CN117398348A

CN117398348A - Pickering emulsion for efficiently loading virus particles, and preparation method and application thereof

Info

Publication number: CN117398348A
Application number: CN202311416578.0A
Authority: CN
Inventors: 孙逊; 郭兆飞; 伍福华; 扈锐; 杜广盛
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-10-30
Filing date: 2023-10-30
Publication date: 2024-01-16

Abstract

The invention provides a Pickering emulsion delivery system and a preparation method thereof. The method selects the soluble protein with good biocompatibility and inorganic particles as solid emulsifying agent, and simultaneously selects the oil phase which can be metabolized by living beings as emulsion inner core, thus constructing Pickering emulsion which can carry AAV virus particles with high efficiency, obviously improves AAV transfection efficiency and adaptive immune response level of AAV vaccine, and can play the dual functions of adjuvant and delivery system.

Description

Pickering emulsion for efficiently loading virus particles, and preparation method and application thereof

Technical Field

The invention relates to a Pickering emulsion for efficiently loading virus particles, and a preparation method and application thereof, and belongs to the technical field of biological medicines.

Background

Currently, adeno-associated virus AAV has been involved in the field of vaccines, and can express antigens in vivo for long periods of time, inducing long-term immune responses. However, AAV has strong selectivity and weak capability of infecting cell lines, and adenovirus needs to be assisted to infect so as to realize effective infection, thus greatly restricting the application of AAV. Thus, there is a need to increase the infection efficiency of adeno-associated virus AAV-infected cells.

Emulsions stabilized by the use of colloidal particles instead of surfactant molecules are known as pickering emulsions, which have the following advantages: (1) The dosage of the colloid particles serving as the emulsifier is low compared with that of the surfactant, so that the production cost of the vaccine can be reduced; (2) The emulsion system has less surfactant component, and is more friendly to the body and the environment; (3) The colloid particles act as an emulsifier, and the heat energy required by the solid particles to break loose the two-phase interface is higher, so that the stability of the emulsion system is stronger; (4) The Pickering emulsion has high surface roughness, and is more favorable for interaction with cells. However, at present, most pickering emulsions are generally prepared by using solid particles or oil phases which are not biocompatible, so that the application of the pickering emulsions in the field of biological medicine is limited. For example, CN101445580a discloses a method for preparing a polyethylene/silica core-shell result composite material by emulsion polymerization, which uses silica nanoparticles as solid particles, but contains a catalyst dissolved by ethyl acetate and toluene, and finally ethylene is added to obtain a polyethylene/silica emulsion, wherein the application of silica and toluene in the system is limited clinically, so that the emulsion cannot be directly applied to the field of biological medicine.

It would therefore be of great interest in the art to provide a delivery system that increases AAV infection efficiency and maximizes AAV vaccine potential. Here, we based on the pickering emulsion system, select the soluble protein with good biocompatibility and inorganic particles as solid emulsifier together, and select the oil phase that can be metabolized biologically as the emulsion kernel at the same time, construct the pickering emulsion delivery system that can improve AAV transfection efficiency and improve adaptive immune response.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a Pickering emulsion delivery system capable of efficiently loading virus particles, and a preparation method and application thereof. The pickering emulsion adopts the soluble protein and the inorganic particles as solid emulsifying agents, so that the stability is good and the safety is high; and the emulsion can efficiently load virus particles due to the rough surface and the excellent particle adsorption capacity, and has stronger capability of infecting cells and activating adaptive immune response.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a pickering emulsion employing both soluble proteins and inorganic particles as solid emulsifiers, the pickering emulsion comprising an oil phase, an aqueous phase, viruses, and soluble proteins and inorganic particles, wherein the soluble proteins and inorganic particles are dispersed in the aqueous phase and/or adsorbed at the oil-water interface.

In the invention, the soluble protein and the inorganic particles are used together as a solid emulsifier to play a role in stabilizing emulsion drops, and the average particle size of the inorganic particles is in the order of nanometers to micrometers.

In the invention, the existence of the soluble protein can lead the inorganic particles to be dispersed more uniformly on the oil-water interface, and the effect of stabilizing the oil phase core is better. The nano-micro grade solid particles can play the role of immune adjuvant, and the action mechanism of the adjuvant can be attributed to the following aspects: 1) The nano-microparticles can specifically activate antigen presenting cells and increase the intake of the antigen presenting cells; 2) The nano-microparticles are used for embedding, adsorbing or coupling the antigen, so that the antigen can be released continuously, and the cell absorption and antigen expression time can be prolonged; 3) Part of nano-microparticles (such as chitosan nano-microparticles with positive charges) can realize lysosome escape of antigen through proton pump effect and the like, realize cross presentation of antigen and promote cellular immune reaction of organisms; 4) Some nanoparticles may also recruit inflammatory cells, thereby facilitating the interaction between the antigen and antigen presenting cells.

Therefore, the Pickering emulsion is prepared by adopting the solid particles to replace the surfactant, so that not only can the negative influence of the surfactant on the vaccine preparation be avoided, but also the more comprehensive, obvious and durable immune protection effect can be obtained through the immune synergistic effect of the solid particles and the oil-in-water emulsion. The Pickering emulsion does not contain a surfactant, avoids the influence of the surfactant on antigens, has good safety and stability, and can be used for different vaccination approaches of vaccines.

