A pH / H2O2 dual-responsive hydrogel, its preparation method and application

Abstract

The present application relates to a kind of pH / H2O2 dual-response drug-loaded hydrogel and its preparation method and application, by ε-polylysine derivative as antitumor active ingredient, with oxidized dextran through Schiff base reaction crosslinking rapid gelation. While using 2-formylphenyl boronic acid covalently package CD73 small molecule inhibitor APCP. ε-polylysine derivative has oncolytic activity, can induce ICD and promote the infiltration of immune cells in tumor cells. CD73 small molecule inhibitor APCP can block extracellular CD39-CD73-adenosine pathway, reduce the level of immunosuppressive metabolite adenosine in tumor tissue, so as to activate the infiltrated immune cells, restore its anti-tumor function. The hydrogel of ε-polylysine derivative and APCP co-delivery synergistically inhibits the effect of tumor growth, and its curative effect is greater than the treatment effect of single drug, and is superior to positive control drug oxaliplatin (OXA). The pH / H2O2 dual-response hydrogel is rapidly gelled, and has good biological safety under mild reaction conditions.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer materials technology, specifically to a pH / H2O2 dual-response hydrogel, its preparation method, and its applications. Background Technology

[0002] Cancer is a growing and serious public health problem worldwide, leading to high morbidity, high mortality, and a severe economic burden. Currently, first-line cancer treatments, including chemotherapy, radiotherapy, and immunotherapy, have improved patient outcomes to some extent. However, these treatments still have limitations; for example, chemotherapy and radiotherapy often lead to treatment resistance and severe side effects. In recent years, immunotherapy has been extensively and deeply researched, bringing new hope to cancer treatment; however, the overall clinical response rate of immunotherapy is low. Therefore, there is an urgent need to explore new cancer treatment strategies to improve the effectiveness of immunotherapy.

[0003] Oncolytic peptides contain a high proportion of hydrophobic amino acid residues and a certain number of positive charges. They can adsorb onto the negatively charged tumor cell membrane surface through electrostatic interactions, insert into the tumor cell membrane, and cause disruption of cell membrane integrity, ultimately leading to immunogenic cell death (ICD) in tumor cells. ICD causes tumor cells to secrete adenosine triphosphate (ATP), recruiting antigen-presenting cells (APCs) into the tumor immune microenvironment (TME) to initiate an anti-tumor immune response. However, the ATP released extracellularly is rapidly metabolized into adenosine, which has immunosuppressive effects, via the CD39-CD73 pathway, resulting in impaired function of infiltrating CD8+ T cells and natural killer (NK) cells. Blocking the extracellular CD39-CD73-adenosine pathway with immunomodulators can amplify the immune response induced by tumor ICD and thus inhibit tumor metastasis.

[0004] Although oncolytic peptides have been proven to kill tumor cells and trigger immune responses, their large-scale in vivo application, similar to most peptide drugs, is still limited by factors such as poor stability, low bioavailability, cumbersome synthesis steps, and high cost. To overcome these shortcomings in existing technologies, this invention simulates oncolytic peptides to prepare an ε-polylysine derivative with antitumor activity. Summary of the Invention

[0005] This invention provides a pH / H2O2 dual-responsive injectable hydrogel, its preparation method, and its application, aiming to solve the aforementioned technical problems. The hydrogel uses an ε-polylysine derivative as the antitumor active ingredient and gel backbone. It rapidly gels by cross-linking with aldehyde-functionalized dextran via a Schiff base reaction to form imine dynamic bonds. Simultaneously, 2-formylphenylboronic acid is introduced to form borate ester bonds, covalently encapsulating one of the CD73 small molecule inhibitors: APCP, AB680, and PSB-12379. This reverses the immunosuppressive microenvironment of tumor tissue and synergistically inhibits tumor growth.

[0006] This invention is achieved through the following technical solution:

[0007] An injectable antitumor hydrogel with the following structural composition: Ax-ran-By-ran-Ca-ran-Lb-D.

[0008] Where Ax is as shown in Equation A; By is as shown in Equation B; Ca is as shown in Equation C; Lb is as shown in Equation L; and D is as shown in Equation III:

[0009]

[0010] Where 1≤x≤15;

[0011]

[0012] Where 5≤y≤30;

[0013]

[0014] Where 2.5 ≤ a ≤ 6.5;

[0015]

[0016] Where 20≤b≤60;

[0017]

[0018] Where 10≤n≤120, m is the oxidized structural unit in n, and the percentage of oxidized structural units is 40%≤m / n≤85%.

