Biological piezoelectric scaffold with tumor glycosylation inhibition synergistic piezoelectric catalytic immunotherapy and preparation method and application thereof

Abstract

The present application relates to a biological piezoelectric scaffold with inhibiting tumor glycosylation synergistic piezoelectric catalytic immunotherapy and its preparation method and application. The biological piezoelectric scaffold comprises: a ROS-responsive hydrogel, and BiFeO3 nanosheets dispersed in the ROS-responsive hydrogel.

Description

TECHNICAL FIELD

[0001] The present application relates to a biological piezoelectric scaffold with high piezoelectric catalytic activity and tumor immune microenvironment regulation function and its related biological applications as a safe and efficient nanomaterial for piezoelectric catalytic immunotherapy of tumors, belonging to the technical field of nanomaterials. BACKGROUND

[0002] Malignant tumor is one of the major diseases threatening human life and health worldwide [1] . Emerging cancer immunotherapy can achieve targeted and sustained attack on cancer cells by activating the body's own immune system, which has great significance for reducing cancer metastasis and recurrence. However, due to low objective response rate (ORR) and drug resistance, only a small number of patients show positive feedback to immunotherapy. Numerous studies have shown that reactive oxygen species (ROS) produced during treatment with different therapies such as photodynamic therapy (PDT), sonodynamic therapy (SDT) or chemical dynamic therapy (CDT) can effectively kill tumor cells, induce immunogenic cell death (ICD) and release tumor-associated antigens (TAAs), thereby effectively activating the body's anti-tumor immune response. This ROS-based anti-cancer strategy combined with immunotherapy is of great interest due to its good specificity, non-invasiveness and little damage to normal tissues. However, despite this, they are still affected by the severe hypoxia in solid tumors or the limited concentration of H2O2. On the other hand, the body's immune system, while killing tumor cells, also increases the adaptability of tumor cells to immune escape. In particular, tumor cells can disguise themselves by synthesizing specific glycan to modify surface-associated protein antibodies and glycosylate them to evade recognition and attack by the immune system, leading to the failure of immunotherapy. Therefore, developing a safe and efficient immune activation strategy and effectively regulating the tumor immune suppressive microenvironment (ITME) to improve the effectiveness of immunotherapy is a key problem that needs to be solved. SUMMARY

[0003] To this end, the present application provides a biological piezoelectric scaffold with high piezoelectric catalytic activity and tumor immune microenvironment regulation function, as well as a preparation method and application thereof.

[0004] In one aspect, the present application provides a biological piezoelectric scaffold for inhibiting tumor glycosylation and synergistic piezoelectric catalytic immunotherapy, comprising: a ROS-responsive hydrogel, and BiFeO3 nanosheets dispersed in the ROS-responsive hydrogel.

[0005] In the present application, the BFO nanosheets with high piezoelectric catalytic activity can break through the limitations of TME and continuously catalyze H2O to generate a large amount of ROS to kill tumor cells under the action of US, while significantly activating the body's anti-tumor immune response.

[0006] Preferably, the ROS-responsive hydrogel, and the BiFeO3 nanosheets and glucose / mannose analogs dispersed in the ROS-responsive hydrogel.

[0007] In the present application, the ROS generated in the piezoelectric catalysis process of BiFeO3 nanosheets can trigger the degradation of the hydrogel, realize the controllable and sustained release of glucose / mannose analogs (such as 2-DG), and thus achieve precise and efficient inhibition of tumor glycosylation, further reverse the immunosuppressive tumor microenvironment, enhance the piezoelectric catalytic immunotherapy of tumors, and achieve the purpose of effectively eliminating primary tumors, inhibiting tumor metastasis and recurrence.

[0008] Preferably, the ROS-responsive hydrogel is obtained by reacting a high molecular polymer, a ROS-responsive crosslinking agent and deionized water.

[0009] Preferably, the high molecular polymer is polyvinyl alcohol (PVA).

