CN107200788A - A kind of quaternary phosphonium chitosan and its application as vaccine immunity adjuvant - Google Patents
A kind of quaternary phosphonium chitosan and its application as vaccine immunity adjuvant Download PDFInfo
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- CN107200788A CN107200788A CN201710329824.7A CN201710329824A CN107200788A CN 107200788 A CN107200788 A CN 107200788A CN 201710329824 A CN201710329824 A CN 201710329824A CN 107200788 A CN107200788 A CN 107200788A
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- quaternary phosphonium
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 54
- 125000005496 phosphonium group Chemical group 0.000 title claims abstract description 35
- 229960005486 vaccine Drugs 0.000 title claims abstract description 28
- 239000002671 adjuvant Substances 0.000 title claims abstract description 18
- 230000036039 immunity Effects 0.000 title claims abstract description 10
- 239000000427 antigen Substances 0.000 claims abstract description 28
- 102000036639 antigens Human genes 0.000 claims abstract description 28
- 108091007433 antigens Proteins 0.000 claims abstract description 28
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 6
- -1 propyl group tri-phenyl-phosphorus bromides Chemical class 0.000 claims abstract description 6
- 238000003756 stirring Methods 0.000 claims abstract description 6
- 150000003839 salts Chemical class 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims abstract description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
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- 229910000073 phosphorus hydride Inorganic materials 0.000 claims 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
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Abstract
The invention discloses a kind of quaternary phosphonium chitosan and its application as vaccine immunity adjuvant, the preparation method of the quaternary phosphonium chitosan comprises the following steps：Chitosan and 1 hydroxybenzotriazole are added in solvent, ice bath reaction, then adjust pH value to 4.8 5.5；Add 3 carboxylic propyl group tri-phenyl-phosphorus bromides, stirring is to being completely dissolved；1 ethyl (3 dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is dissolved in solvent, is added in above-mentioned reaction system, precipitates product after reaction, is dried, dialysis obtains quaternary phosphonium chitosan after freeze-drying.The present invention is used as immunologic adjuvant using quaternary phosphonium chitosan first; quaternary phosphonium chitosan can promote intake of the DC2.4 cells to antigen, promote Spleen cell proliferation, higher levels of antigen-specific IgG antibody potency, IFN γ, IL 4 and IL 10 are induced, therefore there is in vaccine therapy field important application value.
Description
Technical field
The invention belongs to biomedical materials field, it is related to a kind of quaternary phosphonium chitosan and its as vaccine immunity adjuvant
Using.
Background technology
Vaccine inoculation is one of invention most great in generally acknowledged modern times physianthropy development history, is still the mankind so far
Prevent, resist and control the most effectual way of communicable disease, such as influenza, hepatitis, pulmonary tuberculosis.
Originally vaccine is made up of complete pathogen or its lysate, and immunogenicity is good, but can cause ill symptomses and
Infection.Therefore, some safer vaccines are researched and developed, including DNA, subunit and recombinant vaccine.But these are new
The vaccine of type still has the shortcomings that immunogenicity is relatively low, immune response inducing is weaker, targeting is weak.Therefore, explore and research and develop and use
It is significant with the technology for strengthening efficacy of vaccines, design is favorably improved immunogenicity, improves security, enhancing body spy
The immunologic adjuvant of specific immunological response turns into top priority.
From nineteen twenty-five, immunologic adjuvant (Immunoadjuvant) just causes the extensive concern of researchers and dense emerging
Interest.Immunologic adjuvant is that a class can help delivery of antigens, active antigen presenting cell (APC) and the activation for triggering immune cell
With the non-specific immunostimulating molecule of differentiation.
Preferable immunologic adjuvant must possess following characteristics：1. security is good；2. strengthen the immune of weak immunogene vaccine
Originality；3. promoting immune contact, strengthen the immune response of body；4. reduce antigen inoculation dosage and inoculation times；5. quickening is exempted from
The speed of epidemic disease response and extension duration etc..