[0019] The amino group of formula L reacts with the aldehyde group of formula D, and the molar ratio of the amino group of formula L to the aldehyde group of formula D is 1:1.5-3.0.

[0020] The overall structure of the hydrogel is shown in Formula I. The ε-polylysine derivative with the structure described in Formula II is used as the antitumor active ingredient and gel skeleton, and is mixed with the aldehyde-functionalized dextran shown in Formula III in a weakly alkaline aqueous medium to obtain the hydrogel.

[0021]

[0022] Among them, 1≤x≤15, 5≤y≤30, 2.5≤a≤6.5, and 20≤b≤60.

[0023]

[0024] Among them, 12≤z≤18, 1≤x≤15, and 5≤y≤30.

[0025] Furthermore, the aldehyde-functionalized dextran has the structure shown in Formula III:

[0026]

[0027] Where 10≤n≤120, m is the oxidized structural unit in n, and the percentage of oxidized structural units is 40%≤m / n≤85%.

[0028] Furthermore, the APCP has the structure shown in Formula IV:

[0029]

[0030] Furthermore, the AB680 has the structure shown in Formula V:

[0031]

[0032] Furthermore, the PSB-12379 has the structure shown in Formula VI:

[0033]

[0034] On the other hand, the present invention also provides a method for preparing the above-mentioned pH / H2O2 dual-responsive injectable hydrogel, which specifically includes the following steps:

[0035] First, ε-polylysine derivatives, 2-formylphenylboronic acid, and APCP were dissolved in a weakly alkaline aqueous medium, and then an aldehyde-functionalized dextran solution was added and mixed to obtain a drug-loaded hydrogel material.

[0036] Further, the mass ratio of the ε-polylysine derivative to the aldehyde-functionalized dextran is 1:g-1.32; the molar ratio of amino groups in the ε-polylysine derivative to aldehyde groups in the aldehyde-functionalized dextran is 1:1.5-3; and the mass ratio of the ε-polylysine derivative to APCP is 1:0.15-0.35.

[0037] Further, the solvent is weakly alkaline water, physiological saline, or a buffer solution; wherein the pH of the weakly alkaline water is 7.4-8.5; the mass-volume concentration of the aldehyde-functionalized dextran is 7%-20%; the mass-volume concentration of the ε-polylysine derivative is 7%-20%; and the mixing temperature is 15-37°C.

[0038] Furthermore, the aldehyde-functionalized dextran is obtained by oxidizing natural polysaccharide dextran with periodate in a solvent under light-protected, room-temperature conditions to obtain the polymer shown in Formula II; wherein the solvent is water and the periodate is a sodium or potassium salt.

[0039] Thirdly, the present invention also provides the application of the above-mentioned pH / H2O2 dual-responsive injectable hydrogel, which can induce tumor cell lysis and death and trigger ICD, while also reversing the immunosuppressive microenvironment of tumor tissue and activating an anti-tumor immune response to synergistically inhibit tumor growth. The pH / H2O2 dual-responsive injectable hydrogel has at least one of the following uses:

[0040] (1) Inducing ICD in tumorigenesis;

[0041] (2) Promotes the maturation of dendritic cells;

[0042] (3) Increases T cell infiltration in the tumor microenvironment;

[0043] (4) Upregulate the proportion of NK cells;

[0044] (5) Reverse the tumor immunosuppressive microenvironment.

[0045] Advantages of this invention:

[0046] 1) This invention provides a pH / H2O2 dual-responsive injectable hydrogel, its preparation method, and its application. The hydrogel is rapidly gelled by cross-linking ε-polylysine derivatives as antitumor active ingredients with oxidized dextran via a Schiff base reaction. Simultaneously, 2-formylphenylboronic acid is used to covalently encapsulate the CD73 small molecule inhibitor APCP. The ε-polylysine derivative has oncolytic activity, inducing ICD in tumor cells and promoting immune cell infiltration. The CD73 small molecule inhibitor APCP blocks the extracellular CD39-CD73-adenosine pathway, reducing the level of adenosine, an immunosuppressive metabolite in tumor tissue, thereby activating infiltrated immune cells and restoring their antitumor function. The hydrogel co-delivered with ε-polylysine derivatives and APCP synergistically inhibits tumor growth, exhibiting greater efficacy than single-drug therapy and superior to the positive control drug oxaliplatin (OXA).

