Use of crystalline organic polymer nanoplatelets as immunoadjuvants and an immunological preparation

Abstract

This invention relates to the field of immunology, providing the application of crystalline organic polymer nanosheets as immune adjuvants and an immunomodulatory agent. The invention utilizes crystalline organic polymer nanosheets as immune adjuvants. These nanosheets possess a two-dimensional monolayer sheet morphology and are obtained through self-assembly driven by active crystallization. They can alleviate the toxic side effects caused by the accumulation of inorganic nanoadjuvants in vivo. They exhibit high encapsulation and protection of immunologically active substances, and demonstrate good biosafety, biocompatibility, and biodegradability. Furthermore, crystalline organic polymer nanosheets are easily surface-modified, possess structural diversity, and can achieve controlled release of immunologically active substances through polymer degradation. As a vaccine adjuvant, they can effectively activate specific immune responses by promoting the maturation of dendritic cells (DCs), thereby promoting vaccine development and application in tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and more specifically, to the application of crystallization-driven self-assembly crystalline organic polymer micro / nanosheets with active growth, epitaxial growth, and the ability to stimulate immune cell maturation and activate specific immune responses as a vaccine adjuvant, and to an immunomodulator. Background Technology

[0002] As one of the most important public health achievements in human history, vaccines have become a vital means of combating disease since their discovery, and vaccination is considered one of the most effective methods of disease prevention. In recent decades, vaccines have made significant progress and breakthroughs in clinical and preclinical immunotherapy, which is of great significance for protecting human health.

[0003] Most traditional vaccines (such as inactivated virus vaccines and live attenuated virus vaccines) have limited effective application due to safety risks. With the continuous development of biotechnology, researchers have designed and prepared novel vaccines for disease research. These novel vaccines are safer and have fewer side effects than traditional vaccines, but they suffer from poor immunogenicity. Currently, among various strategies for improving vaccine efficacy, the most popular method is to select appropriate adjuvants. Although adjuvants can promote immune responses, their use is still limited by suboptimal immune responses and various potential toxic side effects.

[0004] Currently, common adjuvants include aluminum adjuvants, manganese adjuvants, silicon adjuvants, zinc oxide, calcium phosphate, gold nanoparticles, and Freund's adjuvants. However, most of these adjuvants are inorganic materials, which have some drawbacks, including difficulty in modification, safety issues due to accumulation, induction of side effects, low antigen efficiency, and inability to induce an effective immune response.

[0005] Therefore, it is necessary to develop safer and more effective vaccine adjuvants or vectors to induce sufficiently strong antigen-specific humoral and cellular immunity. Summary of the Invention

[0006] To address the aforementioned problems and overcome the shortcomings of existing immune adjuvants, such as weak immune response induction and significant toxic side effects, this invention provides the application of crystalline self-assembled organic polymer nanosheets as an immune adjuvant and an immunomodulatory agent. This invention utilizes crystalline self-assembled organic polymer nanosheets as an immune adjuvant, which can stimulate the maturation of BMDCs and other immune cells, thereby activating specific immune responses and tumor immunotherapy, enhancing the body's specific immune response, and exhibiting good biosafety, biocompatibility, and biodegradability.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The application of crystalline organic polymer nanosheets as immune adjuvants, wherein the crystalline organic polymer nanosheets have a nanosheet structure, which is formed by the crystalline polymer extending and crystallizing around the polymer nucleus in a two-dimensional direction through active crystallization-driven self-assembly. The crystalline polymer is a polycaprolactone polymer, including polycaprolactone block copolymers.

[0009] Preferably, the crystalline organic polymer nanosheets have a thickness of less than 20 nm and a length of 1000~2000 nm.

[0010] Preferably, the polycaprolactone block copolymer includes at least one of PCL-PDMA and PCL-PDMAEMA.

[0011] Preferably, the crystalline polymer further includes a PCL homopolymer, which is a mixture of polycaprolactone block copolymer and PCL homopolymer.

[0012] Preferably, the polymer nucleus is made of polycaprolactone block copolymer.

