 Daltons, 800,000 Daltons, 900,000 Daltons, 1 million Daltons, 2 million Daltons, 3 million Daltons, 4 million Daltons, 5 million Daltons, 6 million Daltons, 7 million Daltons, 8 million Daltons, 9 million Daltons, etc., preferably 1-8 million Daltons.

[0027] In the present application, the shape of the solid particles can be spherical, rod-like, spindle-like, disc-like, cubic, peanut-like, amorphous, etc., and the morphology of the solid particles can be smooth surface, porous surface, multi-chambered interior, hollow, single eye, double-faced heteromorphism, etc. Those skilled in the art can optimize and screen through limited processes according to the properties of the water phase, the gas phase and the antigen to obtain particles-stabilized gas bubbles that meet the application requirements.

[0028] In the present application, the surface or interior of the solid particles can also adsorb, couple or embed targeting substances, fluorescent or isotope labels, environment-responsive substances (groups with pH sensitivity, heat sensitivity, biological activity substance sensitivity, etc.), cytokines, antibodies, immunomodulators and other functional substances.

[0029] In the present application, the solid particles can have environmental responsiveness, including but not limited to changes in particle size, shape, antigen release curve, etc. in response to changes in external pH, temperature, etc.

[0030] In the present application, the surface or interior of the solid particles can also adsorb, couple or embed antigens. The solid particles can be prepared by various methods. For example, for poly(lactide-co-glycolide) solid particles, various methods such as solvent evaporation, solvent extraction, precipitation, etc. can be used. For chitosan solid particles, single emulsion method (forming water-in-oil emulsion) combined with chemical crosslinking method (such as using glutaraldehyde dissolved in oil phase for crosslinking) can be used for preparation, and spray drying or precipitation method can also be used for preparation. After preparation, the solid particles can be stored in aqueous solution or buffer solution, or can be lyophilized for use.

[0031] Preferably, the three-phase contact angle between the solid particles, the gas phase and the liquid phase is 45-90°, for example, it can be 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, etc., preferably 60-80°.

[0032] In the present application, the solid particles have hydrophilic-hydrophobic amphiphilicity, and the three-phase contact angle between the solid particles, water and gas is between 45-90°, preferably between 60-80°, which can be adsorbed to the gas-liquid interface between the gas phase and the water phase. For different water-gas systems, solid particles with different hydrophilic-hydrophobic properties can be selected to stabilize the gas bubbles, and the surface of the solid particles can be modified, coated or grafted to obtain appropriate hydrophilic or hydrophobic properties (or particle wettability).

[0033] Preferably, the average particle size of the solid particles is 1 nm to 5 μm, for example, it can be 1 nm, 10 nm, 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, 200 nm, 400 nm, 600 nm, 800 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, etc., preferably 5 nm to 3 μm.

[0034] Preferably, the span value of the particle size distribution coefficient of the solid particles is less than 1.0, for example, it can be 0.99, 0.98, 0.96, 0.94, 0.92, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, etc.

[0035] Preferably, the solid particles account for 0.1-20% of the total mass of the aqueous phase, for example, it can be 0.1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, etc., preferably 0.5-10%.

[0036] Preferably, the number concentration of the nanobubbles is 10 2 -10 10 / mL, for example, it can be 10 2 / mL, 10 4 / mL, 10 5 / mL, 10 6 / mL, 10 7 / mL, 10 8 / mL, 10 9 / mL, 10 10 / mL, etc., preferably 10 4 -10 9 / mL.

[0037] In the present application, the particle-stabilized nanobubbles can include, but are not limited to, the following adjuvants: stimulators of pattern recognition receptors (such as Toll-like receptors, RIG-1, and NOD-like receptors (NLR), such as CpG motif-containing oligonucleotides or double-stranded RNA or oligonucleotides containing a palindromic series or oligonucleotides containing a poly(dG) sequence), mineral salts (such as alum, alum combined with single phospholipid (monphosphoryllipid, MPL) A of enteric bacteria (such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri) or alum specifically combined with MPL A of the above-mentioned bacteria (AS04)), MPL, saponins (such as QS-21, Quil-A, iscoMs, iscomatrix TM ​), liposomes and liposomal formulations (e.g. AS01), synthetic or specially prepared microparticles and microcarriers (e.g. outer membrane vesicles (OMV) derived from the outer membrane of N. gonorrheae, Chlamydia trachomatis and other bacteria), specifically modified or prepared peptides (e.g. muramyl dipeptide), aminoalkyl amino glucoside 4-phosphates (e.g. RC529), or proteins (e.g. bacterial toxoids or toxin fragments), alternative pathogen associated molecular patterns (PAMPS), small molecule immunopotentiators (SMIPs), cytokines and chemokines. Cytokines include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF), interferons (e.g. interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), interferon-gamma (IFN-gamma), etc.), interleukins (e.g. interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18)), fetal liver tyrosine kinase 3 ligand (Flt3L), or tumor necrosis factor-alpha (TNF-alpha), etc.

[0038] In the present application, the particle-stabilized nanomicrobubbles are stabilized by solid particles, free of oil phase and surfactant, the solid particles can be adsorbed on the gas-liquid interface in single or multiple layers to stabilize the bubbles, and the solid particles can be cross-linked or not cross-linked.

[0039] The particle-stabilized nanomicrobubbles provided by the present application have a shell layer with obvious particle structure, and gaps exist between the particles, the size of the gaps depends on the size of the particles and the stacking mode and thickness, which is different from the overall shell structure of microcapsules or vesicles with a shell layer composed of polymers, lipids or macromolecules.

[0040] The particle-stabilized nanomicrobubbles provided by the present application can be dried by removing the water phase, and the drying method can be freeze-drying, heating, supercritical drying, spraying, microwave, air-drying or other drying methods, and the dried nanomicrobubbles can be in a dispersed or aggregated state.

[0041] The particle-stabilized nanomicrobubbles provided by the present application can be used as vaccine adjuvants, and the immunization methods include intramuscular injection, subcutaneous injection, inhalation through respiratory tract, intraperitoneal injection, nasal immunization, ocular immunization, oral immunization, mucosal immunization and transdermal immunization.

[0042] In a second aspect, the present application provides a preparation method of the nanomicrobubbles according to the first aspect, and the preparation method comprises the following steps:

[0043] (1) dispersing solid particles in an aqueous phase to obtain a solid particle dispersion liquid;

[0044] (2) introducing a gas phase into the solid particle dispersion liquid obtained in step (1) and dispersing again to make the solid particles adhere to the gas-liquid interface between the gas phase and the aqueous phase, thereby obtaining the nano-micro bubbles.

[0045] In the present application, the particle-stabilized nano-micro bubbles can be prepared by various methods.

[0046] Specifically, the particle-stabilized nano-micro bubbles in the present application can be prepared by the following methods, but are not limited to the following preparation methods.

[0047] In the present application, the particle-stabilized nano-micro bubbles can be prepared by dispersing solid particles in an aqueous phase first, then introducing a gas into the aqueous phase, and dispersing by homogenization, ultrasonic, hand shaking, etc. to realize the adsorption of solid particles on the gas-liquid interface.

[0048] Those skilled in the art can select appropriate dispersion methods and specific operation parameters according to the properties of the aqueous phase and solid particles used and their own experimental equipment conditions.