[0047] 2) This pH / H2O2 dual-responsive injectable hydrogel forms rapidly under mild reaction conditions and exhibits good biocompatibility. In vivo, the hydrogel allows small-molecule APCP drugs to remain at the tumor site for an extended period, acting as a reservoir and enhancing the therapeutic effect. Attached Figure Description

[0048] Figure 1 These are images showing the drug-loaded hydrogel (OP@APCP gel) prepared in Example 1 of this invention before and after gelation.

[0049] Figure 2 This is a characterization diagram of the injectability of the drug-loaded hydrogel (OP@APCP gel).

[0050] Figure 3 To examine the microstructure of the blank hydrogel (OP gel) and the drug-loaded hydrogel (OP@APCP gel) using scanning electron microscopy.

[0051] Figure 4 This is a curve showing the viscosity of the hydrogel as a function of shear rate.

[0052] Figure 5 This is a curve showing the modulus of the hydrogel as a function of frequency.

[0053] Figure 6 This is a curve showing the change in the modulus of the hydrogel over time.

[0054] Figure 7 The curves show the modulus changes over time for the blank adhesive and the drug-loaded adhesive.

[0055] Figure 8 This is a scanning electron microscope image showing the results of hydrogel disrupting tumor cell membranes.

[0056] Figure 9 This is a diagram showing the results of hydrogel-induced release of ATP from tumor cells into the extracellular space.

[0057] Figure 10 This is an analysis of the results of drug-loaded hydrogels inhibiting the conversion of ATP to adenosine (ADO).

[0058] Figure 11 This is a statistical graph showing the body weight of mice in different drug administration groups.

[0059] Figure 12 This is a statistical graph showing the tumor volume of a mouse breast cancer model under different treatment conditions.

[0060] Figure 13 A statistical graph showing the tumor quality of mice in different drug administration groups.

[0061] Figure 14 Flow cytometry image of DC cells (CD80+CD86+) in the tumor microenvironment.

[0062] Figure 15 for Figure 11 The bar chart shows the statistical analysis.

[0063] Figure 16 This is a flow cytometry image of NK cells in the tumor microenvironment.

[0064] Figure 17 for Figure 13 The bar chart shows the statistical analysis. Detailed Implementation

[0065] Comparative Example 1

[0066] Preparation of blank hydrogel (OP gel):

[0067] Weigh out 10 mg of ε-polylysine derivative (z = 15, x = 4.5, y = 10), 0.67 mg of 2-formylphenylboronic acid, and 6.58 mg of oxidized dextran (m = 120, n = 93). Dissolve the ε-polylysine derivative and 2-formylphenylboronic acid in PBS (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. ε-polylysine derivative solution and oxidized dextran solution were mixed in different ratios (10% (w / v) solution was mixed at a ratio of 1:1.2 or 1:0.87, and 15% (w / v) solution was mixed at a ratio of 1:0.84, 1:0.79 or 1:0.58). Oxidized dextran solution was added dropwise to the mixed solution containing ε-polylysine derivative, and the mixture was vortexed for 10 minutes. After the mixture was homogeneous, OP gel was obtained.

[0068] Example 1

[0069] Preparation of drug-loaded hydrogel (OP@APCP gel):

[0070] Weigh out 10 mg of ε-polylysine derivative (z = 15, x = 4.5, y = 10), 0.67 mg of 2-formylphenylboronic acid, 1.66 mg of APCP, and 6.58 mg of oxidized dextran (m = 120, n = 93). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. ε-polylysine derivative solutions and oxidized dextran solutions were mixed in different ratios (10% (w / v) solutions were mixed at a ratio of 1:1.2 or 1:0.87, and 15% (w / v) solutions were mixed at a ratio of 1:0.84, 1:0.79, or 1:0.58). The oxidized dextran solution was added dropwise to the mixed solution containing the ε-polylysine derivative, and the mixture was vortexed for 10 min to ensure homogeneity. The resulting OP@APCP gel (x=4.5, y=10, a=3, b=21) was obtained by functionalizing the amino and aldehyde groups of the ε-polylysine derivative dextran with an aldehyde group ratio of N:C = 1:1.5, 1:2, or 1:3.