[0013] Specifically, the method for preparing the crystalline organic polymer nanosheets is as follows: polymer nanorods are obtained through active crystallization-driven self-assembly; the polymer nanorods are broken to obtain short rod-shaped polymer crystal nuclei; and the crystalline polymer is extended and crystallized around the polymer crystal nuclei in a two-dimensional direction through active crystallization-driven self-assembly to form a planar nanosheet structure, thereby obtaining crystalline organic polymer nanosheets.

[0014] In one specific embodiment, the polymer nucleus is made of PCL-PDMA copolymer, and the crystalline polymer is a mixture of PCL homopolymer and PCL-PDMA copolymer. The PCL homopolymer is used to increase the crystallization force, accelerate the crystallization rate, and maintain a high crystallization efficiency. The preparation method specifically includes the following steps:

[0015] Step 1: Weigh PCL-PDMA powder, dissolve it in anhydrous ethanol, heat it in an oil bath at 70°C for 3 hours and then cool it. The solution contains molten amorphous micelles, free polymer single chains, and a small amount of crystalline micelles. The polymer single chains grow epitaxially on the crystalline micelles. After heating and cooling, one-dimensional crystalline organic polymer nanorod micelles are formed.

[0016] Step 2: Dilute the organic polymer nanorods obtained in Step 1, and then sonicate them in dry ice and acetone to obtain short organic polymer nanorods.

[0017] Step 3: Using the organic polymer nanorods obtained in Step 2 as crystal nuclei, add them to the mixed system prepared by PCL homopolymer and PCL-PDMA copolymer. Immediately after addition, shake vigorously to obtain crystalline organic polymer nanosheets.

[0018] In another specific embodiment, the crystalline polymer is a mixture of PCL homopolymer and PCL-PDMAEMA copolymer, and the resulting crystalline organic polymer nanosheets are positively charged crystalline organic polymer nanosheets. The preparation method specifically includes the following steps:

[0019] Step 1: Weigh PCL-PDMA powder, dissolve it in anhydrous ethanol, heat it in an oil bath at 70°C for 3 hours and then cool it. The solution contains molten amorphous micelles, free polymer single chains, and a small amount of crystalline micelles. The polymer single chains grow epitaxially on the crystalline micelles. After heating and cooling, one-dimensional crystalline organic polymer nanorod micelles are formed.

[0020] Step 2: Dilute the organic polymer nanorods obtained in Step 1, and then sonicate them in dry ice and acetone to obtain short organic polymer nanorods.

[0021] Step 3: Using the organic polymer nanorods obtained in Step 2 as crystal nuclei, add them to the mixed system prepared by PCL homopolymer and PCL-PDMAEMA copolymer. Immediately after addition, shake vigorously to obtain crystalline organic polymer nanosheets.

[0022] In this method, the prepared positively charged crystalline organic polymer nanosheets carry a positive charge in a neutral pH environment. Therefore, these nanosheets can attract and encapsulate antigens through electrostatic interactions and escape from inclusion bodies / lysosomes through the proton sponge effect, thereby promoting cross-presentation and cellular immunity.

[0023] The present invention also provides an immunomodulatory preparation comprising an antigen and an immunoadjuvant, wherein the immunoadjuvant is a crystalline organic polymer nanosheet used in the above-described application; the mass ratio of the immunoadjuvant to the antigen is 100:10~100.

[0024] Specifically, the antigen can be an antigen protein, ovalbumin OVA, or a tumor disease-related specific antigen (E7, MUC1), etc.

[0025] The present invention also provides a method for preparing the above-mentioned immunomodulatory agent, wherein the immunomodulatory agent is obtained by mixing the antigen and the immunoadjuvant in a solvent. Specifically, it includes the following steps:

[0026] Step 1: Dissolve the antigen and the crystalline organic polymer nanosheets in solvents to prepare corresponding solutions.

[0027] Step 2: Stir the antigen solution and crystalline organic polymer nanosheets thoroughly at room temperature to prepare an immunomodulator.

[0028] Specifically, the solvent in step 1 above is any one of water, PBS, HEPES, or physiological saline.