The pickering emulsion delivery system described in the present invention comprises a soluble protein and at least one inorganic particle. The soluble protein in the Pickering emulsion has biocompatibility, including endogenous protein and exogenous protein.

Preferably, the endogenous protein is albumin, hemoglobin, thyroxine transporter and/or ferritin; preferably, the exogenous protein is further preferably a protein antigen, including recombinant proteins, microbial and/or plant-derived protein antigens; preferably, the albumin comprises natural serum albumin and/or synthetic serum albumin, and further preferably any one or a combination of at least two of human serum albumin, bovine serum albumin or murine serum albumin; preferably, the protein is modified, including any one or a combination of at least two of hydrophilic modification, hydrophobic modification, metal ion biomineralization, coating, or grafting modification; preferably, the protein is Mn ²⁺ Further preferably, the mineralized protein is Mn ²⁺ Mineralized albumin.

The inorganic particles in the Pickering emulsion have biocompatibility, and are selected from any one or a mixture of at least two of aluminum salt, calcium salt, polysaccharide, barium salt and transition metal salt, and preferably the aluminum salt is aluminum hydroxide or/and aluminum phosphate; preferably, the calcium salt is calcium phosphate or/and calcium carbonate; preferably, the barium salt is barium sulfate; preferably, the transition metal salt is ferric hydroxide, manganese phosphate or/and zinc biphosphate. The so-called "aluminium hydroxide" is generally an aluminium oxyhydroxide salt, usually an aluminium solution (mostly AlCl ₃ Or AlK (SO) ₄ ) ₂ ) Mixing with sodium hydroxideAnd then dewatering the suspension under hydrothermal conditions. It generally exhibits different degrees of crystallization depending on the different production conditions. Aluminum oxyhydroxide is represented by the formula AlO (OH), which is mixed with other aluminum compounds, such as aluminum hydroxide Al (OH) ₃ Is distinguished by the Infrared (IR) spectrum, in particular at 1070cm ^-1 The absorbent band is present at 3090-3100cm ^-1 There is a strong spike. Aluminum hydroxide is in the typical fibrous form, and the pI of aluminum hydroxide adjuvants is generally about 11, i.e., the adjuvant itself has a positive surface charge at physiological pH. At pH7.4, the adsorption capacity of the aluminum hydroxide is 1.8-2.6 mg protein/mg Al ³⁺ Between them. Because of the differences between different batches of commercial aluminum hydroxide, the current standard is Alhydrogel produced in Denmark, which has a colloidal particle size of 3.07 μm. The so-called "aluminum phosphate" is typically an aluminum hydroxy phosphate, which also typically contains a small amount of sulfate (i.e., aluminum hydroxy phosphate sulfate). Aluminum phosphates are usually prepared by reacting aluminum salts (mostly AlCl ₃ Or AlK (SO) ₄ ) ₂ ) The solution is mixed with an alkaline solution of trisodium phosphate, or an aluminum salt is mixed with a phosphate solution, and then precipitated with sodium hydroxide to obtain a granular form in an amorphous state. Typical diameters of these particles after adsorption of any antigen are 0.5 to 20 μm (e.g., about 5 to 10 μm). At pH7.4, the adsorption capacity of aluminum phosphate is 0.7-1.5 mg protein/mg Al ³⁺ Between them.

Among the various calcium salts, calcium phosphate is preferred as the solid particles of the present invention. Various adjuvant forms of calcium phosphate have been reported, and any of these forms may be used in the present invention. The adjuvant may form irregularly shaped flakes of about 10nm by 150nm in size with needle-like particles of about 20-30nm in diameter. The shape of the solid particles in the invention can be various shapes such as sphere, rod, spindle, disk, cube, peanut shape or amorphous shape, the shape of the solid particles can be various shapes such as smooth surface, porous surface, multiple cavities in the interior, hollow or monocular shape, and the like, and the person skilled in the art can optimize and screen the emulsion according to the used oil-water phase and antigen property through a limited process so as to obtain the Pickering emulsion meeting the application requirements.

The average particle size of the emulsion droplets in the pickering emulsion described in the present invention is 50nm to 100. Mu.m, for example, 100nm, 300nm, 600nm, 1. Mu.m, 30. Mu.m, 80. Mu.m, 100. Mu.m, and the average particle size of the emulsion droplets in the pickering emulsion is selected to match the tissue to which it acts, preferably in the range of 100nm to 10. Mu.m.

Preferably, the pickering emulsion has an oil-water two-phase volume ratio of 1: (1-100), for example, may be 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1: 35. 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, or 1:100, etc., preferably 1: (2-50).

Preferably, the soluble protein is present in the aqueous phase in a mass concentration of 0.1 to 20wt%, for example 0.1wt%, 0.5wt%, 5wt%, 6wt%, 7wt%, 10wt%, 15wt% or 20wt%, preferably 0.5 to 10wt%, further preferably 1 to 8wt%, the mass concentration of the soluble protein in the aqueous phase being the ratio of the mass of the soluble protein divided by the sum of the masses of the soluble protein and the aqueous phase.

