A method for the synthesis of spherical polymer nanoparticles
By synthesizing spherical polymer nanoparticles through the polymerization reaction of ascorbic acid, ethylenediamine, and glyoxal, the problems of antigen delivery and B cell activation of nanoparticle vaccine adjuvants in subunit vaccines were solved, achieving highly efficient enhancement of immune response.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JILIN UNIVERSITY
- Filing Date
- 2023-10-08
- Publication Date
- 2026-06-23
AI Technical Summary
Existing nanoparticle vaccine adjuvants are inefficient at delivering antigens and activating antigen-presenting cells in subunit vaccines, resulting in insufficient strength, breadth, and duration of the immune response.
Spherical polymer nanoparticles were synthesized by polymerization reaction using ascorbic acid, ethylenediamine, and glyoxal as monomers. These nanoparticles serve as novel vaccine adjuvants, enabling efficient antigen delivery and specific activation of B cells.
It achieves efficient antigen delivery and B cell activation, enhancing the strength, breadth, and persistence of the immune response, and has advantages in biosafety and low cost.
Smart Images
Figure CN117603417B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of vaccine technology, specifically a method for synthesizing spherical polymer nanoparticles. Background Technology
[0002] The discovery and widespread use of vaccines is a crucial means for humanity to combat disease. Prevention is the primary method for controlling infectious diseases, and vaccination is the most effective measure. Traditional vaccines include live attenuated vaccines, inactivated vaccines, and toxoid vaccines, which have achieved large-scale industrial production and administration. However, traditional vaccines often have limitations in terms of success rate and duration of protection, especially against certain pathogens. With the continuous development of science and technology, subunit vaccines have gradually become a focus of scientific research. Subunit vaccines consist of antigens and adjuvants. Antigens are the effective components of the immune response, containing certain components derived from pathogenic bacteria, parasites, or viruses, and are generally highly purified proteins or synthetic peptides. Because the antigen alone in a subunit vaccine is insufficient to produce high immunogenicity, non-immunogenic materials called adjuvants are usually incorporated into the vaccine formulation to improve the immune response and enhance vaccine efficacy. Potential adjuvants range from synthetic small molecule compounds to complex natural extracts, particulate materials, and nanomaterials.
[0003] The main functions of adjuvants are immune stimulation and antigen delivery. Immune stimulation refers to the adjuvant's role in promoting the generation of antigen and co-stimulatory signals by targeting Toll-like receptors and other pattern recognition receptors, leading to the maturation and activation of antigen-presenting cells and thus enhancing adaptive immune responses. On the other hand, adjuvants can increase the bioavailability of antigens during their presentation to lymph nodes or other antigen-presenting cells, thereby improving the effective pickup of antigens by immune organs and tissues in vivo. Excellent and appropriate adjuvants can significantly enhance the strength, breadth, and persistence of immune responses in preclinical and clinical studies.
[0004] Effective immune responses to pathogens induced by subunit vaccines depend primarily on antibodies produced by effector B cells. Germinal centers are hubs for B cell clonal expansion, somatic hypermutation, affinity selection, and the production of high-affinity antibodies. However, satisfactory antigen delivery to lymph nodes and activation of antigen-presenting cells remain major challenges for subunit vaccines. Presenting protein antigens on nanoparticle surfaces is a highly effective approach because nanoparticles can more efficiently transport and accumulate in different compartments of lymph nodes, and multivalent antigens presented on nanoparticle surfaces can more effectively participate in and cross-link with low-affinity B cell receptors on B cells. Furthermore, B cells can be used instead of dendritic cells for antigen presentation to initiate T cell responses, thereby forming germinal centers.
[0005] Reported nanoparticles can now be broadly classified into two categories: self-assembled protein nanoparticles, such as ferritin, heat shock proteins, or the E2 subunit of pyruvate dehydrogenase complexes, and synthetic nanoparticles, such as lipid-based, inorganic, and polymeric nanoparticles. Both types of nanoparticles exhibit enhanced immunogenicity compared to soluble antigens, offering protection against a variety of diseases. However, these nanoparticle-based vaccines typically require immunization with additional adjuvants, such as AddaVax, ISCOMATRIX, and Sigma adjuvant systems, the mechanisms by which these adjuvants and nanovaccines induce germinal center responses remain poorly understood. Therefore, it is crucial to design ideal adjuvants capable of simultaneously achieving efficient antigen delivery and activating antigen-presenting cells to specifically target variable and complex pathogens, resulting in potent, broad, and durable immune responses. Summary of the Invention
[0006] The purpose of this invention is to provide a method for synthesizing spherical polymer nanoparticles to solve the problems mentioned in the background art.