[0029] The present invention has the following beneficial effects:

[0030] This invention provides the application of crystalline organic polymer nanosheets as an immune adjuvant. These nanosheets possess a nanosheet structure, formed by the two-dimensional crystalline growth of a crystalline polymer around a polymer nucleus through active crystallization-driven self-assembly. These crystalline organic polymer nanosheets are easily modified and can carry immune components such as antigens and agonists. Compared to existing vaccine adjuvants, they exhibit good biocompatibility, biosafety, and biodegradability. Furthermore, the crystalline organic polymer nanosheets, formed through crystallization-driven and active crystallization-driven self-assembly, have controllable morphology, uniform size, and relatively stable structure, exhibiting a high antigen loading rate. They can also achieve effective specific immune responses, more effectively promoting antigen-specific humoral and cellular immune responses, including significantly increased antigen-specific IgG antibody titers and BMDC maturation, CD8+... + T cells and CD4 + T cells have great potential for activating specific immune responses, tumor immunotherapy, and vaccine development. Crystalline organic polymer nanosheets have important application value in the field of immunotherapy as vaccine adjuvants. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 TEM images of the nanorods, crystal seeds, and nanosheets CDSA-P prepared in Example 1;

[0033] Figure 2 The preparation method of the positively charged nanosheets prepared in Example 2 and the TEM image of the obtained CDSA-CP;

[0034] Figure 3 The image shows the epitaxial growth results of the positively charged nanosheets CDSA-CP prepared in Example 2.

[0035] Figure 4 TEM image of the CDSA-S nanospheres prepared in Example 3;

[0036] Figure 5 The image shows the laser confocal results of BMDCs uptake of CDSA-S from polymer nanospheres;

[0037] Figure 6 The image shows the laser confocal results of BMDCs uptake of the polymer nanosheet CDSA-P.

[0038] Figure 7 Flow cytometry results of BMDCs uptake of polymer nanosheets CDSA-P and polymer nanospheres CDSA-S.

[0039] Figure 8 The figure shows the in vitro maturation results of positively charged nanosheets CDSA-CP, CDSA-P, and CDSA-S nanospheres on BMDCs over a 24-hour period.

[0040] Figure 9 The graph shows the in vivo maturation results of positively charged nanosheets CDSA-CP, CDSA-P, and CDSA-S on skin immune cells over a 48-hour period.

[0041] Figure 10 The image shows the in vivo maturation results of positively charged nanosheets CDSA-CP, CDSA-P, and CDSA-S on immune cells in lymph nodes over a 48-hour period.

[0042] Figure 11 The loading rate of OVA adsorbed by positively charged nanosheets CDSA-CP, CDSA-P, and CDSA-S nanospheres;

[0043] Figure 12 The graph shows the change in zeta potential after OVA adsorption on positively charged nanosheets CDSA-CP. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore only examples, and should not be used to limit the scope of protection of the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are all within the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0045] Crystallization-driven self-assembly (CDSA) and active crystallization-driven self-assembly (ADSA) are novel polymer assembly processes. CDSA is a crucial component of polymer self-assembly, using crystalline and semi-crystalline polymers as nucleating segments and crystallization as the driving force to form complex zero-dimensional, one-dimensional, two-dimensional, and multi-dimensional assemblies. ADSA, on the other hand, is a technique that enables the solution-phase self-assembly of semi-crystalline block copolymers in a controllable and modular manner. Poly(caprolactone)-poly(N,N-dimethylacrylamide) (PCL-PDMA) is a novel semi-crystalline organic polymer nanomaterial with good biocompatibility, biosafety, and biodegradability. Its surface is easily modified, allowing for effective uptake by cells, and the release of its "cargo" can be controlled through degradation. Studies have found that uniformly sized and morphologically controllable two-dimensional organic polymer nanosheets can be obtained through CDSA and ADSA. These organic polymer nanosheets, when co-incubated with immune cells, can be effectively uptaken by cells, further stimulating their maturation.

[0046] Based on this, the present invention provides the application of crystalline organic polymer nanosheets as an immune adjuvant. When the crystalline organic polymer nanosheets bind to the antigen, they can be designed as a novel vaccine to activate a specific immune response, achieve immunotherapy against tumors, and ultimately achieve an effective anti-tumor effect.