[0010] The ROS-responsive crosslinking agent is N1,N1,N3,N3-tetramethylpropane-1,3-diammonium (TSPBA).

[0011] Preferably, the mass ratio of the high molecular polymer and the ROS-responsive crosslinking agent is (2-1):1, preferably 1:1.

[0012] Preferably, the mass ratio of the high molecular polymer and the deionized water is 1:(10-50).

[0013] Preferably, the ROS-responsive hydrogel has a porous structure, the pore size distribution is 500nm-2μm, and the porosity is 15-20%.

[0014] Preferably, the crystal phase of the BFO nanosheet is an oblique hexagonal phase, the morphology is square, the side length is 300-400nm, and the thickness is 5-10nm.

[0015] The mass of the BiFeO3 nanosheet is 10-15wt% of the mass of the ROS-responsive hydrogel, preferably 12.5wt%. With the increase of the BiFeO3 nanosheet, the anti-tumor performance is enhanced, but excessive BiFeO3 nanosheet will damage the properties of the hydrogel.

[0016] Preferably, the mass of the 2DG is 5-10wt% of the mass of the ROS-responsive hydrogel, preferably 6.5wt%.

[0017] In another aspect, the present application provides a preparation method of a biological piezoelectric scaffold, comprising:

[0018] adding BiFeO3 nanosheets and glucose / mannose analogs into a high molecular polymer aqueous solution and mixing to obtain a mixed solution;

[0019] (2) mixing the ROS-responsive crosslinker aqueous solution and the mixed solution to react to obtain the biological piezoelectric scaffold.

[0020] Preferably, in step (1), the concentration of the high molecular polymer aqueous solution is 0.02-0.1 g / mL.

[0021] The mass ratio of the BiFeO3 nanosheet and the high molecular polymer is (100-500) mg: 1000 mg.

[0022] The mass ratio of the glucose / mannose analog and the high molecular polymer is (0-200) mg: 1000 mg, preferably 100 mg: 1 g, preferably (5-50) mg: 1 g.

[0023] Preferably, in step (2), the concentration of the ROS-responsive crosslinker aqueous solution is 1-1.5 g / mL.

[0024] The mass ratio of the high molecular polymer and the ROS-responsive crosslinker is (2-1): 1, preferably 1: 1.

[0025] Preferably, in step (2), the temperature of the reaction is 20-25℃, and the reaction time is 30 minutes-2 hours.

[0026] In another aspect, the application provides a use of the biological piezoelectric scaffold in the preparation of a tumor piezoelectric catalytic immunotherapy drug.

[0027] The application has the following beneficial effects:

[0028] In the application, the biological piezoelectric scaffold has high piezoelectric catalytic activity, i.e., the BFO nanosheet with high piezoelectric catalytic activity can break through the limitation of TME and continuously catalyze H2O to generate a large amount of ROS to kill tumor cells under the action of US, while significantly activating the body's anti-tumor immune response. On the other hand, the ROS generated in the piezoelectric catalytic process can trigger the degradation of the hydrogel, realizing the controllable and sustained release of 2-DG, so as to realize the precise and efficient inhibition of tumor glycosylation and further reverse the ITME. Finally, through effective regulation of the ITME, the piezoelectric catalytic immunotherapy of the tumor is enhanced, achieving the purpose of effectively eliminating the primary tumor and inhibiting tumor metastasis and recurrence. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 TEM image of the piezoelectric catalyst BFO synthesized in Example 1;

[0030] Figure 2 SEM image of the hydrogel piezoelectric scaffold in Example 1;

[0031] Figure 3 EPR spectra of DBG with different oxygen contents after US irradiation, wherein the abscissa is the magnetic field (G);

[0032] Figure 4 Drug release results of the DBG biopiezoelectric scaffold in Example 1 under the action of ultrasound (with / without US irradiation), wherein the abscissa is time (h) and the ordinate is the released 2DG (%);

[0033] Figure 5 Statistical results of the binding rate of tumor cells to plant agglutinin after the DBG biopiezoelectric scaffold in Example 1 was treated by ultrasound irradiation with different concentrations;

[0034] Figure 6 Confocal images of live / dead tumor cells after treatment under different conditions in Example 1. DETAILED DESCRIPTION

[0035] The present application is further illustrated by the following examples, which should not be construed as limiting the present application.

