Preparation method and application of cerium-doped nitrogen carbide-based bionic nano-enzyme capable of absorbing near-infrared light

Abstract

The invention provides a preparation method and application of cerium-doped nitrogen carbide-based bionic nano-enzyme capable of absorbing near-infrared light, and relates to the technical field of biomedicine, and the technical key points are as follows: the preparation method comprises the following steps: S1, weighing a nitrogen-containing precursor and a cerium-containing precursor, and calcining under the protection of nitrogen to obtain CeCNs; s2, dispersing the CeCNs powder obtained in the step S1 into water, and performing ultrasonic treatment; s3, extracting an A549 cell membrane, combining the A549 cell membrane with CeCNs in proportion, performing ultrasonic treatment, and then performing centrifugation, precipitation and resuspension on the mixture to obtain the MCeCNs bionic nano-enzyme. The cerium-doped nitrogen carbide-based bionic nano-enzyme constructed in the invention has good biocompatibility and biological metabolism, is beneficial to application in the field of biomedicine, and can absorb near-infrared light; the bionic nano-enzyme constructed by the invention is mainly synthesized by high-temperature calcination, ultrasonic treatment and cell membrane wrapping, the preparation method is simple, and the bionic nano-enzyme has good repeatability and good industrial production prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a preparation method and application of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption. Background Art

[0002] In recent years, although significant progress has been made in enzyme engineering technology, the industrial application of natural enzymes still faces three technical bottlenecks: First, the low expression level of endogenous enzymes in organisms leads to high costs for separation and purification; second, the inherent structural vulnerability of enzyme proteins makes them prone to irreversible inactivation in non-physiological temperatures, extreme pH, or organic solvent systems; third, it is difficult to achieve chemical modification and functional expansion. It is worth noting that nanozymes, as a new generation of artificial enzyme mimicking systems, are nano-catalytic materials constructed through ordered molecular self-assembly technology. They not only perfectly retain the stereoselectivity of bioenzymes but also possess the unique physical and chemical properties of nano materials. However, poor tumor targeting and the unique tumor microenvironment still limit the efficacy of nanozymes.

[0003] Currently, carbon nitride (CNs) with unique structures and properties has shown excellent performance in the field of photocatalytic water splitting, but its biomedical applications face two technical barriers: First, its intrinsic light absorption is limited to the ultraviolet light region (λ < 450 nm), resulting in a tissue penetration depth of less than 1 mm, which cannot meet the requirements for deep tumor treatment; second, traditional CNs lack intelligent responsiveness to the tumor microenvironment (TME), restricting its applications in the biomedical field. It is worth noting that a tumor-targeted intelligent nanozyme system with near-infrared (700 - 1350 nm) absorption and multi-enzyme activities has not been developed yet. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem of the lack of a tumor-targeted intelligent nanozyme system with near-infrared light absorption and multi-enzyme activities in the prior art.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A preparation method of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption, comprising the following steps:

[0007] S1: Weigh a nitrogen-containing precursor and a cerium-containing precursor and calcine them under nitrogen protection to obtain CeCNs;

[0008] S2: Disperse the CeCNs powder obtained in S1 in water and use ultrasonic treatment;

[0009] S3: Extract A549 cell membranes, combine the A549 cell membranes with CeCNs in proportion, use ultrasonic treatment, and then centrifuge, precipitate, and resuspend the mixture to obtain the MCeCNs biomimetic nanozyme.

[0010] Preferably, in S1, the nitrogen-containing precursor is melamine, the cerium-containing precursor is Ce(NO3)3, the calcination temperature is 200 - 700 °C, the time is 3 - 7 hours, and the heating rate is 3 - 10 °C / min.

[0011] Preferably, in S2, the ultrasonic treatment time is 1.5 h and the ultrasonic power is 300 W.

[0012] Preferably, in S3, the mass ratio of A549 cell membrane to CeCNs is 1:(0.5 - 10).

[0013] Preferably, in S3, the ultrasonic treatment time is 6 - 12 h, and the centrifugation conditions after ultrasonic treatment are: 13400 g, 10 min.

[0014] This application also provides a cerium-doped carbon nitride-based bionic nanozyme with near-infrared light absorption, which is prepared by using the above-mentioned preparation method.

[0015] Preferably, the bionic nanozyme takes cerium-doped carbon nitride as the core and has a new bionic nanozyme with OXD, POD, GSHOx and CAT catalytic activities, oxygen generation and tumor targeting functions.

[0016] This application also provides an application of a cerium-doped carbon nitride-based bionic nanozyme with near-infrared light absorption in the preparation of drugs for treating lung cancer, and the bionic nanozyme is prepared by using the above-mentioned preparation method.

[0017] Preferably, the bionic nanozyme is used as a self-produced oxygen and OXD, POD, GSHOx and CAT nanozyme for the combined treatment of lung cancer.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] (1) The present invention constructs a novel cerium-doped carbon nitride-based bionic nanozyme with near-infrared light absorption, which has good biocompatibility and biodegradability, is conducive to application in the biomedical field, and can absorb near-infrared light. The carbon nitride-based bionic nanozyme MCeCNs constructed by the present invention is mainly synthesized by high-temperature calcination, ultrasonic treatment and cell membrane wrapping. The preparation method of the present invention is simple, has good repeatability, and has good industrial production prospects.

[0020] (2) The carbon nitride-based bionic nanozyme MCeCNs of the present invention can effectively bind to cancer cell membranes and form stable nanoparticles.

[0021] (3) The cell membrane wrapping technology of the carbon nitride-based bionic nanozyme MCeCNs provided by the present invention enhances its targeting to lung cancer, and has good biocompatibility and therapeutic potential.

[0022] (4) The carbon nitride-based biomimetic nanozyme MCeCNs provided by the present invention can generate oxygen to relieve the hypoxic microenvironment of tumors.

[0023] (5) The carbon nitride-based biomimetic nanozyme MCeCNs provided by the present invention have OXD, POD, GSHOx, and CAT nanozyme catalytic activities for the catalytic treatment of tumors. Description of the Drawings

[0024] Figure 1 It is the X-ray diffractometer (XRD) pattern of Ce-doped carbon nitride (CeCNs) in an embodiment of the present invention;

[0025] Figure 2 It is the solid ultraviolet absorption spectrum of CeCNs in an embodiment of the present invention;

[0026] Figure 3 It is the elemental mapping diagram of the carbon nitride-based biomimetic nanozyme MCeCNs in an embodiment of the present invention;

[0027] Figure 4 It is the transmission electron microscope pattern of the carbon nitride-based biomimetic nanozyme MCeCNs in an embodiment of the present invention;

[0028] Figure 5 It is the intracellular oxygen-producing CLSM imaging diagram (400×) of the carbon nitride-based biomimetic nanozyme MCeCNs in an embodiment of the present invention;

[0029] Figure 6 It is the evaluation of the OXD, POD, GSHOx, and CAT nanozyme catalytic performance of the carbon nitride-based biomimetic nanozyme MCeCNs in an embodiment of the present invention;

[0030] Figure 7 It is the crystal violet staining picture of the carbon nitride-based biomimetic nanozyme MCeCNs in an embodiment of the present invention;

[0031] Figure 8 It is the in vivo imaging diagram of the carbon nitride-based biomimetic nanozyme MCeCNs in an embodiment of the present invention. Detailed Embodiments

[0032] The following further describes the present invention in detail with specific embodiments.

[0033] The present application provides a preparation method of a near-infrared light-absorbing cerium-doped carbon nitride-based biomimetic nanozyme, comprising the following steps:

[0034] S1: Calcining a nitrogen-containing precursor and a cerium-containing precursor under nitrogen protection to obtain CeCNs;

[0035] In one embodiment, the nitrogen-containing precursor is melamine and the cerium-containing precursor is Ce(NO3)3;

[0036] The calcination temperature is 200 - 700 °C; the calcination time is 3 - 7 hours; the heating rate is 3 - 10 °C / min.

[0037] S2: Disperse the CeCNs powder obtained in S1 in water and perform ultrasonic treatment to improve the dispersibility and activity of the material;

[0038] In one embodiment, the ultrasonic treatment time is 1.5 h and the ultrasonic power is 300 W.

[0039] S3: Extract the A549 cell membrane and wrap the extracted A549 cell membrane on the surface of the CeCNs obtained in S2 to obtain the final biomimetic nanozyme MceCNs.

