Hydrogel as well as preparation method and application thereof

Abstract

The invention discloses hydrogel as well as a preparation method and application thereof, and belongs to the technical field of medicines. The hydrogel disclosed by the invention is prepared from the following raw materials: mPEG-PLGA, a chemotactic factor and an immunologic adjuvant. The hydrogel provided by the invention can construct an artificial lymph node structure near a tumor, further drains peripheral initial T cells and DC cells in the tumor to be enriched in the hydrogel, and the enriched immune cells are further mature and differentiated in the artificial lymph node, so that the specific response to tumor antigens is achieved, and an immune effect aiming at tumor cells is generated; meanwhile, the hydrogel provided by the invention shows the generation of a novel tumor ectopic vaccine, not only keeps the specificity of individually treated tumors, but also has the universality of a universal vaccine, and a unique vaccine does not need to be designed for each patient; and the method has wide applicability. The hydrogel provided by the invention is conventional in raw materials, simple in preparation method and beneficial to actual production and application.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a hydrogel and a preparation method and application thereof. Background Art

[0002] The removal of paratumoral lymph nodes in patients with malignant tumors is an important means to prevent further spread and metastasis of tumors in clinical practice. Lymph nodes are one of the important components of the lymphatic system, with the functions of filtering, storing and generating lymphocytes. Lymph node resection may affect certain functions of the immune system, leading to reduced antigen presentation and T cell response capabilities.

[0003] 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 specific immune responses 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 immunosuppression of the tumor microenvironment, and the heterogeneity of tumor cells all pose challenges to the effectiveness of tumor vaccines.

[0004] Traditional tumor vaccine technology is often designed and constructed for tumor-associated antigens. Due to their poor specificity, tumor-specific immune response rates are often low. Therefore, scientists have also developed tumor vaccines based on the prediction of tumor neoantigens. Tumor neoantigens have better immunogenicity, but their complex antigen prediction process limits their further development. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a hydrogel that can be effectively applied to tumor immunomodulation and a preparation method and application thereof.

[0006] To achieve the above object, in a first aspect of the present invention, the present invention provides a hydrogel, wherein the raw materials of the hydrogel include mPEG-PLGA, chemokines and immune adjuvants.

[0007] The inventors of the present application have found that by selecting a suitable hydrogel polymer mPEG-PLGA and loading chemokines and immune adjuvants in it, the three work together to achieve the enrichment of tumor DC cells and peripheral initial T cells, and achieve the purpose of constructing an ectopic tumor vaccine in vivo; specifically, mPEG-PLGA can provide the hydrogel with suitable shear viscosity, viscoelasticity and phase transition temperature, so that it can be suitable for introduction to the side of the tumor, and can form good compatibility with chemokines and immune adjuvants; chemokines can not only help the formation of lymphoid structures but also induce DC cells to be enriched in hydrogels; immune adjuvants can stimulate the maturation of DC cells drained into the hydrogel. That is, the present invention provides a hydrogel with excellent coordination effects between components by using materials science means, so that an artificial lymphoid structure can be constructed near the tumor through a hydrogel system, and then the peripheral initial T cells and DC cells in the tumor are enriched in the hydrogel, and the enriched immune cells further mature and differentiate in the artificial lymph nodes, so as to achieve a specific response to tumor antigens and produce an immune effect against tumor cells.

[0008] As a preferred embodiment of the hydrogel of the present invention, 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.

[0009] As a preferred embodiment of the hydrogel of the present invention, 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.

[0010] As a preferred embodiment of the hydrogel of the present invention, the mass ratio of the mPEG-PLGA, chemokine and immune adjuvant is mPEG-PLGA:chemokine:immune adjuvant=50 mg:110 μg:10 μg.

[0011] 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 within the range of the present invention, the obtained hydrogel has suitable physical properties and can be well introduced into the tumor side, and the obtained 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.

[0012] Exemplarily, 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, 5m: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, 25m: 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, 4 5mg: 30μg: 0.2μg, 45mg: 50μg: 0.2μg, 45mg: 80μg: 0.2μg, 45m: 100μg: 0.2μg , 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, 85m: 100μg: 0.2μg, 85mg: 0.1μg: 10μg, 85mg: 30μg: 10μg, 85mg: 80μ g: 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, 100m: 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. .

[0013] In particular, when the mass ratio of the three is preferably 50 mg:110 μg:10 μg, the comprehensive effect obtained is better.

[0014] As a preferred embodiment of the hydrogel of the present invention, the molar mass of the mPEG-PLGA is 0.5-4 kg / mol.

[0015] Exemplarily, 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.

[0016] The inventors have found that when the molar mass of mPEG-PLGA is further selected to be within the above range, it can ensure that the prepared hydrogel has suitable physical properties and excellent anti-tumor effect.

[0017] As a preferred embodiment of the hydrogel of the present invention, the chemokine is at least one of GM-CSF and VEGF-C.

