Antigen delivery carrier with deformability as well as preparation method and application thereof

Abstract

The invention relates to an antigen delivery carrier with deformability and a preparation method and application thereof.The antigen delivery carrier with deformability is of a water-in-oil-in-water type emulsion structure, an inner water phase is turbid liquid containing electropositive nano-particles, an oil phase is an oily substance containing a lipophilic emulsifier, and the pH value of the emulsion structure is 7-8. And the outer water phase is a solution containing a hydrophilic emulsifier. The antigen delivery carrier provided by the invention has deformability, the contact area can be increased through deformation when the antigen delivery carrier is in contact with cells, meanwhile, the electropositive particles in the internal water phase can efficiently adsorb or embed antigens and improve the bioavailability of the antigens, and the positive electricity of the particles is utilized to strengthen a proton pump effect and promote lysosome rupture to release the antigens, so that the antigens are released, and the bioavailability of the antigens is improved. The cellular immune response effect is greatly improved, so that the dual immune response of body fluid and cells is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a deformable antigen delivery carrier and a preparation method and application thereof. Background Art

[0002] Infectious diseases are caused by various pathogens, mainly including viruses, bacteria, fungi or parasites. Vaccination is the most effective prevention and control strategy, but conventional vaccine preparations such as aluminum salt adjuvants and oil emulsion adjuvants tend to induce humoral immune responses in the body, and their cellular immune responses are not effective, resulting in the inability to effectively eliminate viruses and achieve cross-protection against multiple heterotypic viruses. Therefore, how to improve the efficiency of cellular immune responses is the key to achieving more efficient and comprehensive immunotherapy effects of vaccine preparations.

[0003] The key to inducing a cellular immune response lies in the efficiency of antigen uptake by antigen-presenting cells and its escape from lysosomes. Vaccination is usually performed by intramuscular or subcutaneous injection. The number of antigen-presenting cells in peripheral tissues is limited, and free antigens cannot fully contact them, making it difficult to achieve effective cell uptake of antigens. In addition, after free antigens are taken up by cells, most of them are processed and presented through the lysosomal pathway, which makes it difficult to effectively enhance the cellular immune response. Therefore, improving the efficiency of antigen uptake by antigen-presenting cells and efficiently escaping from lysosomes is an urgent problem to be solved in generating a cellular immune response. At present, conventional antigen delivery carriers tend to solve the problem from a single level, and it is difficult to achieve the above goals at the same time.

[0004] Therefore, there is an urgent need to provide a product for antigen delivery to improve antigen utilization, while enhancing the levels of humoral and cellular immune responses, and achieving efficient vaccine immune response efficiency. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a deformable antigen delivery carrier and a preparation method thereof, as well as an antigen delivery system for enhancing dual immune responses.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a deformable antigen delivery carrier, which has a water-in-oil-in-water emulsion structure, wherein the inner aqueous phase is a suspension containing positively charged nanoparticles, the oil phase is an oily substance containing a lipophilic emulsifier, and the outer aqueous phase is a solution containing a hydrophilic emulsifier.

[0008] The antigen delivery carrier involved in the present invention is an emulsion preparation, which has a water-in-oil-in-water (W1 / O / W2) structure, and a suspension containing positively charged nanoparticles as an inner aqueous phase, an oily substance containing a lipophilic emulsifier as an oil phase, and a solution containing a hydrophilic emulsifier as an outer aqueous phase, so that the emulsion preparation has deformability, and stress deformation can occur when in contact with cells, changing from a sphere to an oblate spheroid, and the contact site is expanded from a point to a surface, significantly increasing the contact area between the emulsion droplet and the cell membrane, imitating the dynamic interaction between pathogens and cells, and enhancing cell uptake; at the same time, the nanoparticles in the inner aqueous phase can efficiently adsorb or embed antigens, improve their bioavailability, and the positively charged nanoparticles can strengthen the "proton pump effect", prompting the lysosome to swell and rupture rapidly to release internal antigens. Therefore, it can significantly enhance the body's immune response, especially the cellular immune response, and achieve more efficient and comprehensive immune protection against infectious diseases.

[0009] Preferably, the volume ratio of the oil phase to the external water phase is (1-4):(2-11), and the volume ratio of the internal water phase to the oil phase is (1-3):(2-7).

[0010] The volume ratio of the oil phase to the external water phase can be selected from 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 2:3, 2:5, 2:7, 2:9, 2:11, 3:4, 3:5, 3:7, 3:10, 4:5, 4:7, 4:9, 4:11, etc., and the volume ratio of the internal water phase to the oil phase can be selected from 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 2:3, 2:5, 2:7, 3:5, 3:7, etc. Other points within the above numerical range can be selected, which will not be repeated here.

[0011] The present invention controls the volume ratio of the oil phase to the inner water phase and the outer water phase within the above-mentioned specific numerical range, which can further improve the deformability of the emulsion preparation, increase the contact area between the emulsion droplets and the cell membrane, and enhance cell uptake.

[0012] Preferably, in the water-in-oil-in-water emulsion structure, the particle size of the water-in-oil emulsion droplets is 100nm-10μm, for example, it can be 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 900nm, 1μm, 2μm, 3μm, 5μm, 6μm, 7μm, 8μm, 10μm, etc., and the particle size of the water-in-oil-in-water emulsion droplets is 200nm-20μm, for example, it can be 200nm, 300nm, 500nm, 700nm, 800nm, 1μm, 2μm, 4μm, 6μm, 8μm, 10μm, 12μm, 14μm, 16μm, 18μm, 20μm, etc. Other point values ​​within the above numerical range can be selected, which will not be repeated here one by one.

