CN115737797A - High-affinity Siglec-1 ligand-modified glucan nano vaccine and preparation method and application thereof - Google Patents
High-affinity Siglec-1 ligand-modified glucan nano vaccine and preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the technical field of biological medicine and disease prevention and treatment, and particularly relates to a glucan nano vaccine modified based on a high-affinity Siglec-1 ligand, and a preparation method and application thereof. The Protein Antigen (PA) and the immune stimulant (Rd) in the glucan nano vaccine form ethoxy oxidized with azide modified part through imine bondRadical acetalized dextran nanoparticles (Oxi-Ace-Dex-Az NPs) were bound. Then mixing the obtained NPs with TCC Sia-LacNAc-DBCO promotes azide-alkyne cycloaddition reaction by stress to produce TCC Sia-Ace-Dex-PA-Rd NPs can actively target and deliver protein antigens to macrophages. The vaccine has good immune curative effect, very low toxicity and high safety, and can be used for preventing and treating related diseases such as new corona, tumor and the like, thereby having wide application prospect and market value.
Description
Technical Field
The invention belongs to the technical field of biological medicine and disease prevention and treatment, and particularly relates to a glucan nano vaccine modified based on a high-affinity Siglec-1 ligand, and a preparation method and application thereof.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The vaccine of the invention eradicates smallpox and poliomyelitis, also prevents infectious diseases which cause millions of deaths each year, and becomes one of the greatest achievements in human public health history. However, the existing vaccine development strategies have not been successful in preventing most cancers. In addition, the vaccination with the existing vaccine can not completely prevent and cure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More tricky, the emergence of SARS-CoV-2 variants (e.g., deltaDelta B.1.617.2, omicron BA.1-BA.5) has led to a significant reduction in the neutralizing capacity of antibodies produced by existing vaccines (e.g., murray BNT162b 2). Therefore, there is an urgent need to develop a next-generation vaccine against cancer and SARS-CoV-2 and its variants.
Cytotoxic T-lymphocyte (CTL) immunity and humoral immunity are two major mechanisms by which vaccines function. In order to induce potent CTL and IgG responses, vaccines based on antigenic epitopes have been developed, including polypeptides, carbohydrates and glycopeptides. For the design of such vaccines, the primary task is to obtain critical epitopes. However, identifying key epitopes is time consuming and challenging. The development of protein-based vaccines is a straightforward and effective strategy to address the emergent health crisis worldwide, such as SARS-CoV-2 infection. The recombinant protein can be directly used as a vaccine. However, direct administration of proteins often results in an inadequate immune response, which results in poor disease treatment. Nanoparticle (NP) -based vaccines enhance T cell activation by increasing antigen-presenting cell (APC) antigen-presenting efficiency. In addition, antigens rationally arranged on NPs effectively activate B cells to produce high titers of antibodies by cross-linking with B cell receptors on B cells.
As a high potential carrier, acetalized dextran (Ac-Dex) has better performance in enhancing CTL response than the commonly used poly (lactic-co-glycolic acid) (PLGA). In these reported studies, the inventors found that the uptake of Ac-Dex NPs by immune cells is via passive transport, and the effect of inducing T cell immune and humoral immune responses is far from therapeutic.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a glucan nano vaccine modified by a high-affinity Siglec-1 ligand, and a preparation method and application thereof. The invention firstly prepares and obtains an ethoxy acetalized glucan nanoparticle (Oxi-Ace-Dex-Az NPs) based on azide modification and partial oxidation, and obtains a high-affinity Siglec-1 glycosyl ligand 9-N- (4H-thieno [3,2-c ] with active targeting based on the preparation]chromene-2-carbamoyl)-Siaα2-3Galβ1-4GlcNAc( TCC Sia-LacNAc) modified ethoxylated acetalized dextran nano vaccine (C) TCC Sia-Ace-Dex-PA-Rd NPs) containing a SARS-CoV-2 Protein Antigen (PA) and an immune agonist (resiquimod, rd). The experiment proves that the medicine can effectively prevent SARS-CoV-2 virus infection and has low toxicity. The present invention has been completed based on the above results.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided an ethoxylated acetalized dextran nanoparticle with partially oxidized azide groups (Oxi-Ace-Dex-Az NPs) that can be used as a universal platform for the delivery of protein antigens and adjuvants, said nanoparticle having the schematic structure as follows:
in a second aspect of the present invention, there is provided a method for synthesizing the above nanoparticles, wherein the synthetic route is as follows:
specifically, the synthesis method comprises the following steps:
s1, dissolving glucan in dimethyl sulfoxide, adding CDI-TEG-Azide and 4-Dimethylaminopyridine (DMAP), and reacting under the protection of inert gas; purifying to obtain azido modified glucan (Dex-Az) A1;
s2, dissolving the polymer A1 obtained in the S1 in water, and then adding sodium periodate; stirring for reaction, and purifying to obtain white solid, namely azide-modified partially oxidized dextran (Oxi-Dex-Az) A2;
s3, mixing the polymer A2 prepared in the step S2 with dimethyl sulfoxide DMSO, adding p-toluenesulfonic acid pyridine and 2-ethoxy propylene, stirring to react at room temperature, adding a quenching agent to quench the reaction, and purifying the product to obtain an azide-modified partially oxidized acetalized dextran (Oxi-Ace-Dex-Az) polymer A3;
s4, dissolving the polymer A3 obtained in the S3 in dichloromethane, adding the dichloromethane into aqueous poly (vinyl alcohol) (PVA), and performing low-temperature ultrasonic treatment to obtain a primary emulsion A4;
and S5, continuously adding the emulsion A4 obtained in the S4 into the aqueous PVA, stirring, and purifying to obtain the azide-modified partially-oxidized acetalized glucan nano-particles (Oxi-Ace-Dex-Az NPs).
In a third aspect of the present invention, the nanoparticles are used for preparing active targeting drugs.
The fourth aspect of the invention provides a dextran nano vaccine with active targeting and based on high-affinity Siglec-1 ligand modification TCC Sia-Ace-Dex-PA-Rd, at least having a high affinity Siglec-1 ligand linked to said nanoparticle together with a Protein Antigen (PA) and an immune agonist (e.g. ranimod);
the structure is as follows:
specifically, the Protein Antigen (PA) and the immune agonist (Rd) form imine bond combination through the reaction of the amino group of the Protein Antigen (PA) and the aldehyde group in the azide-modified partially oxidized acetalized glucan nanoparticle (Oxi-Ace-Dex-Az NPs); then the obtained NPs are mixed with 9-N- (4H-thieno [3,2-c ]]chromene-2-carbamoyl) -Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta Pro-DBCO (named as TCC Sia-LacNAc-DBCO) by azidoyne cycloaddition to give TCC Sia-Ace-Dex-PA-Rd NPs; among them, high affinity Siglec-1 ligands TCC Sia-LacNAc can actively target sialic acid binding immunoglobulin-like lectin 1 (Siglec-1, cd169) on macrophages.