Preferably, the inorganic particle size distribution coefficient PDI value is lower than 1.0, and the PDI value of the inorganic particles is obtained by dynamic light scattering.

Preferably, the mass concentration of the inorganic particles in the aqueous phase is 0.1 to 20wt%, for example 0.1wt%, 0.5wt%, 5wt%, 6wt%, 7wt%, 10wt%, 15wt% or 20wt%, preferably 0.5 to 10wt%, further preferably 1 to 8wt%, the mass concentration of the inorganic particles in the aqueous phase being the ratio of the mass of the inorganic particles divided by the sum of the mass of the inorganic particles and the aqueous phase.

The oil phase is preferably a mixture of one or both of squalene and tocopherol. Squalene is a triterpene compound, the English name of which is squarene, the molecular structure of which is thirty-carbon fifty-hydrogen isoprene, and the molecular formula of which is: 2,3,10,15,19,23-hexamethyl-2, 6,10,14,18, 22-tetracosahexene, CAS:111-02-4, molecular mass: 410.72 it may be derived from animal, plant extracts or chemical syntheses. Squalene is a metabolizable oil because it is an intermediate product of cholesterol biosynthesis (Merk index, version 10, accession number 8619). This is a naturally secreted lipid of all higher organisms, including humans (found in sebum). Emulsions containing squalene (containing surfactants) exhibit excellent and large immunopotentiation effects in animal experiments and clinical experiments.

The tocopherol is alpha-tocopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate). Alpha-tocopherol can act to enhance immune responses in vaccines against elderly patients, such as patients older than 60 years of age or older. The tocopherols present include a variety of tocopherols, including alpha, beta, gamma, delta, epsilon, zeta, etc., preferably alpha-tocopherol, especially DL-alpha-tocopherol. Preferably, the oil phase is mutually incompatible with water, and may also include other metabolized oils.

In order to make the oil-in-water emulsion suitable for vaccine or pharmaceutical formulations, the oil phase of the oil-in-water emulsion in the present invention is a metabolizable oil. The term "metabolized oil" means well known in the art. "metabolizable" can be defined as "capable of transformation by metabolism" (interpreted by the medical dictionary of Dorland, w.b. sanders, inc. 25 th edition (1974)).

Exemplary metabolizable oils may be any vegetable, fish, animal or synthetic oil that is non-toxic to the recipient and convertible by metabolism, including but not limited to any one or a combination of at least two of soybean oil, miglitol (Miglyo 1812), medium chain oil, fish oil, vitamin E succinate, vitamin E acetate, safflower oil, corn oil, sea buckthorn oil, linseed oil, peanut oil, tea oil, sunflower oil, almond oil, coix seed oil, evening primrose oil, sesame oil, cottonseed oil, castor oil, canola oil, ethyl oleate, oleic acid, ethyl linoleate, isopropyl laurate, isopropyl myristate, ethyl butyrate, ethyl lactate, caprylic triglyceride, or capric triglyceride. Nuts, seeds, and grains are common sources of vegetable oils.

The aqueous phase of the oil-in-water emulsion in the present invention is preferably any one or a combination of at least two of water for injection, phosphate buffer, citric acid buffer or Tris buffer. Such as a combination of water for injection and phosphate buffer, a combination of citrate buffer and Tris buffer, a combination of water for injection, phosphate buffer and citrate buffer, a combination of Tris buffer, water for injection, phosphate buffer, citrate buffer or Tris buffer.

Preferably, the pH of the phosphate buffer, citrate buffer or Tris buffer is independently 5.0 to 8.1, for example 5.2,5.4,5.6,5.8,6,6.2,6.4,6.6,6.8,7,7.2,7.4,7.6,7.8 or 8, preferably 6.0 to 8.0.

The aqueous phase also comprises medicinal auxiliary substances such as pH regulator or/and buffer, preferably any one or a combination of at least two of 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 or gelatin. Such as sodium acetate and sodium lactate, sodium chloride and potassium chloride, calcium chloride, human serum albumin and essential amino acids, nonessential amino acids, L-arginine hydrochloride, sucrose and anhydrous D-trehalose, mannitol, mannose, starch and gelatin, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochloride, sucrose, anhydrous D-trehalose, mannitol, mannose, starch and gelatin.

Pharmaceutical additives may also be included in the pickering emulsion of the present invention, including, for example, any one or a combination of at least two of diluents, stabilizers or preservatives.

The pickering emulsion of the present invention may also include the following adjuvants, but is not limited to: stimulators of pattern recognition receptors (e.g., toll-like receptors, RIG-1 and NOD-like receptors (NLR), such AS CPG motif-containing oligonucleotides or double stranded RNAs or palindromic sequence-containing oligonucleotides or poly (dG) sequence-containing oligonucleotides), monophosphoryl lipid a (MPLA) of enterobacteria (e.g., escherichia coli, minnesota salmonella, salmonella typhimurium, or shigella flexneri), sapogenins adjuvants, liposomes and liposome formulations (e.g., AS 01), synthetic or specially prepared microparticles and microcarriers (e.g., neisseria gonorrhoeae), bacterial Outer Membrane Vesicles (OMVs) of chlamydia trachomatis and other bacteria origin, polysaccharide (e.g., chitosan), selectable pathogen-associated molecular patterns (PAMPS), small Molecule Immunopotentiators (SMIPs), cytokines and chemokines.