[0007] To achieve the above objectives, the present invention provides the following technical solution:
[0008] A method for synthesizing spherical polymer nanoparticles, the method being based on a polymerization reaction using ascorbic acid, ethylenediamine and glyoxal as monomers;
[0009] Includes the following steps:
[0010] Step S100: Dissolve the polymer stabilizer in the reaction solvent to obtain a clear and transparent mixed solution, and stir for half an hour;
[0011] Step S200: Add glyoxal to the mixed solution from step S100 and stir for half an hour;
[0012] Step S300: Add ascorbic acid to the mixed solution from step S200 and stir for half an hour until the solution becomes clear and transparent again.
[0013] Step S400: Add ethylenediamine to the mixed solution from step S300, open the container and stir for half an hour. The solution color gradually changes from light yellow to brownish-yellow and wine red.
[0014] Step S500: Transfer the solution obtained in step S400 into the reaction vessel and place the reaction vessel into an oven;
[0015] Step S600: Transfer the solution and solid in the reaction vessel of step S500 to a centrifuge tube, centrifuge at 18,000 rpm for 5 minutes, pour off the supernatant, retain the solid at the bottom of the centrifuge tube, and disperse it again with ethanol.
[0016] Step S700: Repeat step S600 three times. The solid obtained in the last step is placed in an oven and dried overnight to obtain the target spherical polymer nanoparticles.
[0017] Based on the above technical solutions, the present invention also provides the following optional technical solutions:
[0018] In one alternative: the polymer stabilizer is polyvinylpyrrolidone and the amount of polyvinylpyrrolidone used is 25-150 mg.
[0019] In one alternative: the reaction solvent is a mixed solution of water and ethanol, wherein the total volume of water and ethanol is 30 mL and the volume ratio of water to ethanol is 1 / 29-9 / 29.
[0020] In one alternative: the amount of glyoxal is greater than 60-160 μL; the amount of ethylenediamine is 7.5-45 μL; and the amount of ascorbic acid is 10-45 mg.
[0021] In one alternative: the reactor in step S500 is made of polytetrafluoroethylene.
[0022] In one alternative: the temperature of the oven in step S500 is 60-160 ℃ and the reaction time is greater than 8 hours under the condition of 60-160 ℃.
[0023] In one alternative: the temperature of the oven in step S500 is 160 °C and the reaction time is 24 hours.
[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0025] In this invention, a novel polymer was designed by copolymerizing ascorbic acid, ethylenediamine, and glyoxal. Detailed variable control was implemented, and the mechanism was explored and preliminarily explained. Due to its controllable size, variable surface chemistry, reliable biocompatibility, effective and selective activation of B cells, environmental friendliness, and low-cost preparation, this polymer is a promising candidate for immunological research and applications. It holds great potential as an adjuvant in the construction of subunit vaccines against various pathogens.
[0026] In this invention patent, a novel polymer nanoparticle was successfully synthesized using ascorbic acid as a raw material, ethylenediamine as an initiator and linker, and glyoxal as a crosslinking agent. This nanoparticle achieves efficient antigen delivery and specific activation of B cells, making it a novel and promising ideal vaccine adjuvant. Attached Figure Description
[0027] Figure 1This is a diagram illustrating the copolymerization mechanism of ascorbic acid, ethylenediamine, and glyoxal according to the present invention.
[0028] Figure 2 This is a scanning electron microscope image of the polymer nanoparticles prepared according to Example 1 of the present invention.
[0029] Figure 3 This is a high-angle annular dark-field image scanning transmission electron microscope image and elemental distribution spectrum in Embodiment 1 of the present invention.
[0030] Figure 4 This is a scanning electron microscope image of the polymer nanoparticles prepared in Example 2 of the present invention.
[0031] Figure 5 This is a scanning electron microscope image of the polymer nanoparticles prepared in Example 3 of the present invention.
[0032] Figure 6 This is a scanning electron microscope image of the polymer nanoparticles prepared in Example 4 of the present invention.
[0033] Figure 7 This is a scanning electron microscope image of the polymer nanoparticles prepared in Example 5 of the present invention.
[0034] Figure 8 This presents in vitro data on the activation of B cells by MHC II as a surface marker for immune cell activation, which is the basis of this invention.
[0035] Figure 9 This presents in vitro data on the activation of B cells by CD69, a surface marker of which is activated, as described in this invention. Detailed Implementation
[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments listed are merely illustrative and not intended to limit the scope of the invention. Any obvious modifications or alterations made to this invention do not depart from its spirit and scope.