It should be noted that the Protein Antigen (PA) can be adjusted according to the target protein, such as selecting a suitable protein antigen for a specific tumor cell, or selecting a suitable new coronaviruse antigen (RBD protein or N protein) for example, the high affinity Siglec-1 ligand and the immune agonist can be properly adjusted and replaced, and based on the inventive concept of the present application, the adjustment and replacement are clearly within the protection scope of the present application without creative effort.
In a fifth aspect of the present invention, a synthetic method of the dextran nano vaccine is provided, wherein the synthetic route is as follows:
specifically, the synthesis steps are as follows:
s1, adding a Protein Antigen (PA) into a buffer solution containing azide-modified partially oxidized acetalized dextran nanoparticles (Oxi-Ace-Dex-Az NPs); reacting to obtain a mixture A5;
s2, adding Rasimethide (R848, rd) and Rasimethide into the mixture A5 obtained in the step S1 TCC Sia-LacNAc-DBCO, stirring to obtain a mixture A6;
s3, purifying the mixture A6 obtained in the step S2 to obtain the product TCC Sia-Ace-Dex-PA-Rd NPs。
In the step S2, the TCC Sia-LacNAc-DBCO, high affinity Siglec-1 ligand 9-N- (4H-thieno [3,2-c ]]chromene-2-carbamoyl) -Neu5Ac (Sia) α 2-3Gal β 1-4GlcNAc β pro-DBCO, having the formula:
the synthetic route is as follows:
the sixth aspect of the invention provides an application of the glucan nano vaccine in preparation of a medicament for preventing and/or treating diseases.
The disease may include any disease, particularly related diseases causing immune system problems (e.g., low or high immune state) and thus may be prevented and/or treated by artificially enhancing or suppressing the immune function of the body. The disease may be in particular a tumor and a new coronary pneumonia.
In a seventh aspect of the present invention, there is provided a method for tumor prevention and/or treatment, the method comprising: administering the dextran nano-vaccine to a subject.
In an eighth aspect of the present invention, there is provided a method for preventing and/or treating a new coronary pneumonia, the method comprising: administering the dextran nano-vaccine described above to a subject.
The beneficial technical effects of the technical scheme are as follows:
the technical scheme provides a novel crown recombinant protein vaccine prepared based on azide-modified partially oxidized acetalized dextran nanoparticles. Wherein the Protein Antigen (PA) and the immune agonist (Rd) are formed by aminimide bond and azide modification of partially oxidized acetalized glucanNanoparticles (Oxi-Ace-Dex-Az NPs) were bound. Then the obtained NPs and 9-N- (4H-thieno [3, 2-c)]chromene-2-carbamoyl) -Neu5Ac (Sia) α 2-3Gal β 1-4GlcNAc β Pro-DBCO (referred to as TCC Sia-LacNAc-DBCO) by azidoyne cycloaddition to give TCC Sia-Ace-Dex-PA-Rd NPs can actively target and deliver protein antigens to macrophages. The action mechanism determines that the new crown recombinant protein vaccine prepared based on the azide-modified partially oxidized acetalized dextran nanoparticles provided by the technical scheme can effectively prevent SARS-CoV-2 virus infection, and has very low toxicity.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 shows a new crown recombinant protein vaccine prepared based on azide-modified partially oxidized acetalized dextran nanoparticles provided by the invention TCC And (3) the experimental result of CTL killing caused by the Sia-Ace-Dex-N-Rd NPs and the control group in mice.
FIG. 2 shows a new crown recombinant protein vaccine prepared based on azide-modified partially oxidized acetalized dextran nanoparticles provided by the present invention TCC The Sia-Ace-Dex-N-Rd NPs and a control group cause anti-N protein IgG antibody titer experiment results in mice.
FIG. 3 shows that the new crown recombinant protein vaccine prepared based on the azide-modified partially oxidized acetalized dextran nanoparticle provided by the invention TCC The result of the experiment on the anti-N protein IgG antibody titer caused by the Sia-Ace-Dex-N-Rd NPs and the control group in rabbits.
FIG. 4 shows a new crown recombinant protein vaccine prepared based on azide-modified partially oxidized acetalized dextran nanoparticles provided by the present invention TCC Sia-Ace-Dex-RBD-Rd NPs and TCC Sia-Ace-Dex-RBD-Rd NPs+ TCC and the Sia-Ace-Dex-N-Rd NPs combined vaccine group induces the anti-RBD protein IgG antibody titer experiment result in a mouse body.
FIG. 5 shows that the new crown recombinant protein vaccine prepared based on the azide-modified partially oxidized acetalized dextran nanoparticle provided by the present invention TCC Sia-Ace-Dex-N-Rd NPs and TCC Sia-Ace-Dex-RBD-Rd NPs+ TCC and (3) a CTL killing experiment result caused by the Sia-Ace-Dex-N-Rd NPs combined vaccine group in a mouse body.
FIG. 6 shows a novel crown recombinant protein vaccine prepared from an azido-modified partially oxidized acetalized dextran nanoparticle TCC And the Sia-Ace-Dex-RBD-Rd NPs and the control group cause anti-RBD protein IgG antibody titer experiment results in rabbits.
FIG. 7 shows a novel crown recombinant protein vaccine prepared based on azide-modified partially oxidized acetalized dextran nanoparticles provided by the present invention TCC And (3) neutralizing the euvirus capability experiment result of the serum obtained after the Sia-Ace-Dex-RBD-Rd NPs and a control group immunize rabbits.
FIG. 8 shows a novel crown recombinant protein vaccine prepared from an azido-modified partially oxidized acetalized dextran nanoparticle TCC And (3) after the Sia-Ace-Dex-RBD-Rd NPs are used for immunizing the rabbit, detecting whether the rabbit organ generates a tissue lesion experiment result or not.
FIG. 9 is a chart of hydrogen spectra characterized by A1 in example 1 of the present invention.
FIG. 10 is a hydrogen spectrum characterized by A2 in example 1 of the present invention.
FIG. 11 is a hydrogen spectrum characterized by A3 in example 1 of the present invention.
FIG. 12 is a hydrogen spectrum characterized by Compound 1 in example 2 of the present invention.
FIG. 13 is a carbon spectrum representation of Compound 1 of example 2 of the present invention.
FIG. 14 is a hydrogen spectrum characterized by Compound 2 of example 2 of the present invention.
FIG. 15 is a carbon spectrum representation of Compound 2 of example 2 of the present invention.