[0049] Among them, the mixing of gas phase and aqueous phase can be selected from microfluidization, homogenization, ultrasonic, spraying, microjet, microchannel emulsification, membrane emulsification, stirring, oscillation, hand shaking, etc. According to different needs, the mixing method can be preferably microfluidization, microchannel, membrane emulsification, etc. which can obtain bubbles with uniform particle size distribution, or can be preferably microjet, homogenization, stirring, oscillation, etc. which are convenient for large-scale preparation. It should be clear that different gas-water phase mixing methods will inevitably affect the particle size, stability, etc. of the obtained bubbles, and will also affect the final immune effect.

[0050] As mentioned in one embodiment of the present application, compared with mechanical stirring, the use of membrane emulsification method can obtain bubbles with more uniform particle size distribution and smaller particle size, and can obtain higher antibody level. Those skilled in the art should determine the appropriate mixing method and specific operation parameters according to the properties of the gas-water phase and solid particles used, the required particle size range of the prepared bubbles, etc.

[0051] Preferably, the dispersion uses any one or a combination of at least two of homogenization, ultrasonic, stirring, oscillation or vortex.

[0052] Preferably, in step (2), the dispersion method comprises any one or a combination of at least two of microfluidics, homogenization, ultrasonication, spray, microjet, microchannel, membrane emulsification, stirring, shaking or hand mixing, preferably any one or a combination of at least two of microfluidics, microchannel or membrane emulsification, and further preferably membrane emulsification.

[0053] Preferably, the specific parameters of the microfluidics are that the inlet hole diameter is 100 nm-20 μm, for example, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 4 μm, 6 μm, 8 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, 20 μm, etc., and the hole spacing is more than 5 times the inlet hole diameter, for example, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, etc.

[0054] Preferably, the specific parameters of the microchannel are that the size of the microchannel is 100 nm-30 μm, for example, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 4 μm, 6 μm, 8 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, 20 μm, 25 μm, 30 μm, etc.

[0055] Preferably, the specific parameters of the membrane emulsification are that the membrane hole diameter is 1-100 μm, for example, 1 μm, 2 μm, 4 μm, 6 μm, 8 μm, 10 μm, 20 μm, 40 μm, 60 μm, 80 μm, 100 μm, etc.

[0056] In a third aspect, the present application provides a nano-micro particle, which is obtained by drying the nano-micro bubble of the first aspect.

[0057] In a fourth aspect, the present application provides a use of the nano-micro bubble of the first aspect or the nano-micro particle of the third aspect in preparing a vaccine adjuvant.

[0058] The particle-stabilized nano-micro bubble as the vaccine adjuvant of the present application can be used in humans, livestock, poultry and aquatic animals; the particle-stabilized nano-micro bubble can also be used as a drug delivery or controlled release system; the solid particles, gas phase and aqueous phase in the particle-stabilized nano-micro bubble can be independently packaged.

[0059] The particles can be mixed immediately before use, or two or three of them can be mixed in advance. The particle-stabilized nanobubbles can be packaged separately from the antigen, mixed immediately before use or within a short time interval (usually within 1 hour, including 1 hour) and inoculated into the same site, or mixed in advance and packaged, and directly used during immunization.

[0060] The immunization or administration method of the particle-stabilized nanobubbles of the present application includes, but is not limited to, intravenous injection, intraspinal injection, intramuscular injection, subcutaneous injection, intradermal injection, respiratory tract spray or inhalation, intraperitoneal injection, respiratory tract spray or inhalation, intraperitoneal injection, nasal immunization, ocular immunization, oral immunization, mucosal immunization, transdermal immunization, and the most preferred immunization method is intramuscular injection, subcutaneous injection, and mucosal immunization.

[0061] The particle-stabilized nanobubbles of the present application can be used in combination with other physical or chemical methods, including but not limited to external stimulation or intervention means such as ultrasound, magnetic field, light, etc.

[0062] In a fifth aspect, the present application provides an immunogenic composition, which comprises the nanobubbles of the first aspect and at least one antigen, or the nanoparticle of the third aspect and at least one antigen.

[0063] The antigen includes, but is not limited to, human antigen, non-human animal antigen, plant antigen, bacterial antigen, fungal antigen, viral antigen, parasitic antigen, or tumor antigen.

[0064] The antigen can be obtained from, but is not limited to, chicken embryo culture, cell culture, carrier body fluid, organ or tissue purification and separation, recombinant gene expression, synthesis, including but not limited to attenuated vaccine, inactivated vaccine, split vaccine, subunit vaccine, polysaccharide conjugate vaccine, recombinant vaccine, DNA vaccine, etc.

[0065] Preferably, the antigen is adsorbed on the surface of the nanobubbles or the nanoparticles, or the antigen is embedded in the interior of the nanobubbles or the nanoparticles.

[0066] The antigen is added to the water phase of the nanobubbles, so that it is adsorbed on the surface of the nanobubbles or the nanoparticles, or the antigen is embedded in the interior of the nanobubbles or the nanoparticles.

[0067] Preferably, the antigen is adsorbed on the surface of the nanobubbles or the nanoparticles, and the adsorption rate is more than 70%, for example, it can be 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc.

[0068] Preferably, the antigen is embedded in the interior of the nanobubble or the nanomicro particle, and the embedding rate is 70% or more, for example, 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc.

[0069] Compared with the prior art, the present application has the following beneficial effects:

[0070] (1) The present application uses solid particles as bubble stabilizers to prepare water-in-air type monodisperse nanobubbles. The bubbles have a high specific surface area, which is conducive to the adsorption of antigens. When the bubbles contact cells, they can deform flexibly to increase the contact area, which is conducive to the uptake of cells. Different response characteristics of particles can be used to stabilize the bubbles, giving the bubbles corresponding response characteristics.

[0071] (2) The system described in the present application does not contain surfactants and oil phases, avoiding the influence of surfactants and oil phases on antigens and the body. The product has good safety and stability, and can be used for different vaccination routes of vaccines.

[0072] (3) The system described in the present application can contain functional gases or functional particles, such as functional gases such as nitric oxide and oxygen, and functional particles such as magnetic responsiveness and pH responsiveness. Combined with external regulation such as ultrasound and magnetic field, the immune effect is further improved.

[0073] (4) The particles described in the present application can activate macrophages and promote the interaction of macrophages with T and B cells, thereby having a specific stimulating effect on lymphocytes.

[0074] (5) The particles used in the present application can adsorb or embed antigens, increase the surface area of antigens, and make the antigens easy to be phagocytosed by macrophages.

[0075] (6) The nanobubbles described in the present application can also cause a slight inflammatory reaction at the injection site, recruit inflammatory cells, stimulate the secretion of inflammatory factors, and activate the immune response.

[0076] (7) The present application selects specific particles, such as pH-sensitive chitosan particles, which can achieve lysosomal escape of antigens after adsorbing or embedding antigens, thereby enhancing cellular immune response. BRIEF DESCRIPTION OF DRAWINGS

[0077] Figure 1A is a schematic diagram of the structure of a bubble;

[0078] Figure 1B is a schematic diagram of the structure of a foam.