[0013] Preferably, the positively charged nanoparticles have a potential of 5-40 mV, such as 5 mV, 6 mV, 7 mV, 8 mV, 9 mV, 10 mV, 12 mV, 15 mV, 16 mV, 18 mV, 20 mV, 23 mV, 25 mV, 27 mV, 29 mV, 30 mV, 32 mV, 35 mV, 37 mV, 39 mV, 40 mV, etc., and a particle size of 10-900 nm, such as 10 nm. , 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, etc. Other point values ​​within the above numerical range can be selected and will not be described here one by one.

[0014] Preferably, the polydispersity index PDI of the positively charged nanoparticles is less than 0.3, for example 0.01, 0.02, 0.05, 0.07, 0.08, 0.1, 0.15, 0.18, 0.2, 0.3, etc. Other points within the above numerical range can be selected and will not be described here one by one.

[0015] Preferably, the positively charged nanoparticles include chitosan and / or chitosan derivative nanoparticles.

[0016] The chitosan nanoparticles or chitosan derivative nanoparticles involved in the present invention can be prepared by the known technical methods in the technical field or the methods disclosed in the prior art, such as ion crosslinking method, nanoprecipitation method, thermal curing method, single-phase gel spheronization method, etc.

[0017] Preferably, the chitosan derivative comprises chitosan hydrochloride and / or chitosan quaternary ammonium salt.

[0018] Preferably, the molecular weight of the chitosan is 20-1000kDa, for example 20kDa, 50kDa, 100kDa, 300kDa, 500kDa, 600kDa, 800kDa, 1000kDa, etc., and the degree of deacetylation is 80-95%, for example 80%, 85%, 90%, 95%, etc. Other points within the above numerical range can be selected and will not be described here one by one.

[0019] Preferably, the oily substance is a pharmaceutical grade oily substance, including any one of squalene, tocopherol or ethyl linoleate, or a combination of at least two thereof.

[0020] Squalene is an acyclic triterpene composed of 6 isoprene groups. Its chemical name is 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexene and its molecular formula is C 30 H 50 Squalene is found in many plants and animals, and is particularly abundant in shark liver. Squalene-based emulsions are one of the most widely used vaccine adjuvants, which can enhance immune responses and prolong immune response time.

[0021] Tocopherol is a fat-soluble vitamin, which is composed of four tocopherol homologues, namely α-, β-, γ-, and δ-tocopherol, among which α-tocopherol has the strongest activity. As a vaccine component, it participates in regulating the expression of certain chemokines and cytokines, such as chemokine 2 (CCL2), chemokine 3 (CCL3), interleukin 6 (IL-6), chemokine 1 (CXCL1), etc., which can enhance the uptake of antigens by antigen-presenting cells and increase the number of innate immune cells recruited in the draining lymph nodes.

[0022] Linoleic acid ethyl ester is a fatty acid ethyl ester compound, which is produced by the reaction of linoleic acid and ethanol. Its chemical formula is C 20 H 36 O2 has good antioxidant properties and can reduce the damage of oxidative stress to cells.

[0023] Preferably, the lipophilic emulsifier includes any one of sorbitan trioleate, polyoxyethylene stearate, polyoxyethylene fatty alcohol ether, PO-500 or Arlacel P135, or a combination of at least two thereof.

[0024] Preferably, the mass fraction of the lipophilic emulsifier in the oil phase is 1-20%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 13%, 15%, 16%, 18%, 19%, 20%, etc. Other points within the above numerical range can be selected and will not be described here one by one.

[0025] Preferably, the hydrophilic emulsifier includes any one of Tween 80, Tween 85, poloxamer, polyoxyethylene monopalmitate, polyoxyethylene castor oil or polyoxyethylene alkyl ether, or a combination of at least two thereof.

[0026] Preferably, the mass fraction of the hydrophilic emulsifier in the external aqueous phase is 1-30%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 13%, 15%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, etc. Other points within the above numerical range can be selected and will not be described here one by one.

[0027] Preferably, the pH values ​​of the external aqueous phase and the internal aqueous phase are independently selected from 5.5-8.5, for example, 5.5, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.1, 7.3, 7.5, 7.7, 7.8, 7.9, 8.0, 8.2, 8.3, 8.5, etc. Other points within the above numerical range can be selected and will not be described one by one here. 6.0-8.0 is more preferred.

[0028] Preferably, the solutions of the external aqueous phase and the internal aqueous phase are independently selected from any one of purified water, water for injection, 0.9% saline, phosphate buffer, citrate buffer or Tris buffer, or a combination of at least two thereof.

[0029] Preferably, the solution of the external aqueous phase is any one of phosphate buffer, citrate buffer or Tris buffer, or a combination of at least two of them.

[0030] Preferably, the solution of the inner aqueous phase is any one of purified water, water for injection, 0.9% saline or phosphate buffer, or a combination of at least two of them.

[0031] In the present invention, the Young's modulus of the antigen delivery carrier is 0.2-50MPa, for example, 0.2MPa, 0.4MPa, 0.5MPa, 0.6MPa, 0.8MPa, 1MPa, 3MPa, 5MPa, 7MPa, 9MPa, 10MPa, 12MPa, 13MPa, 15MPa, 16MPa, 18MPa, 20MPa, 22MPa, 23MPa, 25MPa, 27MPa, 29MPa, 30MPa, 32MPa, 34MPa, 36MPa, 38MPa, 40MPa, 41MPa, 43MPa, 45MPa, 46MPa, 48MPa, 49MPa, 50MPa, etc. Other point values ​​within the above numerical range can be selected, which will not be repeated here one by one.