Common immunologic adjuvant has two kinds of aluminium adjuvant and Freund's adjuvant.Aluminium adjuvant is that the first FDA ratifies to control for clinic
People's adjuvant for the treatment of, but its inducing cellular immune ability is weaker, and local inflammation can be triggered.And mainly application is Freund's adjuvant
Thing vaccine adjuvant, toxic side effect is larger.Therefore research and develop new, safety and effectively inducing cell and humoral immunity can exempt from
Epidemic disease adjuvant is significant.
Chitosan-phospholipid complex is due to good biocompatibility, biodegradability, bioadhesive, immune
The advantages of stimulating activity, sustained-release and controlled release, targeting, turn into the study hotspot of immunologic adjuvant in world wide in recent years.However,
Chitosan it is water-soluble poor, be dissolved only in diluted acid, this severely limits its biomedicine field application.
The content of the invention
The problem of in order to overcome existing Chitosan-phospholipid complex poorly water-soluble, primary and foremost purpose of the invention is to provide
A kind of quaternary phosphonium chitosan, is reacted using acid amides and synthesized, and it is water-soluble good, can be dissolved in water and physiological saline.
In order to overcome existing immunologic adjuvant inducing cellular immune ability weaker and the larger defect of toxic side effect, the present invention
Another object be to provide above-mentioned quaternary phosphoniums chitosan as the application , quaternary phosphonium chitosans of vaccine immunity adjuvant as epidemic disease
Seedling immunologic adjuvant can improve body specific immune response.
The purpose of the present invention is achieved through the following technical solutions：
A kind of preparation method of quaternary phosphonium chitosan, comprises the following steps：
(1) chitosan (CS) and I-hydroxybenzotriazole (HOBt) are added in water/dimethyl sulfoxide (DMSO) mixed liquor, ice bath
Under the conditions of stir 2-3h, then adjust the pH value of mixture to 4.8-5.5；Add 3- carboxylic propyl group tri-phenyl-phosphorus bromides
(CTPB), stirring is to being completely dissolved；
(2) 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) is dissolved in water/diformazan
In base sulfoxide mixed liquor, in the reaction system for being then added to step (1), react 1-2 days, product acetone/diethyl ether mixed liquor
Precipitated, then by precipitated product be dried in vacuo 1-2 days after be dissolved in ultra-pure water, and in ultra-pure water dialyse 3-4 days, freeze do
Quaternary phosphonium chitosan (NPCS) is obtained after dry；
In above-mentioned steps, the sugar unit of chitosan, HOBt, CTPB, EDCHCl mol ratio are 1:2:1:2；
In described water/dimethyl sulfoxide (DMSO) mixed liquor, the volume ratio of water and dimethyl sulfoxide (DMSO) is preferably (1-2):1；
In described acetone/diethyl ether mixed liquor, the volume ratio preferably 2 of acetone and ether:1.
The quaternary phosphonium chitosan as made from the above method, its water solubility is improved, and can be dissolved in water and physiological saline.
Due to good biocompatibility and immunostimulatory activity, while water solubility is improved, above-mentioned quaternary phosphoniums
Chitosan can be used as vaccine immunity adjuvant, can significantly improve body specific immune response；
The mass ratio of , quaternary phosphoniums chitosan and antigen is 2 in described vaccine:1；
The preferred ovalbumin of described antigen (OVA), vaccine therapy immunization wayses are injected for leg muscle.
Chitosan has original application potential in immunologic adjuvant field, but its poor water solubility, which limits it, answers
With.Therefore, small molecule quaternary alkylphosphonium salt on chitosan graft, water solubility is substantially improved.
Quaternary phosphonium chitosan is used for vaccine therapy as adjuvant has following advantage：1. abundance, preparation are simple, raw
Thing security is good；2. good water solubility, is easy to the production of immune vaccine preparation to prepare；3. bioadhesive is good, and with abundant
Positive charge, can efficiently wrap up negatively charged antigen, and adhere to negatively charged cell membrane by electrostatic interaction, make to resist
It is former more effectively to be recognized, absorb by antigen presenting cell (APC)；4. nano-scale, it is easy to recognized and absorbed by APC, it is easy to quilt
Cell swallows, and is conducive to inducing the immune response of body；5. antigen can be effectively protected from internal acid, alkali, protease etc.