[0079] Figure 2A is a schematic diagram of the structure of a particle-stabilized bubble;

[0080] Figure 2BThis is a schematic diagram of the structure of a stable bubble.

[0081] Figure 3A A schematic diagram of the structure of a particle-stabilized bubble;

[0082] Figure 3B This is a schematic diagram of a microcapsule or vesicle with an integral shell.

[0083] Figure 4 This is an optical micrograph of the particle-stabilized bubbles prepared in Example 1.

[0084] Figure 5 This is a particle size distribution diagram of the stable bubbles prepared in Example 1.

[0085] Figure 6 This is a stained image of a tissue section from the injection site in a mouse.

[0086] Figure 7 Comparison of the effects of different samples on the amount of antigen phagocytosed by antigen-presenting cells. Detailed Implementation

[0087] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the specific embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0088] Figure 1A This is a schematic diagram of the bubble structure; Figure 1B Figure 1 shows a schematic diagram of the structure of a bubble. A bubble is a two-phase system in which the gas phase is dispersed within a continuous phase such as water or oil. The gas phase within the bubble is dispersed in the continuous phase as small gas clusters, forming individual, independent suspended gas clusters. There is no interconnection between the bubbles, unlike foam, which is interconnected and aggregated. The invention prepares a type of particle-stabilized nanobubble without surfactants or an oil phase. Figure 1A As shown.

[0089] Figure 2A This is a schematic diagram of the structure of a stable bubble. Figure 2BFigure 1 is a schematic diagram of the structure of a particle-stabilized bubble. As shown in Figure 1, the particle-stabilized bubble (PSB) refers to a bubble stabilized by a nanomicro-particle with suitable hydrophilicity and hydrophobicity. The PSB has higher stability than the surfactant-stabilized bubble (SSB) because the particle has higher desorption energy at the gas-liquid interface. Meanwhile, the PSB has various advantages, such as the ability to select different particles to control the properties of the bubble, the absence of surfactant, and the reduction of toxic side effects. These advantages make the PSB have broad application prospects, especially in the biological and medical fields with high safety requirements. The present application prepares a nanomicro-bubble stabilized by a particle, free of surfactant and oil phase, as shown in Figure 1. Figure 2A

[0090] Figure 3A Figure 1 is a schematic diagram of the structure of a particle-stabilized bubble. Figure 3B Figure 2 is a schematic diagram of the structure of a microcapsule or vesicle with an integral shell. As shown in Figure 2, the particle-stabilized nanomicro-bubble provided by the present application has a shell with a clear particle structure, and there are gaps between the particles, the size of the gap depends on the size of the particles and the stacking mode and thickness. The shell layer is different from the integral shell structure of the microcapsule or vesicle composed of a polymer, a lipid or a macromolecule.

[0091] The sources of the components in the following examples are as follows:

[0092]

[0093]

[0094] The instruments used in the following examples are as follows:

[0095]

[0096]

[0097] The following methods are used for performance characterization in the following examples:

[0098] (1) Particle or particle-stabilized nanomicro-bubble particle size distribution determination:

[0099] ​The particle size distribution of the micron-sized particles or the particles-stabilized bubbles was determined by a laser particle size analyzer. The specific determination steps were as follows: 5 mg of the micron-sized particles was added into 50 mL of deionized water, and was ultrasonically dispersed for 5 min, or 50 mL of the particles-stabilized bubbles was taken, and the particle suspension or the particles-stabilized bubbles was added into a sample cell, and was determined by a laser particle size analyzer (Malvern Instruments, United Kingdom Coulter Co., USA).

[0100] The particle size distribution of the nano-sized particles or the particles-stabilized bubbles was determined by a Zeta potential and particle size analyzer. The specific determination steps were as follows: 1 mg of the nano-sized particles was added into 10 mL of deionized water, and was ultrasonically dispersed for 5 min, or 2 mL of the particles-stabilized bubbles was taken, and the particle suspension or the particles-stabilized bubbles was added into a sample cell, and was determined by a Zeta potential analyzer (Zeta Potential Analyzer, Brookhaven Instruments Corporation).

[0101] The uniformity of the particles or the particles-stabilized nanobubbles was represented by a span value of a particle size distribution. The span calculation formula was as follows: the smaller the value, the more uniform the particle size.

[0102] Span = (d 90 -d 10 ) / d 50 (1)

[0103] In formula (1), d 10 , d 50 and d 90 were particle sizes when cumulative volumes of the particles were 10%, 50% and 90%, respectively.

[0104] The morphology of the particles was observed by a scanning electron microscope. 1 mg of the particles was weighed, and was added into 10 mL of deionized water, and was ultrasonically dispersed for 5 min. 1 mL of the suspension was taken, and was dropped on an aluminum foil, and was evenly spread on the aluminum foil, and was naturally dried. The aluminum foil was pasted on a sample table by conductive glue, and was sprayed with gold under vacuum conditions (appropriate spraying conditions were selected according to the sample properties), and was observed by a scanning electron microscope.

[0105] The contact angle of the particles was determined by a contact angle measuring instrument. The nano-particle dispersion liquid was evenly coated on a glass slide, and was placed in a clean place for air drying, and then the contact angle measuring instrument was used to measure the three-phase contact angle of the particles, water and air.

[0106] The morphology of the particles-stabilized nanobubbles was observed by an optical microscope. A small amount of the particles-stabilized bubbles was taken, and was dropped on a glass slide, and was observed under an optical microscope.

[0107] The stability of the particle-stabilized nanobubbles was detected by centrifugation: 5 mL of the particle-stabilized nanobubbles were taken and added into a 15 mL centrifuge tube, and then centrifuged at 1000 g for 3 min, and the layering was observed.

[0108] (2) Determination of the embedding rate and the loading of the antigen in the particles:

[0109] 10 mg of the antigen-loaded particle lyophilized powder was accurately weighed, and the microspheres were completely degraded by using a suitable method (for example, for polylactic acid microspheres, NaOH solution or acetonitrile was added to degrade the microspheres; for chitosan microspheres, dilute hydrochloric acid was added to degrade the microspheres). After the particles were completely degraded, the degradation solution was neutralized with NaOH or hydrochloric acid to pH = 7, and then diluted to 2 mL. The antigen content was determined by using a BCA kit or a micro-BCA kit or other suitable detection method.

[0110] The embedding rate of the antigen was calculated according to the following formula:

[0111] Embedding rate = (measured amount of the antigen in the particles / the amount of the antigen added during actual preparation) x 100%

[0112] The loading of the antigen on the particles was calculated according to the following formula:

[0113] Loading = (measured amount of the antigen in the particles / the mass of the measured particles).

[0114] (3) Determination of the adsorption rate and the loading of the antigen adsorbed on the particles:

[0115] The particle suspension after the antigen adsorption was centrifuged to obtain the supernatant (the centrifugation conditions were selected according to the size and density of the particles), and the antigen concentration in the supernatant was measured to indirectly calculate the amount of the antigen adsorbed on the surface of the particles. The antigen content was determined by using a BCA kit or a micro-BCA kit or other suitable detection method.

[0116] The adsorption rate of the antigen was calculated according to the following formula:

[0117] Adsorption rate = (antigen concentration before adsorption - antigen concentration in the supernatant after adsorption) / antigen concentration before adsorption x 100%

[0118] The loading of the antigen on the particles was calculated according to the following formula:

[0119] Loading = measured amount of the antigen on the particles / mass of the measured particles.