[0018] Preferably, the chemokine is a mixture of GM-CSF and VEGF-C.

[0019] 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.

[0020] 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 further preferably 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.

[0021] As a preferred embodiment of the hydrogel of the present invention, the immune adjuvant is at least one of MPLA and R848.

[0022] The inventors have found that when the immune adjuvant of the present invention is selected, it can better stimulate the DC cells drained to the lymph nodes to mature and then present tumor antigens, accurately activate the production of tumor-specific T cells, and achieve a more excellent anti-tumor effect.

[0023] In the second aspect of the present invention, the present invention provides a method for preparing the hydrogel, wherein the method comprises: dissolving mPEG-PLGA in a PBS solution, and then adding a chemokine and an immune adjuvant and vortexing to obtain the hydrogel.

[0024] As a preferred embodiment of the preparation method of the present invention, the dissolving is 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.

[0025] Exemplarily, the dissolution is at a temperature below 30°C, the phase transition temperature of mPEG-PLGA, and the stirring speed can be 150rpm, 200rpm, 220rpm, 240rpm, or 250rpm; the time can be 30min, 35min, 40min, 45min, 50min, 55min, or 60min.

[0026] Preferably, the mass volume ratio of the mPEG-PLGA to the PBS solution is (50-200) mg / mL.

[0027] Exemplarily, the mass volume ratio of the mPEG-PLGA to the 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.

[0028] Preferably, the vortexing time is 2-4 hours, and the vortexing speed is 200-600 rpm.

[0029] Exemplarily, the vortex time may be 2h, 2.5h, 3h, 3.5h, 4h, and the vortex speed may be 200rpm, 250rpm, 300rpm, 350rm, 400rpm, 450rpm, 500rpm, 550rpm, 600rpm.

[0030] In the third aspect of the present invention, the present invention provides the use of the hydrogel in preparing ectopic personalized tumor vaccines.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] In the first aspect, the present invention discloses a hydrogel and a preparation method and application thereof. The present invention selects a suitable hydrogel polymer mPEG-PLGA by means of materials science, and loads chemokines and immune adjuvants therein. The three act together to construct an artificial lymphoid structure near the tumor, thereby draining the peripheral initial T cells and DC cells in the tumor to be enriched in the hydrogel, and 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;

[0033] Second aspect: Compared with traditional tumor-associated antigens, the hydrogel provided by the present invention can retain the integrity of the antigen by introducing it into the side of the tumor to form a lymphoid structure, thereby overcoming the problem of tumor heterogeneity; and, compared with the cumbersome antigen prediction required for tumor neoantigens, the hydrogel provided by the present invention demonstrates the generation of a new type of tumor ectopic vaccine, which retains the specificity of tumors for personalized treatment and has the universality of a universal vaccine, without the need to design a unique vaccine for each patient; it has a wide range of applicability;

[0034] The third aspect: The hydrogel raw materials provided by the present invention are conventional and the preparation method is simple, which is conducive to practical production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a slice diagram of the hydrogel prepared in Example 1:

[0036] A is the result of hematoxylin and eosin staining;

[0037] B is the immunofluorescence result. DETAILED DESCRIPTION

[0038] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0039] Example 1

[0040] An embodiment of the present invention provides a hydrogel, and a method for preparing the hydrogel is as follows:

[0041] (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 30° C., the phase transition temperature of mPEG-PLGA, and stirred at a speed of 200 rpm for 45 min to obtain an uncoated hydrogel; wherein the mass volume ratio of mPEG-PLGA to PBS solution was 50 mg / mL.

[0042] (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;

[0043] The sliced ​​image of the hydrogel prepared in Example 1 is as follows: Figure 1 As shown, 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. Figure 1 It can be seen that 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 can be well generated near the tumor, while chemotactic immune cell infiltration can be formed to form a drainage lymph node-like structure.

[0044] Example 2

[0045] The embodiment of the present invention 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.

[0046] Example 3

[0047] The embodiment of the present invention 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.

[0048] Example 4

[0049] The embodiment of the present invention provides a hydrogel, the only difference between the hydrogel and embodiment 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.

[0050] Example 5

[0051] The embodiment of the present invention provides a hydrogel, the only difference between the hydrogel and embodiment 1 is that the mass ratio of GM-CSF, MPLA, VEGF-C and mPEG-PLGA is mPEG-PLGA:GM-CSF:VEGF-C:MPLA=100mg:11μg:110μg:20μg.

[0052] Example 6

[0053] The embodiment of the present invention provides a hydrogel, and the only difference between the hydrogel and the embodiment 1 is that the molar mass of mPEG-PLGA is 2 kg / mol.

[0054] Example 7

[0055] The embodiment of the present invention provides a hydrogel, and the only difference between the hydrogel and the embodiment 1 is that the molar mass of mPEG-PLGA is 4 kg / mol.