[0032] In the present invention, the angle range of the antigen delivery vector when in contact with the cell is 10-70°, for example, it can be 10°, 15°, 20°, 22°, 24°, 26°, 28°, 30°, 33°, 35°, 37°, 38°, 40°, 42°, 44°, 45°, 47°, 50°, 55°, 60°, 70°, etc. Other point values ​​within the above numerical range can be selected and will not be described one by one here.

[0033] In a second aspect, the present invention provides a method for preparing the deformable antigen delivery carrier according to the first aspect, the preparation method comprising:

[0034] Positively charged nanoparticles are dispersed in an inner aqueous phase solution, and then mixed with an oily substance containing a lipophilic emulsifier for homogenous emulsification to obtain an oil-in-water emulsion; and then mixed with an outer aqueous phase solution containing a hydrophilic emulsifier for homogenous emulsification to obtain a water-in-oil-in-water emulsion, thereby obtaining the deformable antigen delivery carrier.

[0035] In a third aspect, the present invention provides an antigen delivery system for enhancing dual immune responses, wherein the antigen delivery system comprises the deformable antigen delivery carrier described in the first aspect and an antigen, wherein the antigen is adsorbed on the surface of positively charged nanoparticles or embedded in the interior of positively charged nanoparticles.

[0036] Preferably, the antigen loading amount is 0.1-10% of the mass of the positively charged nanoparticles, for example 0.1%, 1%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Other points within the above numerical range can be selected and will not be described here one by one.

[0037] The antigen described in the present invention may be a monovalent antigen or a multivalent antigen; the antigen may be derived from, but not limited to, chicken embryo culture, cell culture, purified separation from body fluids, organs or tissues of carriers, recombinant gene expression or chemical synthesis, and the preferred antigen includes, but is not limited to, any one or a combination of at least two of attenuated vaccines, inactivated vaccines, split vaccines, subunit vaccines, polysaccharide conjugate vaccines, recombinant vaccines or DNA vaccines, etc. For example, a combination of attenuated vaccines and inactivated vaccines, a combination of split vaccines and subunit vaccines, a combination of polysaccharide conjugate vaccines and recombinant vaccines, a combination of DNA vaccines and attenuated vaccines, a combination of inactivated vaccines and split vaccines, a combination of subunit vaccines, polysaccharide conjugate vaccines, recombinant vaccines and DNA vaccines.

[0038] In a fourth aspect, the present invention provides a method for using the antigen delivery system for enhancing dual immune responses described in the third aspect, wherein the antigen is administered by injection through immunization, and the administration method includes any one of subcutaneous injection, intramuscular injection, intralymph node injection, intraperitoneal injection, intravenous injection or subpedicular injection.

[0039] In a fifth aspect, the present invention provides use of the antigen delivery vector described in the first aspect or the antigen delivery system for enhancing dual immune response described in the third aspect in the preparation of anti-infectious disease drugs.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The antigen delivery carrier provided by the present invention is deformable and can increase the contact area by deformation when in contact with cells. At the same time, the positively charged particles in the inner water phase can efficiently adsorb or embed antigens to improve their bioavailability, and use the positive charge of the particles to enhance the "proton pump effect", promote the rupture of lysosomes and release of antigens, greatly improve the cellular immune response effect, and thereby achieve the enhancement of both humoral and cellular immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a light microscopic image of the emulsion product No. 1 prepared in Example 2;

[0043] Figure 2 is a comparison chart of Young's modulus of the emulsion product No. 1 prepared in Example 2 and chitosan nanoparticles;

[0044] Figure 3 is a microscopic observation of the contact between the emulsion droplets and the cells in Example 4;

[0045] Figure 4 This is a laser confocal observation diagram of the uptake of antigens in the emulsion by cells in Example 5;

[0046] Figure 5 is a graph showing the serum specific IgG antibody level after inoculation with the emulsion preparation in Example 7;

[0047] Figure 6 This is a result diagram of the activation effect of the emulsion preparation on spleen T cells after inoculation in Example 7;

[0048] Figure 7 This is a graph showing the evaluation results of central memory cells after inoculation of the emulsion preparation in Example 7;

[0049] Figure 8 This is a graph showing the level of IFN-γ secretion by T cells after inoculation of the emulsion preparation in Example 7. DETAILED DESCRIPTION

[0050] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0051] Some of the raw materials involved in the following content are as follows:

[0052]

[0053]

[0054]

[0055] Some of the instruments involved in the following contents are as follows:

[0056]

[0057] Some of the detection methods involved in the following content are as follows:

[0058] (1) Determination of particle size distribution of particles or emulsions:

[0059] The particle size distribution of nano-scale particles or emulsion droplets is measured by a dynamic light scattering particle size analyzer. The specific measurement steps are as follows: 1 mg of nano-scale particles is added to 10 mL of deionized water, and 1 mL is taken and added to the sample pool for detection after ultrasonication for 5 minutes to make it uniformly dispersed; the prepared water-in-oil (W1 / O) emulsion is dispersed in 10 times the volume of the oil phase, and 1 mL is taken and added to the sample pool for detection after mixing evenly; the prepared water-in-oil-in-water (W1 / O / W2) emulsion is dispersed in 10 times the volume of the external water phase, and 1 mL is taken and added to the sample pool for measurement after mixing evenly. The uniformity of particle size is represented by the particle size polydispersity index (PDI) value. The smaller the PDI value, the more uniform the particle size.