Destruction, improves the utilization rate of antigen.
Present invention research is found, when quaternary phosphonium chitosan/antigen vaccine preparation is immune into body, the vaccine delivery system
System can be sustained to antigen, make body constantly receive immunostimulation to induce permanently effective immune response.
The present invention has the following advantages and effect relative to prior art：
1st, quaternary phosphonium chitosan has good biocompatibility, and synthesizes simple, with low cost, economic and environment-friendly.Meanwhile,
The modified quaternary phosphoniums chitosan of the present invention has good water solubility, is easy to prepare bacterin preparation.
2nd, the present invention can promote DC2.4 cells using quaternary phosphonium chitosan as immunologic adjuvant , quaternary phosphoniums chitosan first
Intake to antigen, promotes Spleen cell proliferation, induce higher levels of antigen-specific IgG antibody potency, IFN-γ, IL-4 and
IL-10, therefore there is in vaccine therapy field important application value.
Brief description of the drawings
The contact angle experiments result figure of Fig. 1 Shi quaternary phosphoniums chitosans and chitosan.
Fig. 2 is intake result figure of the DC2.4 cells to OVA.
Fig. 3 is the result figure of immunohistochemical staining.
Fig. 4 is the measurement result figure of IgG antibody potency.
Fig. 5 is the in-vitro multiplication experimental result picture of splenocyte.
Fig. 6 is the secretion experimental result picture of IFN-γ.
Fig. 7 is IL-12 secretion experimental result picture.
Fig. 8 is IL-4 secretion experimental result picture.
Fig. 9 is IL-10 secretion experimental result picture.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1
A kind of quaternary phosphonium chitosan, is made by following steps：
400mg chitosans (CS) and 664mg HOBt are separately added into 250mL three-necked flasks, 30mL H are added2O/
2.5h is stirred under DMSO (v/v=2/1) mixed liquor, condition of ice bath.PH value is then adjusted to 4.8-5.5.Add 1.052g
CTPB (3- carboxylic propyl group tri-phenyl-phosphorus bromide), stirring is to being completely dissolved.940mg EDCHCl are dissolved in 10mL H2O/DMSO
(v/v=1/1) mixed liquor, is added in flask and stirring reaction 24h.Product acetone/diethyl ether (v/v=2/1) mixed liquor
Precipitated, then will be dissolved in after precipitated product vacuum drying 24h in ultra-pure water, and dialysed 3 days in ultra-pure water, after freeze-drying
Obtain quaternary phosphonium chitosan (NPCS).
The contact angle experiments of chitosan (CS) and quaternary phosphonium chitosan (NPCS) made from the present embodiment：By chitosan (CS)
Solution is made with quaternary phosphonium chitosan (NPCS) made from the present embodiment, drips on sheet glass, spreading even, vacuum drying is made thin
Film, then determined with contact angle measurement (DSA100, KRUSS).
As a result as shown in figure 1, CS contact angle is 74.5 °, and NPCS contact angle is 26.6 °.Contact angle is smaller, then close
Aqueous enhancing, this explanation NPCS hydrophily is better than CS.
Embodiment 2
Above-mentioned quaternary phosphoniums chitosan as vaccine immunity adjuvant application：
First, bacterin preparation is prepared
Using ovalbumin OVA as model antigen, the vaccine of different formulations is prepared by with table 1, and then mouse is carried out
Intramuscular injection.
The bacterin preparation of the intramuscular injection of table 1
2nd, mouse immunization protocol
Intramuscular injection：The female Balb/c mouse of 6-8 weeks are randomly divided into 6 groups, every group 5；Respectively at the 0th, 10,20 days legs
Inject 100 μ L vaccine solutions in portion (per injection injects a half-value dose containing 50 μ g OVA, every leg).After third time is immune 10 days,
Blood is taken, separates and is saved backup at serum, -20 DEG C；Mouse spleen is taken, grinding, resuspension prepare the Spleen cell suspensions of various concentrations,
It is standby.
3rd, the measure of every immune indexes
1.DC2.4 intake of the cell to OVA
DC2.4 cell suspensions (1 × 10 are added into 24 orifice plates5Cells/ holes), in 37 DEG C, 5%CO2 cultures 24h.