[0040] In one embodiment, the mass ratio of the cell membrane to CeCNs is 1:(0.5 - 10), and the ultrasonic treatment time is 6 - 12 hours.

[0041] Based on the above preparation method, the present application provides a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption. The biomimetic nanozyme has a cerium-doped carbon nitride as the core and is a brand-new biomimetic nanozyme with OXD, POD, GSHOx, and CAT catalytic activities, oxygen generation, and tumor targeting functions.

[0042] Specifically, in one embodiment, the biomimetic nanozyme has a light response performance in the near-infrared region and can generate oxygen;

[0043] In one embodiment, the biomimetic nanozyme has OXD, POD, GSHOx, and CAT catalytic activities to promote the catalytic treatment of tumors; moreover, the nanocomposite can achieve active targeted delivery through cell membrane wrapping of cancer cells, achieving the purpose of combined treatment of lung cancer.

[0044] In addition, the present application also provides an application of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption in the preparation of drugs for treating lung cancer. The biomimetic nanozyme is used for the combined treatment of lung cancer through self-produced oxygen and OXD-like, POD, GSHOx, and CAT catalysis.

[0045] The above content is elaborated below in conjunction with specific examples:

[0046] Example 1: Preparation of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption:

[0047] (1) Weigh melamine and cerium nitrate with a mass ratio of 1:1, fully dissolve them in an appropriate amount of ethanol solution, and stir for 1 hour at room temperature. After the obtained solution is dried and collected, the powder is calcined in a tube furnace at 200 - 700 °C for 3 - 7 hours, and the heating rate is 3 - 10 °C / min. The finally obtained calcined product is CeCNs.

[0048] Please refer to Figure 1 and Figure 2 , in this application, Ce element was successfully doped into carbon nitride, and the crystal form of the nanosheets did not change significantly; and the absorption of CeCNs in the NIR region was greatly enhanced.

[0049] (2) Disperse the obtained CeCNs powder in water, sonicate for 1.5 hours to ensure its uniform dispersion, and stir overnight at room temperature.

[0050] (3) Combine the extracted A549 cell membrane with CeCNs in a 1:1 ratio, sonicate for 12 hours, and the sonication power is 300 W. Finally, centrifuge the mixture at 13400 g for 10 minutes, resuspend the precipitate in deionized water to obtain the MCeCNs biomimetic nanozyme.

[0051] In addition, as Figure 3 and Figure 4 shown, the presence of phosphorus element indicates that the A549 cell membrane has also successfully coated the surface of the nanozyme; the above characterizations indicate that MCeCNs has been successfully prepared. After introducing the A549 cell membrane to coat the surface of the nanozyme, the material presents an obvious core - shell structure, forming spherical nanoparticles (NPs).

[0052] Example 2: Verification of oxygen generation by MCeCNs:

[0053] Inoculate A549 cells in a glass - bottom culture dish. After culturing for 24 hours, replace the old medium with different samples. After incubating for 12 hours, completely remove the old medium and add a new medium containing the hypoxia probe [Ru(dpp)3]Cl2 (1 μM). Observe the fluorescence of [Ru(dpp)3]Cl2 in cells using a confocal laser scanning microscope (CLSM).

[0054] Please refer to Figure 5 , as the irradiation time of the 808 nm laser increases, the fluorescence signal in A549 cells treated with MCeCNs gradually weakens, indicating a positive correlation between O2 generation and irradiation time. After 10 minutes of laser irradiation, the hypoxic state of the cells is basically relieved. Thus, it can be obtained that under hypoxic conditions, the fluorescence signal intensity of A549 cells decreases significantly, indicating that oxygen has been successfully generated inside the cells.

[0055] Example 3: Verification that MCeCNs have OXD, POD, GSHOx, and CAT nanozyme catalytic activities:

[0056] The OXD-like, POD-like, GSHOx-like, and CAT catalytic activities of the MCeCNs biomimetic nanozyme were evaluated separately using kits according to the steps in the instructions.

[0057] Please refer to Figure 6 , from Figure 6 it can be seen that the carbon nitride-based biomimetic nanozyme MCeCNs has OXD, POD, GSHOx, and CAT nanozyme catalytic activities.

[0058] Example 4: Verification of the inhibition of tumor cell proliferation by MceCNs

[0059] The A54 cells treated with 50 μg / mL of MCeCNs were fixed with 4% paraformaldehyde and gently rinsed 3 times with PBS to remove the fixative; then stained with 0.1% crystal violet staining solution at room temperature in the dark for 20 - 30 minutes to ensure sufficient cell staining; after staining, rinsed repeatedly with deionized water until the background was transparent, air-dried naturally, and photographed under a microscope.

[0060] Please refer to Figure 7 , from Figure 7 it can be seen that the carbon nitride-based biomimetic nanozyme MCeCNs prepared in the present invention significantly inhibited the proliferation of tumor cells after 808 nm laser irradiation.

[0061] Example 5: Mouse experiment verification

[0062] The RhB-labeled biomimetic nanozyme MCeCNs prepared in Example 1 of the present invention at 3 mg / kg was injected into the in-situ lung cancer mouse model through the tail vein. After 6 h of tail vein injection, analysis was performed using a live imaging system.

[0063] Please refer to Figure 8 , from Figure 8 it can be seen that the cerium-doped carbon nitride-based biomimetic nanozyme MCeCNs prepared in the present invention can target the lung cancer site and achieve specific treatment for it.

[0064] In summary, a novel near-infrared light-absorbing cerium-doped carbon nitride-based biomimetic nanozyme was constructed in this application. It has good biocompatibility and biometabolism, which is conducive to its application in the biomedical field. Moreover, the cerium-doped carbon nitride-based biomimetic nanozyme can absorb near-infrared light;

[0065] The cerium-doped carbon nitride-based biomimetic nanozyme MceCNs constructed in this application is mainly synthesized by high-temperature calcination, ultrasonic treatment, and cell membrane encapsulation. The preparation method is simple, has good reproducibility, and has good prospects for industrial production.

Claims

1. A preparation method of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption, characterized in that: It includes the following steps: S1: Weigh a nitrogen-containing precursor and a cerium-containing precursor, and calcine them under nitrogen protection to obtain CeCNs; S2: Disperse the CeCNs powder obtained in S1 in water and use ultrasonic treatment; S3: Extract the A549 cell membrane, combine the A549 cell membrane with CeCNs in proportion, use ultrasonic treatment, and then centrifuge, precipitate and resuspend the mixture to obtain the MCeCNs biomimetic nanozyme.

2. The preparation method of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption according to claim 1, characterized in that: In S1, the nitrogen-containing precursor is melamine, the cerium-containing precursor is Ce(NO3)3, the calcination temperature is 200 - 700 °C, the time is 3 - 7 hours, and the heating rate is 3 - 10 °C / min.

3. The preparation method of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption according to claim 2, wherein: In S2, the ultrasonic treatment time is 1.5 h and the ultrasonic power is 300 W.

4. The preparation method of a cerium-doped carbon nitride-based bionic nanozyme with near-infrared light absorption according to claim 3, characterized in that: In S3, the mass ratio of the A549 cell membrane to CeCNs is 1:(0.5 - 10).

5. The preparation method of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption according to claim 4, characterized in that: In S3, the ultrasonic treatment time is 6 - 12 h, and the centrifugation conditions after ultrasonic treatment are: 13400 g, 10 min.

6. A cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption, characterized in that: It is prepared by using the preparation method described in any one of claims 1 - 5.

7. A cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption according to claim 6, characterized in that: The biomimetic nanozyme has Ce-doped carbon nitride as the core and is a brand-new biomimetic nanozyme with OXD, POD, GSHOx and CAT catalytic activities, oxygen generation and tumor targeting functions.

8. Use of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption in the preparation of a drug for treating lung cancer, characterized in that: The biomimetic nanozyme is prepared by using the preparation method described in any one of claims 1 - 5.

9. Use of a cerium-doped carbon nitride-based biomimetic nanozyme with near-infrared light absorption in the preparation of a drug for treating lung cancer, characterized in that: The biomimetic nanozyme, as an oxygen-producing and OXD, POD, GSHOx and CAT nanozyme, is used for the combined treatment of lung cancer.

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