CN113995834A - Vaccine based on cyclodextrin grafted chitosan, preparation method and application - Google Patents
Vaccine based on cyclodextrin grafted chitosan, preparation method and application Download PDFInfo
- Publication number
- CN113995834A CN113995834A CN202111316793.4A CN202111316793A CN113995834A CN 113995834 A CN113995834 A CN 113995834A CN 202111316793 A CN202111316793 A CN 202111316793A CN 113995834 A CN113995834 A CN 113995834A
- Authority
- CN
- China
- Prior art keywords
- cyclodextrin
- chitosan
- muc1
- grafted chitosan
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- 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
- A61K39/385—Haptens or antigens, bound to carriers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
-
- 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
Abstract
The invention discloses a vaccine based on cyclodextrin grafted chitosan (CS-g-CD), a preparation method and application thereof, belonging to the field of biomedical engineering. The cyclodextrin grafted chitosan provided by the invention has better solubility, and the modified cyclodextrin provides a host-guest binding site, so that the cyclodextrin is easier to be used for interaction with antigen molecules, a high-stability self-assembled supramolecular vaccine preparation is formed, better in-vivo delivery and antigen storage are facilitated, and the immunostimulation capability is further facilitated to be improved. The vaccine preparation prepared by the 'subject-object' self-assembly mode can be used as a platform for vaccine development and is suitable for wider vaccine development.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to a vaccine based on cyclodextrin grafted chitosan, a preparation method and application.
Background
Chitosan (CS) is a cationic polysaccharide consisting of beta (1-4) -linked glucosamine and N-acetylglucosamine, derived from chitin, one of the most abundant polymers in nature, and obtained by chemical deacetylation. Chitosan has good biocompatibility and biodegradability, and is widely applied to the fields of medicines, foods, chemical industry, cosmetics and the like. Chitosan is also considered a potential vaccine adjuvant, enhancing both humoral and cellular immunity. Chitosan has different amount of-NH according to the degree of deacetylation2Exposed, thereby generating a rich positive charge. This cationic polysaccharide has the advantage of being readily attached to negatively charged cell surfaces by electrostatic interactions, which may play an important role in antigen uptake and presentation. The positive charge of chitosan may also enhance the adjuvanticity of chitosan when mixed with antigen, including antigen protection and antigen depot formation. Furthermore, D-glucosamine repeat units-NH of the C-2 position of the element2Group protonation also increases the solubility of chitosan, which is critical for adjuvants. In addition to these advantages, chitosan as a vehicle has specific immunostimulatory activity. It has been demonstrated that chitosan induces type I Interferon (IFN) secretion by the cGAS-STING pathway and NLRP3 inflammasome, leading to Dendritic Cell (DC) activation and Th1 cellular immunity. Following coating of the lysosome with chitosan, protonation of the amino groups causes a proton sponge effect, which induces endosomal escape and promotes cross presentation of antigens, which is particularly important for priming CD8+ killer T cells following interaction with Major Histocompatibility Complex (MHC) class I molecules.
However, chitosan can only be dissolved under acidic conditions, but is poorly soluble at neutral pH or physiological conditions, which is a major drawback that hinders its biomedical applications. Since the Variable Number Tandem Repeat (VNTR) of mucin 1(mucin 1, MUC1) of tumor cells has poor immunogenicity and T cell independence, how to construct an effective vaccine structure to improve the immunogenicity of MUC1 is a great challenge in developing MUC1 vaccines.
Disclosure of Invention
In order to overcome the defects in the related art, the invention aims to provide a vaccine adjuvant of cyclodextrin grafted chitosan and application thereof in preparing vaccines, wherein the chitosan is modified by cyclodextrin, so that the dissolving capacity of the chitosan can be enhanced, and the vaccine adjuvant can be used for self-assembly of 'subject and object', and is suitable for wider development of biological medicines; thereby enhancing the immunogenicity of the vaccine and improving the stability and solubility of the vaccine preparation.
The specific technical scheme is as follows:
in one aspect of the invention, a vaccine based on cyclodextrin grafted chitosan is provided, wherein the cyclodextrin grafted chitosan is used as a vaccine adjuvant or a vaccine carrier, and the cyclodextrin grafted chitosan is linked with chitosan through cyclodextrin in a covalent bonding mode.
In an alternative embodiment of the invention, when the cyclodextrin-grafted chitosan is used as a vaccine adjuvant or a vaccine carrier, an antigenic molecule, an immunostimulant or a drug forms a supramolecular structure with the hydrophobic central cavity of the cyclodextrin-grafted chitosan by self-assembly.
In an alternative embodiment of the present invention, the cyclodextrin grafted chitosan has a structural formula of:
wherein n is a natural number not less than 1.
As a second aspect of the present invention, there is provided a method for preparing a vaccine based on cyclodextrin grafted chitosan, comprising a step of synthesizing cyclodextrin grafted chitosan, a step of solid phase synthesis of a MUC1 polypeptide compound, and a step of assembling the cyclodextrin grafted chitosan and the MUC1 polypeptide compound into nanoparticles.