[0036] In the present disclosure, the innovative point of the biopiezoelectric scaffold with high piezocatalytic activity lies in the construction method of the piezocatalytic scaffold and the means of regulating the tumor immunosuppressive microenvironment based on glycosylation inhibition.

[0037] Specifically, the composition of the biopiezoelectric scaffold of the present application includes ROS-responsive hydrogel, BiFeO3(BFO) nanosheets, and glucose / mannose analogs (such as 2-deoxyglucose (2-DG, etc.). In the present application, the piezocatalyst BFO nanosheets and 2-DG are co-loaded with the help of responsive hydrogel as a carrier, and are injected in situ to the tumor site.

[0038] Among them, the loading of BFO nanosheets and 2-DG in ROS-responsive hydrogel can significantly enhance its residence time at the tumor site.

[0039] Among them, the BFO nanosheets are square in morphology, and the particle size is 300-400 nm. 2-DG is a small molecule drug.

[0040] In an embodiment of the present application, a modified preparation process is used to synthesize BFO nanosheets with high piezoelectric catalytic activity as piezoelectric nanocatalysts. Then, the BFO and 2-DG are co-loaded in a responsive hydrogel to form a biological piezoelectric scaffold (DBG). The following exemplary preparation process of the biological piezoelectric scaffold for safe and efficient piezoelectric catalytic immunotherapy of tumors is provided.

[0041] BiFeO3(BFO) nanosheets are prepared. Specifically, a hydrothermal reaction is first used, followed by annealing to obtain BFO nanosheets.

[0042] Bi source, iron source, and solvent are mixed and the pH is adjusted to 10-11, then stirred and centrifuged to collect the precipitate. The obtained precipitate is placed in an alkaline aqueous solution and subjected to a hydrothermal reaction at 60-80°C for 12-24 hours to obtain a red powder. The solvent can be deionized water, ethylene glycol, methanol, etc. Ammonia is used to adjust the pH. The solute in the alkaline aqueous solution is selected from at least one of NaOH, ammonia, and potassium hydroxide, and the concentration can be 1-10 mol / L. As an example of preparing a red powder, 3 mmol Bi(NO3)3·5H2O is added to 100 mL of ethylene glycol solution and stirred vigorously until completely dissolved. Then 2.5 mmol FeCl3·6H2O and 100 mL of deionized water are added. Next, the pH of the mixed solution is adjusted to 10-11 with NH3·H2O and stirring is continued for 1 h. After stirring, the precipitate is collected by centrifugation, 20 mL of NaOH solution (5 mol / L) is added, and then the solution is transferred to a reaction kettle and heated to 80°C for 12 h.

[0043] The red powder is washed with ethanol and deionized water several times and vacuum dried at 60°C.

[0044] The red powder is annealed at 400-600°C (e.g., 500°C) in an air atmosphere for 1-2 h (e.g., 2 h) to obtain BiFeO3nanosheets (BFO). After annealing, the oblique hexagonal phase structure with high piezoelectric properties is obtained, and the unannealed one has no obvious piezoelectric properties.

[0045] The high molecular polymer and deionized water are mixed at 20-25°C until completely transparent and cooled to room temperature to obtain a high molecular polymer aqueous solution. As an example, 1 g of PVA is added to 20 mL of deionized water and stirred at 95°C until completely transparent and cooled to obtain a PVA aqueous solution.