In a second aspect, the present invention provides a method for preparing a pickering emulsion according to the first aspect, wherein the pickering emulsion delivery system according to the present invention may be prepared by a variety of methods. Specifically, the pickering emulsion of the present invention may be prepared by the following method, but is not limited to the following preparation method.

The pickering emulsion of the present invention may be prepared by dispersing soluble protein and inorganic particles in an aqueous phase and then mixing the oil phase and the aqueous phase. The soluble protein can be directly and uniformly dispersed in the water phase, and the dispersion mode of the inorganic particles can be selected from various modes such as oscillation, stirring, ultrasonic and the like so as to realize good dispersion of the inorganic particles in the water phase. As long as good dispersion of the particles in the aqueous phase can be realized, the adopted dispersion mode does not have obvious influence on the properties of the Pickering emulsion, and the proper dispersion mode and specific operation parameters can be selected according to the properties of the used aqueous phase and solid particles and experimental equipment of the aqueous phase and solid particles. The mixing of the oil phase and the water phase can be carried out in a plurality of modes such as micro-fluidic, homogenization, ultrasonic, syringe double-pushing emulsification, spraying, micro-jet, micro-channel, membrane emulsification, stirring, oscillation, inversion or hand-shaking mixing. According to different requirements, the mixing mode can be preferably microfluidic, micro-channel or membrane emulsification and other modes capable of obtaining emulsion with uniform particle size distribution, and can also be preferably microfluidic, syringe double-push emulsification, homogenization, stirring or oscillation and other mixing modes convenient for large-scale preparation.

In a third aspect, the present invention also provides a viral vaccine, wherein the viral antigen is adsorbed on the surface of the pickering emulsion. The virus particles need only be incubated with the prepared emulsion at room temperature. The antigen may be derived from, but not limited to, chicken embryo culture, cell culture, purified and isolated from carrier body fluids, organs or tissues, recombinant gene expression or chemical synthesis, preferably the antigen includes, but is not limited to, any one or a combination of at least two of an attenuated vaccine, an inactivated vaccine, a split vaccine, an adenovirus vaccine or an adeno-associated virus vaccine, and the like.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the soluble protein and the inorganic particles are simultaneously used as the emulsifying agent for the first time to prepare the Pickering emulsion, wherein the existence of the soluble protein is favorable for the inorganic particles to be more uniformly distributed on the surface of the emulsion, so that the stability is good and the biological safety is high; secondly, after the emulsion is simply incubated with adeno-associated virus (AAV) at room temperature, high-efficiency entrapment can be realized, and the transfection efficiency of the AAV is obviously improved; furthermore, the Pickering emulsion can be used as an effective vaccine adjuvant, obviously improves the capability of AAV vaccine for inducing adaptive immune response, and has the potential of improving the vaccine onset speed, enhancing the vaccine immunogenicity and prolonging the vaccine protection period.

Drawings

FIG. 1 shows a divalent manganese ion (Mn ²⁺ ) Morphological schematic and transmission electron microscopy of Human Serum Albumin (HSA) before and after biomineralization (HSA-Mn).

FIG. 2 is a schematic form and a transmission electron microscope of Pickering emulsion (HSA/Algel-squarene, HANE; HSA-Mn/Algel-squarene, HMANE) prepared by jointly emulsifying squalene (squarene) with HSA or HSA-Mn and commercial aluminum gel (2% Algel).

Fig. 3 is a graph of particle size of pickering emulsion (HANE, HMANE) over time.

Fig. 4 is a schematic view of morphology of pickering emulsion (HANE, HMANE) before and after AAV adsorption and a transmission electron microscopy.

Fig. 5 is a graph showing the detection result of AAV adsorption encapsulation efficiency of pickering emulsion (HANE, HMANE).

FIG. 6 shows the results of infection of HEK293 cells after AAV adsorption by Pickering emulsion (HANE, HMANE).

FIG. 7 shows the results of infection of DC2.4 cells after AAV adsorption by Pickering emulsion (HANE, HMANE).

FIG. 8 is a graph showing the results of detection of AAV-POH-loaded Pickering emulsion (HANE-AAV-POH, HMANE-AAV-POH) induced humoral immune response.

Fig. 9 is a graph of mouse survival over time for AAV-POH-loaded pickering emulsions (HANE-AAV-POH, HMANE-AAV-POH) on a sepsis model.

FIG. 10 is a graph showing the results of measuring the activation ability of Pickering emulsion (HANE, HMANE) vector to STING pathway.

Detailed Description

The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present invention and do not represent or limit the scope of the invention, which is defined by the claims.