In an alternative embodiment of the present invention, the synthesis step of cyclodextrin grafted chitosan comprises:
dissolving beta-cyclodextrin in double distilled water to obtain a suspension; then slowly adding p-toluenesulfonyl chloride, and stirring the mixture at room temperature; a further 20% w/v NaOH solution was added to the suspension and after 6 hours the residue was removed by filtration; adjusting the pH of the filtrate to 7 with dilute hydrochloric acid, and storing the solution at 4 ℃ to obtain a precipitate; filtering the precipitate, recrystallizing, and freeze-drying to obtain a product A named as beta-CD-OTs;
dissolving chitosan CS in 1% (v/v) acetic acid to obtain a chitosan solution; adding N, N-dimethylformamide solution of B-CD-OTs into chitosan solution, and obtaining cyclodextrin grafted chitosan with different grafting degrees according to different dosages of chitosan CS and beta-CD-OTs;
the mixture is refluxed and reacted for 24 hours at the temperature of 100 ℃, and then is dialyzed for 3 days by deionized water; finally, the solution is freeze-dried to obtain cotton-like powder of cyclodextrin grafted chitosan.
In an alternative embodiment of the present invention, the solid phase synthesis step of MUC1 polypeptide compound comprises: and (3) sequentially connecting amino acids from the C end to the N end on a solid-phase resin carrier, and finally cutting the amino acids from the solid-phase carrier to obtain the MUC1 polypeptide compound.
In alternative embodiments of the invention, the MUC1 polypeptide compound comprises MUC1, ada-ACP-MUC1, or ada-ACP-MUC1 (Tn).
In an alternative embodiment of the present invention, the step of assembling the cyclodextrin grafted chitosan and the MUC1 polypeptide compound into nanoparticles comprises: completely dissolving cyclodextrin grafted chitosan in an acetic acid solution, adjusting the pH value of the solution to 4.5-6 by using an NaOH solution, respectively adding MUC1, ada-ACP-MUC1 or ada-ACP-MUC1(Tn), stirring at room temperature, adding a sodium tripolyphosphate solution, stirring at room temperature, and dialyzing for 3d by using a dialysis bag to respectively obtain CS-g-CD/MUC1 nanoparticles, CS-g-CD/ada-ACP-MUC1 nanoparticles or CS-g-CD/ada-ACP-MUC1(Tn) nanoparticles.
In an alternative embodiment of the invention, the cyclodextrin is alpha, beta, gamma cyclodextrin and derivatives thereof, preferably beta-cyclodextrin and derivatives thereof.
In an alternative embodiment of the invention, wherein the antigenic molecule is selected from the group consisting of a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a fungal antigen or a parasitic antigen.
The vaccine based on cyclodextrin grafted chitosan provided by the invention can be used for preparing a medicament for preventing or treating cancers.
Has the advantages that:
(1) compared with the traditional chitosan adjuvant or chitosan carrier, the cyclodextrin grafted chitosan provided by the invention has better solubility.
(2) The modified cyclodextrin provides a 'subject-object' binding site, is easier to be used for interaction with antigen molecules, forms a high-stability self-assembled supramolecular vaccine preparation, and is beneficial to better in-vivo delivery and antigen storage, thereby being beneficial to improving the immunostimulation capacity.
(3) The vaccine preparation prepared by the 'subject-object' self-assembly mode can be used as a platform for vaccine development and is suitable for wider vaccine development.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the synthetic route of chitosan grafted cyclodextrin.
FIG. 2(a), (b), (c) and (d) are respectively the nuclear magnetic resonance H spectra, 400MHz and CD of chitosan, cyclodextrin grafted chitosan product 1, cyclodextrin grafted chitosan product 2 and cyclodextrin grafted chitosan product 33COOD/D2O。
Fig. 3 is an FTIR spectrum of chitosan, β -cyclodextrin, o-p-toluenesulfonyl- β -cyclodextrin, chitosan-grafted cyclodextrin (DS ═ 16.69%).
Figure 4 is a vaccine design of MUC1 grafted with cyclodextrin to chitosan.
FIG. 5 is a TEM image of a transmission electron microscope: (A) chitosan nanoparticles; (B) CS-g-CD/ada-ACP-MUC1(Tn) nanoparticles.
Fig. 6 shows the results of mouse immunization: (A) antibody titers in mouse sera at different days; (B) curve of OD value and dilution of ELISA detection of sera of groups 1-3 mice on day 35; (C) the levels of different antibody subtypes in serum; (D) competitive ELISA experiments with several different MUC1 polypeptides, the inhibition rate of the polypeptides against antibodies in serum.