[0046] In the high molecular polymer aqueous solution, different amounts of 2-DG and BFO nanosheets are added to obtain a series of mixed solutions (or referred to as solution A). In an optional embodiment, the ROS-responsive hydrogel can load different amounts of BFO nanosheets and 2-DG. Preferably, the ratio of BFO nanosheets to 2-DG is 100 mg of 2-DG and 200 mg of BFO nanosheets per 2 g of hydrogel raw materials. More preferably, the feeding ratio of BFO nanosheets, 2-DG and hydrogel is: 100 mg of 2-DG and 200 mg of BFO are added to 1 g of PVA and 1 g of TSPBA crosslinking agent. Alternatively, the BFO feeding value can also be 100 and 500 mg. Alternatively, the 2-DG feeding value can also be 200 mg.

[0047] The ROS-responsive crosslinking agent and deionized water are mixed to obtain a ROS-responsive crosslinking agent aqueous solution (or referred to as solution B). As an example, 1 g of ROS-responsive crosslinking agent N1, N1, N3, N3-tetramethylpropane-1, 3-diammonium (TSPBA) is added to 20 mL of deionized water to obtain solution B.

[0048] After mixing solution A (for example, 2 mL) and solution B (for example, 2 mL) and reacting for a certain period of time, a ROS-responsive gel biopiezoelectric scaffold loaded with different amounts of BFO nanosheets and glucose / mannose analogues is obtained. The reaction can be carried out at room temperature, and the reaction time is generally 10-30 minutes

[0049] The preparation process of the present application is simple, pollution-free, high-yield, low-cost, high-efficiency, and the obtained biopiezoelectric scaffold has good stability, which is conducive to the specific delivery and controllable release of BFO nanosheets and 2-DG at the tumor site, in-situ generation of ROS and precise inhibition of tumor glycosylation to achieve efficient and safe piezoelectric catalytic immunotherapy of tumors, which is one of the tumor treatment schemes with extremely promising application prospects.

[0050] The following further examples are further illustrated to explain the present application. It should also be understood that the following examples are only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application. Some non-essential improvements and adjustments made by those skilled in the art according to the above content of the present application all belong to the protection scope of the present application. The specific process parameters in the following examples are only one example in the appropriate range, i.e. those skilled in the art can select within the appropriate range according to the description herein, and are not limited to the specific values in the following examples.

[0051] Example 1

[0052] (1) 3 mmol Bi(NO3)3·5H2O was added into 100 mL ethylene glycol solution and stirred vigorously until completely dissolved. Then 2.5 mmol FeCl3·6H2O and 100 mL deionized water were added. Next, the pH value of the mixed solution was adjusted to 10-11 with NH3·H2O and the stirring was continued for 1 h. After the stirring was completed, the precipitate was collected by centrifugation, 20 mL NaOH solution (5 mol / L) was added, and then the solution was transferred to a reaction kettle and heated to 180℃ for 48 h to obtain a red powder. The red powder was washed with ethanol and deionized water several times and dried under vacuum at 60℃;

[0053] (2) The dried red powder was annealed at 500℃ for 2 h in an air atmosphere to obtain BiFeO3nanosheets (BFO);

[0054] (3) 1 g of PVA was added to 20 mL of deionized water and stirred at 95℃ until completely transparent. After cooling, 100 mg of 2-DG and 200 mg of BFO nanosheets were added respectively to obtain solution A;

[0055] (4) 1 g of ROS-responsive crosslinking agent N1,N1,N3,N3-tetramethylpropane-1,3-diammonium (TSPBA) was added to 20 mL of deionized water to obtain solution B;

[0056] (5) Then 2 mL of A solution and 2 mL of B solution were taken and mixed, and the ROS-responsive gel biopiezoelectric scaffold was obtained after reaction at room temperature (25℃) for 10 min. The mass of BiFeO3nanosheets in the ROS-responsive gel biopiezoelectric scaffold was 5 wt% of the mass of the ROS-responsive hydrogel. The mass of 2DG was 2.5 wt% of the mass of the ROS-responsive hydrogel.