FIG. 16 is a hydrogen spectrum characterized by Compound 4 in example 2 of the present invention.
FIG. 17 is a carbon spectrum representation of Compound 4 of example 2 of the present invention.
FIG. 18 is a hydrogen spectrum of compound 6 characterized in example 2 of the present invention.
FIG. 19 is a carbon spectrum graph of Compound 6 from example 2 of the present invention.
FIG. 20 is a hydrogen spectrum characterized by Compound 7 in example 2 of the present invention.
FIG. 21 is a carbon spectrum of Compound 7 in example 2 of the present invention.
FIG. 22 shows a compound of example 2 of the present invention TCC A hydrogen spectrum characterized by Sia-LacNAc-DBCO.
FIG. 23 shows a compound of example 2 of the present invention TCC A carbon spectrum characterized by Sia-LacNAc-DBCO.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As previously mentioned, acetalized dextran (Ac-Dex) has been shown to be superior to the commonly used polylactic-co-glycolic acid (PLGA) in enhancing CTL responses. In these reported studies, the uptake of Ac-Dex NPs by immune cells was via passive transport, and the effect of inducing T-cell and humoral immune responses was far from therapeutic.
In view of the above, in an exemplary embodiment of the present invention, there is provided an acetalized dextran nanoparticle with partially oxidized azide groups (Oxi-Ace-Dex-Az NPs) as a versatile platform for delivery of protein antigens and adjuvants, the nanoparticle having the following schematic structure:
in another embodiment of the present invention, there is provided a method for synthesizing the above nanoparticles, wherein the synthetic route is as follows:
specifically, the synthesis method comprises the following steps:
s1, dissolving glucan in dimethyl sulfoxide, adding CDI-TEG-Azide and 4-dimethylaminopyridine DMAP into the solution, and reacting in an inert atmosphere; purifying to obtain azido modified glucan (Dex-Az) A1;
s2, dissolving the polymer A1 obtained in the S1 in water, and then adding sodium periodate; stirring for reaction, and purifying to obtain white solid, namely azide-modified partially oxidized dextran (Oxi-Dex-Az) A2;
s3, mixing the polymer A2 prepared in the step S2 with dimethyl sulfoxide (DMSO), adding p-toluenesulfonic acid pyridine and 2-ethoxy propylene, stirring the reaction solution at room temperature for reaction, adding a quenching agent for quenching reaction, and purifying the product to obtain an azide-modified partially oxidized acetalized glucan (Oxi-Ace-Dex-Az) polymer A3;
s4, dissolving the polymer A3 obtained in the S3 in dichloromethane, adding the dichloromethane into aqueous poly (vinyl alcohol), and performing low-temperature ultrasonic treatment to obtain a primary emulsion A4;
and S5, continuously adding the emulsion A4 obtained in the S4 into the aqueous PVA, stirring, and purifying to obtain the azide-modified partially-oxidized acetalized glucan nano-particles (Oxi-Ace-Dex-Az NPs).
In the step S1, the molecular weight of the glucan is controlled to 9000-11000g mol -1 ，
The mass ratio of the glucan to the CDI-TEG-Azide to the 4-dimethylaminopyridine is 1; the reaction conditions for the reaction under an inert atmosphere (such as argon) are specifically a reaction at room temperature for 1 to 3 days, preferably 2 days;
the specific purification method comprises the following steps: dialyzing the reaction solution, and changing water for 4-5 times; the solution was then lyophilized.
In the step S2, the mass ratio of the sodium periodate to the polymer A1 is 0.1 to 0.5, preferably 0.22; the stirring reaction is specifically stirring reaction at room temperature for 1-10h, preferably 5h; the specific purification method comprises the following steps: dialyzing the reaction solution to remove small molecules, and freeze-drying the solution to obtain the final product.
In the step S3, the mass-to-volume ratio of the polymer A2, the pyridine p-toluenesulfonate, and the 2-ethoxypropene is 1 to 5mL, preferably 1 g; the quenching agent can be triethylamine; the purification method specifically comprises the steps of adding the reaction solution into water to separate out a precipitate, centrifuging the precipitate (such as centrifuging at 12000rpm for 20 min) to obtain the precipitate, and washing and freeze-drying the precipitate to obtain the product.
In step S4, the mass-to-volume ratio of the polymer A3 to the aqueous poly (vinyl alcohol) is 10mg, preferably 10mg; the molecular weight of the poly (vinyl alcohol) in the aqueous poly (vinyl alcohol) is 13-23kg mol -1 The content of the poly (vinyl alcohol) is controlled to be 1-5 percent, w/w; preferably 3%;
the low-temperature ultrasound can be specifically carried out for 0.5-5min by adopting ice bath ultrasound;
in the step S5, the molecular weight of the poly (vinyl alcohol) in the aqueous poly (vinyl alcohol) is 13 to 23kg mol -1 The content of the poly (vinyl alcohol) is controlled to be 1-5 percent, w/w; preferably 3%; the stirring is carried out for 1-5h, preferably 3h at room temperature, and the purification method comprises centrifuging (such as centrifuging at 12000rpm for 20 min) to obtain precipitate, washing with water, and lyophilizing.
In another embodiment of the present invention, the nanoparticles are used for preparing active targeting drugs.
The medicament can be a vaccine, and specifically can be a nano vaccine.
Hair brushIn still another embodiment of the invention, a dextran nano-vaccine based on high affinity Siglec-1 ligand modification with active targeting is provided TCC Sia-Ace-Dex-PA-Rd NPs, which are at least formed by connecting high-affinity Siglec-1 ligand, protein Antigen (PA) and an immune agonist Rasimotene to the nanoparticles;
the structure is as follows:
specifically, the Protein Antigen (PA) and the immune agonist (Rd) are combined with the azide modified partially oxidized acetalized glucan nanoparticle (Oxi-Ace-Dex-Az NPs) through imine bond formation; then the obtained nano particles are mixed with 9-N- (4H-thieno [3,2-c ]]chromene-2-carbamoyl) -Neu5Ac (Sia) alpha 2-3Gal beta 1-4GlcNAc beta Pro-DBCO (referred to as TCC Sia-LacNAc-DBCO) to produce TCC Sia-Ace-Dex-PA-Rd NPs; among them, high affinity Siglec-1 ligands TCC Sia-LacNAc can actively target sialic acid binding immunoglobulin-like lectin 1 (Siglec-1, cd169) on macrophages.
It should be noted that the Protein Antigen (PA) can be adjusted according to the target protein, such as selecting a suitable protein antigen for a specific tumor cell, or selecting a suitable new coronaviruses antigen (RBD/N) for example, and the high affinity Siglec-1 ligand and the immune agonist can be appropriately adjusted and substituted, which are clearly within the scope of the present application without creative efforts based on the inventive concept of the present application.