[0037] Please see Figure 1This invention provides a method for synthesizing spherical polymer nanoparticles. This method is based on a polymerization reaction using ascorbic acid, ethylenediamine, and glyoxal as monomers. The method includes the following steps: Step S100: Dissolving a polymer stabilizer in a reaction solvent to obtain a clear and transparent mixed solution, and stirring for half an hour; Step S200: Adding glyoxal to the mixed solution from Step S100, and stirring for half an hour; Step S300: Adding ascorbic acid to the mixed solution from Step S200, and stirring for half an hour until the solution becomes clear and transparent again; Step S400: Adding ethylenediamine to the mixed solution from Step S300... In the liquid, the container is left open and stirred for half an hour. The solution color gradually changes from slightly yellow to brownish-yellow, and then gradually turns wine red. Step S500: Transfer the solution obtained in step S400 into a reaction vessel and place the reaction vessel in an oven. Step S600: Transfer the solution and solid in the reaction vessel from step S500 into a centrifuge tube, centrifuge at 18,000 rpm for 5 minutes, pour off the supernatant, retain the solid at the bottom of the centrifuge tube, and disperse it again with ethanol. Step S700: Repeat step S600 three times. The solid obtained in the last step is placed in an oven to dry overnight to obtain the target spherical polymer nanoparticles. Example 1
[0038] 125 mg of polyvinylpyrrolidone with a molecular weight of 40,000 (Mw) was dispersed in a mixture of 25 mL of ethanol and 5 mL of water, sonicated until completely dissolved, and stirred at room temperature for half an hour.
[0039] Then add 100 μL of glyoxal aqueous solution (37% wt) and stir at room temperature for half an hour.
[0040] Then add 25 mg of oxidized oxalic acid solid powder and stir at room temperature for half an hour.
[0041] Then add 30 μL of anhydrous ethylenediamine solution and stir at room temperature for half an hour in an open container.
[0042] The above solution was transferred into a 40 ml polytetrafluoroethylene reaction vessel, and then the reaction vessel was placed in a 160 ℃ oven and kept there for 24 hours.
[0043] The reacted solution and solid were transferred into centrifuge tubes and centrifuged at 18,000 rpm. The residual solid was then washed with ethanol, and the process was repeated three times. Finally, the solid was dried overnight in an oven at 65 °C to obtain a dark brown powder sample, which is the novel polymer nanoparticle.
[0044] Based on the control of product variables and the analysis of intermediate products, please refer to the possible polymerization mechanism diagram. Figure 1 .
[0045] Scanning analysis of the product from Example 1 revealed that it consists of spherical nanoparticles with a diameter of approximately 680 nm. Please refer to the scan image for the product. Figure 2 .
[0046] High-angle annular dark-field scanning transmission electron microscopy (STEM) images and elemental distribution spectral analysis of the product of Example 1 revealed it to be a polymer with carbon as the main skeletal element and uniformly distributed nitrogen and oxygen elements. (See [link to relevant documentation]). Figure 3 .
[0047] Example 2
[0048] Compared with Example 1, the difference in the synthesis method of spherical polymer nanoparticles in this example is that the amount of glyoxal used is adjusted to 60 μL, 80 μL, 100 μL, 120 μL, 140 μL and 160 μL in sequence.
[0049] The various sample products prepared in Example 2 were subjected to scanning analysis. For product scanning data, please refer to [link to sample analysis]. Figure 4 Among them, as the amount of glyoxal increases, the diameter of the product nanoparticles gradually increases, from 260 nm in diameter (…). Figure 4 a) Initially, it could eventually reach 1300 nm ( Figure 4 f).
[0050] Example 3
[0051] Compared with Example 1, the difference in the synthesis method of spherical polymer nanoparticles in this example is that the amount of ethylenediamine is adjusted sequentially to: 7.5 μL, 15 μL, 22.5 μL, 30 μL, 37.5 μL and 45 μL.
[0052] Scanning analysis was performed on the various sample products prepared in Example 3. For product scanning data, please refer to [link to relevant documentation]. Figure 5 When the amount of ethylenediamine used was small (7.5 μL), the product obtained was spherical nanoparticles of uneven size. Figure 5 a) As the amount of ethylenediamine increases, the diameter of the product nanoparticles gradually decreases, from 950 nm ( Figure 5 b) Initially, it could eventually be reduced to 250nm ( Figure 5 f).
[0053] Example 4
[0054] Compared with Example 1, the difference in the synthesis method of spherical polymer nanoparticles in this example is that the amounts of ascorbic acid, ethylenediamine, and glyoxal are increased or decreased proportionally based on the mass of ascorbic acid; wherein the mass of ascorbic acid is 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, and 45 mg, respectively.
