Hydrogel and preparation method therefor and use thereof

Abstract

The present application belongs to the technical field of medicine. Disclosed are a hydrogel, and a preparation method therefor and the thereof. The raw materials of the hydrogel comprise mPEG-PLGA, a chemokine and an immunoadjuvant. The hydrogel can be used to construct an artificial lymph node-like structure in the vicinity of the tumor, whereby naive T cells in the draining periphery and DC cells within the tumor are enriched in the hydrogel, and the enriched immune cells are further matured and differentiated in the artificial lymph node, thereby achieving a specific response to the tumor antigen, and generating an immune effect against tumor cells. In addition, the hydrogel shows the generation of a novel ectopic vaccine against tumors, which has both the specificity of personalized tumor treatment and the universality of a universal vaccine, that is, the vaccine does not need to be uniquely designed for each patient and has wide applicability. Moreover, the hydrogel has conventional raw materials and a simple preparation method, both of which are beneficial to practical production and application.

Description

A hydrogel and its preparation method and application

[0001] Related cross-references

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on July 3, 2023, with application number "202310801549.X" and public name "A hydrogel, its preparation method and application", the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application relates to the field of medical technology, and in particular to a hydrogel and a preparation method and application thereof. Background Art

[0004] Removal of adjacent lymph nodes in patients with malignant tumors is an important clinical approach to prevent further tumor spread and metastasis. Lymph nodes are a crucial component of the lymphatic system, filtering, storing, and producing lymphocytes. However, lymph node removal may affect certain immune system functions, leading to decreased antigen presentation and T cell responsiveness.

[0005] Tumor vaccines are an anti-tumor therapy that uses tumor antigens to stimulate the body's own immune protection mechanism to achieve specific killing of tumor cells. Compared with other therapies, tumor vaccines that use tumor antigens to train the immune system to produce a specific immune response to kill tumors are a key strategy for actively reshaping the anti-tumor immune state. However, most clinical trials of tumor vaccines have been terminated due to poor results. Currently, only one tumor vaccine based on dendritic cells (DC cells) has been approved by the FDA. The complexity of vaccine design, the immunosuppressive nature of the tumor microenvironment, and the heterogeneity of tumor cells all pose challenges to the effectiveness of tumor vaccines.

[0006] Traditional tumor vaccine technologies are often designed and constructed based on tumor-associated antigens. However, due to their poor specificity, tumor-specific immune responses are often low. Therefore, scientists have also developed tumor vaccines based on the prediction of tumor neoantigens. Tumor neoantigens have better immunogenicity, but the complex antigen prediction process has limited their further development.

[0007] Summary of the Invention

[0008] The embodiments of the present application disclose a hydrogel that can be effectively applied to tumor immunomodulation, and its preparation method and application, with the aim of overcoming the deficiencies of the above-mentioned prior art.

[0009] In order to achieve the above objectives, the present application discloses a hydrogel in a first aspect, wherein the raw materials of the hydrogel include mPEG-PLGA, chemokines and immune adjuvants.

[0010] The inventors of this application have discovered that by selecting a suitable hydrogel polymer, mPEG-PLGA, and simultaneously loading it with chemokines and immune adjuvants, the three interact to enrich tumor DCs and peripheral naive T cells, achieving the goal of constructing an ectopic tumor vaccine in vivo. Specifically, mPEG-PLGA provides the hydrogel with suitable shear viscosity, viscoelasticity, and phase transition temperature, making it suitable for introduction into the vicinity of tumors, and also provides good compatibility with chemokines and immune adjuvants. Chemokine not only facilitates the formation of lymphoid-like structures but also induces DCs to accumulate within the hydrogel. The immune adjuvant stimulates the maturation of DCs that have been drained into the hydrogel. In other words, by utilizing materials science, this application provides a hydrogel with excellent synergistic effects between its components, enabling the construction of artificial lymphoid-like structures near tumors through the hydrogel system. This allows the accumulation of peripheral naive T cells and DCs within the tumor within the hydrogel. The enriched immune cells further mature and differentiate within the artificial lymph nodes, achieving a specific response to tumor antigens and generating an immune response against tumor cells.