[0060] Micron-sized particles and droplet size were detected by laser particle size analyzer. The specific operation was as follows: 5 mg of particles were dispersed in 5 mL of deionized water, ultrasonicated for 5 min to make them uniformly dispersed, and then mixed and placed in the sample pool for detection; 1 mL of the prepared water-in-oil-in-water (W1 / O / W2) emulsion was added dropwise to the sample pool for detection. The uniformity of particle size is represented by the particle size distribution coefficient (Span) value. The smaller the value, the more uniform the particle size.

[0061] (2) Potential detection of nanoparticles:

[0062] The potential detection of nanoparticles is carried out using a dynamic light scattering particle size analyzer. The specific measurement steps are as follows: 1 mg of nanoparticles is added to 10 mL of deionized water, and after ultrasonication for 5 minutes to make them evenly dispersed, 1 mL is taken and added to the potential cell for measurement.

[0063] (3) Morphological observation and deformation characterization of emulsion:

[0064] The morphology of the emulsion was observed under an optical microscope. A small amount of the emulsion was taken up with a pipette, dropped onto a glass slide, and observed under an optical microscope.

[0065] The deformability of the emulsion was first measured using an atomic force microscope. A circle was drawn on a positively charged glass slide with a hydrophobic pen, and the emulsion was dropped into the circle and allowed to stand for 5 minutes. The excess sample was washed off with deionized water and then tested. The spring constant was 0.915 N / m, and the Young's modulus of the obtained indentation curve was calculated using the Sneddon model.

[0066] Secondly, the interaction between the emulsion and the cell can be observed for characterization. The fluorescent dye-labeled emulsion was co-incubated with mouse bone marrow-derived dendritic cells (BMDCs) and observed using a multi-mode single-molecule localization detection microscope. When the emulsion was in full contact with the cell, it was photographed and analyzed after taking 20 photos. The angle formed when the emulsion contacts the cell refers to the angle between the tangent of the air-emulsion interface at the intersection of air, emulsion and cell membrane passing through the emulsion and the cell membrane-emulsion boundary.

[0067] (4) Determination of antigen loading rate:

[0068] Accurately measure 1 mL of the antigen-loaded emulsion and place it in a 300 kDa dialysis tube. Accurately measure 10 mL of deionized water and place the dialysis tube in deionized water. Suspend overnight at 4 °C and take the solution outside the dialysis bag the next day. Also set up a group of emulsions without antigens of the same volume as a blank control. Use a micro-BCA kit or other appropriate detection methods for determination.

[0069] The antigen loading rate was calculated according to the following formula:

[0070] Loading rate = (antigen amount before loading - antigen amount in the solution outside the dialysis bag) / antigen amount before loading × 100%.

[0071] Preparation Example 1

[0072] Preparation of chitosan nanoparticles by ionic crosslinking method:

[0073] Weigh 0.3g chitosan (molecular weight of 20,000, deacetylation degree 90%) with an electronic balance and dissolve it in 3.0mL 1mol / L acetic acid solution, add deionized water to make up to 10mL. Take 1mL chitosan solution and dilute it to 1mg / mL with deionized water, adjust the pH to 4.5 with sodium acetate, add 9.02mL of 0.75mg / mL sodium tripolyphosphate solution dropwise under magnetic stirring conditions (500rpm, 30min), and the drop rate is 30mL / h. Centrifuge the obtained particle suspension at 10000g for 30min, discard the supernatant, resuspend it with deionized water and centrifuge it, repeat 3 times. Freeze dry to obtain chitosan nanoparticles (product No. 1 in Table 1). The average particle size of the particles is 137nm, the PDI value of the particles is 0.122, and the potential of the particles is 22.5mV. The surface of the prepared particles is smooth and the spherical shape is regular when observed by cold field emission scanning electron microscopy.

[0074] In this preparation example, a series of chitosan nanoparticles were prepared using different raw materials and process conditions. The preparation steps were similar to those described above (variables are shown in Table 1, and other conditions were the same). The specific process parameters and characterization results are shown in Table 1 (products No. 2-11 in Table 1).

[0075] Table 1

[0076]

[0077] Preparation Example 2

[0078] Preparation of chitosan nanoparticles by nanoprecipitation method:

[0079] Weigh 0.3g chitosan (molecular weight of 50,000, deacetylation degree 90%) with an electronic balance and dissolve it in 2mL 1mol / L acetic acid solution, add deionized water to make up to 30mL. After fully dissolved, slowly drip 4mol / L NaOH solution to adjust the pH of the chitosan solution to about 6.5 to obtain a micron-sized chitosan particle suspension. Take 5mL of the suspension in a 50mL centrifuge tube, homogenize (12000rpm, 3min) and ultrasonic crush (power 30%, 1min) respectively under ice-water bath conditions, and the desired chitosan nanoparticles (product No. 1 in Table 2) can be prepared. The average particle size of the particles is 108nm, the PDI value of the particles is 0.134, and the potential of the particles is 18.4mV. The surface of the prepared particles is smooth and spherical and regular when observed by cold field emission scanning electron microscope.

[0080] In this preparation example, a series of chitosan nanoparticles were prepared using different raw materials and process conditions. The preparation steps were similar to those described above (variables are shown in Table 2, and other conditions were the same). The specific process parameters and characterization results are shown in Table 2 (products No. 2-11 in Table 2).

