CN111569061A - Nano material for nucleic acid vaccine enhancer - Google Patents
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
Abstract
The invention provides a nano material for a nucleic acid vaccine enhancer, which is characterized by being prepared by the following method: (1) preparation of solution A: mixing CaCl2Dissolving with Tris buffer in water; (2) preparing a solution B: mixing HEPES buffer solution with water; (3) dropwise adding the solution B into the solution A, and stirring to obtain CP nanoparticle precipitate; (4) and (4) dropwise adding sodium pamidronate into the AB mixed solution obtained in the step (3), and dropwise adding human gamma globulin to obtain the nano material. The nanometer material of the invention obviously improves the transfection efficiency of the nucleic acid vaccine to target cells, increases antigen expression, promotes the maturation of antigen presenting cells so as to establish effective immune response and realize the enhancement or synergy function of the nucleic acid vaccine.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a nano material for a nucleic acid vaccine enhancer.
Background
The nucleic acid vaccine is prepared through introducing exogenous gene (DNA or RNA) encoding certain antigen protein into animal or human body cell, synthesizing antigen protein via the expression system of host cell, and inducing the host to produce immune response to the antigen protein for preventing and treating diseases. The nucleic acid vaccine has many advantages compared with the traditional vaccine, and the existing nucleic acid vaccine enters the clinical stage and is applied to the market at present. However, how to make the nucleic acid vaccine reach the target cell smoothly after entering the body and express and present the antigen protein effectively is a bottleneck that hinders the rapid development and accelerated application of the nucleic acid vaccine.
At present, only a very small amount of nucleic acid vaccines (2%) can freely enter cells after entering an organism, but fewer vaccines which can reach target cells (antigen presenting cells) exist, so that the nucleic acid vaccines are large in immune dosage, large in side effect, high in cost and not ideal in immune effect. After the nucleic acid vaccine immunizes an organism, the antigen protein must be effectively expressed in the antigen presenting cells, the antigen is processed and presented, and the cell maturation immune reaction is completed, but if the nucleic acid vaccine enters the immature antigen presenting cells, the expression of the antigen protein still can not be realized, so that the low immune efficiency is caused. Many developed nucleic acid vaccines cannot be finally used in clinic, but because the vaccine is designed, a nucleic acid vaccine enhancer is not provided to improve the transfection efficiency of the nucleic acid vaccine enhancer on effective cells, an effective immune response cannot be established, and finally, further development and popularization and application of the vaccine are abandoned carelessly.
Although there are some rare studies to enhance immune response by using nanomaterial adjuvant nucleic acid vaccines, they are called vaccine adjuvants and present only a certain link to antigen. The nucleic acid vaccine reinforcing agent is different from an adjuvant, and comprises the steps of protecting a nucleic acid vaccine, promoting the nucleic acid vaccine to enter antigen presenting target cells, improving the maturity of the antigen presenting target cells, enhancing the transcription expression and the antigen processing and presentation of protein, and comprehensively improving the efficiency of the nucleic acid vaccine.
There is no report of nucleic acid vaccine enhancing (efficacy) agent at present. The method makes up the blank, can remarkably improve the transfection efficiency of the nucleic acid vaccine to target cells, increases antigen expression, promotes the maturation of antigen presenting cells so as to establish effective immune response, and realizes the enhancement or synergy function of the nucleic acid vaccine.
Disclosure of Invention
The present invention aims at overcoming the demerits of available technology, and provides one kind of nanometer material for nucleic acid vaccine intensifier, which can send nucleic acid vaccine into target cell and raise the expression and presentation of antigen protein effectively to raise immune reaction in several steps.
In order to achieve the purpose, the invention adopts the technical scheme that: a nanometer material for nucleic acid vaccine enhancers is prepared by the following steps:
(1) preparation of solution A: adding CaCl2Dissolving with Tris buffer in water;
(2) preparing a solution B: mixing HEPES buffer solution with water;
(3) dropwise adding the solution B into the solution A, and stirring to obtain CP nanoparticle precipitate;
(4) and (4) dropwise adding sodium pamidronate into the AB mixed solution obtained in the step (3), and dropwise adding human gamma globulin to obtain the nano material.
Preferably, CaCl2The concentration of (A) is 1.0-2.0M; the concentration of the Tris buffer is 10mM, and the pH value is 10.
Preferably, the HEPES buffer comprises the following components: 200 to 280mM NaCl, 10 to 15mM Na2HPO4And 24 to 50mM HEPES. The HEPES is 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid.
Preferably, the concentration of the Pamidronate sodium (Pamidronate) is 0.1-5 mg/mL.
Preferably, the concentration of the human gamma globulin is 0.04-0.8 ug/mL.