[0011] As an optional implementation of the example of the present application, the mass ratio of the mPEG-PLGA, chemokine and immune adjuvant is mPEG-PLGA:chemokine:immune adjuvant=(5-100)mg:(0.1-125)μg:(0.2-20)μg.

[0012] As an optional implementation of the example of the present application, the mass ratio of the mPEG-PLGA, chemokine and immune adjuvant is mPEG-PLGA:chemokine:immune adjuvant=(40-60) mg:(90-115) μg:(5-15) μg.

[0013] As an optional implementation of the example of the present application, the mass ratio of the mPEG-PLGA, chemokine and immune adjuvant is mPEG-PLGA:chemokine:immune adjuvant=50 mg:110 μg:10 μg.

[0014] The inventors have found that the mass ratio of mPEG-PLGA, chemokines, and immune adjuvants in the hydrogel not only affects the physical properties of the hydrogel itself, such as viscoelasticity and shear viscosity, but also affects the enrichment effect of the product on immune cells after it is introduced into the tumor side. When the mass ratio is selected within the range of the present invention, the resulting hydrogel has appropriate physical properties and can be well introduced into the tumor side, and the resulting product can effectively enrich immune cells, thereby achieving a good anti-tumor effect. In particular, the mass ratio of the three is further preferably (40-60) mg: (90-115) μg: (5-15) μg.

[0015] For example, the mass ratio of the three can be 5mg:0.1μg:0.2μg, 5mg:30μg:0.2μg, 5mg:50μg:0.2μg, 5mg:80μg:0.2μg, 5mg:100μg:0.2μg, 5mg:0.1μg:10μg, 5mg:30μg:10μg, 5mg:80μg:10μg, 5mg:100μg:10μg, 5mg:0.1μg:20μg, 5mg:30μg:20μg, 5mg:80μg:20μg, 5mg:100μg:20μg, 25mg:0.1μg:0.2μg, 25mg:30μg:0.2μg, 25mg:50 μg:0.2μg, 25mg: 80μg: 0.2μg, 25mg: 100μg: 0.2μg, 25mg: 0.1μg: 10μg, 25mg: 30μg: 10μg, 25mg: 80μg: 10μg, 25mg: 100μg: 10μg, 25mg: 0.1μg: 20μg, 25m g: 30μg: 20μg, 25mg: 80μg: 20μg, 25mg: 100μg: 20μg, 45mg: 0.1μg: 0.2μg, 45mg: 30μg: 0.2μg, 45mg: 50μg: 0.2μg, 45mg: 80μg: 0.2μg, 45mg: 100μg: 0.2μ g, 45mg: 0.1μg: 10μg, 45mg: 30μg: 10μg, 45mg: 80μg: 10μg, 45mg: 100μg: 10μg, 45mg: 0.1μg: 20μg, 45mg: 30μg: 20μg, 45mg: 80μg: 20μg, 45mg: 100μg: 20 μg, 85mg: 0.1μg: 0.2μg, 85mg: 30μg: 0.2μg, 85mg: 50μg: 0.2μg, 85mg: 80μg: 0.2μg, 85mg: 100μg: 0.2μg, 85mg: 0.1μg: 10μg, 85mg: 30μg: 10μg, 85mg: 80 μg:10μg, 85mg: 100μg: 10μg, 85mg: 0.1μg: 20μg, 85mg: 30μg: 20μg, 85mg: 80μg: 20μg, 85mg: 100μg: 20μg, 100mg: 0.1μg: 0.2μg, 100mg: 30μg: 0.2μg, 10 0mg: 50μg: 0.2μg, 100mg: 80μg: 0.2μg, 100mg: 100μg: 0.2μg, 100mg: 0.1μg: 10μg, 100mg: 30μg: 10μg, 100mg: 80μg: 10μg, 100mg: 100μg: 10μg, 100mg: 0.1μg:20μg, 100mg:30μg:20μg, 100mg:80μg:20μg, 100mg:100μg:20μg. .