Example 1

Preparation of divalent manganese ion mineralized albumin particles based on albumin the specific preparation method is as follows (fig. 1-a):

by utilizing the natural biological template characteristic of albumin, divalent manganese ions can be orderly arranged on the surface of the protein in an alkaline environment to finish biomineralization, and the specific steps are as follows: weigh 0.2695g MnCl ₂ ·4H ₂ Adding 15ml of ultrapure water into a 50ml flask, and dissolving under stirring; HSA solution (200 mg/ml,5 ml) was added thereto and stirred at room temperature for 5min; adjusting the pH to about 10.00 with 1M sodium hydroxide; transferring to an oil bath pot at 34 ℃ and stirring for reaction for 2h; collecting a sample, and dialyzing for two days by a 10kDa dialysis bag; filtering with 0.45 or 0.22 μm filter head, lyophilizing to collect powder; and accurately quantifying the manganese element content in the mineralized powder by utilizing ICP-OES.

Example 2

A divalent manganese ion mineralized thyroxine transporter particle was prepared based on thyroxine transporter (MSA), specifically prepared as follows:

by utilizing the biological template characteristics of thyroxine transporter MSA and HSA, divalent manganese ions can be orderly arranged on the surface of protein in an alkaline environment to finish biomineralization, and the specific steps are as follows: weigh 0.2695g MnCl ₂ ·4H ₂ Adding 15ml of ultrapure water into a 50ml flask, and dissolving under stirring; MSA solution (200 mg/ml,5 ml) was added and stirred at room temperature for 5min; adjusting the pH to about 10.00 with 1M sodium hydroxide; transferring to an oil bath pot at 34 ℃ and stirring for reaction for 2h; collecting a sample, and dialyzing for two days by a 10kDa dialysis bag; filtering with 0.45 or 0.22 μm filter head, lyophilizingCollecting powder; and accurately quantifying the manganese element content in the mineralized powder by utilizing ICP-OES.

Example 3

The bivalent manganese ion mineralized protein is prepared based on recombinant protein pcrV protein (pcrV protein is pseudomonas aeruginosa cell membrane surface protein), and is specifically prepared as follows:

the divalent manganese ions can be orderly arranged on the surface of the protein in an alkaline environment to finish biomineralization, and the specific steps are as follows: weigh 0.2695g MnCl ₂ ·4H ₂ Adding 15ml of ultrapure water into a 50ml flask, and dissolving under stirring; adding pcrV solution (200 mg/ml,5 ml), and stirring at room temperature for 5min; adjusting the pH to about 10.00 with 1M sodium hydroxide; transferring to an oil bath pot at 34 ℃ and stirring for reaction for 2h; collecting a sample, and dialyzing for two days by a 10kDa dialysis bag; filtering with 0.45 or 0.22 μm filter head, lyophilizing to collect powder; and accurately quantifying the manganese element content in the mineralized powder by utilizing ICP-OES.

Example 4

The morphology of HSA-Mn prepared in example 1 is observed by a transmission electron microscope, and the specific steps are as follows: 1mg of lyophilized HSA-Mn was dissolved in 1ml of sterile water for injection, and a proper amount of the suspension was dipped in a capillary tube, placed on a copper mesh coated with a carbon film, allowed to stand for a while, and the excess solution was sucked off with a filter paper. Then dipping 2% phosphotungstic acid solution by using a capillary tube, standing for dyeing for 1-2min, sucking the dye liquor by using filter paper, drying under an infrared lamp, and finally observing the dye liquor in a transmission electron microscope.

The morphology of HSA-Mn under electron microscope is shown in FIG. 1b, and the morphology is in the form of a cube.

Example 5

Preparation of pickering emulsion HANE based on albumin HSA co-emulsifying squalene (squarene) with commercial aluminum gel (2% Algel) (FIG. 2-a); preparation of Pickering emulsion HMANE based on HSA-Mn co-emulsifying squalene (squarene) with commercial aluminum gel (2% Algel) (FIG. 2-b); the preparation method comprises the following steps:

30mgHSA is dissolved in 2943 mu l of sterile water for injection, 27 mu l of commercial aluminum glue and 30 mu l of squalene are added after the complete dissolution, and the mixture is evenly dispersed by 120-150W ultrasonic for 3min, thus obtaining HANE; 30mgHSA-Mn is dissolved in 2943 mu l of sterile water for injection, 27 mu l of commercial aluminum glue and 30 mu l of squalene are added after the complete dissolution, and the mixture is evenly dispersed by 120-150W ultrasonic for 3min, thus obtaining the HMANE. And respectively diluting the prepared HANE and HMANE by 50-100 times, taking 1ml of diluted emulsion, adding the diluted emulsion into a sample cell, measuring by using a Markov particle size meter, and characterizing the morphology by a transmission electron microscope.

Wherein, the particle size and morphology of HANE and HMANE are shown in figures 2-c and 2d, the particle size of hydration is maintained at about 300nm, and the particle size and morphology of HMANE are white spherical. The particle size change of HANE and HMANE during storage at 4deg.C is shown in figure 3, and the result shows that the Pickering emulsion prepared by the invention has good stability, albumin or Mn ²⁺ Mineralized albumin and inorganic particles can obtain the pickering emulsion with good long-term stability.