[0120] (4) Animal experiment determination:

[0121] Balb / c mice used in the experiment were provided by Vantian Lihua Company. The immunization steps are basically as follows: first, the mice were randomly divided into groups, and more than 6 mice in each group were used for the experiment. The mice were grouped and immunized according to the specific instructions of the examples. Before vaccination, 200 μL of blood was taken and immediately centrifuged at 12,000 rpm for 5 min to separate the serum, and the IgG antibody level was determined. The IgG antibody level at this time was taken as the initial value, and then the mice were immunized. After immunization, blood was taken from the eyes or tail tips of the mice at regular intervals, 200 μL each time, and the IgG antibody level was determined. Two weeks later, the second immunization was performed, and the mice were sacrificed 35 days later. Blood was taken and the IgG antibody level was determined (for influenza vaccine, hemagglutination titer (HI) was also determined). Mouse spleen cells were taken for culture, and enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of IL-4 and IFN-γ cytokines in the supernatant of mouse spleen cell culture,

[0122] Example 1

[0123] Preparation of particle-stabilized nanomicro-bubbles using PLGA particles

[0124] (1) Preparation of PLGA particles by nanometer precipitation method: 0.30 g of PLGA (LA: GA is 50:50, molecular weight is 110,000 daltons) was accurately weighed by an electronic balance, dissolved in 1 mL of acetone, and the solution was added to 20 mL of water solution (containing 1 wt% of PVA (alcoholysis degree is 99%, viscosity is 5.0 mPa·s)) at a speed of 1 drop per second by using a 9-gauge needle. The solution was stirred at 25°C overnight, centrifuged at 20,000 g for 20 min, the supernatant was discarded, 5 mL of deionized water was added to the precipitate, and the precipitate was dispersed by ultrasonic, centrifuged at 20,000 g for 5 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain PLGA particles, which were stored in a refrigerator at 4°C. The average particle size of the prepared PLGA particles was 226 nm, the Span was 0.535, and the three-phase contact angle was 61°. Scanning electron microscopy observation showed that the surface of the prepared particles was smooth and spherical.

[0125] (2) Preparation of particle-stabilized nanomicro-bubbles: 0.50 g of PLGA particles was accurately weighed by an electronic balance and added to 40 mL of deionized water. The mixture was ultrasonically dispersed for 1 min to obtain a water phase suspension containing dispersed particles. The pH value of the water phase was 6.5. 1 mL of the above suspension was taken and placed in a 15 mL centrifuge tube, which was sealed with a sealing film. Eight fluoropropane gas was filled into the centrifuge tube at a flow rate of 40 mL / min by using a syringe, and the original air in the centrifuge tube was discharged. Particle-stabilized bubbles were prepared by homogenization (25,000 rpm, 2 min).

[0126] The light microscope photograph and particle size distribution graph of the particle-stabilized bubbles are shown in Figure 4-5 As shown in the light microscope photograph and particle size distribution graph of the particle-stabilized bubbles, the particle-stabilized bubbles were spherical and uniform in size.Figure 4-5 As shown, the granule prepared by the present application has good bubble dispersion stability and regular spherical shape. The average particle size of the bubble is 12.5±0.2 μm, and the Span is 0.721±0.112. The bubble can be stored at room temperature for 1 month without stratification, and can be stored at 4° for more than 3 months without stratification.

[0127] Example 2

[0128] Preparation of granule-stabilized nanobubbles using poly-PLGA granules

[0129] Other PLA-based granules can also be used to prepare granule-stabilized bubbles, and the preparation steps are the same as those in Example 1, with the only difference being the specific process parameters as follows:

[0130] The specific process parameters and results are shown in Table 1 below:

[0131] Table 1

[0132]

[0133]

[0134]

[0135] Example 3

[0136] Preparation of granule-stabilized nanobubbles using chitosan-coated alginate granules

[0137] (1) Preparation of chitosan-coated alginate granules using rapid membrane emulsification technology: First, mix petroleum ether (boiling range 60-90°C) and liquid paraffin at a volume ratio of 2:1, and add 4wt.% emulsifier Span 80 to the mixed organic phase. The above mixture is used as the oil phase, and the water phase is a sodium alginate aqueous solution (1.0wt%). 2mL of the water phase is emulsified with 60mL of the oil phase under the action of homogenization (3600rpm, 1min) to form a pre-emulsion, which is then added to a rapid membrane emulsification tank under nitrogen pressure (1MPa). The pre-emulsion passes through an SPG membrane (membrane pore size 1.4μm) for 5 cycles to obtain a relatively uniform emulsion. A CaCl2solution (5mol / L, 12mL) is used as a curing agent, which is dispersed into the oil phase (24mL) under ultrasonic treatment (120W, 1min) to form a fine emulsion. The fine emulsion is mixed with the above-obtained uniform emulsion and stirred at 37°C for 5h (250rpm) to cure the emulsion and obtain colloidal granules. The granules are washed with petroleum ether, ethanol, and water three times each to obtain alginate granules.

[0138] The chitosan coating step of the alginate particles includes: first dispersing 1 g of alginate particles into a 0.7 wt% chitosan acetic acid solution (20 mL, chitosan molecular weight of 800,000 Dalton, deacetylation degree of 90%), after stirring for 1 h (200 rpm), the particles are washed with acetic acid buffer solution (pH 4 and pH 5.5) and deionized water to obtain chitosan-coated alginate particles. The particles can also be coated multiple times, the steps are: dispersing the coated colloidal particles (1 g) into a 0.5 wt% sodium alginate aqueous solution (20 mL), stirring for 1 h (200 rpm), washing once with deionized water, and then dispersing the particles into a 0.7 wt% chitosan acetic acid solution (20 mL) and stirring for 1 h (200 rpm). Then the particles are washed with acetic acid buffer solution (pH 4 and pH 5.5) and deionized water to obtain double-coated chitosan-alginate particles. Repeat the above steps multiple times to obtain chitosan-coated alginate particles. In this application, the alginate particles are coated three times, the average particle size of the particles is 457 nm, the Span of the particles is 0.839, and the three-phase contact angle is 45°. The surface of the prepared particles is rough and the spherical shape is regular.

[0139] (2) Preparation of particle-stabilized nanobubbles: 1.22 g of chitosan-coated alginate particles were accurately weighed using an electronic balance and added to 10 mL of phosphate buffer solution. The mixture was ultrasonicated for 1 min to disperse the particles uniformly, obtaining a water phase suspension containing dispersed particles. The pH value of the water phase was 8.0. 1 mL of the above suspension was taken and placed in a 15 mL centrifuge tube, which was sealed with sealing film. Sulfur hexafluoride gas was filled into the centrifuge tube at a flow rate of 40 mL / min using a syringe, and the original air in the centrifuge tube was discharged. Particle-stabilized nanobubbles were prepared by ultrasonication (400 W, 2 min). The particle-stabilized nanobubbles were well dispersed and had a regular spherical shape. The average particle size of the nanobubbles was 15.6 μm, and the Span was 0.807. The nanobubbles could be stored stably at room temperature for 14 days without separation, and could be stored stably at 4° for more than 1 month without separation.