[0016] In particular, when the mass ratio of the three is preferably 50 mg:110 μg:10 μg, the comprehensive effect obtained is better.

[0017] As an optional implementation of the embodiment of the present application, the molar mass of the mPEG-PLGA is 0.5-4 kg / mol.

[0018] Illustratively, the molar mass of the mPEG-PLGA may be 0.5 kg / mol, 1 kg / mol, 1.5 kg / mol, 2 kg / mol, 2.5 kg / mol, 3 kg / mol, 3.5 kg / mol, or 4 kg / mol.

[0019] The inventors have found that when the molar mass of mPEG-PLGA is further selected to be within the above range, the prepared hydrogel can have suitable physical properties and excellent anti-tumor effect.

[0020] As an optional implementation of the embodiment of the present application, the chemokine is at least one of GM-CSF and VEGF-C.

[0021] Preferably, the chemokine is a mixture of GM-CSF and VEGF-C.

[0022] More preferably, in the mixture of GM-CSF and VEGF-C, the mass ratio of GM-CSF to VEGF-C is GM-CSF:VEGF-C=1:10.

[0023] The inventors have found that when the chemokine is selected as at least one of GM-CSF and VEGF-C, and especially when the chemokine is a mixture of the two, the resulting product can better help the formation of lymphoid structures and induce the enrichment of DC cells in the hydrogel, thereby achieving a better anti-tumor effect.

[0024] As an optional implementation of the example of the present application, the immune adjuvant is at least one of MPLA and R848.

[0025] The inventors have found that when the immune adjuvant of the present invention is selected, it can better stimulate the maturation of DC cells draining to the lymph nodes and then present tumor antigens, accurately activate the production of tumor-specific T cells, and achieve a more excellent anti-tumor effect.

[0026] The second aspect of the present application discloses a preparation method, which comprises: dissolving mPEG-PLGA in a PBS solution, then adding a chemokine and an immune adjuvant and vortexing to obtain any of the hydrogels described above.

[0027] As an optional implementation of the embodiment of the present application, the dissolution is carried out by stirring at a rotation speed of 150-250 rpm for 30-60 min at a temperature below the phase transition temperature of mPEG-PLGA of 30°C.

[0028] Illustratively, the dissolution is at a temperature below the phase transition temperature of mPEG-PLGA of 30°C, and the stirring speed can be 150rpm, 200rpm, 220rpm, 240rpm, or 250rpm; and the time can be 30min, 35min, 40min, 45min, 50min, 55min, or 60min.

[0029] Preferably, the mass volume ratio of the mPEG-PLGA to the PBS solution is (50-200) mg / mL.

[0030] Illustratively, the mass volume ratio of the mPEG-PLGA to PBS solution may be 50 mg / mL, 70 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0031] Preferably, the vortexing time is 2-4 hours, and the vortexing speed is 200-600 rpm.

[0032] Exemplarily, the vortex time may be 2h, 2.5h, 3h, 3.5h, or 4h, and the vortex speed may be 200rpm, 250rpm, 300rpm, 350rm, 400rpm, 450rpm, 500rpm, 550rpm, or 600rpm.

[0033] The third aspect of the present application discloses the use of any of the above-mentioned hydrogels in the preparation of ectopic personalized tumor vaccines.

[0034] Compared with the prior art, this application has the following beneficial effects:

[0035] First, the present application discloses a hydrogel, its preparation method, and application. By utilizing materials science to select a suitable hydrogel polymer, mPEG-PLGA, and simultaneously loading it with chemokines and immune adjuvants, the three work together to construct an artificial lymphoid structure near the tumor, thereby attracting peripheral naive T cells and DC cells within the tumor to be enriched in the hydrogel. The enriched immune cells further mature and differentiate in the artificial lymph nodes, thereby achieving a specific response to tumor antigens and generating an immune effect against tumor cells.