The OVA of Cy5.5 marks is mixed with DMEM basal mediums and NPCS solution respectively, and 500 μ L are then added per hole, with DMEM bases
Culture medium is blank control, and every group three parallel, in 37 DEG C, 5%CO2 cultures 6h.Digest and collect cell, then washed with PBS
Wash and be resuspended.Pass through the intake situation of flow cytomery DC2.4 cells in vitro to antigen.
As a result as shown in Fig. 2 compared with negative control, it is thin that 1mg/mL NPCS/OVA bacterin preparation significantly increases DC2.4
Intake of the born of the same parents to antigen.This explanation is in the presence of NPCS, the significant increase of intake of the DC2.4 cells to antigen, and this is favourable
In DC2.4 cell captures and the more antigens of submission, so as to induce stronger immune response.
2. immunohistochemical assay
It is divided into 4 groups (n=4), and with bacterin preparations (every μ g OVA of mouse 50, every leg half-value dose) different 100 μ L
Carry out intramuscular immunisation.2 or 7 days after immune, mouse is implemented to be euthanized, and separating spleen of performing the operation, it is fixed in 10% formaldehyde,
FFPE, and it is cut on the coated slide of polylysine 4 μm of slabs.It is immunized according to the specification of manufacturer
Histochemical stain.
As a result as shown in figure 3, the bacterin preparation containing NPCS and Freund's adjuvant 2nd day and the 7th day can be after immune
Substantial amounts of antigen is detected in the spleen of immune mouse, and was only detected at the 7th day in the spleen for the mouse being immunized with negative control
To a small amount of antigen.With reference to fluorescent vital imaging results, ImmunohistochemistryResults Results are further illustrated in the help of NPCS, antigen from
Injection site is sustained and is gradually transferred in spleen, so as to increase the antigen utilizability in spleen, and then induces more effective
Immune response.
The measure of 3.IgG antibody titers
96 orifice plates are coated with 10 μ g/mL OVA antigen coats liquid, 4 DEG C of coatings are stayed overnight.Then PBS-T board-washings 1 time,
The confining liquid that 200 μ L are added in every hole is placed on 37 DEG C of shaking table and is incubated 1h.Then PBS-T board-washings are used 3 times, adds 100 μ L
1h is incubated on Mouse serum dilutions, the shaking table for being placed in 37 DEG C.Then with PBS-T board-washings 3 times, and 50 μ L are added in all holes
The anti-mouse IgG secondary antibodies of HRP marks, 1h is incubated on 37 DEG C of shaking table.Then PBS-T board-washings are used 4 times, 100 μ L is added per hole
TMB nitrite ions, at room temperature lucifuge be incubated 15min.100 μ L terminate liquid is then added per hole.Existed immediately with ELIASA
Absorption value is detected at 450nm.
As a result as shown in figure 4, compared with negative control, 1mg/mL NPCS and positive control have pole significant difference (p<
0.001).This explanation NPCS can effectively strengthen the humoral immune response of body, lift IgG secretion level.
4. Spleen cell proliferation is tested
In 96 orifice plates, add 100 μ L splenocyte suspensions (2 × 10 per hole6Cells/mL), 100 μ L egg white egg is added respectively
White solution (concentration is 100 μ g/mL, RPMI1640 basic culture solutions) or RPMI1640 basic culture solutions (blank control), every group 3
It is individual parallel.37 DEG C, 5%CO2Cultivate after 72h, add 20 μ L CCK-8 solution to every hole, 96 orifice plates are incubated in incubator
4h, the absorbance at 450nm is determined with ELIASA.
As a result as shown in figure 5, the Spleen cell proliferation index under negative control group effect is about 1.2, by contrast,
The Spleen cell proliferation index that NPCS/OVA is stimulated reaches 1.5 or so, suitable with positive control, in the presence of extremely significant sex differernce.This
Show that NPCS can be effectively facilitated Spleen cell proliferation, so as to strengthen immune response.