FIG. 7 shows the expression levels of cytokines IFN-. gamma.and IL-6 in the sera of immunized mice: (a) expression levels of IFN- γ in sera from several groups of mice; (b) expression levels of IL-6 in serum of several groups of mice.
Fig. 8 shows the ELISA detection of sTn-specific antibody titers in the sera of immunized mice: (A) curve of OD value versus dilution; (B) and (3) the titer of the sTn-specific antibody in the serum of the group 1-4 mice.
Detailed Description
The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.
It should be noted that tumor-associated antigens expressed in large amounts on tumor cells are important targets for the development of anti-cancer vaccines and immunotherapies. In TAA, MUC1 is a highly glycosylated protein and is considered the second most promising target for the development of therapeutic vaccines. The extracellular portion of MUC1 has a Variable Number Tandem Repeat (VNTR) of 20 amino acid core sequence (HGVTSAPDTRPAPGSTAPA, amino acid sequence). Tumor-associated carbohydrate antigens T, Tn, sTn, etc. are usually expressed in MUC 1.
The degree of cyclodextrin grafting is defined as: the number of sugar monomers grafted with cyclodextrin is a percentage of the total number of sugar monomers.
The sources of reagents used in the examples of the present invention are commercially available except where otherwise specified.
Example 1: synthesis of Cyclodextrin grafted Chitosan (CS-g-CD)
The synthetic route is shown in figure 1. Dissolving beta-cyclodextrin (50.0g) in 200mL of double distilled water to obtain a suspension; then p-toluenesulfonyl chloride (15.0g) was slowly added, and the mixture was stirred at room temperature. After addition of 20% w/v NaOH solution (50mL) to the suspension for 6 hours, the residue was removed by filtration. The filtrate was adjusted to pH 7 with dilute hydrochloric acid, and the solution was left at 4 ℃ overnight to obtain a precipitate. Filtering the precipitate, recrystallizing to obtain precipitate, and freeze-drying the precipitate to obtain the beta-CD-OTs.
Chitosan CS (1.00g) was dissolved in 1% (v/v) acetic acid (80 mL). Adding beta-CD-OTs (1.00-5.00g) in N, N-dimethylformamide (DMF, 40mL) to the chitosan solution, and obtaining cyclodextrin grafted chitosan with different grafting degrees according to different proportions. The reaction mixture was refluxed at 100 ℃ for 24 hours and dialyzed against deionized water for 3 days. Finally, the solution was freeze-dried to obtain a cotton-like powder of cyclodextrin grafted chitosan (CS-g-CD) (FIG. 1).
The structural characterization of cyclodextrin grafted chitosan (CS-g-CD) is carried out, and a nuclear magnetic resonance H spectrum (figure 2) shows that cyclodextrin is successfully grafted to chitosan, and 3 kinds of cyclodextrin grafted chitosan (product 1, product 2 and product 3) with different grafting degrees are obtained according to different proportions of reaction substrates.
The grafting degrees of the products 1-3 are respectively 3.01%, 10.7% and 16.69%.
Further, the results of detection by fourier transform infrared spectroscopy are shown in fig. 3. From the infrared spectrum, 1594.39cm for beta-CD-OTs-1Is a C ═ C extension on the aryl benzene ring, 1156.06cm-1Is an S ═ O extension of the sulfonyl group, indicating successful synthesis of substituted p-toluenesulfonyl cyclodextrins. As for CS-g-CD, 1651.79cm-1 and 1359.57cm, respectively-1C ═ O and C — O stretching vibrations corresponding to the acetylglucosamine group on CS; 1148.24cm-1 and 1075.18cm-1 correspond to C-O-C on CS. The new peak at 949.95cm-1 is characteristic of the alpha-pyranyl oscillation of beta-CD, demonstrating successful grafting of cyclodextrin onto chitosan.
Example 2: synthesis of MUC1 polypeptide Compound
The MUC1 polypeptide compound is synthesized by a solid-phase synthesis strategy proposed by R.Bruce Merrifield, the process is that amino acids are connected in sequence from C end to N end on a solid-phase resin carrier, and finally the polypeptide is obtained after the amino acids are cut from the solid-phase resin carrier.