Compared with the prior art, the invention has the following beneficial effects:
1. the nucleic acid vaccine nano enhancer has cell selectivity, can be specifically combined with antigen presenting cells, and has the cell targeting property (antigen presenting cells) of nucleic acid vaccine delivery.
2. The cell membrane is a phospholipid bilayer, and an electronic dense band with the thickness of about 2.5nm is respectively arranged at the inner side and the outer side of the membrane, so that the characteristics of the cell membrane are not beneficial to most of nucleic acid fragments or plasmids to enter cells, and the probability of nucleic acid entering the cells is greatly reduced. The nucleic acid vaccine nano reinforcing agent is a nano particle with good physical and chemical compatibility, can effectively establish bridge connection between nucleic acid and cell membranes, greatly improve the contact probability of the nucleic acid and the cell membranes and the efficiency of entering cytoplasm and cell nucleus, and can stimulate the phagocytic function and cell maturation of antigen presenting cells and promote the immune reaction of the nucleic acid vaccine.
3. After the nanoparticles carrying the nucleic acid vaccine are phagocytized by antigen presenting cells, the pH value in a phagocytosis lysosome can be changed, and then the nucleic acid is protected from being degraded and destroyed by nuclease in the phagocytosis lysosome.
4. The DNA can be transcribed and translated only after entering the cell nucleus, and finally the antigen protein is expressed. The nucleic acid vaccine nano enhancer disclosed by the invention has the capability of improving the high-efficiency entry of the nucleic acid vaccine into cell nucleus.
5. Immature antigen-presenting cells, even after taking up nucleic acid material and entering the nucleus, are unable to present the relevant expressed antigen, and only after these cells are mature, can achieve efficient presentation of the antigen to T or B cells, inducing an immune response. The nucleic acid vaccine nano enhancer disclosed by the invention can obviously promote the conversion of immature antigen presenting cells to mature antigen presenting cells, so that the immune efficacy of the nucleic acid vaccine is effectively improved.
Drawings
FIG. 1 is a transmission electron microscope image of the MCP nanoparticles of the present invention.
FIG. 2 is a graph showing the efficiency of uptake of 4-hour macrophage RAW264.7 to CP-Cy5, Liposome-Cy5, MCP-Cy 5.
FIG. 3 is a diagram showing the in vitro delivery efficiency of MCP nanoparticles after pGFP transfection to macrophages, wherein A is the percentage of positive cells and B is the result of flow cytometry.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Example 1
The embodiment provides a nano material for a nucleic acid vaccine enhancer, which is prepared by the following method:
(1) preparation of solution A: 0.42mL of 2.0M CaCl2Dissolving in 2mL of Trisbuffer with 0.84mL of pH 10 and 10mM in 2mL of ddH2O is in;
(2) preparing a solution B: 0.43mL of HEPES buffer and 0.3mL of ddH2O mixing; wherein the HEPES buffer solution contains 280mM NaCl and 15mM Na2HPO4And 50mM HEPES.
(3) Dropwise adding the solution B into the solution A, and stirring for 30 minutes at 500rpm to obtain CP nanoparticle precipitate;
(4) and (3) dropwise adding 0.1mg/mL Pamidronate sodium (Pamidronate) into the AB mixed solution obtained in the step (3), dropwise adding 0.04ug/mL Human gamma-globuline (Human gamma-globuline), and stirring for 30 minutes to obtain the nano material.
Example 2
The only difference between this example and example 1 is that the concentration of pamidronate in this example is 1 mg/mL.
Example 3
The only difference between this example and example 1 is that the concentration of pamidronate in this example is 3 mg/mL.
Example 4
The only difference between this example and example 1 is that the concentration of pamidronate in this example is 5 mg/mL.
Example 5
The only difference between this example and example 1 is that the human gamma globulin concentration in this example is 0.08 mg/mL.
Example 6
The only difference between this example and example 1 is that the human gamma globulin concentration in this example is 0.15 mg/mL.
Example 7
The only difference between this example and example 1 is that the human gamma globulin concentration in this example is 0.3 mg/mL.
Example 8
The only difference between this example and example 1 is that the human gamma globulin concentration in this example is 0.6 mg/mL.
Example 9
The only difference between this example and example 1 is that the human gamma globulin concentration in this example is 0.8 mg/mL.
Example 10
Scanning MCP nano-particles prepared by the method by an electron microscope, and placing the nano-particles in ddH2Diluted to a final concentration of 0.1mg/mL in O, and subjected to Transmission Electron Microscopy (TEM) imaging, which is a 7700 electron microscope from Hitachi, Inc. As shown in FIG. 1, FIG. 1A shows that MCP nanoparticles are circular spheres of about 100nm, and a plurality of particles are aggregated together, while FIG. 1B shows that the particles are protein-modified to have a particle size of about 180nm, and thus have good dispersibility.