In still another embodiment of the present invention, the present invention relates to a novel corona recombinant protein vaccine prepared based on azide-modified partially oxidized acetalized dextran nanoparticles TCC Sia-Ace-Dex-N (SARS-CoV-2 nucleocapsid protein) -Rd NPs- TCC The Sia-Ace-Dex-RBD (SARS-CoV-2 spike protein receptor structure domain RBD,321-591 aa) -Rd NPs generate stronger T cell immune response in mice and rabbits by single and combined useShould be combined with high titers of IgG antibodies, and the antibodies induced from the rabbits efficiently neutralized authentic SARS-CoV-2 virus infected Vero E6 cells.
In a specific aspect, the present invention provides a method for producing, TCC inoculation of Sia-Ace-Dex-N-Rd NPs alone caused a strong N protein specific cytotoxic T lymphocyte killing (CTL) response and high titers of anti-N protein IgG antibodies, TCC after single inoculation of Sia-Ace-Dex-RBD-Rd NPs, high-titer anti-RBD IgG antibodies are induced in mice and rabbits, and the combination of the two vaccines can generate higher anti-RBD IgG antibody titer than that of the vaccine TCC Sia-Ace-Dex-N-Rd NPs produce stronger N protein specific CTL responses.
Obviously, the nano-vaccine may further comprise any other pharmaceutically acceptable non-pharmaceutical active ingredients, especially those used in vaccines, such as commonly used carriers, excipients, diluents, etc., which may be included as well known in the art and which can be determined by one of ordinary skill in the art to meet clinical standards. The pharmaceutical composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup and spray, external preparations, suppositories and sterile injectable solutions according to a usual method, and is not particularly limited.
In another embodiment of the present invention, there is provided a method for synthesizing the dextran nano vaccine, wherein the synthetic route is as follows:
specifically, the synthesis steps are as follows:
s1, adding a Protein Antigen (PA) into a buffer solution containing azide-modified partially oxidized acetalized dextran nanoparticles (Oxi-Ace-Dex-Az NPs); reacting to obtain a mixture A5;
s2, adding Rasimethide (R848, rd) and Rasimethide into the mixture A5 obtained in the step S1 TCC Sia-LacNAc-DBCO, stirring to obtain a mixture A6;
s3, purifying the mixture A6 obtained in the step S2 to obtain the compound TCC Sia-Ace-Dex-PA-Rd NPs。
Wherein, in the step S1, the buffer solution can be PBS buffer solution (0.1M, pH7.4);
the mass ratio of the nanoparticle to the protein antigen is 10 to 5, preferably 5; the reaction is carried out for 1 to 10 hours at room temperature, and preferably for 5 hours.
In the step S2, the Rasimotent, TCC The mass ratio of Sia-LacNAc-DBCO to the nanoparticles in the step S1 is 1-4; preferably 1; the stirring is in particular overnight at room temperature.
The above-mentioned TCC Sia-LacNAc-DBCO, high affinity Siglec-1 ligand 9-N- (4H-thieno [3,2-c ]]chromene-2-carbamoyl) -Neu5Ac (Sia) α 2-3Gal β 1-4GlcNAc β pro-DBCO, having the formula:
the synthetic route is as follows:
s2-1, adding MgCl 2 Adding a compound 1, UDP-galactose into a Tris-HCl buffer solution, adding beta 1, 4-galactosyltransferase (Lgtb) into a reaction system, and reacting and purifying to obtain a product 2;
s2-2, adding the product 2 obtained in the step S2-1 into a solution containing MgCl 2 Compound 3 and cytidine-5' -triphosphate were added to the Tris-HCl buffer of (1), and then CMP sialic acid synthetase (NmCSS) and α 2, 3-sialyltransferase (PmST 1) were added to the reaction solution; after reaction, a white solid is obtained after purification, namely a product 4;
s2-3, dissolving the product 4 obtained in the step S2-2 in water, and then adding a compound 5 dissolved in Tetrahydrofuran (THF); adding triethylamine into the reaction solution, stirring at room temperature for reaction, and completely purifying the reaction to obtain a white solid product 6;
s2-4, dissolving the product 6 obtained in the step S2-3 in water, and then adding Pd/C and MeOH; the reaction mixture is reacted in H 2 Stirring under the atmosphere until the reaction is complete; then filtering to remove Pd/C, and concentrating to obtain a product 7;
s2-5, dissolving a product 7 obtained in the step S2-4 in water, adding a compound 8 dissolved in THF, and adding triethylamine into a reaction mixture; stirring at room temperature until the reaction is complete, purifying the mixture after solvent removal by column chromatography (BioGel P-2 gel column chromatography) to obtain white solid as product TCC Sia-LacNAc-DBCO。
Wherein in the step S2-1, the mass ratio of the compound 1, UDP-galactose and galactosyltransferase is 50-80, preferably 59.4 2 In Tris-HCl buffer (g) of MgCl 2 The concentration is controlled to be 10-50mM, preferably 20mM; the reaction is carried out in particular at 30-40 ℃ and preferably 37 ℃ overnight at a speed of 80-150rpm and by thin layer chromatography (developer: etOAc/MeOH/H) 2 O/HOAc = 12); the purification method specifically comprises the step of subjecting the reaction residue to BioGel P-2 gel column chromatography (H) 2 O elution), DEAE-Sepharose (0.02M NaCl solution elution), bioGel P-2 gel second purification.
In the step S2-2, the molar mass ratio of the product 2, the compound 3, the cytidine-5' -triphosphate, the NmCSS and the PmST1 is 0.1mmol:40-50mg:80-100mg:0.1-0.5mg:0.1-0.5mg, preferably 0.1mmol:46.2mg:91.5mg:0.3mg:0.38mg; the reaction is carried out in particular at 30-40 ℃ and preferably 37 ℃ overnight at a speed of 80-150rpm and by thin layer chromatography (developer: etOAc/MeOH/H) 2 O/HOAc = 9); the purification method comprises removing the enzyme precipitate by centrifugation (8000 rpm/min, 30 min) after standing on ice for 0.5 h; the supernatant was concentrated and subjected to BioGel P-2 gel column chromatography (using H) 2 O elution), DEAE-Sepharose (elution with 0.05M NaCl solution) and BioGel P-2 gelPerforming gel column chromatography to obtain a white solid, namely a product 4;
in the step S2-3, the mass-to-volume ratio of the product 4, the compound 5 and triethylamine is 100mg:50-80mg:100 μ L, preferably 100mg:64mg:100 mu L, and the stirring reaction time is controlled to be 1-3h, preferably 1.5h; the purification method comprises removing solvent from the reaction solution by rotary evaporation and purifying by BioGel P-2 gel column chromatography.