MUC1, adamantane-modified MUC1(ada-ACP-MUC1), and adamantane-modified MUC1(Tn) (ada-ACP-MUC1(Tn)) polypeptide compounds (the polypeptide structures are shown in FIG. 4). Wherein ACP is a 6-aminocaproic acid connecting arm, Tn is glycosylation modification, and the specific synthesis method comprises the following steps:
1. swelling the resin, weighing Fmoc-Ala-OH Wang resin (0.05mmol) with a certain load, placing the Fmoc-Ala-OH Wang resin in a 10mL polypeptide solid phase synthesis tube, adding 6mL N, N-Dimethylformamide (DMF) solution, swelling for 3h, draining the solvent, and washing 3 times respectively with DMF and DCM;
2. since the first amino acid at the Fmoc-protected N-terminus was previously supported by the Wang resin used, the Fmoc protecting group had to be removed to expose the amino group. Taking a 20% piperidine (Pip, solvent is DMF) solution as a removal reagent, adding 6mL of the solution into a synthesis tube, shaking for 15min, pumping out the solution, washing with DMF once, adding 6mL of the solution, shaking for 10min, pumping out the solution, washing the resin with DMF for 6 times, and finally pumping out the washing solution;
3. amino acid coupling, wherein the coupling operation uses Fmoc protected amino acid as a raw material, O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) as a condensing agent, 1-Hydroxybenzotriazole (HOBT) as a racemization preventing agent and N, N-Diisopropylethylamine (DIPEA) as an acid binding agent, and the reaction is carried out in a DMF solution. The specific method is that 3.0eq amino acid, 3.0eq HBTU, 3.0eq HOBT, 6.0eq DIPEA (1 eq bare amino on resin) is dissolved in 6ml DMF, then added into a synthesis tube, vibrated for 2h, if glycosyl amino acid is needed for condensation, changed into 2.0eq glycosyl amino acid, 2.0eq.2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), 2.0 eq.N-hydroxy-7-azobenzotriazol (HOAT), 4.0eq DIPEA vibrated for 4 h. Then pumping the solution, washing the resin with DMF for 3 times, and finally pumping the washing solution;
4. and 2, sequentially circulating the steps 3 until the synthesis of the polypeptide modified by the adamantane is finished. In the synthesis of the ada-ACP-MUC1 and ada-ACP-MUC1(Tn) polypeptides, after the synthesis of the polypeptide sequences is finished, the coupling of 6-aminocaproic acid (namely an ACP connecting arm) is continued, and then, the modification of N-terminal adamantane (ada) is performed by using adamantanecarboxylic acid and using a general amino acid coupling strategy. After each step of Fmoc removal and amino acid coupling, ninhydrin detection can be adopted, a small amount of drained resin is added into a 1.5mL centrifuge tube, ninhydrin working solution is added into the centrifuge tube according to the volume of solution A, solution B and solution C being 2: 3: 1, heating is carried out in boiling water for 2min, if exposed amino groups exist, the resin is blue, and if not, the resin is colorless or yellow;
5. and (3) performing polypeptide cleavage, washing the synthesized resin for 3 times respectively by using DMF (dimethyl formamide) and DCM (DCM), drying by suction, performing cleavage by using 1mL of a cleavage reagent per 100mg of the resin, wherein the ratio of the cleavage reagent is trifluoroacetic acid to triisopropylsilane to water is 95: 2.5 (TFA: TLS: H2O is 95: 2.5), placing the resin in the cleavage reagent, stirring for 2H, filtering to remove the resin, adding 10 times of glacial ethyl ether in volume into the residual liquid, precipitating, and centrifuging for 5min at the speed of 10000r min-1 in a centrifuge to remove the supernatant so as to obtain the crude peptide. If the crude peptide contains glycosyl amino acid, dissolving the crude peptide in 5% hydrazine hydrate and reacting for 3h to remove acetyl protecting group on hydroxyl;
6. purification of crude peptide using water acetonitrileDissolving with 50: 50 mixed solvent, filtering with 0.22 μm filter membrane, separating by semi-preparative HPLC, and freeze drying the collected components to obtain pure polypeptide. Mobile phase a used for HPLC was water + 0.1% TFA and mobile phase B was acetonitrile plus 0.1% TFA. The semi-preparation liquid phase method of the polypeptide 1 and the polypeptide 2 comprises the following steps: flow rate 4 mL/min-1The acetonitrile content is increased from 22 percent to 35 percent in 20 min; the polypeptide 3 semi-preparation liquid phase method comprises the following steps: flow rate 4 mL/min-1The acetonitrile content increased from 27% to 40% within 20 min. The purity of the target polypeptide is then detected using analytical HPLC, and the analytical liquid phase method is: flow rate 1 mL/min-1The acetonitrile content increased from 10% to 90% within 20 min. The target product is identified by mass spectrometry means.
A series of MUC1 polypeptides were synthesized. The ada part of the synthesized ada-ACP-MUC1 and ada-ACP-MUC1(Tn) polypeptide compounds can form a stable structure of self-assembly of a 'host-guest' with a hydrophobic central cavity of beta-cyclodextrin grafted chitosan (CS-g-CD).
Example 3 Assembly of Cyclodextrin-grafted Chitosan and MUC1 polypeptide Compound (i.e., MUC1 antigen) into nanoparticles
This example constructed a vaccine formulation of MUC1 grafted with cyclodextrin to chitosan as shown in figure 4. The cyclodextrin grafted chitosan used in this example was product 3 with a higher degree of grafting, which was 16.69%.