Example 10 pDNA Loading Rate of MCP nanoparticles
Preparation of CP-pGFP and MCP-pGFP
DNA samples were first premixed in solution A and then mixed with solution b the synthesized nanoparticles were centrifuged at 15000 × g for 10min, the supernatant was collected for DNA loading determination, and then 1 ml of ddH was added2O Wash particles the suspension was centrifuged at 15000 × g for 5min, the supernatant carefully removed, finally the sample was redispersed in ddH2Further testing was performed in O or cell culture medium.
Each sample was fixed with 10 μ g/ml pGFP, and the CP nanoparticles and MCP nanoparticles were adjusted in number to achieve 5%, 10%, 20% (wt%) loading. Each sample was collected in a new 1.5mL tube and centrifuged at 15,000g for 10 minutes. The supernatant was collected and the unloaded pGFP residues quantified using Nanodrop 1000(Thermo scientific). Gel retention analysis was used to confirm the binding of pDNA to the nanoparticles. The positive control was plasmid prepared at 10. mu.g/ml water. For each sample and positive control, a mix of 20 μ L of supernatant was taken 4 μ L of 6 × load buffer and loaded with 1% agarose gel GelRed dye. Electrophoresis was performed using PowerPac Basic (BIO-RAD), 80V electrophoresis for 1 hour, and gel images were collected by ChemiDoc MP (BIO-RAD).
Example 11 uptake efficiency of macrophages into MCP nanoparticles.
50nM dsDNA-Cy5 was loaded, CP NPs-dsDNA-Cy5(CP-Cy5) and MCP NPs-dsDNA-Cy5(MCP-Cy5) were synthesized. Liposomes (Oligofectamine) were prepared using 50nM dsDNA-Cy5TMReagent, ThermoFisher) -Cy 5. Quantitative detection of CP-Cy5, using FACS (CytoFLEX, Beckman Coulter),Cell uptake by MCP-Cy5 and Liposome-Cy5 RAW264.7 cells were seeded in 24-well plates at a density of 5 × 104/well, cultured in a wet incubator at 37 ℃ and 5% CO2 for 24h, and then cultured with the addition of 10. mu.g/ml MCP-Cy5 for 4h, after which the medium was discarded and the cells were washed 3 times with PBS, trypsinized, collected by centrifugation, fixed in 4% PFA solution for FACS analysis, while Liposome-Cy5 and CP-Cy5 were used as control groups, following the same protocol, the results are shown in FIG. 2, with marked differences in the uptake efficiency of MCP nanoparticles by macrophages compared to CP-Cy5 and Liposome-Cy5 groups.
Example 12 model of in vitro DNA vaccine delivery to macrophages
We used pGFP as a functional model to evaluate the delivery efficiency of MCP nanoparticles in vitro. The pDNA was verified in the U2OS cell line using a flow cytometer. In particular, the percentage of GFP-positive cells after transfection (FIG. 3A) and the mean fluorescence intensity MFI (FIG. 3B) were used for evaluation. The transfection efficiencies of liposomes (lipome) -pGFP, electroporation (electroporation), CP-pGFP, MCP-pGFP were compared. Each transfection system was incubated with 1.0. mu.g/mL pGFP for 48 hours. Both MCP transfection efficiencies (percentage and MFI) were significantly higher compared to liposome and electrotransformation methods. And also significantly higher than similar Calcium Phosphate (CP) particles. These results show that MCP particles have superior ability to deliver DNA vaccines.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. A nanomaterial for a nucleic acid vaccine enhancer, characterized in that the nanomaterial is prepared by the following method:
(1) preparation of solution A: adding CaCl2Dissolving with Tris buffer in water;
(2) preparing a solution B: mixing HEPES buffer solution with water;
(3) dropwise adding the solution B into the solution A, and stirring to obtain CP nanoparticle precipitate;
(4) and (4) dropwise adding sodium pamidronate into the AB mixed solution obtained in the step (3), and dropwise adding human gamma globulin to obtain the nano material.
2. The nanomaterial of claim 1, wherein the CaCl is2The concentration of (A) is 1.0-2.0M; the concentration in the Trisbuffer is 10mM, and the pH value is 10.
3. The nanomaterial of claim 1, wherein the HEPES buffer comprises the following components: 200 to 280mM NaCl, 10 to 15mM Na2HPO4And 24 to 50mM HEPES.
4. The nanomaterial of claim 1, wherein the concentration of the pamidronate sodium is from 0.1 to 5 mg/mL.
5. The nanomaterial of claim 1, wherein the concentration of human gamma globulin is from 0.04 to 0.8 ug/mL.
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