In the step S2-4, the mass-to-volume ratio of the product 6, pd/C and MeOH is 100mg:50mg:1-5mL, preferably 100mg:50mg:2mL.
In the step S2-5, the mass-to-volume ratio of the product 7, the compound 8 and triethylamine is 80-100mg:50-70mg:80-100 μ L, preferably 94.4mg:60mg:100 mu L of the solution; stirring and reacting for 0.2-1h at room temperature.
In the step S3, the specific purification method includes obtaining a precipitate by centrifugation (12000rpm, 20min), washing with water, performing ultrafiltration with water, and lyophilizing.
In another embodiment of the present invention, the dextran nano-vaccine is used for preparing a medicament for preventing and/or treating diseases.
The disease may include any disease, particularly related diseases causing immune system problems (e.g., low or high immune state) and thus may be prevented and/or treated by artificially enhancing or suppressing the immune function of the body. In one embodiment of the invention, the disease may be in particular tumor and neocoronary pneumonia; wherein tumors are used in the present invention as known to those skilled in the art, which include benign tumors and/or malignant tumors. Benign tumors are defined as cellular hyperproliferation that fails to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs). Examples of such malignancies include solid tumors and hematological tumors. And is not particularly limited herein.
In another embodiment of the present invention, there is provided a method for tumor prevention and/or treatment, the method comprising: administering the dextran nano-vaccine described above to a subject.
In another embodiment of the present invention, there is provided a method for preventing and/or treating a new coronary pneumonia, the method comprising: administering the dextran nano-vaccine described above to a subject.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
An azido-modified partially oxidized acetalized dextran nanoparticle (Oxi-Ace-Dex-Az NPs) and a preparation method thereof are prepared.
The structure and the preparation method are as follows:
the preparation of Oxi-Ace-Dex-Az NPs comprises the following steps:
step one, 1g of glucan (Dex, molecular weight: 9000-11000g mol) -1 ) Dissolved in 10mL of ultra-dry dimethyl sulfoxide, and then 0.7g of synthesized CDI-TEG-Azide and 1g of DMAP were added. The reaction was carried out at room temperature for 2 days under argon protection. Then the reaction solution was dialyzed, during which time water was changed 4-5 times. And then the solution is freeze-dried to obtain the azide modified glucan (Dex-Az) polymer A1.
Step two, dissolve polymer A1 (1 g) obtained in step one in 10mL water. Then 0.22g of sodium periodate was added. After stirring the reaction at room temperature for 5h, the reaction solution was dialyzed to remove small molecules. The white solid obtained by subsequent freeze-drying is the azide-modified partially oxidized dextran (Oxi-Dex-Az) polymer A2.
Step three, the polymer A2 (1 g) obtained in step two and DMSO (10 mL) were added to the flask. And p-toluenesulfonic acid pyridine (15.6 mg) and 2-ethoxypropene (3.2 mL) were added. After the reaction mixture was stirred at room temperature for 3 hours, triethylamine (1 mL) was added to quench the reaction. Then, the reaction solution was dropped into 100mL of ultrapure water to precipitate. Centrifuging at 12000rpm for 20min, washing the precipitate with ultrapure water (50 mL × 2), and lyophilizing to obtain azide-modified partially oxidized acetalized dextran (Oxi-Ace-Dex-Az) polymer A3.
Step four, 20mg of the polymer A3 obtained in step three was dissolved in 1mL of methylene chloride. It was added dropwise to 2mL of aqueous poly (vinyl alcohol) (PVA, molecular weight: 13-23kg mol) -1 3% w/w), and subjected to ultrasonic treatment in ice bath for 1min to obtain a primary emulsion A4.
Step five, the emulsion A4 obtained in step four was added to water (10 mL) of PVA at 0.3% (w/w). Stirring for 3h at room temperature to remove dichloromethane in the emulsion, centrifuging (12000rpm, 20min), washing the obtained precipitate with ultrapure water (3X 20 mL) for three times, and freeze-drying to obtain the azide-modified partially-oxidized acetalized dextran nanoparticles (Oxi-Ace-Dex-Az NPs).
Wherein the structure of the CDI-TEG-Azide required in the step one is as follows:
the CDI-TEG-Azide synthesis steps are as follows:
into a flask were charged triazo triethylene glycol (0.5 g, 2.85 mmol), molecular sieves (0.6 g, molecular sieves, etc.),
) And tetrahydrofuran THF (8 mL). Under the protection of argon, 1' -carbyldiimidazole (CDI, 32g, 3.2mmol) was added. After stirring and reacting for 30min, filtering to remove the molecular sieve. The solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate (50 mL), washed with ammonium chloride (10%, 50 mL), then brine (50 mL), and dried over anhydrous sodium sulfate. After removal of the solvent by rotary evaporation, CDI-TEG-Azide (0.7g, 2.6mmol, 91%) was obtained.
Example 2
A high affinity Siglec-1 ligand (9-N- (4H-thieno [3, 2-c) based on the nanoparticles described in example 1, modified to actively target sialic acid binding immunoglobulin-like lectin 1 (Siglec-1, CD169) on macrophages, was prepared]chromene-2-carbamoyl)-Neu5Ac(Sia)α2-3Galβ1-4GlcNAc is called TCC Sia-LacNAc), a new crown protein antigen (RBD protein or N protein) and an immune agonist Rasimethide (R848, rd), and the dextran nano vaccine with active targeting property based on high affinity Siglec-1 ligand modification comprises vaccine I (I) TCC Sia-Ace-Dex-N-Rd NPs) and vaccine II (B: (B) TCC Sia-Ace-Dex-RBD-Rd NPs) and a preparation method thereof.