CS-g-CD and several different MUC1 antigens were further assembled into nanoparticles, namely: preparing cyclodextrin grafted chitosan (product 3, CS-g-CD) into a 1mg/mL solution with acetic acid, adjusting the pH of the solution to 4.5 with a 0.1M NaOH solution, and stirring for 1 h; subsequently, the above polypeptide compounds MUC1, ada-ACP-MUC1 and ada-ACP-MUC1(Tn) were added thereto, and the mixture was stirred at room temperature, and after adding a sodium tripolyphosphate solution (1mg/mL, 0.29mL) thereto and stirring at room temperature for 1 hour, the mixture was dialyzed for 3 days using a 7000Da dialysis bag to obtain nanoparticles. The method comprises the following specific steps:
CS-g-CD/MUC 1: CS-g-CD was dissolved in 1% acetic acid to prepare a 1mg/mL solution, and 2.9mL of the solution was taken, and pH was adjusted to 4.5 with 0.1M NaOH. Subsequently, 3.8mg of MUC1 was added, a sodium tripolyphosphate solution (1mg/mL, 0.29mL) prepared with deionized water was added to the reaction system, and after stirring at room temperature for 1 hour, the deionized water was dialyzed with a 7000Da dialysis bag for 3d to obtain nanoparticles.
CS-g-CD/ada-ACP-MUC 1: CS-g-CD was dissolved in 1% acetic acid to prepare a 1mg/mL solution, and 2.9mL of the solution was taken, and pH was adjusted to 4.5 with 0.1M NaOH. Subsequently, 4.0mg of ada-ACP-MUC1 was added, stirred at room temperature overnight, and a sodium tripolyphosphate solution (1mg/mL, 0.29mL) prepared with deionized water was added to the reaction system, stirred at room temperature for 1h, and dialyzed against deionized water for 3d with a 7000Da dialysis bag to obtain nanoparticles.
CS-g-CD/ada-ACP-MUC1 (Tn): CS-g-CD was dissolved in 1% acetic acid to prepare a 1mg/mL solution, and 2.9mL of the solution was taken, and the pH was adjusted to 4.5 with 0.1m naoh. Subsequently, 4.2mg of ada-ACP-MUC1(Tn) was added thereto, and stirred at room temperature overnight, and a sodium tripolyphosphate solution (1mg/mL, 0.29mL) prepared with deionized water was added to the reaction system, stirred at room temperature for 1h, and dialyzed against deionized water for 3d with a 7000Da dialysis bag, to obtain nanoparticles.
Nanoparticles are generated spontaneously by ionic gels between positively charged amino groups of CS-g-CD and negatively charged sodium Tripolyphosphate (TPP) as a cross-linker.
Example 4: characterization of nanoparticles
The nanoparticles were characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS), see fig. 5, table 1. According to the DLS results, the sizes of the nanoparticles of group 1, group 2 and group 3 were 91.28nm, 97.79nm and 129.0nm, respectively. These dimensions are significantly increased compared to the dimensions of CS-g-CD nanoparticles without MUC1 (80.71 nm). The zeta potential of the CS-g-CD nanoparticles was 23.7mv, which is mainly due to protonation of the CS amino group, while the zeta potentials of groups 1-3 were positive, which indicates that the nanoparticles can be stably distributed in solution. The TEM characterization further confirms the morphology of the nanoparticles, and shows that the particle size of the nanoparticles is about 100nm, and the morphology is irregular spherical. Nanoparticles ranging in size from 20 to 200 nanometers are easily engulfed by dendritic cells and can be directly targeted to lymph nodes.
TABLE 1 size and potential of nanoparticle vaccines
Example 5 vaccine formulation immune Activity Studies of MUC1 with Cyclodextrin grafted Chitosan
To evaluate the in vivo immunogenicity of the vaccine, three groups of C57BL/6J mice were used for studies of humoral and cellular immunity. The CS-g-CD nanoparticles containing the MUC1 antigen prepared in the above way are further emulsified with Freund's complete adjuvant to be used as a vaccine preparation for mouse immunization. On days 1, 7, 14, 21 and 28, each mouse was inoculated with 100 μ L of emulsion of 15nmol MUC1 antigen by subcutaneous injection (s.c.). Antisera were prepared from mouse blood collected through the femoral vein on days 0, 7, 14, 21, 28, and 35 for analysis of antibodies and cytokines. The titer of MUC1 specific antibodies was determined by enzyme-linked immunosorbent assay (ELISA) using the corresponding MUC1-HSA conjugate as capture reagent. Typically, antigen-coated ELISA plates were treated with blocking buffer and then incubated with half-log serial dilutions of mouse antiserum from 1: 300 to 1: 218700 in PBS. After washing, HRP-linked goat anti-mouse (H + L), IgG1, IgG2b, IgG2c, IgG3, and IgM secondary antibodies were added to the plates, respectively. Finally, the plate was reacted with TMB solution and then detected by a microplate reader at a wavelength of 450 nm. The antibody titer was calculated from a curve of light intensity (OD) against the number of serum dilutions, and the dilution at which the OD value reached 0.2 was defined as the antibody titer.