[0057] Figure 1 The TEM image of the piezocatalyst BFO synthesized in Example 1 shows its relatively regular square sheet structure;

[0058] Figure 2 The SEM image of the hydrogel piezoelectric scaffold formed by adding 100 mg of 2-DG and 200 mg of BFO to 1 g of PVA and 1 g of TSPBA crosslinking agent in Example 1 shows that it has a good network structure which is conducive to the loading of BFO and 2-DG;

[0059] Figure 3 The EPR spectrum of DBG after US irradiation under different oxygen contents shows that DBG can generate a large amount of ROS under US action under both anoxic conditions (2% oxygen content) and normal oxygen content (15% oxygen content), which can be used to trigger subsequent drug release and piezocatalytic therapy;

[0060] Figure 4 Results of drug release of the DBG biopiezoelectric scaffold under ultrasound in Example 1. It can be seen from the results that the DBG can controllably and continuously release 2-DG under ultrasound for subsequent inhibition of tumor glycosylation;

[0061] Figure 5 Results of the binding rate of tumor cells to plant agglutinin after treatment of the DBG biopiezoelectric scaffold with different concentrations of ultrasound irradiation in Example 1. The results show that the DBG biopiezoelectric scaffold can significantly inhibit tumor glycosylation to reduce the binding efficiency of cell surface proteins to plant agglutinin;

[0062] Figure 6 Confocal images of live / dead tumor cells after treatment under different conditions in Example 1. The results show that the DBG-mediated piezoelectric catalytic treatment of tumors can efficiently kill tumor cells.

Claims

1. A biopiezoelectric scaffold, characterized by, include: The invention relates to a ROS-responsive hydrogel, comprising BiFeO3 nanosheets and 2-deoxyglucose dispersed within the ROS-responsive hydrogel. The ROS-responsive hydrogel is a porous structure obtained by reacting polyvinyl alcohol, N1,N1,N3,N3-tetramethylpropane-1,3-diammonium, and deionized water. The mass ratio of polyvinyl alcohol to N1,N1,N3,N3-tetramethylpropane-1,3-diammonium is (2-1):

1. The mass of the BiFeO3 nanosheets is 4%-10% of the mass of the ROS-responsive hydrogel, and the mass of the 2-deoxyglucose is 2%-5% of the mass of the ROS-responsive hydrogel.

2. The biopiezoelectric scaffold of claim 1, wherein, The mass ratio of polyvinyl alcohol to deionized water is 1:(10-50).

3. The biopiezoelectric scaffold of claim 1, wherein, The ROS-responsive hydrogel has a pore size distribution of 500 nm to 2 μm and a porosity of 15% to 20%.

4. The bio-piezoelectric scaffold of claim 1, wherein, The BiFeO3 nanosheets have an italic hexagonal crystal phase, a square morphology, a side length of 300–400 nm, and a thickness of 5–10 nm.

5. A method of producing a biological piezoelectric scaffold according to any one of claims 1-4, characterized by, include: (1) BiFeO3 nanosheets and 2-deoxyglucose were added to an aqueous solution of polyvinyl alcohol and mixed to obtain a mixed solution; (2) The N1, N1, N3, N3-tetramethylpropane-1,3-diammonium aqueous solution and the mixed solution prepared in step (1) are mixed and reacted to obtain the bio-piezoelectric scaffold.

6. The production method according to claim 5, characterized by, In step (1), the concentration of the polyvinyl alcohol aqueous solution is 0.02-0.1 g / mL; the mass ratio of BiFeO3 nanosheets to polyvinyl alcohol is (100-500) mg: 1000 mg; and the mass ratio of 2-deoxyglucose to polyvinyl alcohol is (100-200) mg: 1000 mg.

7. The preparation method according to claim 5, characterized in that, In step (2), the concentration of the aqueous solution of N1, N1, N3, N3-tetramethylpropane-1,3-diammonium is 1 to 1.5 g / mL.

8. The preparation method according to claim 5, characterized in that, In step (2), the reaction temperature is 20-25°C and the reaction time is 30 minutes to 2 hours.

9. The use of a bio-piezoelectric scaffold as described in any one of claims 1-4 in the preparation of tumor piezoelectric catalytic immunotherapy drugs.

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