[0036] Second, compared to traditional tumor-associated antigens, the hydrogels provided herein form lymphoid structures adjacent to tumors, thereby preserving the integrity of the antigens and overcoming the problem of tumor heterogeneity. Furthermore, compared to the tedious antigen prediction required for neoantigens, the hydrogels provided herein demonstrate the generation of a novel ectopic tumor vaccine, which retains the tumor specificity of personalized treatment while possessing the universality of a universal vaccine, eliminating the need to design a unique vaccine for each patient. This approach has broad applicability.

[0037] Third aspect: The hydrogel raw materials provided in this application are conventional and the preparation method is simple, which is conducive to actual production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0039] FIG1 is a cross-sectional view of the hydrogel prepared in Example 1: A is the result of hematoxylin and eosin staining; B is the result of immunofluorescence. DETAILED DESCRIPTION

[0040] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below in conjunction with specific embodiments.

[0041] Example 1

[0042] The present invention provides a hydrogel, and the preparation method of the hydrogel is as follows:

[0043] (1) mPEG-PLGA with a molecular weight of 0.5 kg / mol was added to a PBS solution, and the system was placed at a temperature below the phase transition temperature of mPEG-PLGA of 30°C and stirred at a speed of 200 rpm for 45 minutes to obtain an uncoated hydrogel; wherein the mass volume ratio of mPEG-PLGA to PBS solution was 50 mg / mL.

[0044] (2) adding GM-CSF, MPLA, and VEGF-C to the uncoated hydrogel, and then vortexing and mixing to obtain a hydrogel; wherein the mass ratio of GM-CSF, MPLA, VEGF-C, and mPEG-PLGA is mPEG-PLGA:GM-CSF:VEGF-C:MPLA=50 mg:10 μg:100 μg:10 μg;

[0045] Among them, the cross-section of the hydrogel prepared in Example 1 is shown in Figure 1, where A is the result of hematoxylin and eosin staining, and B is the result of immunofluorescence; the red fluorescence represents lymphatic endothelial cells, and the blue fluorescence represents the cell nucleus. As can be seen from Figure 1, on the side of the tumor close to the hydrogel implantation, there is more red fluorescence, representing the generation of more lymphatic endothelial cells, that is, the hydrogel near the tumor can be well generated, while chemotactic immune cell infiltration is formed to form a structure similar to a draining lymph node.

[0046] Example 2

[0047] The present embodiment provides a hydrogel, the only difference between the hydrogel and Example 1 is that the mass ratio of GM-CSF, MPLA, VEGF-C and mPEG-PLGA is mPEG-PLGA:GM-CSF:VEGF-C:MPLA=40mg:8.5μg:85μg:5μg.

[0048] Example 3

[0049] The present embodiment provides a hydrogel, the only difference between the hydrogel and Example 1 is that the mass ratio of GM-CSF, MPLA, VEGF-C and mPEG-PLGA is mPEG-PLGA:GM-CSF:VEGF-C:MPLA=60mg:10.4μg:104μg:15μg.

[0050] Example 4

[0051] The present embodiment provides a hydrogel, the only difference between the hydrogel and Example 1 is that the mass ratio of GM-CSF, MPLA, VEGF-C and mPEG-PLGA is mPEG-PLGA:GM-CSF:VEGF-C:MPLA=5mg:0.1μg:1μg:0.2μg.

[0052] Example 5

[0053] The present embodiment provides a hydrogel, the only difference between the hydrogel and Example 1 is that the mass ratio of GM-CSF, MPLA, VEGF-C and mPEG-PLGA is mPEG-PLGA:GM-CSF:VEGF-C:MPLA=100 mg:11 μg:110 μg:20 μg.

[0054] Example 6

[0055] This embodiment of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 is that the molar mass of mPEG-PLGA is 2 kg / mol.

[0056] Example 7

[0057] This embodiment of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 is that the molar mass of mPEG-PLGA is 4 kg / mol.