Fig. 6(a) shows the antibody titers of antisera collected from groups 1-3 of mice on different days, with the antigen-specific antibody titers increasing with increasing number of immunizations and reaching the highest levels after the fifth booster needle. As can be seen from fig. 6(B), the antibody titer reached 18550 on day 35 in group 3, which is much higher than the other two groups, indicating that the group 3 vaccine had the best immunogenicity. The subclasses of antibodies produced in groups 1-3 were analyzed with reference to FIG. 6(C), and each group induced high antibody titers of IgG2b, IgG2C, and IgG3, indicating that vaccines using CS-g-CD as a vector successfully induced T cell-mediated immunity. T cell immunity may be caused by CS-g-CD vectors, which are likely to induce STING immune activity and induce sufficient cross-presentation when antigens are presented by APCs. In addition, the antibody titers in group 2 were higher than in group 1, indicating that the host-guest interaction between the antigen and CS-g-CD increased the immunogenicity of the vaccine. To further confirm the specificity of the antibodies, a competition ELISA was performed using the corresponding MUC1 peptide as an inhibitor against the antigen coated on the plate. The results in FIG. 6(D) show that MUC1 antigen can successfully recognize and inhibit vaccine-induced group 1-3 antibodies.
To further investigate T cell mediated immune responses, serum expression levels of IFN-. gamma.and IL-6 were analyzed by cytokine ELISA. In general, IFN- γ is a Th 1-type cytokine with important immunomodulatory properties that can proliferate and differentiate lymphocyte populations, activate NK cells and enhance antigen presentation. IL-6, a Th2 type cytokine, improves innate and adaptive immunity, mediates aspects of B cell and T cell responses, and promotes antibody production. As shown in fig. 7, group 3 produced the highest level of IFN- γ compared to groups 1 and 2, indicating that the vaccine constructs of group 3 had the strongest Th 1-type immune responses. This is caused by the specific "host-guest" self-assembly structure between the antigen and CS-g-CD in group 3, further indicating that the "host-guest" inclusion of CS-g-CD is important for inducing strong Th1 immunity. However, IL-6 levels were similar in groups 1 and 3, but higher than in group 2. This may be influenced by a number of factors, including the CFA adjuvant, the antigen and the CS-g-CD vector.
Comparative example: immune activity study of vaccine of sTn-BSA-ada and cyclodextrin grafted chitosan
According to the prior art route [ chem.commun., 2020, 56, 1395, DOI: 10.1039/d0cc05263a ], preparing a conjugate of tumor-associated carbohydrate antigen sTn and Bovine Serum Albumin (BSA) as a carrier protein, modifying an adamantane structure ada (namely sTn-BSA-ada) on the conjugate, coupling 4.3 sTn antigens on each BSA molecule according to mass spectrometry, and coupling 5 ada chemical structures on average. CS-g-CD with different grafting degrees (the grafting degrees are respectively 3.01 percent, 10.7 percent and 16.69 percent) is fully dissolved in an acetic acid solution, then the pH value is adjusted to 4.5 by 0.1M NaOH, and the solution is stirred for 1h to prepare the solution with the concentration of the CS-g-CD being 1 mg/mL; subsequently, 3.0mg of the prepared sTn-BSA-ada conjugate was added, respectively, and stirred at room temperature at 200rpm overnight to form nanoparticles. The obtained nanoparticle solution is mixed with Freund's complete adjuvant (volume ratio is 1: 1) and used as a vaccine for mouse immunization. The immunization dose of mice was 3 μ g of sTn saccharide antigen per mouse per injection.
Set up 4 groups of mice immunization experiments: the first group is sTn-BSA conjugate; the second group is vaccines prepared by sTn-BSA-ada and CS-g-CD (3.01 percent of grafting degree); the third group is vaccine prepared by sTn-BSA-ada and CS-g-CD (10.7 percent of grafting degree); the fourth group was a vaccine prepared from sTn-BSA-ada and CS-g-CD (16.69% grafting).
On days 1, 7, 14 and 21, each mouse was inoculated with 100 μ L of emulsion containing 3 μ g of sTn antigen by subcutaneous injection (s.c.). Antisera were prepared from mouse blood collected through the femoral vein on days 0, 7, 14, 21, and 28 for analysis of antibodies. The sTn-specific antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using the sTn-HSA conjugate as capture reagent.
The sTn-specific antibody titer results are shown in FIG. 8. As can be seen from the results, the titers of sTn-specific antibodies in the sera of the second, third and fourth groups of mice were all higher than the titer of the first group of sTn-BSA control group. The results show that CS-g-CD has an enhancement effect on the immunogenicity of sTn-BSA-ada, and the specific antibody titer of sTn is improved. Meanwhile, it can be seen that the antibody titer of the second group was the highest, and with the increase of the grafting degree, the antibody titer was somewhat decreased. This result indicates that the higher the degree of grafting of cyclodextrin, the higher the "host-guest" inclusion ability of the antigen substance, but the more the immunostimulating ability of chitosan itself is affected, and thus it is necessary to select an appropriate degree of grafting of cyclodextrin.