[0140] Example 4

[0141] Preparation of particle-stabilized nanobubbles using monomethoxy polyethylene glycol-lactic acid copolymer (PELA) porous particles

[0142] (1) Preparation of PELA porous particles by phase separation-solvent removal method: 100 mg of PELA (molar ratio of mPEG:PLA is 1:19, average molecular weight is 40 kDa) was dissolved in 7.5 mL of acetone, 7.5 mL of anhydrous ethanol solution was added, and the above solution was dropped into (1 drop / s) deionized water (90 mL, containing 10 g / L SDS) under rapid stirring (750 rpm), and after the dropping was completed, the stirring was continued for 24 h (750 rpm). After washing with deionized water for 5 times, the precipitate was suspended in 10 mL of deionized water and stored as a PELA particle suspension.

[0143] 200 μL of dichloromethane was added to 3 mL of deionized water and mixed uniformly in an ultrasonic cell crusher (400 W, 60 s) as a swelling agent. The above 2 mL of PELA particle suspension was added to the solvent agent, and the magnetic stirring (250 rpm) was maintained for 30 min, and after standing for 1 h, it was quickly put into liquid nitrogen for rapid freezing, and stored at -20°C to volatilize the organic solvent to obtain PELA particles with porous structure, the average particle size of the particles was 78.55 nm, the Span was 0.331, and the three-phase contact angle was 55°, and the particles were spherical structure with porous surface.

[0144] (2) Preparation of particle-stabilized nanobubbles: 3.51 g of PELA particles was accurately weighed by an electronic balance and added to 100 mL of water for injection, and ultrasonicated for 2 min to disperse uniformly, to obtain a water phase suspension dispersed with particles. The pH value of the water phase was 7.0. The above 30 mL of suspension was placed in a 50 mL centrifuge tube and sealed with sealing film. Nitrogen was filled into it at a flow rate of 40 mL / min by using a syringe to discharge the original air in the centrifuge tube. Particle-stabilized nanobubbles were prepared by a rapid membrane emulsification method (Shirasu Porous Glass (SPG) microporous membrane with a pore size of 25 μm, transmembrane pressure of 200 KPa, and passing through the membrane three times). The particle-stabilized nanobubbles were well dispersed and regular spherical. The average particle size of the nanobubbles was 17.3 μm, and the Span was 0.727. The nanobubbles were dried by supercritical drying, and the average particle size of the dried nanobubbles was 12.7 μm, and the Span was 0.568.

[0145] Example 5

[0146] Preparation of particle-stabilized nanobubbles using polylactic acid (PLA) particles embedded with antigens

[0147] (1) PLA particles embedding HBsAg were prepared by rapid membrane emulsification combined with solvent evaporation method: 200 mg PLA was dissolved in 4.0 mL ethyl acetate, 0.4 mL 5% (w / v) HBsAg was added, and primary emulsification was performed by ultrasonic cell disruptor under ice water bath condition (power 12%, time 15 s). Then the primary emulsion was poured into 200 mL aqueous solution containing 1.0 wt.% PVA (external water phase) with magnetic stirring for pre-emulsification (300 rpm, 50 s). After pre-emulsification, the pre-emulsion was poured into the storage tank of the rapid membrane emulsification device, and the pre-emulsion was pressed through the SPG membrane (pore size 5.2 μm) by nitrogen gas with a pressure of 300 KPa to obtain the emulsion. The emulsion was poured into 800 mL 0.9 wt.% NaCl aqueous solution (solidification solution) with magnetic stirring at 500 rpm for 10 min to solidify the microspheres. The solidified microspheres were washed three times by centrifugation (4000 r / min, 5 min) with deionized water, and finally freeze-dried to obtain the finished product. The average particle size of the particles was 2.32 μm, the Span was 0.496, the three-phase contact angle was 85°, and the particles were spherical in structure with smooth surface. The embedding rate of the antigen was 90%, and the antigen loading of the particles was 0.09 mg antigen / g microspheres.

[0148] (2) Preparation of particle-stabilized nanobubbles: 7.22 g of PLA particles were accurately weighed by an electronic balance and added to 40 mL of injectable water, and vortexed for 5 min to disperse uniformly to obtain a water phase suspension with particles dispersed therein. The pH value of the water phase was 7.0. 30 mL of the above suspension was taken and placed in a 100 mL beaker and sealed with a sealing film. Ten fluorobutane was filled into the beaker at a flow rate of 40 mL / min by a syringe to expel the original air in the centrifuge tube. Particle-stabilized nanobubbles were prepared by magnetic stirring (500 rpm, 5 min). The particle-stabilized nanobubbles were well dispersed and regular in spherical shape. The average particle size of the nanobubbles was 15.8 μm, and the Span was 0.793. The nanobubbles could be stably stored at room temperature for 1 month without separation, and could be stably stored at 4° for more than 6 months without separation.

[0149] Example 6

[0150] Preparation of particle-stabilized nanobubbles using chitosan particles

[0151] (1) Chitosan particles were prepared by rapid membrane emulsification combined with temperature solidification method:

[0152] Aqueous phase preparation: A certain amount of chitosan (molecular weight of 50,000 Dalton, degree of deacetylation of 80%) was dissolved in 9 mL of acetic acid solution (0.1 mol / L) to obtain a chitosan acetic acid solution under magnetic stirring until it was fully dissolved; another certain amount of glycerophosphate sodium was dissolved in 1 mL of deionized water. After the chitosan acetic acid solution and the glycerophosphate sodium solution were respectively incubated at 4°C for 10 min, the glycerophosphate sodium solution was slowly added to the chitosan acetic acid solution, and they were mixed uniformly under magnetic stirring (300 rpm, 10 min). The solution was centrifuged at 20,000 rpm to remove insoluble impurities, and the supernatant was reserved as the aqueous phase. The concentration of chitosan in the aqueous phase was 3.5 wt.%, and the concentration of glycerophosphate sodium in the aqueous phase was 10.0 wt.%.

[0153] Oil phase preparation: Oil-soluble emulsifier PO-500 was added to a mixture of 60 mL of liquid paraffin and petroleum ether (boiling range of petroleum ether: 60-90°C) (volume ratio: 5:7) to prepare an oil phase. The concentration of PO-500 in the oil phase was 4 wt.%, and the mixture was stirred until it was completely dissolved. The mixture was incubated at 4°C for 10 min.

[0154] Emulsion preparation: At 4°C, 2 mL of the aqueous phase was mixed with 50 mL of the oil phase, and a pre-emulsion was prepared by homogenizing the mixture at 6,000 rpm for 1 min. The obtained pre-emulsion was quickly poured into a pre-emulsion reservoir of a rapid membrane emulsification device, and the pre-emulsion was rapidly passed through an SPG microporous membrane (pore size: 2.8 μm) under a nitrogen pressure of 5.0 MPa to obtain a W / O emulsion with uniform particle size. The obtained emulsion was used as the pre-emulsion and was passed through the SPG microporous membrane again under a nitrogen pressure of 5.0 MPa. The emulsification process was repeated five times, and a W / O emulsion with uniform particle size was finally obtained. The emulsification process took about 10 min. After the emulsification was completed, the W / O emulsion was placed in a water bath at 35°C and was allowed to solidify under mechanical stirring (200 rpm) for 1 h. After the solidification reaction was completed, the chitosan particles were obtained by centrifugation at 10,000 rpm and washing with petroleum ether, ethanol, and deionized water in sequence. The average diameter of the particles was 870 nm, the Span value was 0.487, the three-phase contact angle was 70°, and the particles had a loose and porous spherical structure.