[0058] Example 8

[0059] An embodiment of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 being that GM-CSF, MPLA, R848 and VEGF-C are added to the uncoated hydrogel, wherein the mass ratio of MPLA to R848 is 1:1, i.e., mPEG-PLGA:GM-CSF:VEGF-C:MPLA:R848=50 mg:10 μg:100 μg:5 μg:5 μg.

[0060] Example 9

[0061] This embodiment of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 is that mPEG-PLGA:GM-CSF:VEGF-C:MPLA=50 mg:55 μg:55 μg:10 μg.

[0062] Example 10

[0063] This embodiment of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 is that MPLA is replaced by R848.

[0064] Comparative Example 1

[0065] The comparative example of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 is that GM-CSF and VEGF-C are added to the uncoated hydrogel, wherein the mass ratio of GM-CSF, VEGF-C and mPEG-PLGA is mPEG-PLGA:GM-CSF:VEGF-C=50 mg:10 μg:100 μg.

[0066] Comparative Example 2

[0067] The comparative example of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 is that MPLA is added to the uncoated hydrogel, wherein the mass ratio of MPLA to mPEG-PLGA is mPEG-PLGA:MPLA=50 mg:10 μg.

[0068] Comparative Example 3

[0069] The comparative example of the present application provides a hydrogel, the only difference between the hydrogel and Example 1 is that PCL-b-PEG-b-PCL replaces mPEG-PLGA.

[0070] Effect Example 1

[0071] The effects of the hydrogels prepared in Examples 1-10 and Comparative Examples 1-3 on lymphocyte drainage were tested in this application, specifically comprising the following steps:

[0072] To construct 4T1 subcutaneous tumors in mice, 6-8 week old female mice were selected and divided into 13 groups, with 10 mice in each group. 500,000 4T1 cells were inoculated subcutaneously on the right side of the mice. When the tumors grew to 100 mm, the mice were resected and the tumors were resected. 3 100 μL of the hydrogel prepared in Examples 1-10 and Comparative Examples 1-3 was implanted 1-2 mm adjacent to the tumor. CD45-PE (Biolegend) was then used to label immune cells. B cells were labeled with B220-FITC (Biolegend). T cells were labeled with CD3-FITC (Biolegend). CD8-PC7 (Biolegend) and CD4-APC (Biolegend) were then used to distinguish between different CD4 and CD8 cells. DC cells were stained with CD11c-FITC (Biolegend). Macrophages were stained with CD11b-FITC (Biolengend) and F4 / 80-APC (Biolegend). Flow cytometry was used for analysis. The number of cells enriched after 3 days is shown in Table 1.

[0073] Table 1

[0074] As can be seen from Table 1, the technical solution of the present application can significantly enrich paratumoral lymphocytes. It can be seen from Examples 1-5 that when the mass ratio of the components in the hydrogel is changed, the product's lymphocyte enrichment effect is affected; it can be seen from Examples 1 and 6-7 that the molar molecular weight of mPEG-PLGA also affects the enrichment of lymphocytes; it can be seen from Examples 1, 8 and 10 that the type of immune adjuvant also affects the performance of the product. When MPLA or R848 in the present application is further preferred, the recruitment effect is better; it can be seen from Examples 1 and 9 that the type of activating factor also affects the performance of the product. When the mass ratio of GM-CSF and VEGF-C is GM-CSF: VEGF-C = 1:10, the performance is better; it can be seen from Example 1 and Comparative Examples 1-3 that whether the mPEG-PLGA in the present application is replaced or the activating factor or immune adjuvant in the present application is lacking, the performance of the obtained product is significantly reduced, and almost no lymphocyte enrichment effect is reflected.