[0071] Example 2

[0072] Weigh 10 mg of ε-polylysine derivative (z = 15, x = 4.5, y = 10), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 93) (8.77 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 10, a = 3, b = 36).

[0073] Example 3

[0074] Weigh 10 mg of ε-polylysine derivative (z = 15, x = 4.5, y = 10), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 93) (13.15 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 10, a = 3, b = 54).

[0075] Example 4

[0076] Weigh 10 mg of ε-polylysine derivative (z = 15, x = 4.5, y = 10), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 80) (7.98 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 10, a = 3, b = 28).

[0077] Example 5

[0078] Weigh 10 mg of ε-polylysine derivative (z = 15, x = 4.5, y = 10), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 80) (10.65 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 10, a = 3, b = 37.5).

[0079] Example 6

[0080] Weigh 10 mg of ε-polylysine derivative (z = 17, x = 4.5, y = 8), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 93) (7.55 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 8, a = 3, b = 23).

[0081] Example 7

[0082] Weigh 10 mg of ε-polylysine derivative (z = 17, x = 4.5, y = 8), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 93) (10.07 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 8, a = 3, b = 30).

[0083] Example 8

[0084] Weigh 10 mg of ε-polylysine derivative (z = 17, x = 4.5, y = 8), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 93) (15.11 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 8, a = 3, b = 46).

[0085] Example 9

[0086] Weigh 10 mg of ε-polylysine derivative (z = 17, x = 4.5, y = 8), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 80) (9.17 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 8, a = 3, b = 24).

[0087] Example 10

[0088] Weigh 10 mg of ε-polylysine derivative (z = 17, x = 4.5, y = 8), 2-formylphenylboronic acid (0.67 mg), APCP (1.66 mg), and oxidized dextran (m = 120, n = 80) (12.22 mg). Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and APCP in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare 10% (w / v) and 15% (w / v) solutions. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@APCP gel (x = 4.5, y = 8, a = 3, b = 32).

[0089] Example 11

[0090] Preparation of drug-loaded hydrogel (OP@AB680 gel):

[0091] Weigh out 10 mg of ε-polylysine derivative, 0.67 mg of 2-formylphenylboronic acid, 2.35 mg of AB680, and 6.58 mg of oxidized dextran. Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and AB680 in PBS buffer (pH 7.4) to prepare a 10% (w / v) solution. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare a 10% (w / v) solution. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@.

[0092] AB680 gel.

[0093] Example 12

[0094] Preparation of drug-loaded hydrogel (OP@PSB-12379gel):

[0095] Weigh out 10 mg of ε-polylysine derivative, 0.67 mg of 2-formylphenylboronic acid, 2.61 mg of PSB-12379, and 6.58 mg of oxidized dextran. Dissolve the ε-polylysine derivative, 2-formylphenylboronic acid, and PSB-12379 in PBS buffer (pH 7.4) to prepare a 10% (w / v) solution. Similarly, dissolve the oxidized dextran in PBS buffer (pH 7.4) to prepare a 10% (w / v) solution. Add the oxidized dextran solution dropwise to the mixed solution containing the ε-polylysine derivative, vortex for 10 min, and mix thoroughly to obtain OP@PSB-12379 gel.

[0096] Experimental Example 1

[0097] Comparative Example 1 and Example 1: Comparison images of the hydrogels before and after gelation at room temperature (see reference). Figure 1 As shown, the prepared hydrogel has lost its fluidity and reached a macroscopic gel state. Its injectability characterization results are as follows: Figure 2 As shown, this verifies that the hydrogel does indeed possess good injectability. Scanning electron microscopy was used to observe the prepared OP gel and OP@APCP gel, respectively. Specifically, the two hydrogel samples were freeze-fixed, freeze-fractured, and the brittle fracture surfaces were sputter-coated with gold. The microstructure was then observed using scanning electron microscopy. (Refer to...) Figure 3 As shown, the results indicate that the three-dimensional network structure inside the hydrogel provides a basis for loading diverse drugs. After loading drugs onto the hydrogel ( Figure 3 (Right in the image) The hydrogel pores become denser, but its internal microstructure remains basically unchanged, indicating that the drug loading has no significant effect on the formation of the hydrogel skeleton.