[0047] Preparation Example 1

[0048] The preparation of crystalline organic polymer nanorods, short crystalline organic polymer nanorods, and crystalline organic polymer nanosheets specifically includes the following steps:

[0049] Step 1: Weigh 10 mg of PCL-PDMA powder and dissolve it in 2 ml of anhydrous ethanol. Heat the solution in an oil bath at 70°C for 3 hours and then cool it. The solution contains molten amorphous micelles, free polymer single chains, and a small amount of crystalline micelles. Using the crystalline micelles as nuclei, the polymer single chains grow epitaxially on them. After heating and cooling, one-dimensional crystalline organic polymer nanorod micelles are formed.

[0050] Step 2: Dilute the organic polymer nanorods obtained in Step 1, and then sonicate them (60w) for 20 minutes in dry ice and acetone to obtain short organic polymer nanorods (as seed crystals).

[0051] Step 3: Using the short organic polymer nanorods obtained in Step 2 as seed crystals, add a mixed monomer solution prepared from PCL homopolymer and PCL-PDMA copolymer. Immediately after addition, shake vigorously to obtain crystalline organic polymer nanosheets, denoted as CDSA-P.

[0052] Transmission electron microscopy (TEM) was performed on the crystalline organic polymer nanorods, short crystalline organic polymer nanorods, and crystalline organic polymer nanosheets prepared in Preparation Example 1. Their structures are as follows: Figure 1 As shown.

[0053] Preparation Example 2

[0054] like Figure 2 As shown, the preparation of another type of positively charged crystalline organic polymer nanosheets includes the following steps:

[0055] Step 1: Weigh 10 mg of PCL-PDMA powder and dissolve it in 2 ml of anhydrous ethanol. Heat the solution in an oil bath at 70°C for 3 hours and then cool it. The solution contains molten amorphous micelles, free polymer single chains, and a small amount of crystalline micelles. Using the crystalline micelles as nuclei, the polymer single chains grow epitaxially on them. After heating and cooling, one-dimensional crystalline organic polymer nanorod micelles are formed.

[0056] Step 2: Dilute the organic polymer nanorods obtained in Step 1, and then sonicate them (60w) for 20 minutes in dry ice and acetone to obtain short organic polymer nanorods (as seed crystals).

[0057] Step 3: Using the short organic polymer nanorods obtained in Step 2 as seed crystals, add a mixed monomer solution prepared from PCL homopolymer and PCL-PDMAEMA copolymer. Immediately after addition, shake vigorously to obtain crystalline organic polymer nanosheets, denoted as CDSA-CP.

[0058] The crystalline organic polymer nanosheets CDSA-CP prepared in Preparation Example 2 were subjected to transmission electron microscopy (TEM) analysis, and their structure is as follows: Figure 2 As shown.

[0059] The positively charged nanosheets CDSA-CP prepared in Example 2 were formed by epitaxial growth, and the results are as follows: Figure 3 As shown.

[0060] Preparation Example 3

[0061] This embodiment describes a method for preparing crystalline organic polymer nanospheres, which includes the following steps:

[0062] Step 1: Weigh a certain amount of PCL / PCL-PDMA mixed monomer solution and add 2 ml of tetrahydrofuran (THF) to the solution to obtain the reaction solution.

[0063] Step 2: Slowly add 3 ml of deionized water to the reaction solution obtained in Step 1, and sonicate for 30 seconds after the addition is complete.

[0064] Step 3: The reaction solution obtained in step 2 is then magnetically stirred at 1000 rpm for 24 hours at room temperature. The resulting nanospheres are denoted as CDSA-S.

[0065] The crystalline organic polymer nanospheres CDSA-S prepared in Example 3 were subjected to transmission electron microscopy (TEM) testing, and their structure is as follows: Figure 4 As shown.

[0066] Performance Test 1

[0067] The uptake of crystalline organic polymer nanosheets CDSA-P and crystalline organic polymer nanospheres CDSA-S by BMDC cells at time points of 2h, 8h, and 24h was investigated.