[0081] Table 2

[0082]

[0083]

[0084] Preparation Example 3

[0085] Preparation of chitosan quaternary ammonium salt nanoparticles by thermal curing method:

[0086] Weigh 0.18g chitosan quaternary ammonium salt and dissolve it in 2.5mL 0.1mol / L lactic acid. Under ice bath conditions, add 1mL 0.4g / mL α-β-GP solution to the above solution, crosslink for 5min, and form a hydrogel as the aqueous phase. Liquid paraffin and petroleum ether are mixed in a volume ratio of 1:2, and 6.0wt.% PO-500 is added as the oil phase. The prepared oil phase is poured into a high-pressure homogenizer, and the aqueous phase is slowly added to the oil phase, homogenized (homogenizing pressure 7500psi, homogenizing time 6min), and a uniform emulsion is obtained. The emulsion is then placed in a conical flask and solidified for 3h at 37°C. The solidified emulsion was added with petroleum ether with a boiling range of 60-90 and a boiling range of 30-60 respectively for centrifugal washing (2000 rpm, 3 min), and the obtained product was added with 10 mL of 20% isopropanol, and vacuumed in a 37°C water bath until the volume in the bottle was reduced by half, and then washed three times with deionized water, and freeze-dried to obtain chitosan quaternary ammonium salt nanoparticles.

[0087] Characterization shows that the average particle size of chitosan quaternary ammonium salt nanoparticles is 780nm, the PDI value of the particles is 0.167, and the potential of the particles is 13.8mV. Cold field emission scanning electron microscopy is used to observe that the surface of the prepared particles is rough and the spherical shape is regular.

[0088] Preparation Example 4

[0089] Chitosan hydrochloride nanoparticles were prepared by single-phase gel spheronization method:

[0090] 0.3 g of chitosan hydrochloride was weighed and dissolved in 8 mL of deionized water, and 2 mL of 0.7 g / mL α-β-GP solution was added dropwise under ice bath conditions, cross-linked for 5 min, and the hydrogel was solidified at 37 ° C for 30 min. Then, ultrasonication (power 20%, 2 min) was performed using an ultrasonic crusher to obtain a suspension of chitosan hydrochloride nanoparticles, which was freeze-dried to obtain chitosan quaternary ammonium salt nanoparticles.

[0091] The characterization showed that the average particle size of the chitosan quaternary ammonium salt nanoparticles was 474 nm, the PDI value of the particles was 0.233, and the potential of the particles was 9.4 mV. The surface of the prepared particles was smooth and the spherical shape was regular when observed by cold field emission scanning electron microscopy.

[0092] Example 1

[0093] In this example, the nanoparticles prepared in Preparation Examples 1-4 were used to prepare a water-in-oil (W1 / O) emulsion:

[0094] An electronic balance was used to accurately weigh a certain amount of chitosan nanoparticles prepared in Preparation Example 2 (Product No. 1) and dispersed in 2 mL of deionized water. After uniform dispersion, the mixture was added to 3 mL of squalene containing 5 wt% Arlacel P135 and homogenized and emulsified using an IKA homogenizer (10000 rpm, 8 min) to obtain a water-in-oil (W1 / O) emulsion (Product No. 1 in Table 3). The prepared water-in-oil (W1 / O) emulsion had a particle size of 807 nm and a PDI of 0.122.

[0095] This embodiment also uses different particle raw materials and preparation processes to prepare a series of water-in-oil (W1 / O) emulsions, and the preparation steps are similar to those described above (variables are shown in Table 3, and other conditions are the same), and the specific parameters and characterization results are shown in Table 3 (products No. 2-11 in Table 3).

[0096] Table 3

[0097]

[0098]

[0099] In Table 3, the preparation method of PLGA nanoparticles is as follows: 0.5g PLGA (LA:GA is 50:50, molecular weight is 70,002 Daltons) is accurately weighed using an electronic balance and dissolved in 10mL dichloromethane as the oil phase, and a 2.5% polyvinyl alcohol aqueous solution is used as the water phase. The oil phase is dispersed in the water phase, homogenized for 6min, the speed is 12000rpm, and stirred overnight at room temperature. Centrifuge at 15000g for 30min, discard the supernatant, add 5mL deionized water to the precipitate, ultrasonically disperse, and wash repeatedly 3 times. The precipitate is freeze-dried to obtain PLGA particles. The average particle size of the particles is 102nm, the PDI value of the particles is 0.123, and the potential of the particles is -18.9mV. The surface of the prepared particles is smooth and spherical and regular using a cold field emission scanning electron microscope.

[0100] The aluminum particles were obtained by ultrasonic dispersion (power 30%, 1 min) using InvivoGen aluminum adjuvant. The average particle size of the particles was 358 nm, the PDI value of the particles was 0.324, and the potential of the particles was 52 mV.

[0101] Example 2

[0102] In this example, the water-in-oil (W1 / O) emulsion prepared in Example 1 was used to prepare a water-in-oil-in-water (W1 / O / W2) emulsion:

[0103] 10 wt% of water-soluble surfactant Tween 80 was added to 2 mL of phosphate buffer, and the mixture was stirred and mixed to obtain the external aqueous phase. 1 mL of No. 1 water-in-oil (W1 / O) emulsion in Example 1 was added to the external aqueous phase, and magnetic stirring was performed (500 rpm, 6 min) to obtain a water-in-oil-in-water (W1 / O / W2) emulsion (No. 1 product in Table 4).

[0104] Emulsion light microscopy images Figure 1 As shown, the particle size of the emulsion is 1.8 μm, the PDI is 0.121, and the Young's modulus of the emulsion is 0.35 MPa. Figure 2 As shown, it is 1 / 120 times that of the chitosan nanoparticles (Product No. 1 in Preparation Example 2), indicating that the force required to compress the emulsion is relatively low and the emulsion has good deformation ability.