Example 6

Preparing a pickering emulsion MANE based on thyroxine transporter MSA and commercial aluminum gel emulsified squalene (squarene); the preparation method of the Pickering emulsion MMANE based on MSA-Mn and commercial aluminum gel (2% Algel) jointly emulsifying squalene (squarene) comprises the following steps:

30mg of MSA is dissolved in 2943 mu l of sterile water for injection, 27 mu l of commercial aluminum glue and 30 mu l of squalene are added after the MSA is fully dissolved, and the MSA is evenly dispersed by 120-150W ultrasonic for 3min, thus obtaining MANE; 30mg of MSA-Mn is dissolved in 2943 mu l of sterile water for injection, 27 mu l of commercial aluminum glue and 30 mu l of squalene are added after the complete dissolution, and the mixture is uniformly dispersed by 120-150W ultrasonic for 3min, thus obtaining the MMANE. And respectively diluting the prepared MANE and MMANE by 50-100 times, taking 1ml of diluted emulsion, adding the diluted emulsion into a sample cell, and measuring by using a Markov particle size meter.

The MANE, MMANE particle sizes and PDI distribution are shown in table 1.

TABLE 1 Pickering emulsion particle size and PDI distribution based on MSA and Algel

Example 7

The preparation method of the Pickering emulsion (HANE-AAV, HMANE-AAV) carrying viruses based on adeno-associated virus (AAV) comprises the following steps:

and (3) sucking a proper amount of AAV virus liquid into the low adsorption EP tube, adding a blocking Buffer to 500 mu L, then adding 500 mu L of pre-prepared HANE or HMANE, swirling for 30s, and incubating for 30min at room temperature with a room temperature horizontal shaking table at 150rpm to obtain HANE-AAV (or HMANE-AAV).

Wherein, the morphology of HANE-AAV and HMANE-AAV under an electron microscope is shown as figure 4, and AAV viruses are uniformly distributed on the surface of spherical particles, which shows that the albumin or Mn prepared by the invention ²⁺ The Pickering emulsion of mineralized albumin and inorganic particles instead of surfactant can absorb AAV virus well, and can be used as a virus delivery system.

Example 8

The present embodiment is used for detecting the encapsulation efficiency of pickering emulsion (fine, HMANE) on AAV, and the specific method is as follows:

during high-speed centrifugation, the oil phase (squalene) with smaller density tends to be distributed on the upper layer of the system, while the solid emulsifier of the pickering emulsion tends to be distributed on the oil-water interface, wherein the solid emulsifier mainly plays a role of adsorbing antigen, so that the encapsulation rate of the emulsion to the antigen can be measured by detecting the content of the antigen in the lower water phase. Since AAV content can be directly and accurately determined by qPCR, the obtained HANE-AAV or HMANE-AAV is centrifuged for 10min at 12000g at 4deg.C, and emulsion droplets are accumulated and float on the upper layer, while the water phase containing free AAV is on the lower layer. After removing the lower liquid and digesting the free nucleic acid with DnaseI, qPCR titer was performed, and parallel operation was performed using the same dose of free virus as an internal reference. The calculation formula of the obtained encapsulation efficiency is as follows:

encapsulation efficiency= [1-1/2 (Cq ^Formulations -Cq ^{Internal reference} )]×100％

The Cq preparation represents the response value of the preparation amplification, and the Cq internal reference represents the response value of the control amplification.

The results are shown in fig. 5, where the pickering emulsion after centrifugation separated into a compact milky upper oil phase and a clear and clear lower aqueous phase without significant precipitation. Subsequently, qPCR detection shows that the Cq value of the water phase at the lower layer of the emulsion is increased by more than 3 cycles compared with that of a free group, and the encapsulation efficiency of HANE to AAV is 96.18 +/-0.11 percent, the encapsulation efficiency of HMANE to AAV is 89.56 +/-0.09 percent, so that the Pickering emulsion prepared by the invention has good encapsulation efficiency to viruses, the encapsulation efficiency is more than 89 percent, and the emulsion is an excellent viral vector.

Example 9

This example was used to examine the effect of pickering emulsions (HANE, HMANE) on AAV infection of HEK293 cells, as follows:

AAV mediates endocytosis mainly through sialic acid receptors on the cell surface, so that the ability of free AAV to infect cells will be significantly reduced after HEK293 cells are treated with sialidases; meanwhile, if the ability of infected cells does not show a decrease after the emulsion group adsorbs AAV, it is indicated that the emulsion can change the way of AAV into cells. Cells were plated using 24 well plates, the cell concentration was set to 1X 10≡5 cells/well, the MOI was set to 1E6, and the sialidase-treated group and the sialidase-untreated group were simultaneously set for incubation at 37℃for 48-72h. AAV1-mCherry (expressing red fluorescent protein mCherry) was selected as the subject.

As shown in fig. 6, the emulsion can significantly enhance the transfection efficiency of AAV1 for HEK293 cells, wherein the fine can be increased by 2.23-fold, and the HMANE can be increased by 2.89-fold; secondly, the HMANE group is significantly better than the HANE group, demonstrating that manganese-mineralized albumin is beneficial to promote AAV1 transfection; furthermore, after sialidase (neuroaminidase) treatment of cells, the infection efficiency of free AAV1 was reduced by 1-fold, while the two emulsion groups appeared to be unaffected by sialidases, indicating that the participation of the emulsion did alter the infection pathway of AAV1, contributing to an increase in the infection efficiency of AAV.