[0068] Primary cells were isolated from the bone marrow of 6-8 week old female C57 mice and induced to culture until day 7. BMDCs were diluted with complete culture medium to a concentration of 5 × 10⁻⁶. 5 Single-cell suspensions of 0.5 ml per cell were cultured in 24-well plates containing cell spreaders at a volume of 0.5 ml per well. After 24 hours of incubation to allow cell adhesion, CDSA-P nanosheets and CDSA-S nanospheres at a final concentration of 20 μg / ml were added and incubated for 2 h, 8 h, and 24 h, respectively. The suspensions were aspirated, and the cells were gently washed twice with PBS. The cell spreaders were then removed and mounted with a sealing agent. Finally, the qualitative uptake of the material by BMDCs was observed under a laser confocal microscope.

[0069] Figure 5 This is a diagram showing the laser confocal acquisition results of polymer nanospheres CDSA-S by BMDCs. Figure 6 The image shows the laser confocal uptake results of BMDCs on the polymer nanosheet CDSA-P.

[0070] The results show that: Figure 5-6The laser confocal microscopy results show that, at different time points, BMDCs exhibited significantly better uptake of the polymer nanosheet CDSA-P than the polymer nanosphere CDSA-S, with the uptake gradually increasing over time. The uptake of the polymer nanosheet CDSA-P reached its peak after 24 hours. This indicates that after interacting with cells, the polymer nanosheet CDSA-P can be effectively uptaken by BMDCs, which is beneficial for stimulating BMDC maturation and its potential application as a novel vaccine adjuvant.

[0071] Primary cells were isolated from the bone marrow of 6-8 week old female C57 mice and induced to culture until day 7. BMDCs were diluted with complete culture medium to a concentration of 1×10⁻⁶. 6 Single-cell suspensions of 1 cell per 1 ml were cultured in flow cytometry tubes at a volume of 1 ml per tube for 24 hours. Then, CDSA-P nanosheets and CDSA-S nanospheres at a final concentration of 30 μg / ml were added and incubated for 2 h, 8 h, and 24 h, respectively. Cells were first centrifuged and collected (350 g, 5 min, 4 °C). The precipitated cells were washed once with 1 ml PBS, centrifuged again, and resuspended in 250 μl FACS buffer. The quantitative uptake of the material by BMDCs was determined using a flow cytometer.

[0072] Figure 7 The flow cytometry results of BMDCs uptake of polymer nanosheets CDSA-P and polymer nanospheres CDSA-S are consistent with the results of laser confocal microscopy.

[0073] The results showed that at any time point (2h, 8h, 24h), the percentage of uptake of polymer nanospheres CDSA-S by BMDCs was less than 5%, while the percentage of uptake of polymer nanosheets CDSA-P by BMDCs was higher than 20%, even reaching 60%. Similarly, regarding uptake intensity, at 2h, 8h, and 24h, the uptake intensity of polymer nanosheets CDSA-P by BMDCs was higher than that of polymer nanospheres CDSA-S. These results, based on uptake percentage and intensity, indicate that polymer nanosheets CDSA-P are more effectively promoted by BMDCs, thereby facilitating cell maturation or antigen uptake. Therefore, the sheet-like structure has the ability to promote cellular uptake. In conclusion, the combined action of polymer nanosheets CDSA-P and BMDCs can significantly promote cellular uptake of polymer nanosheets CDSA-P, increasing the likelihood of immune cell activation.

[0074] Performance Test 2

[0075] This test is a study on the in vitro maturation of BMDCs by the prepared crystalline organic polymer nanosheets and crystalline organic polymer nanospheres over a 24-hour period.

[0076] Primary cells were isolated from the bone marrow of 6-8 week old female C57 mice and induced to culture until day 7. BMDCs were diluted with complete culture medium to a concentration of 10 × 10⁻⁶. 5 Single-cell suspensions of 1 cell per 1 ml were cultured in flow cytometry tubes. Crystalline organic polymer nanosheets, positively charged nanosheets, and nanospheres at a final concentration of 40 μg / ml were added and incubated for 24 h, respectively. The cells were centrifuged (350 g, 5 min, 4 °C), and the cell pellet was collected and washed once with PBS. The cells were then incubated with Aqua and Fc block on ice for 20 min, followed by washing once with FACS buffer. The cell pellets were then incubated with Anti-CD11c, Anti-MHC II, Anti-CD80, and Anti-CD86 on ice for 20 min, followed by washing once with FACS buffer. The resulting cell pellet was resuspended in 250 μL of FACS buffer. Finally, the expression levels of the cell surface co-stimulatory factors CD80 and CD86 were detected by flow cytometry. During this process, spleens were collected and temporarily stored in complete culture medium, gently ground and filtered to obtain single-cell suspensions for spleen cell staining.