[0105] This embodiment also adopts different preparation processes to prepare a series of water-in-oil-in-water (W1 / O / W2) emulsions, and the preparation steps are similar to the above (variables are shown in Table 4, and other conditions are the same). The specific parameters and characterization results are shown in Table 4 (products No. 2-11 in Table 4), among which product No. 10 is an oil-in-water emulsion prepared by aluminum particles that cannot form a stable water-in-oil-in-water (W1 / O / W2) emulsion.

[0106] Table 4

[0107]

[0108]

[0109] Example 3

[0110] This example prepares an antigen-loaded water-in-oil-in-water (W1 / O / W2) emulsion:

[0111] 0.18g of chitosan hydrochloride nanoparticles in Preparation Example 4 were accurately weighed using an electronic balance and dispersed in 2mL of phosphate buffer. 1mg / mL of OVA aqueous solution was added to the above solution at a volume ratio of 1:1 to obtain an inner aqueous phase W1 loaded with OVA. 2mL of the above inner aqueous phase was then added to 3mL of tocopherol containing 10% PO-500 and homogenized (speed 7200rpm, 8min) to obtain an oil-in-water (W1 / O) emulsion. 15% Tween 80 was added to 2mL of citric acid buffer, stirred and mixed to obtain an outer aqueous phase, and the oil-in-water (W1 / O) emulsion was added to the outer aqueous phase, and magnetic stirring (500rpm, 6min) was performed to prepare a water-in-oil-in-water (W1 / O / W2) emulsion (Product No. 1 in Table 5).

[0112] Characterization showed that the particle size of the emulsion was 1.6 μm, the PDI was 0.119, the Young's modulus of the emulsion was 0.4 MPa, and the embedding rate of OVA was 92.36%.

[0113] This example also uses different raw materials and processes to prepare a series of antigen-loaded water-in-oil-in-water (W1 / O / W2) emulsions, and the preparation steps are similar to those described above (variables are shown in Table 5, and other conditions are the same), and the specific parameters and characterization results are shown in Table 5 (Products No. 2-11 in Table 5).

[0114] Table 5

[0115]

[0116]

[0117] Example 4

[0118] This example evaluates the deformability of water-in-oil-in-water (W1 / O / W2) emulsions when in contact with cells:

[0119] 0.1 g of chitosan nanoparticles (product No. 1) in Preparation Example 2 was accurately weighed using an electronic balance and dispersed in 2 mL of water for injection. 1 mg of Cy5 dye was added to the chitosan nanoparticle suspension, and mixed overnight at 4 ° C in the dark to prepare an inner water phase. 3 mL of squalene was accurately measured with a pipette and added to DiI with a final concentration of 1 μM / mL and mixed for 30 min, and then 0.15 g of Arlacel P135 was added to obtain an oil phase. The inner water phase and the oil phase were mixed and homogenized (speed 12000 rpm, 6 min) to obtain an oil-in-water (W1 / O) emulsion. 10% Tween 85 was added to 2 mL of citric acid buffer, and the mixture was stirred to obtain an outer water phase. The oil-in-water (W1 / O) emulsion was added to the outer water phase, and magnetic stirring was performed (500 rpm, 6 min) to prepare a water-in-oil-in-water (W1 / O / W2) emulsion. The particle size was characterized to be 1.6 μm.

[0120] The water-in-oil-in-water (W1 / O / W2) emulsion was added to a Petri dish with mouse bone marrow-derived dendritic cells (BMDCs) attached to the bottom and incubated for 3 h. After washing twice with phosphate buffer, the dish was fixed with 4% paraformaldehyde for 5 min, and the cytoskeleton was stained with FITC-labeled phalloidin. The contact between the emulsion droplet and the cell was then observed using a multi-mode single-molecule localization detection microscope. Figure 3 As shown, compared with chitosan particles (particle size of 1.7 μm, and its preparation method refers to Preparation Example 2) with a particle size similar to that of the water-in-oil-in-water (W1 / O / W2) emulsion, the emulsion group underwent obvious deformation when in contact with cells, becoming flat, and the angle between the emulsion droplets and the cells was about 30°.

[0121] Example 5

[0122] This example evaluates the uptake of antigens in a water-in-oil-in-water (W1 / O / W2) emulsion:

[0123] 0.3 g of chitosan nanoparticles (product No. 1) in Preparation Example 2 were accurately weighed using an electronic balance and dispersed in 1 mL of phosphate buffer. 7.5 mg of OVA were accurately weighed using an electronic balance, dissolved in 1 mL of phosphate buffer, and OVA was labeled with Cy5. The labeled OVA and chitosan nanoparticle suspension were mixed evenly to prepare an inner aqueous phase. The inner aqueous phase was then added to 3 mL of squalene containing 5% PO-500 and homogenized (speed 7200 rpm, 6 min) to obtain an oil-in-water (W1 / O) emulsion. 15% Tween 80 was added to 2 mL of phosphate buffer, and the mixture was stirred to obtain an outer aqueous phase. The oil-in-water (W1 / O) emulsion was added to the outer aqueous phase, and magnetic stirring was performed (500 rpm, 6 min) to prepare a water-in-oil-in-water (W1 / O / W2) emulsion.