5.ELISA determines the cytokine levels of splenocyte secretion
In 24 orifice plates, add 750 μ L splenocyte suspensions (2x10 per hole6Cells/mL), 750 μ L ovalbumin is added respectively
Solution (concentration is 100 μ g/mL, RPMI1640 nutrient solutions), 1 hole of every mouse boosting cell.37 DEG C, 5%CO260h is cultivated, is received
Collect cell suspension into 1.5mL EP pipes, 2000r/min centrifuges 5min, collects supernatant, -80 DEG C save backup.Pass through ELISA
Method determines splenocyte IFN-γ, IL-12, IL-10 and IL-4 cytokine levels.
As a result as Figure 6-9, compared with negative control, the IL-12 levels of NPCS/OVA preparations induction splenocyte secretion
Not statistically significant significant difference, and the secretion level of IFN-γ is then significantly increased, with significant difference.In addition,
NPCS/OVA preparations induction IL-4 and IL-10 secretion level is significant to be higher than negative control.In summary, the IFN- of NPCS inductions
γ, IL-4 and IL-10 secretion level substantially increase, and particularly the secretion level of Th1 cell factors (mainly IFN-γ) is slightly
It is micro- to be better than Th2 cell factors (mainly IL-10).
In summary, the water solubility of quaternary phosphonium chitosan (NPCS) produced by the present invention is good, has been made from OVA different
Bacterin preparation, and intramuscular injection is carried out to mouse.As a result show, the preparation based on 1mg/mL NPCS can significantly increase DC2.4
Cell is to the intake of antigen, Spleen cell proliferation, IgG levels and cytokine secretion (IFN-γ, IL-10 and IL-4).This is main
Protections and slow-release capability of the NPCS to antigen are given the credit to, is conducive to DCs intakes and the more antigens of submission, stronger be immunized is produced
Response.In a word, the potential vaccine delivery systems applied to intramuscular immunisation of NPCS.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (7)
1. a kind of preparation method of quaternary phosphonium chitosan, it is characterised in that comprise the following steps：
(1) chitosan and I-hydroxybenzotriazole are added in water/dimethyl sulfoxide (DMSO) mixed liquor, 2- is stirred under condition of ice bath
3h, then adjusts the pH value of mixture to 4.8-5.5；Add 3- carboxylic propyl group tri-phenyl-phosphorus bromides, stirring is to being completely dissolved；
(2) 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate is dissolved in water/dimethyl sulfoxide (DMSO) mixed liquor
In, in the reaction system for being then added to step (1), react 1-2 days, product is precipitated with acetone/diethyl ether mixed liquor, then will
Precipitated product is dissolved in ultra-pure water after being dried in vacuo 1-2 days, and is dialysed 3-4 days in ultra-pure water, and quaternary phosphine is obtained after freeze-drying
Change chitosan；
In above-mentioned steps, sugar unit, I-hydroxybenzotriazole, 3- carboxylic propyl group tri-phenyl-phosphorus bromide, the 1- ethyls-(3- of chitosan
Dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate mol ratio be 1:2:1:2.
2. preparation method according to claim 1, it is characterised in that：In described water/dimethyl sulfoxide (DMSO) mixed liquor, water with
The volume ratio of dimethyl sulfoxide (DMSO) is (1-2):1.
3. preparation method according to claim 1, it is characterised in that：In described acetone/diethyl ether mixed liquor, acetone and second
The volume ratio of ether is 2:1.
4. a kind of quaternary phosphonium chitosan, it is characterised in that：It is to be made as the method described in claim any one of 1-3.
5. quaternary phosphoniums chitosan described in claim 4 is used as the application of vaccine immunity adjuvant.
6. quaternary phosphonium chitosan according to claim 5 is used as the application of vaccine immunity adjuvant, it is characterised in that：Described
The mass ratio of , quaternary phosphoniums chitosan and antigen is 2 in vaccine:1.
7. quaternary phosphonium chitosan according to claim 6 is used as the application of vaccine immunity adjuvant, it is characterised in that：Described
Antigen is ovalbumin.
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| CN116284497A (en) * | 2023-04-03 | 2023-06-23 | 华侨大学 | Preparation method of chitosan quaternary phosphonium salt |
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