The structure and the preparation method are as follows:
TCC Sia-Ace-Dex-N-Rd NPs/ TCC the preparation of the Sia-Ace-Dex-RBD-Rd NPs comprises the following steps:
step one, 2mg of neocoronin antigen (N protein or RBD protein) was added to a PBS (0.1M, pH 7.4) solution containing 10mg of azide-modified partially oxidized acetalized dextran nanoparticles (Oxi-Ace-Dex-Az NPs). Reacting at room temperature for 5h to obtain a mixture A5;
step two, adding 2mg of Rasimethide (Rd) and 2mg of Rasimethide (Rd) into the mixture A5 obtained in the step one TCC Sia-LacNAc-DBCO, and then stirring at room temperature overnight to obtain a mixture A6;
step three, the mixture A6 obtained in the step two is centrifuged (12000rpm, 20min) to obtain a precipitate, and the precipitate is washed 3 times with ultrapure water (3X 20 mL). Then ultra-filtering the obtained granules with ultrapure water, and freeze-drying to obtain the product TCC Sia-Ace-Dex-N-Rd NPs/ TCC Sia-Ace-Dex-RBD-Rd NPs。
Wherein, the invention provides the high affinity Siglec-1 ligand (9-N- (4H-thieno [3,2-c ]) used in the second step]chromene-2-carbamoyl) -Neu5Ac (Sia) alpha 2-3Gal beta 1-4GlcNAc beta pro-DBCO, named as TCC Sia-LacNAc-DBCO) has a structural formula:
TCC the synthesis route of Sia-LacNAc-DBCO is as follows:
TCC the preparation of Sia-LacNAc-DBCO comprises the following steps:
step one, adding MgCl 2 Compound 1 (59.4 mg), UDP-galactose (UDP-Gal, 158.6 mg) was added to Tris-HCl buffer (100 mM, pH 7.5,10 mL) (20 mM). Beta 1, 4-galactosyltransferase (Lgtb, 440. Mu.g) was then added to the reaction. The reaction was kept in a shaker at 37 ℃ and shaken overnight at 100 rpm. By thin layer chromatography (developing solvent: etOAc/MeOH/H) 2 O/HOAc = 12. After the reaction was complete, an equal volume of cold ethanol (EtOH) was added to stop the reaction. The residue was chromatographed on a BioGel P-2 gel column (H) 2 O elution), DEAE-Sepharose (DEAE Sepharose fast flow,0.02M NaCl solution elution), and BioGel P-2 gel for the second purification to obtain product 2;
step two, adding the product 2 (46.6 mg, 0.1mmol) obtained in the step one into MgCl 2 Tris-HCl buffer (100 mM, pH 8.5,10 mL) (20 mM), compound 3 (46.2 mg) and cytidine-5' -triphosphate (CTP, 91.5 mg) were added. Then NmCSS (300. Mu.g) and PmST1 (380. Mu.g) were added to the reaction solution. The reaction mixture was kept in a shaker at 37 ℃ and shaken overnight at 100 rpm. By thin layer chromatography (developing solvent: etOAc/MeOH/H) 2 O/HOAc = 9). When the reaction was complete, an equal volume of cold ethanol (EtOH) was added. After standing on ice for 0.5 hour, the enzyme precipitate was removed by centrifugation (8000 rpm/min, 30 minutes). The supernatant was concentrated and subjected to BioGel P-2 gel column chromatography (using H) 2 O elution), DEAE-sepharose (elution with 0.05M NaCl solution) and BioGel P-2 gel column chromatography to obtain white solid, namely product 4;
step three, dissolving the product 4 (100 mg) obtained in the step two in H 2 In the presence of O (2 mL),compound 5 (64 mg) in THF (2 mL) was then added. Then triethylamine (100 μ L) was added to the reaction solution and stirred at room temperature for 1.5 hours. After completion of the reaction, the solvent of the reaction solution was removed by rotary evaporation and purified by BioGel P-2 gel column chromatography to provide product 6 as a white solid;
step four, dissolve product 6 from step three (100 mg) in 2mL water and add Pd/C (50 mg) and MeOH (2 mL). Reaction mixture is reacted in H 2 Stirring under atmosphere until the reaction is complete. Then, pd/C was removed by filtration. Concentrating to obtain a product 7;
step five, dissolve product 7 from step four (94.4 mg) in water (3 mL) and add compound 8 (60 mg) in THF (3 mL). Triethylamine (100. Mu.L) was then added to the reaction mixture. Stirring at room temperature for 0.5 hr to complete reaction, and purifying the mixture with the solvent removed by BioGel P-2 gel column chromatography to obtain white solid as product TCC Sia-LacNAc-DBCO。
Example 3
The application and immune effect of the novel crown recombinant protein vaccine prepared based on the azide-modified partially oxidized acetalized dextran nanoparticles are proved by specific experiments as shown in example 2 TCC Sia-Ace-Dex-N-Rd NPs/ TCC Sia-Ace-Dex-RBD-Rd NPs are taken as an example:
1. the new crown recombinant protein vaccine prepared based on the azide-modified partially oxidized acetalized glucan nanoparticle provided by the invention TCC Analysis of immune effect induced by Sia-Ace-Dex-N-Rd NPs in mice:
c57BL/6 mice were immunized subcutaneously with free N protein and vaccine one (day 0, day 7, respectively) TCC Sia-Ace-Dex-N-Rd NPs). All injected vaccines contained the same dose of N protein (50. Mu.g). Spleens were isolated from uninmmunized C57BL/6 mice at day 14 to prepare single cell suspensions. Half of the spleen cells (2X 10) 7 2 mL) was labeled with 1. Mu.M CFSE and placed in CO 2 Staining was performed for 10 min in a cell incubator. Removing free CFSE by centrifugation at 1600rpm for 3 min to obtain CFSE lo N 219-227 - Spleen cells. The other half of spleen is thinCell (2X 10) 7 2 mL) was labeled with 10. Mu.M CFSE, followed by the N of the LALLLLDRL sequence 219-227 (1μg·mL -1 ) Incubation with cells for 1h to obtain CFSE hi N 219-227 + Spleen cells. Then, CFSE is conducted lo N 219-227 - And CFSE hi N 219-227 + (1:1,0.1mL,2×10 6 ) The mixed cells were injected intravenously through the mouse tail into untreated and vaccine immunized mice. After 1 day, mouse spleen and lymph nodes were prepared as single cell suspensions for FACS analysis.
The results are shown in fig. 1, and compared with the control group, the novel crown recombinant protein vaccine prepared by the azide-modified partially oxidized acetalized dextran nanoparticle provided by the invention (i), (ii) TCC Sia-Ace-Dex-N-Rd NPs) produces a significantly enhanced T cell killing effect in vivo.
On days 0, 14 and 28, C57BL/6 mice were immunized subcutaneously with free N protein and vaccine one TCC Sia-Ace-Dex-N-Rd NPs). Sera were collected at day-1 (pre-immunization) and day 35 for subsequent ELISA experiments to determine anti-N protein IgG antibody titers produced by the vaccine in mice. The results are shown in fig. 2, and compared with the control group, the novel crown recombinant protein vaccine prepared by the azide-modified partially oxidized acetalized dextran nanoparticle provided by the invention (i), (ii) TCC Sia-Ace-Dex-N-Rd NPs) resulted in higher titers of anti-N protein IgG antibody titers in the mice immunized.