[0124] Mouse bone marrow-derived dendritic cell (BMDCs) suspension was spread on the bottom of a Petri dish, and a water-in-oil-in-water (W1 / O / W2) emulsion containing Cy5-OVA was added. After incubation for 12 h, the cells were washed twice with phosphate buffer. After cell permeabilization, the cytoskeleton and cell nucleus were stained with FITC-labeled phalloidin and DAPI, respectively, and then the antigen uptake was observed using laser confocal microscopy. Figure 4 As shown, compared with the simple OVA antigen group, the emulsion group could significantly enhance the cellular uptake of antigen.

[0125] Example 6

[0126] This example evaluates the lysosomal escape of a water-in-oil-in-water (W1 / O / W2) emulsion:

[0127] 0.15g of chitosan quaternary ammonium salt nanoparticles in Preparation Example 3 were accurately weighed using an electronic balance and dispersed in 2mL of phosphate buffer. 7.5mg of OVA was accurately weighed using an electronic balance and dissolved in 2mL of phosphate buffer, and OVA was labeled with Cy5. The labeled OVA was evenly mixed with the chitosan quaternary ammonium salt nanoparticle suspension to prepare an inner aqueous phase. Afterwards, the inner aqueous phase was added to 5mL of squalene containing 3% Arlacel P135 and homogenized (rotating speed 7200rpm, 6min) to obtain a W1 / O emulsion. 10% Tween 80 was added to 2mL of phosphate buffer, and the outer aqueous phase was obtained by stirring and mixing. The W1 / O emulsion was added to the outer aqueous phase, and magnetic stirring (500rpm, 6min) was performed to prepare a W1 / O / W2 emulsion.

[0128] The BMDCs cell suspension was mixed with a water-in-oil-in-water (W1 / O / W2) emulsion containing Cy5-OVA, incubated on a shaker for 8 h, transferred to a Petri dish and allowed to stand for 30 min, washed three times with phosphate buffer, and incubated in the dark for 30 min with Lysotracker Green DND-26 dye. The co-localization of lysosomes and antigens was observed using a laser confocal microscope.

[0129] The experimental results show that the co-localization rate of lysosomes and antigens in the chitosan quaternary ammonium salt nanoparticle emulsion group is 37.9%, the co-localization rate of lysosomes and antigens in the simple chitosan quaternary ammonium salt nanoparticle group (preparation method see Preparation Example 3) is 73.2%, and the co-localization rate of lysosomes and antigens in the PLGA nanoparticle emulsion group (preparation method see Example 1) is 88.7%. It is proved that the emulsion group with positively charged nanoparticles can significantly enhance the lysosomal escape efficiency of antigens and has great potential for enhancing cellular immune response.

[0130] Example 7

[0131] This example evaluates the use of water-in-oil-in-water (W1 / O / W2) emulsions as antigen delivery vehicles:

[0132] The mice were immunized with the emulsion No. 1 prepared according to the method in Example 3. BALB / c mice (6-8 weeks old) were selected for the experiment, and the sex was female mice, provided by Weitong Lihua Company, adapted to the environment for 3 days, and were given free food and water. OVA was used as the model antigen, and the inoculation dose for each mouse was 10 μg. The mice inoculated with PBS were used as blank controls, and the mice inoculated with 10 μg OVA / mouse and 100 μL of commercial emulsion adjuvant MF59 were used as experimental control 1, and the mice inoculated with 10 μg OVA / mouse and 100 μL of purified water were used as experimental control 2. The mice inoculated with 10 μg OVA / mouse and 100 μL of emulsion No. 1 were used as experimental groups, with 6 mice in each group.

[0133] The first and second immunizations were performed on day 0 and day 14, respectively. The mice were killed on day 35, and blood samples were collected to detect the specific IgG antibody levels in the serum of mice in each group. Figure 5 As shown in Figure 2, the emulsion can induce a strong humoral immune response. At the same time, the spleen of mice was cultured to prepare spleen single cell suspension, and the activation of spleen T cells was detected based on flow cytometry. CD69 is a marker of spleen cell activation, such as Figure 6 As shown in Figure 2, the emulsion can significantly enhance the activation of T cells. At the same time, central memory cells (CD44 + CD62L - ) expression, such as Figure 7 As shown, the emulsion can simultaneously promote CD4 + T and CD8 +The expression of central memory cells in T cells suggests that it can induce strong immune memory in the body. In addition, based on ELIspot detection of IFN-γ secretion levels of T cells, such as Figure 8 As shown, the emulsion was able to generate the highest Th1 immune response.

[0134] This example also uses emulsions No. 2-11 prepared by the method in Example 3 to evaluate the immune effect. The operation method is referred to the above content. The specific results are shown in Table 6.

[0135] Table 6

[0136]

[0137] Example 8

[0138] This example evaluates the safety of water-in-oil-in-water (W1 / O / W2) emulsions as antigen delivery vehicles:

[0139] The No. 2 emulsion prepared by the method in Example 3 was immunized to mice. BALB / c mice were administered by subcutaneous injection, intramuscular injection and intraperitoneal injection respectively, and the dosage was 100 μL / , and the mice were administered once on the 0th day and the 14th day respectively. The weight change of mice was weighed on the 0th, 14th and 28th days respectively. As shown in Table 7, the weight growth of mice was normal. Blood was taken at 35 days, and serum was separated by centrifugation at 10000g for 10min. The biochemical indexes in the serum were detected: myocardial index (lactate dehydrogenase LDH), renal function index (urea nitrogen BUN), liver function index (alanine aminotransferase ALT, aspartate aminotransferase AST, alkaline phosphatase ALP). As shown in Table 8, each index was no abnormality with the blank group mouse index, indicating that the emulsion had no obvious toxicity to the main organs and had good safety.