[0155] (2) Preparation of particle-stabilized nanobubbles: 1.00 g of chitosan particles was accurately weighed by an electronic balance and was added to 20 mL of a PBS buffer solution. The mixture was ultrasonically dispersed for 1 min to obtain a water phase suspension containing the particles. The pH value of the water phase was 8.1. 5 mL of the suspension was taken and was placed in a 15 mL centrifuge tube, which was sealed with a sealing film. Eightfluoropropane was filled into the centrifuge tube at a flow rate of 40 mL / min by using a syringe to expel the air in the centrifuge tube.

[0156] The particle-stabilized bubbles were prepared by vortexing (10 min). The particle-stabilized bubbles were well dispersed and spherical. The average size of the bubbles was 13.6 μm and the span was 0.712. The bubbles were dried by freeze-drying. The average size of the dried bubbles was 10.2 μm and the span was 0.534.

[0157] Example 7

[0158] Other chitosan materials can also be used to prepare the particles and the particle-stabilized nanobubbles, and the preparation steps are the same as those in Example 6, except that chitosan materials with different molecular weights and degrees of deacetylation are used, and the oil-water phase composition and the preparation process are changed.

[0159] The specific process parameters and results are shown in Table 2.

[0160] Table 2

[0161]

[0162]

[0163] As shown in Table 2, under the same degree of deacetylation, the smaller the molecular weight, the lower the viscosity, and the smaller the particle size prepared by the same membrane emulsification condition, and the smaller the particle-stabilized nanobubbles prepared. Under the same molecular weight, the higher the degree of deacetylation, the lower the viscosity, and the smaller the particle size prepared by the same membrane emulsification condition, and the smaller the particle-stabilized nanobubbles prepared.

[0164] Example 8

[0165] Preparation of particle-stabilized nanobubbles using chitosan particles adsorbed with antigens

[0166] (1) The preparation method of the particles in this example is the same as that in Example 5, except that the chitosan particles adsorb H5N1 avian influenza split vaccine: (1) accurately weigh 1 g of the prepared chitosan particles, add 10 mL of PBS buffer containing H5N1 avian influenza split vaccine (HA concentration is 150 μg / mL), oscillate (120 rpm, 24 h) at 4°C, centrifuge at 10,000 rpm, and wash with deionized water three times to obtain chitosan particles adsorbed with H5N1 avian influenza split vaccine. The adsorption rate of the antigen is 60%, and the antigen loading on the particles is 900 μg HA / g particles.

[0167] (2) Preparation of particle-stabilized nanobubbles: 1.00 g of the chitosan particles with adsorbed antigens was accurately weighed by an electronic balance and added into 20 mL of PBS buffer solution, and then ultrasonicated for 1 min to make it uniformly dispersed, to obtain a water phase suspension with dispersed particles. The pH value of the water phase was 8.1. 5 mL of the above suspension was taken and placed in a 15 mL centrifuge tube, which was sealed with a sealing film. Eight fluoropropane was filled into the centrifuge tube at a flow rate of 40 mL / min by using a syringe to discharge the original air in the centrifuge tube. Particle-stabilized nanobubbles were prepared by vortex oscillation (10 min). The particle-stabilized nanobubbles were well dispersed and had regular spherical shape. The average particle size of the bubbles was 13.8 μm, and the Span was 0.912. The bubbles could be stably stored for 1 day without stratification at room temperature, and could be stably stored for more than 1 month without stratification at 4°.

[0168] Test Example 1

[0169] Safety evaluation of particle-stabilized nanobubbles

[0170] 1. Vessel irritation test

[0171] Example 1 and Example 3 were respectively injected into the ear vein of a rabbit once a day for 3 days. The results showed that there was no obvious change in the injection site of the ear vein of the rabbit, and the endothelium of the blood vessels 1 cm and 5 cm away from the injection site was continuous and complete under the microscope, and no hyperplasia, swelling, inflammatory cell infiltration, necrosis or thrombosis was observed. It was shown that the product had no obvious irritation to the blood vessels of the ear vein of the rabbit.

[0172] 2. Hemolysis and aggregation test

[0173] The conventional in vitro test tube method (macroscopic observation method) was used to mix Example 1 and Example 3 with 2% red blood cell suspension. The results showed that there was no hemolysis and red blood cell aggregation within 3 hours.

[0174] 3. Muscle irritation test

[0175] Example 1 and Example 3 were respectively used for injection into the quadriceps muscle of a rabbit, 1 mL for each side. The injection site was observed macroscopically and histopathologically after 48 hours. The results showed that the product had no irritation to the quadriceps muscle of the rabbit.

[0176] Test Example 2

[0177] Influence of mixing method of water phase and gas phase in particle-stabilized nanobubbles on antibody titer

[0178] PLGA particles were prepared according to the method in Example 1.

[0179] Preparation of particle-stabilized nanobubbles:

[0180] 2g PLGA particles were accurately weighed by using an electronic balance, and added into 40 mL of citric acid buffer solution. The particles were dispersed uniformly by ultrasonic treatment for 5 min to obtain a water phase suspension containing dispersed particles. The pH value of the water phase was 6.0. The above 40 mL suspension was placed in a 100 mL beaker and sealed with a sealing film. Eight fluoropropane gas was filled into the beaker at a flow rate of 40 mL / min by using a syringe to discharge the original air in the beaker. The particle-stabilized bubbles were prepared by using a rapid membrane emulsification method (membrane pore size of 2.9 μm, membrane pressure of 500 KPa, and passing through the membrane three times) and a mechanical stirring method (500 rpm, 5 min), respectively. The particle-stabilized bubbles prepared by using the two methods were both well dispersed and had regular spherical shape. The average particle size of the bubbles in the emulsion prepared by using the rapid membrane emulsification method was 1.77 μm, and the span was 0.772. The average particle size of the bubbles in the emulsion prepared by using the mechanical stirring method was 14.7 μm, and the span was 0.709.

[0181] The particle-stabilized nanobubbles prepared above were mixed with H5N1 avian influenza split vaccine (hemagglutinin (HA) content of 4.5 μg / 100 μL) (volume ratio of the particle-stabilized nanobubbles to the vaccine was 1:1, and vertical mixing was performed at 20 rpm), and the same hemagglutinin content of split vaccine was used as a control. The Balb / c mice were injected with the vaccines in the left hind leg muscle, and the mice were immunized twice two weeks later, and were sacrificed 35 days later.

[0182] The specific test results are shown in Table 3.

[0183] Table 3

[0184] Antigen type Adjuvant type Emulsion preparation method Serum IgG levels HI levels Split vaccine Example 1 Fast membrane emulsification 480000 2560 Split vaccine Example 1 Mechanical agitation 320000 640 Split vaccine Example 1 Example 1 62000 32

[0185] As shown in the test data in Table 3, compared with the mechanical stirring group, the rapid membrane emulsification group can exert a good adjuvant effect, and high levels of IgG and HI titers are generated in the experimental animals, and there is a significant difference between the adjuvant group and the single antigen injection group.