2. The invention provides a new crown recombinant protein vaccine I (a) prepared based on an azide-modified partially oxidized acetalized glucan nanoparticle TCC Sia-Ace-Dex-N-Rd NPs) in rabbits:
on days 0, 14 and 28, rabbits were immunized subcutaneously with free N protein and vaccine one (C) TCC Sia-Ace-Dex-N-Rd NPs) (N =3 per group). Respectively. Sera were collected at-1, and 35 days to determine anti-N protein IgG antibody titers in rabbit sera. The results are shown in fig. 3, and compared with the control group, the novel crown recombinant protein vaccine prepared from the azide-modified partially oxidized acetalized dextran nanoparticle provided by the present invention: (a) TCC Sia-Ace-Dex-N-Rd NPs) resulted in higher titers of anti-N protein IgG antibody in rabbits.
3. The invention provides a novel crown recombinant protein vaccine prepared from an azide-modified partially oxidized acetalized glucan nanoparticle TCC Analysis of immune effect induced by Sia-Ace-Dex-RBD-Rd NPs in mice:
c57BL/6 mice were immunized subcutaneously with free RBD protein and vaccine two on days 0, 14 and 28 TCC Sia-Ace-Dex-RBD-Rd NPs). Sera were collected at day-1 (pre-immunization) and day 35 for subsequent ELISA experiments to determine the anti-RBD protein IgG antibody titer produced by the vaccine in mice. The results are shown in fig. 4, and compared with the control group, the new crown recombinant protein vaccine prepared by the partially oxidized acetalized dextran nanoparticle based on azide modification provided by the invention is two ( TCC Sia-Ace-Dex-RBD-Rd NPs) produces higher titers of anti-RBD protein IgG antibody.
4. The invention provides a new crown recombinant protein vaccine II (B) prepared based on an azide-modified partially oxidized acetalized glucan nanoparticle TCC Sia-Ace-Dex-RBD-Rd NPs) in vivo immune effect analysis induced in rabbits:
rabbit was immunized subcutaneously with free RBD protein and vaccine two on days 0, 14 and 28 TCC Sia-Ace-Dex-RBD-Rd NPs) (n =3 per group). Respectively. Sera were collected at-1, and 35 days to determine anti-RBD protein IgG antibody titers in rabbit sera. The results are shown in fig. 6, and compared with the control group, the new crown recombinant protein vaccine prepared by the partially oxidized acetalized dextran nanoparticle based on azide modification provided by the invention is two ( TCC Sia-Ace-Dex-RBD-Rd NPs) gave higher titers of anti-RBD protein IgG antibody in rabbits.
5. The invention provides a new crown recombinant protein combined vaccine III (C) prepared based on an azide-modified partially oxidized acetalized glucan nanoparticle TCC Sia-Ace-Dex-N-Rd NPs+ TCC Sia-Ace-Dex-RBD-Rd NPs) in combination with an analysis of the immune effect induced in mice:
respectively on day 0 and day 7C57BL/6 mice are immunized subcutaneously with free N protein, vaccine I and combined vaccine III. All injected vaccines contained the same dose of N protein. Spleens were isolated from uninmmunized C57BL/6 mice at day 14 to prepare single cell suspensions. Half of the spleen cells (2X 10) 7 2 mL) was labeled with 1. Mu.M CFSE and placed in CO 2 Staining was performed for 10 min in a cell incubator. Removing free CFSE by centrifugation at 1600rpm for 3 min to obtain CFSE lo N 219-227 - Spleen cells. The other half of the spleen cells (2X 10) 7 2 mL) was labeled with 10. Mu. MCFSE followed by the N of the LALLLLDRL sequence 219-227 (1μg·mL -1 ) Incubation with cells for 1h to obtain CFSE hi N 219-227 + Spleen cells. After that, the CFSE is connected lo N 219-227 - And CFSE hi N 219-227 + (1:1,0.1mL,2×10 6 ) The mixed cells were injected intravenously through the mouse tail into untreated and vaccine immunized mice. After 1 day, mouse spleen and lymph nodes were prepared as single cell suspensions for FACS analysis.
The results are shown in fig. 5, and compared with the control group and vaccine one, the novel crown recombinant protein combination vaccine three (based on the azide-modified partially oxidized acetalized dextran nanoparticle provided by the invention) is prepared TCC Sia-Ace-Dex-N-Rd NPs+ TCC Sia-Ace-Dex-RBD-Rd NPs) produces a significantly enhanced T cell killing effect in vivo.
We immunized C57BL/6 mice subcutaneously with free RBD protein, vaccine two, and combination vaccine three, again on days 0, 14, and 28. Sera were collected at day-1 (pre-immunization) and day 35 for subsequent ELISA experiments to determine the anti-RBD protein IgG antibody titer produced by the vaccine in mice. The results are shown in fig. 4, and compared with the control group and the vaccine two, the novel crown recombinant protein combination vaccine three (a) prepared by the azide-modified partially oxidized acetalized dextran nanoparticle provided by the invention TCC Sia-Ace-Dex-N-Rd NPs+ TCC Sia-Ace-Dex-RBD-Rd NPs) produces higher titers of anti-RBD protein IgG antibody.
6. The invention provides azide-modified partial oxidation-based condensationNew crown recombinant protein vaccine prepared from hydroformylation glucan nanoparticles II (B) TCC Sia-Ace-Dex-RBD-Rd NPs) on the virus neutralizing capacity:
to determine live virus neutralization titers. Serum antibodies obtained after immunization of rabbits with vaccine two were serially diluted in DMEM containing FBS (2.5%) and mixed with an equal volume of the virus suspension. After 1 hour incubation, the mixture was added to 24-well plates containing Vero E6 cells and incubated for an additional 1 hour. The inoculum was replaced with DMEM containing FBS (2.5%) and carboxymethyl cellulose (0.9%). Plates were fixed with paraformaldehyde (8%) and stained with 0.5% crystal violet after 3 days. Plaque reduction neutralization titers were calculated using the "inhibitor versus normalized response (variable slope)" model in GraphPad (Prism 8.0) software. The cut-off value (geometric mean +3 times geometric standard deviation) was calculated from the negative control. As shown in FIG. 7, the new crown recombinant protein vaccine prepared by the partially oxidized acetalized dextran nanoparticle based on azide modification provided by the invention is II ( TCC Sia-Ace-Dex-RBD-Rd NPs) can effectively neutralize the euvirus.