Example 10

This example was used to examine the effect of pickering emulsions (HANE, HMANE) on AAV-infected DC2.4 cells, as follows:

here we selected to study AAV1 serotypes (AAV 1-mCherry, expressing red fluorescent protein) with lower infection efficiency of DC2.4 cells, if HANE and HMANE were able to enhance the infection efficiency of AAV1 on DC2.4 cells, they would be more able to demonstrate the pro-infectious ability of emulsions. Cells were plated using 24 well plates, the cell concentration was set to 1X 10≡5 cells/well, MOI was set to 1E6, and incubated at 37℃for 48-72h.

As shown in fig. 7, the emulsion groups (both HANE and HMANE) were able to significantly increase infection of DC2.4 cells with AAV1 compared to free AAV1, which hardly infects DC2,4 cells, especially the HMANE group, demonstrating that manganese-mineralized albumin not only increased the infection efficiency of AAV1 (fig. 6), but also altered the selectivity of AAV1 for cells (fig. 7).

Example 11

This example was used to compare the effect of the pickering emulsion, MF-59 adjuvant, and aluminum hydroxide adjuvant provided in example 5 on AAV-POH vaccine (AAV expressing pseudomonas aeruginosa antigen POH-OprI-Hcp1, AAV-POH, using genetic engineering) induced antibodies and protective efficacy on bacterial challenge models. 70 SPF-class female BALB/c mice (6-8 weeks old) were randomly divided into 7 groups, wherein PBS group was used as a negative control group, four groups of Low dose free virus (AAV-POH (Low)), high dose free virus (AAV-POH (High)), MF-59-AAV-POH and 2% Algel-AAV-POH were used as control groups, and AAV-POH-loaded Pickering emulsion (HANE-AAV-POH, HMANE-AAV-POH) was prepared as experimental group according to example 7, 10 groups each, specific immunization information as shown below (Table 2), wherein the injection mode was 100. Mu.l (comprising 50. Mu.l virus solution and 50. Mu.l adjuvant) for the hind limb gastrocnemius muscle injection. The orbital blood of each group of mice was taken at 27 days after immunization, left standing at 37℃for 2h, centrifuged at 10,000g for 10min, and serum was collected and assayed for antibody levels using ELISA. Every other day, a lethal dose of pseudomonas aeruginosa PAO1 was injected via the tail vein, followed by observation of death of the mice every 12h for 14 consecutive days.

TABLE 2 immunization protocol

The results of the antibody test showed (FIG. 8) that the antibody titer of AAV-POH vaccine was increased to a different extent compared to AAV-low, high dose and adjuvant containing vaccine groups (AAV-high, HANE-AAV, HMANE-AAV, MF-59-AAV,2% Alge-AAV), wherein the three groups AAV-high, HANE-AAV, HMANE-AAV were the most balanced, indicating that Th1 and Th2 type responses were induced simultaneously. In substantial agreement with the antibody results, the bacterial challenge results showed that 20%,80%,90%,90%,50% and 70% protection against sepsis, 80%,90%, 50% and 70% protection against sepsis, in particular, the HANE-AAV and HMANE-AAV achieved 90% protection only at 1/5 high dose of virus, compared to PBS, AAV-low, AAV-high, HANE-AAV, HMANE-AAV, respectively, demonstrated excellent resistance to P.aeruginosa infection in vivo by activating strong humoral and cellular immunity (FIG. 9).

Example 12

This example was used to examine the activation ability of pickering emulsion vectors for STING pathways.

DC2.4 cell concentration was adjusted to 1.5X10 ⁵ Each of them was inoculated into a 12-well flat-bottomed cell culture plate at a dose of 1mL per well, and after stabilizing at 37℃for 2 hours, 20. Mu.l of HANE, 20. Mu.l of HMANE, and 2. Mu.g of CDN (i.e., 2,3-cGAMP as a positive control group for stimulating STING activation) were added to each well, followed by further culturing for 22 hours. Cell culture supernatants were collected by centrifugation and assayed by IFN- β ELISA. The results are shown in FIG. 10-a, where the HMANE group induced the highest IFN- β levels.

The THP1-Lucia ISG cells were adjusted to a concentration of 1X 10 ⁵ cells/180. Mu.L were inoculated into a flat bottom 96-well plate at 180. Mu.L per well, HANE 20. Mu.L/HMANE 20. Mu.L, CDN 2. Mu.g, AAV-POH 1.0E10vg were added to each well, and after further culturing for 24 hours, cells in the plate were gently beaten with a row gun to distribute them uniformly, 20. Mu.L of THP1-Lucia ISG cell suspension was pipetted into a black 96-well plate, and 50. Mu.L of QUANTI-Luc substrate was added thereto, and immediately detected with a microplate reader. As shown in FIG. 10-b, the HMANE group can activate the STING-IRF-3 pathway to the maximum extent, and the corresponding highest level reporter gene, lucia ISG, was secreted and superior to the CDN (2, 3-cGAMP) group of classical test dose (5- μg) (FIGS. 10-b and 10-c).