[0075] Effect Example 2

[0076] The present application's effect example uses mice bearing 4T1 breast cancer to conduct in vivo evaluation of the hydrogels prepared in Examples 1-10 and Comparative Examples 1-3, specifically comprising the following steps:

[0077] Hydrogels were prepared using Examples 1-10 and Comparative Examples 1-3. On the 4th day after tumor inoculation, the hydrogels were implanted next to the tumors of subcutaneous 4T1 mice (a total of 140 mice, 10 mice in each group were divided into 14 groups, including a control group, Example 1-10, and Comparative Example 1-3 groups, respectively), and the tumor growth of mice compared to the control group was observed. The growth of the tumors was recorded at 6, 8, 10, 12, 14, 16, and 18, respectively. On the 18th day, the mice were sacrificed, and the lymph nodes, spleen, and tumor tissues of the mice were taken respectively. The lymph nodes of the mice were stained, and B cells, T cells, DC cells, and macrophages were marked, and their changes relative to the control group were analyzed. The tumors were photographed and weighed, and tumors of the same weight were stained for CD3, CD4, and CD8 to analyze the changes in tumor-infiltrating T cells. 4T1 tumor cell lysate was added to the isolated T cells of the spleen to stimulate spleen cells, and tumor-specific T cells were analyzed using ELISPOT; the results obtained are shown in Table 2;

[0078] As can be seen from Table 2, the product obtained by using the technical solution of the present application can effectively increase the infiltration of immune cells in the tumor and stimulate the generation of tumor antigen-specific T cells. As can be seen from Examples 1-5, when the mass ratio of the components in the hydrogel is changed, the proportion of immune cells in the tumor will be affected to a certain extent; as can be seen from Examples 1 and Examples 6-7, the molar molecular weight of mPEG-PLGA will also affect the infiltration of immune cells; as can be seen from Examples 1, 8 and 10, the type of immune adjuvant will also affect the infiltration of immune cells. When MPLA or R848 in the present invention are further preferred, the immune cell infiltration of the tumor is significantly improved; as can be seen from Examples 1 and 9, the type of activating factor affects the infiltration of immune cells. When the mass ratio of the two is further preferred to be GM-CSF:VEGF-C = 1:10, the performance is better; as can be seen from Example 1 and Comparative Examples 1-3, whether the mPEG-PLGA in the present application is replaced or the activating factor or immune adjuvant in the present application is missing, the infiltration effect of the obtained product is significantly reduced.

[0079] Table 2

[0080] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A hydrogel, characterized in that: The raw materials of the hydrogel include: mPEG-PLGA, chemokine and immune adjuvant.

2. The hydrogel according to claim 1, characterized in that The mass ratio of the mPEG-PLGA, chemokine and immune adjuvant is mPEG-PLGA:chemokine:immune adjuvant=(5-100)mg:(0.1-125)μg:(0.2-20)μg.

3. The hydrogel according to claim 2, characterized in that The mass ratio of the mPEG-PLGA, chemokine and immune adjuvant is mPEG-PLGA:chemokine:immune adjuvant=(40-60)mg:(90-115)μg:(5-15)μg.

4. The hydrogel according to claim 3, characterized in that The mass ratio of the mPEG-PLGA, chemokine and immune adjuvant is mPEG-PLGA:chemokine:immune adjuvant=50mg:110μg:10μg.

5. The hydrogel according to claim 1, characterized in that The molar mass of the mPEG-PLGA is 0.5-4 kg / mol.

6. The hydrogel according to claim 1, characterized in that The chemokine is at least one of GM-CSF and VEGF-C.

7. The hydrogel according to claim 1, characterized in that The immune adjuvant is at least one of MPLA and R848.

8. A preparation method, characterized in that: The preparation method is: Dissolve mPEG-PLGA in PBS solution, then add chemokine and immune adjuvant and vortex to obtain the hydrogel according to any one of claims 1 to 7.

9. The preparation method according to claim 8, characterized in that: The dissolution is carried out by stirring at a rotation speed of 150-250 rpm for 30-60 min at a temperature below 30° C., the phase transition temperature of mPEG-PLGA.

10. An application of a hydrogel, characterized in that: The invention comprises the use of the hydrogel as described in any one of claims 1 to 7 in preparing ectopic personalized tumor vaccines.

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