In conclusion, the CS-g-CD has stronger immune stimulation capability and can be used as a vaccine adjuvant or a vaccine carrier, and the host-guest self-assembly structure formed by grafting the cyclodextrin onto the chitosan can enhance the immunogenicity of the vaccine, improve the stability and solubility of the vaccine preparation and have high application value for the development of the vaccine preparation.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The vaccine based on cyclodextrin grafted chitosan is characterized in that the cyclodextrin grafted chitosan is used as a vaccine adjuvant or a vaccine carrier, and the cyclodextrin grafted chitosan is connected with chitosan in a covalent bonding mode through cyclodextrin.
2. The cyclodextrin grafted chitosan-based vaccine of claim 1, wherein when the cyclodextrin grafted chitosan is used as a vaccine adjuvant or a vaccine carrier, an antigenic molecule, an immunostimulant or a drug forms a supramolecular structure with the hydrophobic central cavity of the cyclodextrin grafted chitosan by means of self-assembly.
3. The cyclodextrin grafted chitosan-based vaccine according to claim 1 or 2, wherein the cyclodextrin grafted chitosan has the general structural formula:
wherein n is a natural number not less than 1.
4. The method of any one of claims 1-3, comprising the steps of synthesis of cyclodextrin-grafted chitosan, solid phase synthesis of MUC1 polypeptide compound, and assembly of the cyclodextrin-grafted chitosan and MUC1 polypeptide compound into nanoparticles.
5. The method for preparing a cyclodextrin grafted chitosan-based vaccine as claimed in claim 4, wherein the step of synthesizing cyclodextrin grafted chitosan comprises:
dissolving beta-cyclodextrin in double distilled water to obtain a suspension; then slowly adding p-toluenesulfonyl chloride, and stirring the mixture at room temperature; a further 20% w/v NaOH solution was added to the suspension and after 6 hours the residue was removed by filtration; adjusting the pH of the filtrate to 7 with dilute hydrochloric acid, and storing the solution at 4 ℃ to obtain a precipitate; filtering the precipitate, recrystallizing, and freeze-drying to obtain a product A named as beta-CD-OTs;
dissolving chitosan CS in 1% (v/v) acetic acid to obtain a chitosan solution; adding N, N-dimethylformamide solution of beta-CD-OTs into chitosan solution, and obtaining cyclodextrin grafted chitosan mixtures with different grafting degrees according to different dosages of chitosan CS and beta-CD-OTs;
carrying out reflux reaction on the cyclodextrin grafted chitosan mixture at 100 ℃ for 24 hours, and dialyzing for 3 days by using deionized water; finally, the solution is freeze-dried to obtain cotton-like powder of cyclodextrin grafted chitosan.
6. The method of claim 4, wherein the step of solid phase synthesis of MUC1 polypeptide compound comprises: and (3) sequentially connecting amino acids from the C end to the N end on a solid-phase resin carrier, and finally cutting the amino acids from the solid-phase carrier to obtain the MUC1 polypeptide compound.
7. The method of claim 4 or 6, wherein the MUC1 polypeptide compound comprises MUC1, ada-ACP-MUC1, or ada-ACP-MUC1 (Tn).
8. The method of claim 7, wherein the step of assembling the cyclodextrin grafted chitosan and the MUC1 polypeptide compound into nanoparticles comprises: completely dissolving cyclodextrin grafted chitosan in an acetic acid solution, adjusting the pH value of the solution to 4.5-6 by using an NaOH solution, respectively adding MUC1, ada-ACP-MUC1 or ada-ACP-MUC1(Tn), stirring at room temperature, adding a sodium tripolyphosphate solution, stirring at room temperature, dialyzing for 3d by using a dialysis bag to respectively obtain CS-g-CD/MUC1 nanoparticles, chitosan, and a sodium tripolyphosphate solution, stirring at room temperature, and filtering to obtain CS-g-CD/MUC1 nanoparticles, chitosan, and a sodium tripolyphosphate solution,
CS-g-CD/ada-ACP-MUC1 nanoparticles or CS-g-CD/ada-ACP-MUC1(Tn) nanoparticles.
9. A cyclodextrin-grafted chitosan-based vaccine according to claim 1, wherein said cyclodextrin is alpha, beta, gamma cyclodextrin and derivatives thereof, preferably beta-cyclodextrin and derivatives thereof.