[0055] The various sample products prepared in Example 4 were subjected to scanning analysis. For product scanning results, please refer to [link to sample analysis]. Figure 6 Among them, with the increase of ascorbic acid, ethylenediamine, and glyoxal dosage, the diameter of the product nanoparticles gradually increased from 260 nm. Figure 6 a) Initially, it could eventually reach 1000 nm ( Figure 6 f).
[0056] Example 5
[0057] Compared with Example 1, the difference in the synthesis method of spherical polymer nanoparticles in this example is that the amounts of ascorbic acid, ethylenediamine, and glyoxal are all increased to twice the amounts in Example 1, and the reaction temperatures in the oven are adjusted to 60 ℃, 80 ℃, 100 ℃, 120 ℃, 140 ℃, and 160 ℃.
[0058] The various sample products prepared in Example 4 were subjected to scanning analysis. For product scanning results, please refer to [link to sample analysis]. Figure 7 Because the product could not be collected at low concentrations at lower temperatures, only 50 mg of ascorbic acid and the corresponding optimal amounts of ethylenediamine and glyoxal were used in this set of data. In particular, the product at a reaction temperature of 60 °C required 75 mg of ascorbic acid.
[0059] Property Test
[0060] like Figure 8 and Figure 9 As shown; Figure 8 and Figure 9 In this study, PBS is phosphate-buffered saline, and 1 to 8 are spherical nanoparticles of different sizes obtained by changing the amount of the three raw materials. The average diameters of 1 to 8 are 250 nm, 430 nm, 560 nm, 680 nm, 800 nm, 850 nm, 920 nm and 1000 nm, respectively.
[0061] Primitive B cells (A20) were incubated with particles at 37 °C and 5% CO2 for 24 hours. After centrifugation at 1000 rpm for 5 minutes, the cells were collected and washed three times with phosphate-buffered saline (PBRS), then blocked with CD16 / 32 (101301, BioLegend, San Diego, CA, USA) antibody for 30 minutes, and washed three more times with PBRS. The cells were then stained with anti-MHC II (107606, BioLegend) and anti-CD69 (104522, BioLegend) at room temperature for 30 minutes, washed three times with PBRS, and analyzed by flow cytometry.
[0062] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.
Claims
1. A method for synthesizing spherical polymer nanoparticles, characterized in that, This synthetic method is based on a polymerization reaction using ascorbic acid, ethylenediamine, and glyoxal as monomers; it includes the following steps: Step S100: Dissolve the polymer stabilizer in the reaction solvent to obtain a clear and transparent mixed solution, and stir for half an hour; Step S200: Add glyoxal to the mixed solution from step S100 and stir for half an hour; Step S300: Add ascorbic acid to the mixed solution from step S200 and stir for half an hour until the solution becomes clear and transparent again. Step S400: Add ethylenediamine to the mixed solution from step S300, open the container and stir for half an hour. The solution color gradually changes from light yellow to brownish-yellow and wine red. Step S500: Transfer the solution obtained in step S400 into the reaction vessel and place the reaction vessel into an oven; Step S600: Transfer the solution and solid in the reaction vessel of step S500 to a centrifuge tube, centrifuge at 18,000 rpm for 5 minutes, pour off the supernatant, retain the solid at the bottom of the centrifuge tube, and disperse it again with ethanol. Step S700: Repeat step S600 three times. The solid obtained in the last step is placed in an oven and dried overnight to obtain the target spherical polymer nanoparticles.
2. The method for synthesizing spherical polymer nanoparticles according to claim 1, characterized in that, The polymer stabilizer is polyvinylpyrrolidone, and the amount of polyvinylpyrrolidone used is 25-150 mg.
3. The method for synthesizing spherical polymer nanoparticles according to claim 1, characterized in that, The reaction solvent is a mixed solution of water and ethanol, with a total volume of 30 mL and a volume ratio of water to ethanol of 1 / 29 to 9 / 29.
4. The method for synthesizing spherical polymer nanoparticles according to claim 1, characterized in that, The amount of glyoxal used is greater than 60-160 μL; the amount of ethylenediamine used is 7.5-45 μL; and the amount of ascorbic acid used is 10-45 mg.
5. The method for synthesizing spherical polymer nanoparticles according to claim 1, characterized in that, The reactor in step S500 is made of polytetrafluoroethylene.
6. The method for synthesizing spherical polymer nanoparticles according to claim 5, characterized in that, In step S500, the oven temperature is 60-160 ℃ and the reaction time is greater than 8 hours under the condition of 60-160 ℃.
7. The method for synthesizing spherical polymer nanoparticles according to claim 6, characterized in that, In step S500, the oven temperature is 160 ℃ and the reaction time is 24 hours.