[0140] Table 7

[0141] Blank Group Subcutaneous injection Intramuscular injection Intraperitoneal injection Day 0 18.4±0.3g 17.8±0.6g 17.2±0.5g 17.9±0.5g 14 days 19.3±0.6g 18.3±0.4g 18.5±0.4g 18.7±0.6g 28 days 20.4±0.2g 19.1±0.6g 19.7±0.4g 20.1±0.4g

[0142] Table 8

[0143] Group ALT ALP LDH BUN AST Blank Group 41.2±5.9 213.5±11.7 998.4±110.3 7.2±1.9 79.4±9.2 Subcutaneous injection 40.7±4.4 232.1±10.3 1087.1±106.2 8.6±1.4 83.5±10.7 Intramuscular injection 42.6±6.3 229.6±12.5 1008.6±177.9 8.1±0.7 89.3±11.1 Intraperitoneal injection 41.5±8.7 235.2±19.8 1056.7±127.4 7.4±0.9 80.7±7.9

[0144] From the results in Table 8, it can be seen that each index is no different from that of the blank group mice, indicating that the emulsion has no obvious toxicity to major organs and has good safety.

[0145] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0146] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0147] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A deformable antigen delivery carrier, characterized in that: The deformable antigen delivery carrier has a water-in-oil-in-water emulsion structure, wherein the inner water phase is a suspension containing positively charged nanoparticles, the oil phase is an oily substance containing a lipophilic emulsifier, and the outer water phase is a solution containing a hydrophilic emulsifier.

2. The deformable antigen delivery carrier according to claim 1, characterized in that: The volume ratio of the oil phase to the external water phase is (1-4):(2-11), and the volume ratio of the internal water phase to the oil phase is (1-3):(2-7); Preferably, in the water-in-oil-in-water emulsion structure, the particle size of the water-in-oil emulsion droplets is 100 nm-10 μm, and the particle size of the water-in-oil-in-water emulsion droplets is 200 nm-20 μm.

3. The deformable antigen delivery carrier according to claim 1 or 2, characterized in that: The positively charged nanoparticles have a potential of 5-40 mV and a particle size of 10-900 nm; Preferably, the positively charged nanoparticles include chitosan and / or chitosan derivative nanoparticles; Preferably, the chitosan derivative comprises chitosan hydrochloride and / or chitosan quaternary ammonium salt; Preferably, the molecular weight of the chitosan is 20-1000 kDa, and the degree of deacetylation is 80-95%.

4. The deformable antigen delivery carrier according to any one of claims 1 to 3, characterized in that: The oily substance is a pharmaceutical grade oily substance, including any one of squalene, tocopherol or ethyl linoleate, or a combination of at least two thereof; Preferably, the lipophilic emulsifier includes any one or a combination of at least two of sorbitan trioleate, polyoxyethylene stearate, polyoxyethylene fatty alcohol ether, PO-500 or Arlacel P135; Preferably, the mass fraction of the lipophilic emulsifier in the oil phase is 1-20%.

5. The deformable antigen delivery carrier according to any one of claims 1 to 4, characterized in that: The hydrophilic emulsifier includes any one or a combination of at least two of Tween 80, Tween 85, poloxamer, polyoxyethylene monopalmitate, polyoxyethylene castor oil or polyoxyethylene alkyl ether; Preferably, the mass fraction of the hydrophilic emulsifier in the external water phase is 1-30%; Preferably, the pH values ​​of the external aqueous phase and the internal aqueous phase are independently selected from 5.5-8.5, more preferably 6.0-8.0; Preferably, the solutions of the external aqueous phase and the internal aqueous phase are independently selected from any one of purified water, water for injection, 0.9% saline, phosphate buffer, citrate buffer or Tris buffer, or a combination of at least two thereof.

6. The deformable antigen delivery carrier according to any one of claims 1 to 5, characterized in that: The solution of the external aqueous phase is any one of phosphate buffer, citrate buffer or Tris buffer, or a combination of at least two thereof; Preferably, the solution of the inner aqueous phase is any one of purified water, water for injection, 0.9% saline or phosphate buffer, or a combination of at least two of them.

7. The deformable antigen delivery carrier according to any one of claims 1 to 6, characterized in that: The Young's modulus of the antigen delivery carrier is 0.2-50 MPa; the angle range when the antigen delivery carrier contacts the cell is 10-70°.

8. The method for preparing a deformable antigen delivery carrier according to any one of claims 1 to 7, characterized in that: The preparation method comprises: Positively charged nanoparticles are dispersed in an inner aqueous phase solution, and then mixed with an oily substance containing a lipophilic emulsifier for homogenous emulsification to obtain an oil-in-water emulsion; and then mixed with an outer aqueous phase solution containing a hydrophilic emulsifier for homogenous emulsification to obtain a water-in-oil-in-water emulsion, thereby obtaining the deformable antigen delivery carrier.

9. An antigen delivery system for enhancing dual immune responses, characterized in that: The antigen delivery system comprises the deformable antigen delivery carrier according to any one of claims 1 to 7 and an antigen, wherein the antigen is adsorbed on the surface of the positively charged nanoparticles or embedded in the interior of the positively charged nanoparticles.

10. The antigen delivery system according to claim 9, characterized in that The antigen loading amount is 0.1-10% of the mass of the positively charged nanoparticles; Preferably, the antigen comprises any one of attenuated vaccines, inactivated vaccines, split vaccines, subunit vaccines, polysaccharide conjugate vaccines, recombinant vaccines or DNA vaccines, or a combination of at least two thereof.

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