10. The cyclodextrin grafted chitosan-based vaccine of claim 2, wherein the antigenic molecule is selected from the group consisting of a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a fungal antigen or a parasitic antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111316793.4A CN113995834A (en) | 2021-11-08 | 2021-11-08 | Vaccine based on cyclodextrin grafted chitosan, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111316793.4A CN113995834A (en) | 2021-11-08 | 2021-11-08 | Vaccine based on cyclodextrin grafted chitosan, preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113995834A true CN113995834A (en) | 2022-02-01 |
Family
ID=79928151
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111316793.4A Pending CN113995834A (en) | 2021-11-08 | 2021-11-08 | Vaccine based on cyclodextrin grafted chitosan, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113995834A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115850722A (en) * | 2023-02-22 | 2023-03-28 | 翔鹏(北京)生物科技有限公司 | Preparation method and application of efficient transdermal supramolecular sodium hyaluronate |
| CN116003652A (en) * | 2023-01-04 | 2023-04-25 | 齐鲁工业大学(山东省科学院) | Ginsenoside Rg1 nano hydrogel and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080220030A1 (en) * | 2005-06-02 | 2008-09-11 | Universidade De Santiago De Compostela | Nanoparticles Comprising Chitosan and Cyclodextrin |
| CN108849995A (en) * | 2018-06-12 | 2018-11-23 | 甘肃省科学院生物研究所 | Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski extract nano insecticide and preparation method thereof |
-
2021
- 2021-11-08 CN CN202111316793.4A patent/CN113995834A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080220030A1 (en) * | 2005-06-02 | 2008-09-11 | Universidade De Santiago De Compostela | Nanoparticles Comprising Chitosan and Cyclodextrin |
| CN108849995A (en) * | 2018-06-12 | 2018-11-23 | 甘肃省科学院生物研究所 | Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski extract nano insecticide and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 王惠武等: "甲壳素/壳聚糖接枝共聚反应", 甲壳素/壳聚糖接枝共聚反应, vol. 18, no. 5, pages 2 * |
| 解云天等: "基于环糊精自组装的MUC1纳米金疫苗的制备及免疫活性评价", 中国科技论文在线 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116003652A (en) * | 2023-01-04 | 2023-04-25 | 齐鲁工业大学(山东省科学院) | Ginsenoside Rg1 nano hydrogel and preparation method and application thereof |
| CN115850722A (en) * | 2023-02-22 | 2023-03-28 | 翔鹏(北京)生物科技有限公司 | Preparation method and application of efficient transdermal supramolecular sodium hyaluronate |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hudalla et al. | A self-adjuvanting supramolecular vaccine carrying a folded protein antigen | |
| Wen et al. | Switching the immunogenicity of peptide assemblies using surface properties | |
| CA2152917C (en) | Liposome with oligosaccharide on surface | |
| KR100680014B1 (en) | Imine-forming polysaccharides, preparation thereof and the use thereof as adjuvants and immunostimulants | |
| Dmour et al. | Recent advances on chitosan as an adjuvant for vaccine delivery | |
| CN102333541B (en) | Immunogenic composition | |
| CN113995834A (en) | Vaccine based on cyclodextrin grafted chitosan, preparation method and application | |
| Niederhafner et al. | Glycopeptide dendrimers, Part III—a review: use of glycopeptide dendrimers in immunotherapy and diagnosis of cancer and viral diseases | |
| Zhang et al. | Curdlan sulfate–O-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination | |
| JP2022058911A (en) | Glycopolysialylation of non-blood coagulation protein | |
| CN111249453B (en) | Nano vaccine and preparation method thereof | |
| CN103052402B (en) | Immunogenic composition | |
| Weiss et al. | Immunostimulatory polymers as adjuvants, immunotherapies, and delivery systems | |
| AU2007258550A1 (en) | Liposome-mediated native chemical ligation | |
| US20070031447A1 (en) | Method of isolating biologically active fraction containing clinically acceptable native S-lipopolysaccharides obtained from bacteria producing endotoxic lipopolysaccharides | |
| EP2407178A2 (en) | Monovalent and polyvalent synthetic polysaccharide antigens for immunological intervention in disease | |
| CN105611936B (en) | Immunopotentiating agent | |
| Pogostin et al. | Multidomain peptide hydrogel adjuvants elicit strong bias towards humoral immunity | |
| CN113577255B (en) | Tumor nano vaccine, preparation method and application thereof | |
| Yoon et al. | Enhanced efficacy of immunization with a foot-and-mouth disease multi-epitope subunit vaccine using mannan-decorated inulin microparticles | |
| Hevey et al. | Conjugation Strategies Used for the Preparation of Carbohydrate‐Conjugate Vaccines | |
| Donahue et al. | Synthesis and immunological study of N-Glycan-Bacteriophage Qβ conjugates reveal dominant antibody responses to the conserved chitobiose core | |
| EP3473641A1 (en) | Nucleic acid-antigen peptide conjugate | |
| JP7023460B2 (en) | TLR ligand-immobilized nanoparticles | |
| Yin et al. | Strategies for Synthesizing and Enhancing the Immune Response of Cancer Vaccines Based on MUC1 Glycopeptide Antigens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |