Multifunctional hydrogel based on Au-MnOx nano-enzyme and application thereof

Abstract

The invention discloses multifunctional hydrogel based on Au-MnOx nano-enzyme and application of the multifunctional hydrogel. The multifunctional hydrogel is prepared by mixing the meteor hammer-shaped Au-MnOx nano-enzyme and an agarose solution. The multifunctional hydrogel has a photo-thermal response release characteristic and a radiotherapy sensitization function, the hardness of the hydrogel is reduced and Au-MnOx nano-enzyme is released under 1064nm near-infrared second-zone laser irradiation,-OH can be generated in a tumor microenvironment under catalysis, meanwhile, the local temperature is increased through the photo-thermal effect, and the photo-thermal treatment effect is enhanced; in addition, the Au element with the high atomic number can enhance DNA damage induced by X rays, and radiotherapy sensitization is achieved. In-vivo and in-vitro experiments show that the hydrogel system can efficiently inhibit tumors through a photothermal-radiotherapy synergistic effect, has extremely low toxicity to normal tissues, and provides a new strategy for tumor combined treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a multifunctional hydrogel based on Au-MnOx nanoscale enzyme and application thereof. BACKGROUND

[0002] Radiotherapy (RT) is an important means of clinical treatment of tumors, but its high-dose X-rays can cause irreversible damage to normal tissues. Combining low-dose radiotherapy with new treatment methods is a current research hotspot. High-Z nano materials (such as Au) can be used as radiotherapy sensitizers to enhance X-ray absorption, and photothermal therapy (PTT) uses the near-infrared light-to-heat conversion property to achieve local tumor hyperthermia. However, the tumor targeting efficiency of traditional nano materials through intravenous injection is less than 10%, and the systemic toxicity is high.

[0003] As a macroscopic delivery system, hydrogels have high drug loading rate and stimulus-responsive release characteristics, and can achieve local controlled release of nano materials and reduce system toxicity. Therefore, it is of great significance to develop a hydrogel system with both photothermal response and radiotherapy sensitization functions. SUMMARY

[0004] The application aims to provide a multifunctional hydrogel based on Au-MnOx nanoscale enzyme and application thereof. The hydrogel reduces the hardness and releases Au-MnOx nanoscale enzyme under the irradiation of 1064 nm near-infrared light, which can catalyze the generation of -OH in the tumor microenvironment, improve the local temperature through the photothermal effect, and enhance the X-ray-induced DNA damage by high-atomic-number Au elements, thereby achieving a synergistic effect of photothermal- radiotherapy to efficiently inhibit tumors with very low toxicity to normal tissues, and providing a new strategy for combined tumor therapy. The -OH generated by the Au-MnOx nanoscale enzyme can catalyze the generation of -OH; the local temperature is improved through the photothermal effect; the high-atomic-number Au element can enhance the X-ray-induced DNA damage, thereby achieving a synergistic effect of photothermal- radiotherapy to efficiently inhibit tumors, and the toxicity to normal tissues is very low, thereby providing a new strategy for combined tumor therapy.

[0005] To achieve the above-mentioned purpose, the following technical solutions are adopted in the application: In a first aspect, the application provides a multifunctional hydrogel based on Au-MnOx nanoscale enzyme, which is prepared by mixing streamer-shaped Au-MnOx nanoscale enzyme and agarose solution.

[0006] In the above technical solution, the Au-MnOx nanoscale enzyme is prepared by one-pot method, using and as raw materials to form a nanostructure with uniform distribution of Au-Mn-O elements.

[0007] In the above technical solution, the Au-MnOx nanoscale enzyme has a particle size of 120-160 nm and a negative charge on the surface.

[0008] In the above technical solution, the preparation method of the Au-MnOx nanoscale enzyme is as follows: a solution of is mixed with The solution is mixed, NaOH solution is added under stirring to adjust the pH to 10, and reaction is carried out at 80 DEG C, after the reaction is completed, cooling, centrifugation, and water washing are carried out, and thus the Au-MnOx nanoscale enzyme suspension is obtained.

[0009] In the above technical scheme, the preparation method of the multifunctional hydrogel is that the Au-MnOx nanoscale enzyme is mixed with an agarose solution, heated and stirred, and cooled to room temperature to form the multifunctional hydrogel.

[0010] In the above technical scheme, under the irradiation of a 1064 nm laser, the hardness of the multifunctional hydrogel is reduced, the Au-MnOx nanoscale enzyme is released, and the release rate is greater than or equal to 60% within 5 min.

[0011] In the above technical scheme, under the irradiation of a 1064 nm laser, the hardness of the multifunctional hydrogel is reduced, the Au-MnOx nanoscale enzyme is released, and the release rate is greater than or equal to 60% within 5 min.

[0012] In the above technical scheme, the treatment includes the combined use of the photothermal treatment triggered by the near-infrared second region laser and the low-dose radiotherapy, wherein the laser power density is 0.5 , and the radiotherapy dose is less than or equal to 4 Gy.

[0013] In the above technical scheme, the multifunctional hydrogel is administered by intratumoral injection, and the retention time of the multifunctional hydrogel in the tumor site is greater than or equal to 72 h.

[0014] The multifunctional hydrogel has the photothermal response release characteristic and the radiotherapy sensitization function, under the irradiation of a 1064 nm near-infrared second region laser, the hardness of the hydrogel is reduced and the Au-MnOx nanoscale enzyme is released, the Au-MnOx nanoscale enzyme can catalyze the generation of -OH in the tumor microenvironment , and simultaneously enhances the local temperature through the photothermal effect, and enhances the photothermal treatment effect. In addition, the high-atomic-number Au element can enhance the X-ray-induced DNA damage, and realize the radiotherapy sensitization. In vivo and in vitro experiments show that the multifunctional hydrogel can efficiently inhibit tumors through the synergistic effect of photothermal treatment and radiotherapy, and has very low toxicity to normal tissues, and provides a new strategy for tumor combined treatment. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1A: Transmission electron microscopy image of Au-manganese dioxide. B: Dynamic light scattering diameter of Au-manganese dioxide in different solutions over 7 days. C: Zeta potential of Au-manganese dioxide. D: High-angle annular dark-field scanning transmission electron microscopy image of Au-manganese dioxide and its corresponding elemental mapping with a scale bar of 50 nm. E: X-ray diffraction spectrum of Au-manganese dioxide. F: UV-Vis absorption spectra of tetramethylbenzidine in different reaction systems. G: UV-Vis absorption spectra values of Au-manganese dioxide at 650 nm when reacting with tetramethylbenzidine in different pH solutions for 5 minutes.

[0016] Figure 2 A: UV-Vis-near infrared absorption spectra of Au-manganese oxide. B: Heating and cooling curves of Au-manganese oxide solution under 1064 nm laser irradiation for 3 on / off cycles. C: Scanning electron microscopy image of MNH. D: Infrared thermal images of prepared MNH before and after irradiation.

[0017] Figure 3 A: 4T1 cells were subjected to different treatments and a radiation dose of 4 Gy. B: Representative images of nuclear condensation and DNA fragmentation presented by DAPI and γ-H2AX, respectively, for the indicated treatment conditions. C: Results of colony formation experiments performed on 4T1 cells that received a 4 Gy radiation treatment. D: Quantification of γ-H2AX foci density based on counts of at least 100 cells per treatment group.

[0018] Figure 4 A: Schematic of MNH-based photothermal enhanced radiotherapy. B: Body temperature of mice after laser irradiation. C: Tumor growth curve. D: Changes in tumor weight after different treatment modalities. E: Changes in body weight after different treatment modalities. F: Individual 4T1 tumor volume curve. G: Relative TUNEL and H&E staining analysis of tumor tissue under different treatment modalities with a scale bar of 100 microns. DETAILED DESCRIPTION

[0019] For the purpose of better illustrating the object, technical solutions and advantages of the present application, the present application will be further described in conjunction with specific embodiments. The present application can be implemented in many different forms, and should not be understood as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art, and the present application will be limited only by the claims.

[0020] The test methods or test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are all obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0022] The application provides a multifunctional hydrogel based on Au-MnOx nanoscale enzyme, which is prepared by mixing meteor hammer-shaped Au-MnOx nanoscale enzyme and agarose solution.

[0023] The Au-MnOx nanoscale enzyme is prepared by one-pot method, and is prepared by using raw materials of and under alkaline conditions. The nanoscale enzyme has uniform particle size distribution (120-160 nm), a Zeta potential of -23.3 to -26.8 mV, good stability in water and PBS, a photo-thermal conversion efficiency of 35.44%, and significantly enhanced peroxidase activity under acidic conditions.

[0024] The preparation method of the hydrogel is as follows: the Au-MnOx nanoscale enzyme is mixed with agarose solution, heated to 50-60 DEG C, and then cooled to form a multifunctional nanoscale enzyme hydrogel (MNH) with a three-dimensional network structure. The hydrogel has a suitable hardness at 37 DEG C, and the storage modulus decreases with the increase of temperature. After irradiation with 1064 nm laser (0.5 ) for 5 min, the temperature increases by more than 15 DEG C, and the hardness decreases by more than 50%.

[0025] The hydrogel can be applied to the preparation of a tumor photothermal-radiotherapy synergistic treatment drug, wherein the treatment includes the combination of near-infrared two-region laser triggered photothermal treatment and low-dose radiotherapy, and the laser power density is 0.5 , and the radiotherapy dose is less than or equal to 4 Gy. The hydrogel is administered by intratumoral injection, and the residence time in the tumor site is more than or equal to 72 h. The specific mechanism of the tumor synergistic treatment is as follows: (1) Photothermal effect release: NIR-II laser irradiation softens the MNH, releases the Au-MnOx nanoscale enzyme, and directly kills tumor cells through the photothermal effect (temperature rise of 19 DEG C).

[0026] (2) ROS generation: The released nanoscale enzyme catalyzes the generation of -OH (verified by 652 nm absorption peak) in the tumor microenvironment, and destroys cell biological active substances.

[0027] (3) Radiotherapy sensitization: The high-Z element Au enhances X-ray absorption, promotes ROS generation, and combined with photothermal damage, the DNA damage efficiency reaches 68.2% (only 20.7% for single radiotherapy).

[0028] Example 1 Preparation of hydrogel 1. Experimental process Weigh 0.1g of agarose, add 10mL of deionized water, heat to 95℃ and stir continuously until completely dissolved to form a transparent agarose solution.

[0029] Take 5 mL of the above agarose solution and add 0.05 g of Au-MnOx nanozyme (made from... Figure 1 1 mL of a dispersion (prepared by method A, exhibiting a meteor hammer-like structure) was ultrasonically treated for 3 min to ensure uniform dispersion of Au-MnOx.

[0030] Pour the mixture into a mold and cool at room temperature for 30 minutes. After the solution solidifies, you will get a multifunctional nanoenzyme hydrogel (MNH). Store it away from light for later use.

[0031] 2. Experimental Results Depend on Figure 2 The SEM image of C shows that the prepared MNH exhibits a three-dimensional porous network structure, which can stably encapsulate Au-MnOx; and Figure 2 The D-display shows that the temperature of MNH rises rapidly after irradiation with a 1064nm laser, proving that Au-MnOx is uniformly dispersed and retains its photothermal properties. This indicates that the preparation process can successfully construct MNH with both drug loading capacity and photothermal responsiveness, meeting the requirements of subsequent experiments.

[0032] Example 2: Photothermal Response Release Experiment 1. Experimental Procedure Sample preparation: Accurately weigh MNH containing a known total Au-MnOx loading, place it in a 5mL centrifuge tube, add 3mL of 37℃ PBS (simulating human physiological pH and temperature), and place it in a 37℃ constant temperature water bath for 10min to ensure the system temperature is stable and avoid temperature fluctuations affecting the hydrogel state.

[0033] Photothermal stimulation: Fix the 1064nm laser and adjust the output power density to 0.5. The laser spot completely covers the MNH inside the centrifuge tube. The laser is started and timed, and the irradiation continues for 5 minutes to ensure that the hydrogel fully absorbs the laser energy.

[0034] Separation and Detection: Immediately after irradiation, place the centrifuge tubes in a high-speed centrifuge and centrifuge at 10,000 rpm for 8 minutes to allow the hydrogel residue that has not released Au-MnOx to fully precipitate. Carefully aspirate the supernatant into a new centrifuge tube. Scan the supernatant with a UV-Vis spectrophotometer (wavelength range 400-800 nm) and record the absorbance values ​​corresponding to the characteristic absorption peaks of Au-MnOx.

[0035] Data calculation: According to the pre-drawn Au-MnOx concentration-absorbance standard curve, the release amount of Au-MnOx in the supernatant was calculated, and the 5 min release rate was 65% by "release rate = (release amount / total loading amount) x 100%".

[0036] 2. Experimental results Figure 2 A shows that Au-MnOx has a wide absorption peak in the range of 900-1100 nm, which can efficiently absorb 1064 nm laser; Figure 2 B confirms that it has good photothermal stability, and the heating effect is stable after 3 ON-OFF cycles, and the photothermal conversion efficiency reaches 35.44%, which can provide sufficient heat for hydrogel softening. Figure 2 C The SEM image of MNH shows a three-dimensional cross-linked network structure, which disintegrates after laser irradiation, so the 5 min release rate is 65%. When no laser is irradiated, the release rate of MNH is <5%, which indicates that the release is laser-controllable and can be accurately targeted to reduce drug leakage at non-target sites.

[0037] Example 3 in vivo anti-tumor experiment 1. Experimental process Model construction: Adjust the concentration of 4T1 cells in logarithmic growth phase to 1 x 106 / mL, and subcutaneously inject 0.1 mL of cell suspension after depilation on the right back of female Balb / c mice (6-8 weeks old). Measure the long diameter (L) and short diameter (W) of the tumor with a vernier caliper every day, and calculate the tumor volume according to "tumor volume = (L x W2) / 2". When the tumor reaches 200 mm, randomly divide it into 5 groups (n = 5).

[0038] Grouping and processing: MNH group, MNH+NIR group, MNH+NIR+RT group intratumorally inject 0.1 mL of MNH (containing 1 mg of Au-MnOx), PBS+NIR group inject 0.1 mL of PBS, and RT group does not inject. 24 h later, the MNH+NIR group and the MNH+NIR+RT group are irradiated with 1064 nm laser (0.5 ) for 5 min; then the RT group and the MNH+NIR+RT group receive 4Gy X-ray irradiation.

[0039] Monitoring and observation: Measure the tumor volume and body weight of mice every 2 days, and sacrifice the mice after 15 days. Peel off the tumor and heart, liver, spleen, lung, kidney, make tumor H&E, TUNEL staining sections and organ H&E staining sections, and observe the pathological changes.

[0040] 2. Experimental results From Figure 4C (tumor growth curve), 4D (tumor weight), 4F (individual tumor volume curve) can be known that the tumor volume of the MNH+NIR+RT group is close to 0 after 15 days , which is much better than other groups (PBS+NIR group, RT group, etc. The tumor volume increases significantly). Figure 4 G The H&E staining of the tumor in this group shows a large area of necrosis, and the TUNEL staining of the apoptotic cells is high, which confirms the synergistic treatment effect. Figure 4 E shows that the body weight of mice in each group does not fluctuate significantly, and in general, Figure 4 It shows that this group has no pathological damage, indicating that this treatment is highly effective in inhibiting tumors while being safe and having no significant damage to normal tissues.

[0041] The present application realizes the near-infrared two-zone photothermal triggered radiosensitization synergistic treatment by constructing an Au-MnOx nanoscale enzyme-agarose hydrogel system. The local delivery of the hydrogel solves the problem of low targeting efficiency of nanomaterials, and the photothermal response release mechanism ensures the spatiotemporal controllability of the treatment. The multifunctional properties (photothermal conversion, enzyme catalysis, high Z sensitization) of Au-MnOx synergistically work to achieve high-efficiency tumor inhibition under low-dose radiotherapy, providing a new technical path for precise tumor treatment.

[0042] The hydrogel of the present application can realize the targeted delivery and controlled release of nanomaterials: MNH stays in the tumor site for a long time after intratumoral injection, avoiding the loss of nanomaterials caused by systemic circulation, and the local concentration of drugs is increased by more than 3 times.

[0043] The hydrogel of the present application can realize high efficiency of tumor synergistic treatment: the tumor inhibition rate of the MNH+NIR+RT group is more than 90%, which is significantly higher than that of single therapy (the inhibition rate of single radiotherapy is 35%, and the inhibition rate of single photothermal therapy is 50%).

[0044] The hydrogel of the present application has high safety for tumor treatment: under the therapeutic dose (laser 0.5 , radiotherapy 4Gy), no obvious pathological damage is found in the main organs (heart, liver, spleen, lung, kidney) of mice, and the body weight fluctuation is ≤5%.

[0045] Obviously, the above examples are only examples for the purpose of clear illustration, and are not limitations on the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. Here, it is not necessary or possible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A multifunctional hydrogel based on Au-MnOx nanozymes, characterized in that: The multifunctional hydrogel was prepared by mixing a meteor hammer-shaped Au-MnOx nanozyme with an agarose solution.

2. The multifunctional hydrogel according to claim 1, characterized in that: The Au-MnOx nanozyme was prepared by a one-pot method, with and Using these as raw materials, a nanostructure with a uniform distribution of Au-Mn-O elements is formed.

3. The multifunctional hydrogel according to claim 1, characterized in that: The Au-MnOx nanozyme has a particle size of 120-160 nm and a negatively charged surface.

4. The multifunctional hydrogel according to claim 1, characterized in that: The preparation method of the Au-MnOx nanozyme is as follows: ... Solution and The solutions were mixed, and NaOH solution was added under stirring to adjust the pH to 10. The reaction was carried out at 80℃. After the reaction was completed, the solution was cooled, centrifuged, and washed with distilled water to obtain the Au-MnOx nanozyme suspension.

5. The multifunctional hydrogel according to claim 1, characterized in that: The preparation method of the multifunctional hydrogel is as follows: the Au-MnOx nanozyme is mixed with agarose solution, heated and stirred, and cooled to room temperature to form the multifunctional hydrogel.

6. The multifunctional hydrogel according to claim 1, characterized in that: The hardness of the multifunctional hydrogel decreases under 1064nm laser irradiation, releasing Au-MnOx nanozymes with a release rate of ≥60% within 5 minutes.

7. The use of the multifunctional hydrogel according to any one of claims 1-6 in the preparation of a tumor photothermal-radiotherapy synergistic therapy drug.

8. The application according to claim 7, characterized in that: The treatment comprises a combination of near-infrared II laser-triggered photothermal therapy and low-dose radiotherapy, wherein the laser power density is 0.

5. The radiotherapy dose is ≤4 Gy.

9. The application according to claim 7, characterized in that: The multifunctional hydrogel is administered via intratumoral injection, and its residence time at the tumor site is ≥72 hours after administration.

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