In summary, as a result of the example, the pickering emulsion of the present invention can load AAV virus particles with high efficiency (encapsulation efficiency is greater than 89%), and at the same time, the efficiency of AAV vaccine infection of HEK293T cells, which are susceptible to infection, and DC2.4 cells, which are difficult to infect, is significantly improved, that is, the infection efficiency is improved and the selectivity of AAV1 to cells is changed. Further, the adsorption of AAV by HANE and HMANE can further increase the humoral immune response level of AAV vaccines and maximize protection of mice from lethal doses of bacterial challenges.

The present invention is not limited to the above-described preferred embodiments, but is intended to be limited to the following description, and any modifications, equivalents and variations of the above-described embodiments, which are included in the technical spirit of the present invention, are intended to fall within the scope of the present invention, as long as they are equivalent to the present invention, and are within the scope of the technical spirit of the present invention.

Claims

1. A pickering emulsion delivery system for highly efficient loading of viral particles, wherein the pickering emulsion delivery system comprises an oil phase, an aqueous phase, a virus, and soluble proteins and inorganic particles, wherein the soluble proteins and inorganic particles are dispersed in the aqueous phase and/or adsorbed at the oil-water interface, and the viral particles are adsorbed on the surface of the emulsion.

2. The pickering emulsion delivery system of claim 1, wherein the average particle size of the emulsion droplets in the pickering emulsion is 50nm to 100 μιη, preferably 100nm to 10 μιη; preferably, the pickering emulsion has an oil-water two-phase volume ratio of 1: (1 to 100), preferably 1: (2-50); preferably, the mass concentration of the soluble protein in the aqueous phase is 0.1 to 20wt%, preferably 0.5 to 10wt%, further preferably 1 to 8wt%; preferably, the mass concentration of the inorganic particles in the aqueous phase is 0.1 to 20wt%, preferably 0.5 to 10wt%, further preferably 1 to 8wt%.

3. The pickering emulsion delivery system of any one of claims 1 or 2, wherein the soluble protein comprises an endogenous protein and an exogenous protein, the endogenous protein further preferably being one or more of albumin, hemoglobin, thyroxine transporter, and/or ferritin; the exogenous protein is further preferably a protein antigen, including recombinant proteins, proteins of microbial and/or plant origin; preferably, the soluble protein is albumin, including natural serum albumin and/or synthetic serum albumin, further preferably the albumin is at least one of human serum albumin, bovine serum albumin or murine serum albumin; preferably, the albumin is modified, the modification comprising any one or a combination of at least two of hydrophilic modification, hydrophobic modification, metal ion biomineralization, coating or graft modification.

4. The pickering emulsion delivery system of any one of claims 1-2, wherein the inorganic particles are selected from at least one of aluminum salts, calcium salts, barium salts, transition metal salts; preferably, the aluminum salt is aluminum hydroxide or/and aluminum phosphate; preferably, the calcium salt is calcium phosphate or/and calcium phosphate; preferably, the barium salt is barium sulfate; preferably, the transition metal salt is ferric hydroxide, manganese phosphate or/and zinc biphosphate.

5. The pickering emulsion delivery system of any one of claims 1-2, wherein the oil phase comprises any one or a combination of at least two of squalene, tocols, olive oil, soybean oil, vitamin E, ethyl oleate, oleic acid, ethyl lactate, dimethicone, isopropyl laurate, or capric triglyceride, further preferably any one or a combination of at least two of squalene, tocols, or dimethicone; preferably, the aqueous phase comprises any one or a combination of at least two of purified water, water for injection, phosphate buffer, citrate buffer or Tris buffer; preferably, the pH of the phosphate buffer, citrate buffer or Tris buffer is independently 5.0 to 8.1, preferably 6.0 to 8.0.

6. The pickering emulsion delivery system of any of claims 1-2, wherein the pickering emulsion delivery system further comprises a pharmaceutically acceptable additive; preferably, the pharmaceutical additive comprises any one or a combination of at least two of a diluent, a stabilizer or a preservative.

7. The pickering emulsion delivery system of any of claims 1-2, wherein the aqueous phase and/or oil-water interface further comprises an immunologically active substance; preferably, the immunologically active substance comprises any one or a combination of at least two of CPG, a STING agonist, monophosphoryl lipid A, a saponin or lysozyme.

8. Method for the preparation of a pickering emulsion delivery system according to any of claims 1-8, characterized in that the preparation method comprises the steps of: preparing an aqueous phase suspension containing soluble proteins and inorganic particles, mixing the oil phase suspension with the aqueous phase suspension, and emulsifying to obtain the Pickering emulsion type delivery system.

9. A viral vaccine, characterized in that the viral antigen is adsorbed on the surface of the pickering emulsion of any one of claims 1-8, and the viral antigen is selected from any one or a combination of at least two of an attenuated vaccine, an inactivated vaccine, a split vaccine, an adenovirus vaccine or an adeno-associated virus vaccine; preferably, the immunization regimen of the vaccine comprises any one or a combination of at least two of intravenous injection, spinal cavity injection, intramuscular injection, subcutaneous injection, intradermal injection, respiratory tract injection or inhalation, intraperitoneal injection, nasal administration, ocular administration, oral administration, rectal administration, vaginal administration, topical administration or scalp administration.

10. Use of the pickering emulsion delivery system of any one of claims 1-8 in the preparation of a vaccine and/or medicament.