[0077] As one of the most important antigen-presenting cells (APCs), dendritic cells (DCs) play a crucial role in bridging innate and adaptive immune responses. Immature DCs can phagocytose and process antigens in tissues and peripheral blood. After maturation, they migrate to lymph nodes and present antigens to naive T cells. Mature DCs can express and upregulate co-stimulatory molecules on their surface, such as CD80 and CD86, thereby activating naive CD8 cells. + T cells and naïve CD4 + T cells proliferate and differentiate into effector T cells. Therefore, the in vitro maturation-promoting effects of crystalline organic polymer nanosheets, positively charged nanosheets, and nanospheres on BMDCs were investigated by analyzing the upregulation of CD80 and CD86.

[0078] Figure 8 The results show the in vitro maturation effects of CDSA-S nanospheres, CDSA-P nanosheets, and positively charged CDSA-CP nanosheets on BMDCs over a 24-hour period.

[0079] Results analysis: such as Figure 8 As shown, compared with other groups, the expression of CD80 and CD86 in the positively charged nanosheet CDSA-CP group was higher than that in other groups, indicating that the positively charged nanosheet CDSA-CP can promote DC maturation and upregulate the co-stimulatory molecules on the surface of DCs, which is beneficial to activate cytotoxic T lymphocytes and helper T cells, thereby initiating tumor-specific T cell responses.

[0080] Performance Test 3

[0081] This test investigates the in vivo maturation of immune cells in the skin and lymph nodes over a 48-hour period using the prepared crystalline organic polymer nanosheets CDSA-P and crystalline organic polymer nanospheres CDSA-S.

[0082] Six- to eight-week-old female C57 mice were subcutaneously injected into their backs with CDSA-P nanosheets, CDSA-CP positively charged nanosheets, and CDSA-S nanospheres, respectively. Two days later, the mice were sacrificed, and immunocellular cells were collected from the dorsal skin and lymph nodes for staining. The maturation levels of different types of immunocellular cells were analyzed using flow cytometry. Simultaneously, spleens were collected and temporarily preserved in complete culture medium, gently ground, filtered, and used to obtain a single-cell suspension for spleen cell staining.

[0083] 1. Determination of skin cell maturation levels: Mice were euthanized by cervical dislocation, and dorsal skin was collected and temporarily stored in centrifuge tubes containing complete culture medium. The skin was then transferred to centrifuge tubes containing digestive fluid, minced, and incubated at 37°C with shaking for 1.5 h. The skin was then ground and filtered to obtain a skin cell suspension. The suspension was centrifuged (350 g, 5 min, 4°C), the supernatant was carefully discarded, and the suspension was washed once with PBS. The suspension was incubated with Aqua and Fc block on ice for 20 min, followed by washing once with FACS buffer. The suspension was then incubated with Anti-TCRβ, Anti-CD45, Anti-EpCAM, Anti-MHC II, Anti-CD103, Anti-CD11c, Anti-Ly6G, Anti-CD11b, and Anti-Ly6C on ice for 20 min, followed by washing once with FACS buffer. The cells were then incubated on ice with Anti-F4 / 80 for 20 min, followed by washing once with FACS buffer. The resulting cell pellet was resuspended in 250 μL of FACS buffer. The maturation of different types of immune cells was detected using flow cytometry.