[0186] Test Example 3

[0187] Effect of mixing method of the particle-stabilized nanobubbles and antigens on the generation of antibody titers

[0188] The particle-stabilized nanobubbles prepared according to the method in Example 1 were mixed with H5N1 avian influenza inactivated whole virus vaccine and split vaccine (hemagglutinin (HA) content of 4.5 μg / 100 μL) (volume ratio of the particle-stabilized nanobubbles to the vaccine was 1:1, and different mixing methods were used, including oscillation (5 min), ultrasonic treatment (10 W, 3 min), and homogenization (10000 rpm, 1 min)). The Balb / c mice were injected with the vaccines in the left hind leg muscle, and the mice were immunized twice two weeks later, and were sacrificed 35 days later.

[0189] The specific test results are shown in Table 4:

[0190] Table 4

[0191] Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 320000 720 Example 1 Example 1 Example 1 510000 2580 Example 1 Example 1 Example 1 600000 3600 Example 1 Example 1 Example 1 80000 64 Example 1 Example 1 Example 1 300000 640 Example 1 Example 1 Example 1 480000 2560 Example 1 Example 1 Example 1 580000 3200 Example 1 Example 1 Example 1 62000 32

[0192] The results are shown in Table 4. The granular stable nanobubbles all have good adjuvant effect, and the experimental animals produce high levels of IgG and HI titers, and there is a significant difference between the single antigen injection group. The granular stable nanobubbles mixed by the homogenization method have higher antibody levels.

[0193] Test Example 4

[0194] Effect of granular stable nanobubble and antigen inoculation method on adjuvant effect

[0195] The granular stable nanobubbles prepared according to the method in Example 1 were mixed with hepatitis B surface antigen recombinant vaccine (recombinant Hansenula yeast vaccine, antigen concentration 1.9 mg / mL) (emulsion and vaccine mixed at a volume ratio of 1:1, and homogenized (10000 rpm, 1 min) mixed), and the above vaccine composition was used for muscle, subcutaneous and intraperitoneal injection of Balb / c mice, and two weeks later, the mice were immunized twice, and the mice were sacrificed 35 days later. The results are shown in Table 5. The muscle injection group showed higher humoral antibody levels (IgG) and cytokine secretion levels (IL-4 and IFN-γ).

[0196] The specific test results are shown in Table 5:

[0197] Table 5

[0198] Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 256000 32 11240 Example 1 Example 1 Example 1 184000 28 10020 Example 1 Example 1 Example 1 102400 18 8000 Example 1 Example 1 Example 1 20480 5 2000 Example 1 Example 1 Example 1 14560 4 1540 Example 1 Example 1 Example 1 10240 2 1000

[0199] Test Example 5

[0200] Comparison of adjuvant effect of granular stable nanobubbles with aluminum adjuvant and MF59 adjuvant

[0201] The granular stable nanobubbles, aluminum hydroxide adjuvant and MF59 in Example 1 were mixed with H1N1 influenza A inactivated whole virus vaccine according to a volume ratio of 1:1 to prepare vaccine adjuvant compositions, and the hemagglutinin concentration in the composition was 15 μg / mL, 0.5 mL per dose. Another inactivated whole virus vaccine with a hemagglutinin concentration of 15 μg / mL was used as a control. Equal volumes of the above three vaccine adjuvant compositions and the control vaccine were used for muscle injection immunization of Balb / c mice, and two weeks later, the mice were immunized twice, and the mice were sacrificed 35 days later;

[0202] The immunization results are shown in Table 6.

[0203] Table 6

[0204]

[0205] The results show that the granular stable nanometer micro-bubbles can significantly improve the humoral immune response level and the cellular immune level of the body to the inactivated whole virus vaccine of influenza. The granular stable nanometer micro-bubbles have the same effect as the MF59 in enhancing the humoral immunity, are higher than the aluminum hydroxide adjuvant, and are higher than the vaccine without the adjuvant. In terms of cellular immunity, the granular stable nanometer micro-bubbles are superior to the MF59 and the aluminum hydroxide adjuvant.

[0206] Test Example 6

[0207] Effect of the adding amount of the granular stable nanometer micro-bubbles on the immune effect

[0208] The granular stable nanometer micro-bubbles prepared according to the method in the embodiment 1 are mixed with the EV71 hand-foot-mouth disease inactivated virus vaccine in different proportions of 2:1, 1:1, 1:2 and 1:4 to form vaccine adjuvant compositions, wherein the antigen concentration is 0.05 mg / mL, and each dose is 0.2 mL. The Balb / c mice are immunized by intramuscular injection with the above vaccine adjuvant compositions, and the immunization is performed twice two weeks later.

[0209] The specific test results are shown in Table 7.

[0210] Table 7

[0211]

[0212] The results show that increasing the amount of the adjuvant in the vaccine adjuvant composition can enhance the immune response level of the quantified antigen, and the amount of the adjuvant is positively correlated with the strength of the adjuvant.

[0213] Test Example 7

[0214] Effect of vaccines with different antigen concentrations on the immune effect

[0215] The granular stable nanometer micro-bubbles prepared according to the method in the embodiment 1 are mixed with the H5N1 influenza inactivated whole virus vaccine to form vaccine adjuvant compositions with the hemagglutinin concentration of 37.5 μg / mL, 75 μg / mL and 150 μg / mL, and each dose is 0.1 mL. The Balb / c mice are immunized by intramuscular injection with the above two vaccine adjuvant compositions and two vaccines without the adjuvant with the same hemagglutinin concentration, the immunization is performed twice two weeks later, and the mice are killed 35 days later.

[0216] The specific test results are shown in Table 8.

[0217] Table 8

[0218]

[0219] The results show that the granular stable nanometer micro-bubbles have good adjuvant effect on the H5N1 influenza inactivated whole virus vaccine, and the adjuvant vaccine can obviously improve the immune response intensity in the mouse body compared with the vaccine without the adjuvant with the same hemagglutinin concentration.

[0220] Test Example 8

[0221] Adjuvant effect of granular stable nanometer micro-bubbles for nasal mucosa immunization

[0222] The granular stable nanometer micro-bubbles prepared according to the method in the embodiment 1 are combined with the H7N9 influenza split vaccine to form a vaccine adjuvant composition with the hemagglutinin concentration of 150 μg / mL, and each dose is 0.03 mL. The Balb / c mice are immunized by nasal instillation with the vaccine adjuvant composition and the vaccine without the adjuvant with the same hemagglutinin concentration, and the mice are immunized twice two weeks later, and are killed 35 days later;

[0223] The specific test results are shown in Table 9:

[0224] Table 9

[0225]

[0226] The results show that the granular stable nanometer micro-bubbles have good adjuvant effect on the H7N9 influenza split vaccine, and the adjuvant vaccine can obviously improve the immune response intensity in the mouse serum and mucosa compared with the vaccine without the adjuvant with the same hemagglutinin concentration.