[0098] Experiment Example 2

[0099] The rheological properties of the OP gel and OP@APCP gel in Comparative Example 1 and Example 1 were investigated: the rheological behavior of hydrogels with different shear rates and frequencies and the change of modulus over time were studied using a rheometer.

[0100] Data were collected at a constant temperature and a strain amplitude of 1%, and the change in viscosity with shear rate was observed, with reference to... Figure 4 As shown.

[0101] Data were collected at a constant temperature and a strain amplitude of 1%, and the changes in storage modulus (G') and loss modulus (G") with frequency were observed, with reference to... Figure 5As shown.

[0102] Data were collected at a constant temperature and a strain amplitude of 1%, and the changes in storage modulus (G') and loss modulus (G") over time were observed, with reference to... Figure 6 As shown in Figure 7

[0103] Rheological analysis of the hydrogel was conducted, and the effect of shear rate on the hydrogel viscosity was tested. We found that the viscosity of the hydrogel gradually decreased with increasing shear rate, indicating the shear-thinning property of the hydrogel. At 1% strain, the storage modulus of the gel material was consistently higher than the loss modulus, proving that the hydrogel was successfully prepared and maintained in a highly elastic state.

[0104] Experimental Example 3

[0105] The disruptive effect of OP gel on the cell membrane of 4T1 cells. 2.5 × 10⁻⁶ cells were used. 4 4T1 cells were seeded in 12-well plates and incubated for 24 h. They were then incubated with OP gel at different pH conditions (pH 7.4, pH 6.5). Simultaneously, a blank control group was incubated with an equal volume of culture medium. Culture was terminated after 1 h, 3 h, and 5 h, respectively. After fixation, dehydration, and drying, the cell membrane damage was observed under a scanning electron microscope. (Refer to...) Figure 8 As shown, under acidic conditions, the Schiff base bonds in the hydrogel break more rapidly, leading to the rapid release of ε-polylysine derivatives from the hydrogel to exert their antitumor effect.

[0106] Experiment Example 4

[0107] Evaluation of the ability of drug-loaded hydrogels to induce cell membrane lysis, leading to the release of ATP into the extracellular space. 10 5 Four T1 cells were seeded in 6-well plates and incubated with free APCP, OP@APCP gel, and PBS for 12 hours. ATP production was then characterized using an ATP assay kit. (See reference...) Figure 9 As shown, OP@APCP gel induces the generation of a large amount of ATP.

[0108] Experimental Example 5

[0109] Evaluation of the effect of drug-loaded hydrogels on inhibiting the conversion of ATP to adenosine (ADO). The ε-polylysine derivative in the hydrogel induced a large release of ATP after acting on tumor cells. ATP was then metabolized into immunosuppressive ADO via the CD39-CD73 pathway. The CD73 small molecule inhibitor APCP in the drug-loaded hydrogel could inhibit the conversion of ATP to ADO, thereby reversing the immunosuppressive microenvironment. (Reference) Figure 10 As shown, the cell supernatant produced a large amount of ADO after treatment with OP gel, while treatment with OP@APCP gel reduced ADO to normal levels.

[0110] Experimental Example 6

[0111] In vivo antitumor experiments. Construction of a 4T1 breast cancer model: 4T1 cells in logarithmic growth phase and in good growth condition were digested and collected, and the cell concentration was counted and adjusted to 3 × 10⁻⁶ cells. 6 Cells / mL. 100 μL of cell suspension was injected subcutaneously into mice to establish a mouse breast cancer model. The tumor volume was allowed to grow to approximately 60-100 mm². 3 Mice were randomly divided into 4 groups of 5 mice each (n=5). The groups were as follows: PBS, OXA (oxaliplatin), OP gel, and OP@APCP gel.

[0112] The drug was administered via intratumoral injection. Starting from day 0, the tumor volume of mice was measured every two days using calipers, and the weight of the mice was recorded simultaneously. Treatment ended on day 11, and the mice were sacrificed. The tumors of each group were collected and weighed.

[0113] The changes in body weight of mice in each group were as follows: Figure 11 As shown, there was no significant change in the mouse's weight, indicating that the hydrogel has good biocompatibility.