[0084] 2. Determination of maturation levels of different DC subtypes in lymph nodes: Proximal lymph nodes were collected from mice after cervical dislocation and temporarily stored in centrifuge tubes containing complete culture medium. The lymph nodes were then transferred to centrifuge tubes containing digestive fluid, minced, and digested at 37°C for 1 h. The lymph nodes were then ground and filtered to obtain a lymph node cell suspension. The suspension was centrifuged (350 g, 5 min, 4°C), the supernatant was carefully discarded, and the cells were washed once with PBS. The suspension was incubated with Aqua and Fc block on ice for 20 min, followed by washing once with FACS buffer. The cells were then incubated with Anti-CD80, Anti-CD86, Anti-EpCAM, Anti-MHC II, Anti-CD103, Anti-CD11c, Anti-CD8a, and Anti-CD11b on ice for 20 min, followed by washing once with FACS buffer. The final cell pellet was resuspended in 250 μL of FACS buffer. The maturation of different DC subtypes was detected using flow cytometry.

[0085] Figure 9 The effects of CDSA-S nanospheres, CDSA-P nanosheets, and positively charged CDSA-CP on skin immune cells (CD11c) + DC cells, CD11b + Results of an in vivo maturation study of myeloid cells over a 48-hour period.

[0086] The results show that CD11c of the positively charged nanosheets CDSA-CP group + Dendritic cell (DC) expression was relatively low, while the expression in the nanosphere CDSA-S and nanosheet CDSA-P groups was higher than that in the positively charged nanosheet CDSA-CP group. This indicates that in the positively charged nanosheet CDSA-CP group, after treatment, dendritic cells in the skin migrated towards the lymph nodes. Similarly, CD11b expression in the positively charged nanosheet CDSA-CP group was also higher. + Myeloid cells showed relatively high expression (65%), while the expression of nanospheres CDSA-S and nanosheets CDSA-P was lower, at 40% and 60%, respectively, indicating that immune cells in the skin can be effectively activated after material stimulation. These results suggest that positively charged nanosheets CDSA-CP can significantly induce the maturation of immune cells in the skin, thereby enhancing the body's immune response.

[0087] Figure 10 This study presents the in vivo maturation results of CDSA-S nanospheres, CDSA-P nanosheets, and positively charged CDSA-CP nanosheets on immune cells (Res DCs, Mig DCs) in lymph nodes over a 48-hour period.

[0088] The results showed that the expression of migrating DCs was relatively high in the positively charged nanosheet CDSA-CP group, while the expression in the nanosphere CDSA-S and nanosheet CDSA-P groups was lower than that in the positively charged nanosheet CDSA-CP group, consistent with the results observed in the skin area above. Furthermore, among various subtypes of migrating DCs, the expression proportion of the positively charged nanosheet CDSA-CP group was higher than that in the nanosphere CDSA-S and nanosheet CDSA-P groups. In contrast, the expression levels of resident DCs and their subtypes were relatively similar across groups. In summary, these results indicate that the positively charged nanosheet CDSA-CP significantly promotes the maturation of lymph node DCs, thereby leading to high expression of co-stimulatory molecules (CD80, CD86), which is beneficial for antigen presentation to T lymphocytes and subsequent activation of the immune response.

[0089] Example 1

[0090] Preparation of the vaccine formulation CDSA-CP@OVA: Phosphate-buffered saline (PBS) at pH 7.4 was selected as the solvent. Ovalbumin OVA and positively charged nanosheets were dissolved in the PBS solvent to prepare corresponding solutions. The OVA solution (0.3 mg / ml) was mixed with the positively charged nanosheet CDSA-CP solution (1 mg / ml) and stirred to prepare the vaccine formulation CDSA-CP@OVA.

[0091] Example 2

[0092] Preparation of the vaccine formulation CDSA-P@OVA: Phosphate-buffered saline (PBS) at pH 7.4 was selected as the solvent. Ovalbumin OVA and nanosheet CDSA-P were dissolved in the PBS solvent to prepare corresponding solutions. The OVA solution (0.3 mg / ml) and the nanosheet CDSA-P solution (1 mg / ml) were mixed and stirred to prepare the vaccine formulation CDSA-P@OVA.

[0093] Comparative Example 1

[0094] Preparation of vaccine formulation CDSA-S@OVA: Phosphate-buffered saline (PBS) at pH 7.4 was selected as the solvent. Ovalbumin OVA and nanospheres CDSA-S were dissolved in the PBS solvent to prepare corresponding solutions. The OVA solution (0.3 mg / ml) and the nanosphere CDSA-S solution (1 mg / ml) were mixed and stirred to prepare the vaccine formulation CDSA-S@OVA.