[0227] Test Example 9

[0228] Partial enhancement mechanism of granular stable nanometer micro-bubbles as an immune adjuvant (local inflammatory effect)

[0229] In order to observe the immune responses caused by the three different dosage forms of vaccine adjuvants, MF59, AS03 and the granular stable nanometer micro-bubbles (embodiment 8, the granular stable nanometer micro-bubbles are prepared by using the chitosan particles with adsorbed antigens), the chicken ovalbumin (OVA) is mixed with the three dosage forms of vaccine adjuvants to prepare the adjuvant vaccine composition. The Balb / c mice are immunized by intramuscular injection with the adjuvant vaccine composition in the right hind leg, the mice are killed at different times, the tissue sections of the injection sites of the mice are dyed by the H&E method (as shown in Example 1 , wherein PBS refers to the blank control group, PBS buffer; MF59 and AS03 refer to the groups using MF59 adjuvant and AS03 adjuvant respectively, and PSBs refers to embodiment 8, the granular stable nanometer micro-bubbles are prepared by using the chitosan particles with adsorbed antigens), and the inflammatory responses caused by the three dosage forms of vaccine adjuvants in the body are observed.

[0230] Local tissue section observation showed that all three vaccine adjuvants could induce a certain degree of inflammatory response and recruit inflammatory cells, but the degree of inflammation was mild and subsided after seven days, demonstrating good biocompatibility.

[0231] Experimental Example 10

[0232] Partial enhancement mechanism of particle-stabilized nanobubbles as immune adjuvants (promoting phagocytosis)

[0233] Using mouse macrophage line RAW264.7 as antigen-presenting cells, and OVA as antigen, an adjuvant vaccine composition was prepared by mixing with particulate-stabilized nanobubbles. In vitro cell experiments were conducted to investigate the effect of particulate-stabilized nanobubbles (Example 8, prepared using chitosan particles adsorbed with antigen) on the amount of antigen phagocytosed by antigen-presenting cells.

[0234] The results showed that the adjuvant group significantly increased the amount of antigen phagocytosed by RAW264.7 cells (e.g., Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 Example 1 As shown, PBS refers to the blank control group, PBS buffer; OVA refers to OVA as the antigen alone; CNPs refers to chitosan particles loaded with OVA; and CPSBs+OVA refers to chitosan particles loaded with OVA and stabilized nanobubbles.

[0235] The applicant declares that this invention illustrates the nano-microbubbles, their preparation method, and applications through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

Claims

1. A nano-microbubble, characterized in that, The nano-microbubbles include a gas phase, an aqueous phase, and solid particles; Wherein, the gas phase is the internal dispersed phase, the aqueous phase is the external continuous phase, and the solid particles are attached to the gas-liquid interface between the gas phase and the aqueous phase; The contact angle between the solid particles, the gas phase, and the aqueous phase is 45-90°. The solid particles are amphiphilic, exhibiting both hydrophilic and hydrophobic properties. The nano- and microbubbles are free of oil phase and surfactants; The average particle size of the solid particles is 1 nm-5 µm; The gas phase is octafluoropropane; The solid particles are lactic acid-glycolic acid copolymer or chitosan; The nanobubbles are prepared by the following method, which includes the following steps: (1) Disperse solid particles in the aqueous phase to obtain a solid particle dispersion; (2) Introduce a gas phase into the solid particle dispersion obtained in step (1) and then disperse it so that the solid particles adhere to the gas-liquid interface between the gas phase and the water phase to obtain the nano-micro bubbles.

2. The nano-microbubble according to claim 1, characterized in that, The particle size of the nanobubbles is 10 nm-20 μm.

3. The nano-microbubble according to claim 2, characterized in that, The particle size of the nanobubbles is 100 nm-10 µm.

4. The nano-microbubble according to claim 1, characterized in that, The aqueous phase is a clinically usable aqueous liquid.

5. The nano-microbubble according to claim 4, characterized in that, The aqueous phase is any one or a combination of at least two of the following: purified water, water for injection, aqueous glycerol solution, or aqueous buffer salt solution.

6. The nano-microbubble according to claim 4, characterized in that, The aqueous phase is any one or a combination of at least two of water for injection, phosphate buffer, citrate buffer, or Tris buffer.

7. The nano-microbubble according to claim 5, characterized in that, The pH of the buffer salt solution is 5.0-8.

1.

8. The nano-microbubble according to claim 1, characterized in that, The contact angle between the solid particles, the gas phase, and the aqueous phase is 60-80°.

9. The nano-microbubble according to claim 1, characterized in that, The average particle size of the solid particles is 5 nm-3 µm.

10. The nano-microbubble according to claim 1, characterized in that, The particle size distribution coefficient span value of the solid particles is less than 1.

0.

11. The nano-microbubble according to claim 1, characterized in that, The solid particles account for 0.1-20% of the total mass of the aqueous phase.

12. The nano-microbubble according to claim 11, characterized in that, The solid particles account for 0.5-10% of the total mass of the aqueous phase.

13. The nano-microbubble according to claim 1, characterized in that, The number concentration of the nanobubbles is 10. 2 -10 10 per mL.

14. The nano-microbubble according to claim 13, characterized in that, The number concentration of the nanobubbles is 10. 4 -10 9 per mL.

15. The method for preparing nano- and microbubbles according to any one of claims 1-14, characterized in that, The method for preparing the nano-microbubbles includes the following steps: (1) Disperse solid particles in the aqueous phase to obtain a solid particle dispersion; (2) Introduce a gas phase into the solid particle dispersion obtained in step (1) and then disperse it so that the solid particles adhere to the gas-liquid interface between the gas phase and the water phase to obtain the nano-micro bubbles.

16. The method for preparing nano-microbubbles according to claim 15, characterized in that, In step (1), the dispersion is performed by any one or a combination of at least two of the following: homogenization, ultrasonication, stirring, oscillation or vortexing.

17. The method for preparing nano-microbubbles according to claim 15, characterized in that, In step (2), the dispersion method includes any one or a combination of at least two of the following: microfluidics, homogenization, ultrasound, spraying, microjeting, microchannels, membrane emulsification, stirring, oscillation, or hand-cranking mixing.

18. The method for preparing nano-microbubbles according to claim 17, characterized in that, In step (2), the dispersion method is any one or a combination of at least two of microfluidics, microchannels or membrane emulsification.

19. The method for preparing nano-microbubbles according to claim 18, characterized in that, In step (2), the dispersion method is membrane emulsification.

20. A nanoparticle, characterized in that, The nanoparticles are obtained by drying the nanobubbles according to any one of claims 1-14.

21. The use of nanobubbles according to any one of claims 1-14 or nanoparticles according to claim 20 in the preparation of vaccine adjuvants.

22. An immunogenic composition, characterized in that, The immunogenic composition comprises nanobubbles and at least one antigen as described in any one of claims 1-14, or nanoparticles and at least one antigen as described in claim 20.

23. The composition according to claim 22, characterized in that, The antigen is adsorbed on the surface of the nanobubbles or nanoparticles, or the antigen is embedded inside the nanobubbles or nanoparticles.

24. The composition according to claim 23, characterized in that, The antigen is adsorbed onto the surface of the nanobubbles or nanoparticles, with an adsorption rate of over 70%.

25. The composition according to claim 22, characterized in that, The antigen is embedded inside the nanobubbles or nanoparticles, with an embedding rate of over 70%.

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