7. The invention provides a new crown recombinant protein vaccine I (a) prepared based on an azide-modified partially oxidized acetalized glucan nanoparticle TCC Sia-Ace-Dex-RBD-Rd NPs) in vivo toxicity evaluation analysis:
to evaluate the biotoxicity of the new crown recombinant protein vaccine one prepared based on the azide-modified partially oxidized acetalized dextran nanoparticles provided by the present invention, rabbits were immunized subcutaneously with the vaccine on days 0, 14, and 28. On day 35, three rabbit organs were removed, sectioned and stained with hematoxylin/eosin. As shown in fig. 8, histological analysis showed that no significant damage was observed with these slides. The new crown recombinant protein vaccine prepared by the azide-modified partially oxidized acetalized dextran nanoparticles has lower biological toxicity and can be safely applied.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. An ethoxylated acetalized dextran nanoparticle (Oxi-Ace-Dex-AzNPs) with partially oxidized azide groups, characterized in that the schematic structure of the nanoparticle is as follows:
2. the method for synthesizing the nano-particles as described in claim 1, wherein the synthetic route is as follows:
3. the synthetic method according to claim 2, comprising the specific steps of:
s1, dissolving glucan in dimethyl sulfoxide, adding CDI-TEG-Azide and 4-dimethylaminopyridine DMAP into the solution, and reacting in an inert atmosphere; purifying to obtain azido modified glucan (Dex-Az) A1;
s2, dissolving the polymer A1 obtained in the S1 in water, and then adding sodium periodate; stirring for reaction, and purifying to obtain white solid, namely azide-modified partially oxidized dextran (Oxi-Dex-Az) A2;
s3, mixing the polymer A2 prepared in the step S2 with dimethyl sulfoxide (DMSO), adding p-toluenesulfonic acid pyridine and 2-ethoxy propylene, stirring the reaction solution at room temperature for reaction, adding a quenching agent for quenching reaction, and purifying the product to obtain an azide-modified partially oxidized acetalized glucan (Oxi-Ace-Dex-Az) polymer A3;
s4, dissolving the polymer A3 obtained in the S3 in dichloromethane, adding the dichloromethane into aqueous poly (vinyl alcohol), and performing low-temperature ultrasound to obtain a primary emulsion A4;
and S5, continuously adding the emulsion A4 obtained in the S4 into the aqueous PVA, stirring, and purifying to obtain the azide-modified partially-oxidized acetalized glucan nano-particles (Oxi-Ace-Dex-Az NPs).
4. The method of claim 3, wherein in step S1, the molecular weight of the glucan is controlled to 9000-11000g mol -1 (ii) a The mass ratio of the glucan to the CDI-TEG-Azide to the 4-dimethylaminopyridine is 1; the reaction condition of the reaction under the inert atmosphere is specifically that the reaction is carried out for 1 to 3 days at room temperature;
the specific purification method comprises the following steps: dialyzing the reaction solution, and changing water for 4-5 times; then freeze-drying the solution;
in the step S2, the mass ratio of the sodium periodate to the polymer A1 is 0.1-0.5; the stirring reaction is specifically stirring reaction at room temperature for 1-10h; the specific purification method comprises the following steps: dialyzing the reaction solution to remove small molecules, and freeze-drying the solution to obtain the compound;
in the step S3, the mass-to-volume ratio of the polymer A2 to the p-toluenesulfonic acid pyridine to the 2-ethoxypropylene is 1g; the quenching agent is triethylamine; adding the reaction solution into water to separate out a precipitate, wherein the precipitate is obtained by centrifugation and is obtained by washing and freeze-drying;
in step S4, the mass to volume ratio of the polymer A3 to the aqueous poly (vinyl alcohol) is 10 mg; the molecular weight of the poly (vinyl alcohol) in the aqueous poly (vinyl alcohol) is 13-23kg mol -1 The content of the poly (vinyl alcohol) is controlled to be 1-5 percent，w/w；
Specifically, the low-temperature ultrasound is carried out for 0.5-5min by adopting ice bath ultrasound;
in the step S5, the molecular weight of the poly (vinyl alcohol) in the aqueous poly (vinyl alcohol) is 13 to 23kg mol -1 The content of the poly (vinyl alcohol) is controlled to be 1-5 percent, w/w; and stirring for 1-5h at room temperature, wherein the specific purification method comprises centrifuging to obtain precipitate, washing with water, and lyophilizing.
5. The application of the nanoparticle of claim 1 in preparing drugs with active targeting; the medicament is a vaccine.
6. Active targeting glucan nano vaccine modified based on high-affinity Siglec-1 ligand TCC Sia-Ace-Dex-PA-Rd NPs obtainable by linking at least a high affinity Siglec-1 ligand to a nanoparticle according to claim 1 together with a protein antigen and an immune agonist.
7. The glucan nanoconjugate claimed in claim 6, having the structure:
the protein antigen PA and the immune agonist Rd are combined with the azide modified partially oxidized acetalized glucan nanoparticle Oxi-Ace-Dex-Az NPs through imine bond formation; then the obtained nano particles are mixed with 9-N- (4H-thieno [3,2-c ]]chromene-2-carbamoyl) -Neu5Ac (Sia) alpha 2-3Gal beta 1-4GlcNAc beta Pro-DBCO (referred to as TCC Sia-LacNAc-DBCO) by promoting an azoalkyne cycloaddition reaction to produce TCC Sia-Ace-Dex-PA-Rd NPs。
8. The method for synthesizing the glucan nano vaccine as claimed in claim 6 or 7, wherein the synthetic route is as follows:
specifically, the synthesis steps are as follows:
s1, adding a Protein Antigen (PA) into a buffer solution containing azide-modified partially oxidized acetalized glucan nanoparticles (Oxi-Ace-Dex-Az NPs), and reacting to obtain a mixture A5;
s2, adding Rasimethide (R848, rd) and Rasimethide into the mixture A5 obtained in the step S1 TCC Sia-LacNAc-DBCO, stirring to obtain a mixture A6;
s3, purifying the mixture A6 obtained in the step S2 to obtain the compound TCC Sia-Ace-Dex-PA-Rd NPs。
9. The method of claim 8, wherein in step S1, the buffer solution is PBS buffer (0.1M, pH 7.4);
the mass ratio of the nanoparticles to the protein antigen is 10-5; the reaction is specifically carried out for 1-10h at room temperature;
in the step S2, the Rasimotent, TCC The mass ratio of Sia-LacNAc-DBCO to the nanoparticles in the step S1 is 1-4; the stirring is carried out at room temperature overnight;
the above-mentioned TCC Sia-LacNAc-DBCO, high affinity Siglec-1 ligand 9-N- (4H-thieno [3,2-c ]]chromene-2-carbamoyl) -Neu5Ac (Sia) alpha 2-3Gal beta 1-4GlcNAc beta pro-DBCO, the synthetic route of which is shown below:
in the step S3, the specific purification method comprises the steps of obtaining a precipitate through centrifugation, washing the precipitate with water, performing ultrafiltration by using water, and performing freeze-drying.
10. Use of the glucan nano-vaccine of claim 6 or 7 in the manufacture of a medicament for the prevention and/or treatment of disease; the disease includes tumor and neocoronary pneumonia.
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