[0114] Tumor volume in mice was measured every two days, and tumor growth curves for each group of mice were referenced. Figure 12 As shown, compared with the PBS group, all other groups showed varying degrees of inhibition.

[0115] OXA and OP gel showed moderate tumor growth inhibition effects, with no significant difference between the two groups. OP@APCPgel showed better in vivo antitumor effects than the positive control drug (OXA).

[0116] After treatment, weigh the tumor and refer to the following: Figure 13 As shown, the OP@APCP gel treatment group had the lightest tumor tissue weight and the best treatment effect.

[0117] Experimental Example 7

[0118] Three days after treatment, tumor tissue was removed from mice, cut into small pieces, and incubated with 1640 containing collagenase IV at 37°C for 2 hours. After enzymatic digestion, the tissue was filtered through a filter to obtain a single-cell suspension. Next, according to the instructions, all dendritic cells (DCs) were stained with antibodies against anti-CD11c-APC, anti-CD80-FITC, and anti-CD86-PE, and the proportion of mature DCs was detected by flow cytometry. The results are as follows: Figure 14 and 15As shown, the mature DC rate in PBS-treated mice was approximately 7.96%. After treatment with OXA and OP gel, the proportion of mature DCs increased to 17.3% and 18.5%, respectively, which is related to the ICD effect induced by antitumor drugs. Mice treated with OP@APCP gel had the highest content of mature DCs, at 26.7%, which was 1.5 times that of the positive control group (OXA), indicating that the OP@APCP gel group had the best effect in activating the antitumor immune response. This may be because APCP reduced intratumoral adenosine levels, relieved the inhibition of DCs, and further increased the content of mature DCs.

[0119] Furthermore, adenosine can impair the function of natural killer (NK) cells, hindering their immune response. Following the instructions for use, NK cells were stained with an antibody against anti-NK1.1-Blue, and the proportion of NK cells was detected by flow cytometry. Results are as follows... Figure 16 and 17 As shown, after treatment with OP@APCP gel, the proportion of activated NK cells increased significantly compared to the OP gel group, indicating that the inhibitory effect of adenosine on NK cells was reversed.

Claims

1. A pH / H2O2 dual-responsive hydrogel, characterized in that, The preparation method is as follows: Using ε-polylysine derivatives as antitumor active ingredients and gel backbones, cross-linking with aldehyde-functionalized dextran through Schiff base reaction to generate imine dynamic bonds for rapid gelation, while introducing 2-formylphenylboronic acid to form borate ester bonds, covalently encapsulating CD73 small molecule inhibitors. The CD73 small molecule inhibitor is selected from one of APCP, AB680, and PSB-12379; The ε-polylysine derivative has the structure shown in Formula I: Formula I Among them, 12≤z≤18, 1≤x≤15, 5≤y≤30; The aldehyde-functionalized dextran has the structure shown in Formula II: Formula II Among them, 10≤n≤120, 40%≤m / n≤85%; The hydrogel has the structure shown in Formula III: Formula III Among them, 1≤x≤15, 5≤y≤30, 2.5≤a≤6.5, and 20≤b≤60.

2. The pH / H2O2 dual-response hydrogel according to claim 1, characterized in that, The molar ratio of amino groups to aldehyde groups in the ε-polylysine derivative functionalized dextran is 1:1.5-3.

0.

3. The pH / H2O2 dual-response hydrogel according to claim 1 or 2, characterized in that, The mass ratio of the ε-polylysine derivative to the aldehyde-functionalized dextran is 1:0.65-1.32; the mass ratio of the ε-polylysine derivative to 2-formylphenylboronic acid is 1:0.06-0.

1.

4. A method for preparing the hydrogel according to any one of claims 1-3, characterized in that, Includes the following steps: An ε-polylysine derivative, 2-formylphenylboronic acid, and any one of the antitumor active ingredients selected from APCP, AB680, or PSB-12379 are dissolved in a solvent, and then aldehyde-functionalized dextran is added and mixed to obtain a drug-loaded hydrogel.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the ε-polylysine derivative to APCP is 1:0.15-0.

35.

6. Use of the pH / H2O2 dual-responsive hydrogel according to any one of claims 1-3 in the preparation of an antitumor drug, wherein the tumor is a breast cancer tumor.

Images