[0095] Performance Test 4

[0096] Antigen loading rate of CDSA-CP@OVA was determined as follows: 1 mg / ml of positively charged nanosheets (in PBS at pH 7.4) and 0.3 mg / ml of OVA (in PBS at pH 7.4) were weighed and stirred at room temperature for 3 hours. The positively charged nanosheets loaded with OVA were designated CDSA-CP@OVA. The mixture was centrifuged at 18,000 rpm for 20 minutes to remove unloaded OVA. The supernatant containing excess OVA was collected, and the OVA coating was quantified using a Micro-BCA protein assay kit (Beyotime). The loading efficiency was calculated using...

[0097] x 100%

[0098] Figure 11 The graph shows the loading rates of OVA adsorbed by nanospheres CDSA-S, nanosheets CDSA-P, and positively charged nanosheets CDSA-CP.

[0099] Figure 12 The graph shows the change in zeta potential after OVA adsorption on positively charged nanosheets CDSA-CP.

[0100] The results showed that the loading of OVA on positively charged nanosheets CDSA-CP (reaching 85%) was significantly higher than that on nanospheres CDSA-S and nanosheets CDSA-P (60%). This indicates that positively charged nanosheets CDSA-CP can effectively adsorb OVA and transport it to dendritic cells (DCs), thereby activating the maturation of downstream T cells and effectively promoting the immune response. In conclusion, as a novel immune adjuvant, positively charged nanosheets CDSA-CP can not only stimulate the maturation of immune cells but also deliver antigens to antigen-presenting cells, ultimately triggering a strong immune response after being taken up by the cells.

[0101] Performance Test 5

[0102] Mouse immunization: Six- to eight-week-old female C57 mice (n=5 per group, three groups total) were subcutaneously injected with 100 μL of OVA (denoted as OVA alone), CDSA-CP@OVA, and OVA / Freudelor adjuvant formulation (denoted as FA@OVA). A total of three injections were administered, with one week between each injection. Seven days after the third injection, spleens and serum from the immunized mice were collected for OVA antigen-specific immunoreactivity analysis.

[0103] The results showed that, compared with the negative control and other treatment groups, mice treated with CDSA-CP@OVA induced higher levels of TNF-α and IFN-γ secretion and increased antigen-specific IgG antibody titers.

[0104] The manufacturers of the materials used in the above embodiments, comparative examples, and performance tests are as follows:

[0105] The anhydrous ethanol and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0106] The tetrahydrofuran was purchased from Aladdin Company;

[0107] The culture medium and PBS were purchased from Gibco.

[0108] The sealing agent was purchased from Beyotime Corporation.

[0109] The antibody was purchased from BD FACSAria.

[0110] The digestive fluid was purchased from Roche Pharmaceuticals, Shanghai Co., Ltd.

[0111] The OVA was purchased from damas-beta.

[0112] The BCA was purchased from Beyotime Corporation.

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of crystalline organic polymer nanoplatelets as an immunoadjuvant, characterized in that, The method for preparing the crystalline organic polymer nanosheets is as follows: polymer nanorods are obtained through crystallization-driven self-assembly; the polymer nanorods are broken to obtain short rod-shaped polymer crystal nuclei; and the crystalline polymer is extended and crystallized around the polymer crystal nuclei in a two-dimensional direction through crystallization-driven self-assembly to form a planar nanosheet structure, thereby obtaining crystalline organic polymer nanosheets. The polymer nucleus is PCL-PDMA; The crystalline polymer is a mixture of PCL homopolymer and polycaprolactone block copolymer; The polycaprolactone block copolymer is PCL-PDMA or PCL-PDMAEMA.

2. An immunological preparation, characterized in that, It includes an antigen and an immune adjuvant, wherein the immune adjuvant is a crystalline organic polymer nanosheet as described in claim 1, and the antigen is an OVA antigen.

3. The method of claim 2, wherein the immune preparation is prepared by mixing the antigen and the adjuvant in a ratio of 1 : 1 to 1 : 1000. The antigen and the immune adjuvant are mixed in a solvent to obtain an immune preparation.

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