[0056] Example 8

[0057] An embodiment of the present invention provides a hydrogel, the only difference between the hydrogel and Example 1 is 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, that is, mPEG-PLGA:GM-CSF:VEGF-C:MPLA:R848=50mg:10μg:100μg:5μg:5μg.

[0058] Example 9

[0059] The embodiment of the present invention provides a hydrogel, the only difference between the hydrogel and the embodiment 1 is that mPEG-PLGA:GM-CSF:VEGF-C:MPLA=50mg:55μg:55μg:10μg.

[0060] Example 10

[0061] The embodiment of the present invention provides a hydrogel, and the only difference between the hydrogel and the embodiment 1 is that MPLA is replaced by R848.

[0062] Comparative Example 1

[0063] The comparative example of the present invention 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=50mg:10μg:100μg.

[0064] Comparative Example 2

[0065] The comparative example of the present invention 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=50mg:10μg.

[0066] Comparative Example 3

[0067] The comparative example of the present invention provides a hydrogel, the only difference between the hydrogel and Example 1 is that mPEG-PLGA is replaced by PCL-b-PEG-b-PCL.

[0068] Effect Example 1

[0069] The effects of the hydrogels prepared in Examples 1-10 and Comparative Examples 1-3 on lymphocyte drainage were tested, and the steps were specifically as follows:

[0070] Construction of mouse 4T1 subcutaneous tumors: 6-8 week old female mice were selected and divided into 13 groups, 10 mice in each group. 500,000 4T1 cells were inoculated subcutaneously on the right side of the mice. When the tumor grew to 100 mm 3 100 μL of the hydrogel prepared in Examples 1-10 and Comparative Examples 1-3 were implanted at 1-2 mm adjacent to the tumor; CD45-PE (Biolegend) was then used to label immune cells, B220-FITC (Biolegend) was used to label B cells, CD3-FITC (biolegend) was used to label T cells, and then CD8-PC7 (Biolegend) and CD4-APC (Biolegend) were used to distinguish different CD4 and CD8 cells; DC cells were stained with CD11c-FITC (Biolegend) for analysis; macrophages were stained with CD11b-FITC (Biolengend) and F4 / 80-APC (Biolegend); flow cytometry was used for analysis; the number of cells enriched in 3 days was shown in Table 1.

[0071] Table 1

[0072]

[0073]

[0074] As can be seen from Table 1, the technical solution of the present invention 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 enrichment effect of the product on lymphocytes will be affected; it can be seen from Example 1 and Examples 6-7 that the molar molecular weight of mPEG-PLGA will also affect the enrichment of lymphocytes; it can be seen from Examples 1, 8 and 10 that the type of immune adjuvant will also affect the performance of the product. When MPLA or R848 in the present invention is further preferred, the recruitment effect is better; it can be seen from Examples 1 and 9 that the type of activation factor will also affect 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 it is replacing the mPEG-PLGA in the present invention or lacking the activation factor or immune adjuvant in the present invention, the performance of the obtained product is significantly reduced, and the effect of enriching lymphocytes is almost not reflected.

[0075] Effect Example 2

[0076] The effect example of the present invention uses 4T1 breast cancer-bearing mice to conduct in vivo evaluation on the hydrogels prepared in Examples 1-10 and Comparative Examples 1-3, which specifically includes 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, Examples 1-10, and Comparative Examples 1-3), and the tumor growth of mice was observed compared with that of the control group. The growth of the tumor 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 respectively, and their changes relative to the control group were analyzed. The tumors were photographed and weighed, and tumors of the same weight were taken for CD3, CD4, and CD8 staining 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] Table 2

[0079]

[0080] As can be seen from Table 2, the product obtained by adopting the technical solution of the present invention can effectively increase the infiltration of immune cells in the tumor and stimulate the generation of tumor antigen-specific T cells. It can be seen from Examples 1-5 that 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; it can be seen from Examples 1 and 6-7 that the molar molecular weight of mPEG-PLGA will also affect the infiltration of immune cells; it can be seen from Examples 1, 8 and 10 that the type of immune adjuvant will also affect the infiltration of immune cells. When MPLA or R848 in the present invention is further preferred, the immune cell infiltration of the tumor is significantly improved; it can be seen from Examples 1 and 9 that 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; it can be seen from Example 1 and Comparative Examples 1-3 that whether it is replacing the mPEG-PLGA in the present invention or lacking the activating factor or immune adjuvant in the present invention, the infiltration effect of the obtained product is significantly reduced.

[0081] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

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. The method for preparing the hydrogel according to any one of claims 1 to 7, characterized in that: The preparation method is: mPEG-PLGA was dissolved in PBS solution, and then chemokines and immune adjuvants were added and vortexed to obtain hydrogel.

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. Use of the hydrogel according to any one of claims 1 to 7 in the preparation of ectopic personalized tumor vaccines.

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