Use of non-ros-dependent photoenergy enzymes in photocatalysis and nrf2 activation

Abstract

The application belongs to the field of nanomaterials and biotechnology, and particularly relates to application of non-ROS-dependent photoenzyme in photocatalysis and Nrf2 activation. The preparation method of the non-ROS-dependent photoenzyme is as follows: chloroauric acid is added into a mixed solution of 6-azido-2-thiothymine and sodium hydroxide, and the reaction is stirred at 25-30 DEG C to prepare nanometer photoenzyme; the molar ratio of 6-azido-2-thiothymine to chloroauric acid is (2.1-2.6):1. The non-ROS-dependent nanometer photoenzyme provided by the application has the advantages of simple and efficient synthesis method, ultrahigh photocatalytic activity, non-ROS-dependent catalytic mechanism, excellent biocompatibility, and the ability to realize intracellular light-controlled activation of Nrf2, and solves the problems of ROS-dependent oxidative damage, uncontrollable Nrf2 activation, and over-activation of signal pathways.

Description

Technical Field

[0001] This invention belongs to the fields of nanomaterials and biotechnology, specifically relating to the application of non-ROS-dependent photoenzymes in photocatalysis and Nrf2 activation. Background Technology

[0002] Photocatalysis technology can directly utilize light energy to drive biochemical reactions and achieve optical regulation of the reaction process, showing significant application prospects in biomedical fields such as protein catalytic modification and regulation of cellular physiological activities. Photoenzymes, as a novel photocatalytic system combining light and biocatalysis, possess high efficiency, specificity, and good biocompatibility. However, current research on photoenzymes still mainly relies on expensive and unstable natural enzymes, or requires cumbersome protein modification techniques and complex genetic engineering methods, limiting their widespread application.

[0003] In recent years, nanomaterials with enzyme-mimicking activity, namely nanozymes, have been regarded as powerful alternatives to natural enzymes due to their advantages such as high catalytic activity, structural stability, and low cost. In particular, the emergence of photoresponsive nanozymes has provided possibilities for constructing novel photocatalytic systems. Traditional photoresponsive nanozymes mainly rely on photoexcitation to generate large amounts of reactive oxygen species (ROS) to exert their catalytic function, and have been shown to have good effects in areas such as drug activation, cell signaling pathway regulation, and disease treatment. However, this ROS-dependent catalytic mechanism is prone to causing oxidative damage to cells or interfering with normal physiological processes, posing certain biosafety risks. Furthermore, the catalytic mechanism of photoenzymes essentially relies on photoexcitation to form the enzyme's own intermediate state to drive the reaction, which is fundamentally different from the ROS-dependent catalytic pathway of traditional photoresponsive nanozymes. Therefore, the latter are not strictly photoenzyme mimics. It is evident that developing ROS-independent photoexcited nanozymes can not only fill the current gap in nanozyme research in photoenzyme mimicry but also promises to provide a safer and more controllable new tool for biocatalysis.

[0004] Oxidative stress is a crucial mechanism in the development and progression of many diseases. It causes biomolecular damage and cellular dysfunction through high levels of reactive oxygen species, participating in the processes of inflammation, neurodegenerative diseases, cancer, and other illnesses. In the cellular antioxidant stress system, the Keap1-Nrf2 signaling pathway plays a central regulatory role. Under physiological homeostasis, Keap1, a cysteine-rich protein, maintains Nrf2 at low levels by promoting E3 ubiquitin ligase-mediated proteasome degradation. Under oxidative stress, Keap1 undergoes cysteine ​​residue modification, leading to Nrf2 activation and translocation to the nucleus and induction of target gene expression, thus playing a key role in oxidative stress, metabolism, and innate immunity.

[0005] Based on the above mechanisms, activating Nrf2 has become an important strategy for treating oxidative stress-related diseases, and the development of Nrf2 activators has attracted widespread attention. Currently, several small-molecule Nrf2 activators have been commercialized; however, these compounds generally lack a stimulus-response module, making it difficult to achieve dynamic and spatial control of the Nrf2 activation process. This can easily lead to physiological disturbances due to continuous or excessive activation of the signaling pathway, limiting the safety and precision of their clinical applications. Non-ROS-dependent photoenzymes have gained attention due to their good biosafety and efficient photocatalytic performance; their photoactivation characteristics can provide excellent spatiotemporal controllability for biocatalytic reactions. Therefore, utilizing such photoenzymes to achieve controllable activation of the Nrf2 signaling pathway offers new possibilities for developing next-generation intervention strategies for oxidative stress-related diseases. Summary of the Invention

[0006] The purpose of this invention is to provide the application of non-ROS-dependent photoenzymes in photocatalysis and Nrf2 activation, which solves the problems of ROS dependence leading to aggravated oxidative damage, uncontrollable Nrf2 activation, and overactivation of signaling pathways in the prior art.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] The application of non-ROS-dependent photoenzyme in photocatalysis and Nrf2 activation, the preparation method of the non-ROS-dependent photoenzyme is as follows: chloroauric acid is added to a mixed solution of 6-aza-2-thiothymidine and sodium hydroxide, and the reaction is stirred at 25℃-30℃ to obtain nano-photoenzyme;

[0009] The molar ratio of 6-aza-2-thiothymidine to chloroauric acid is (2.1-2.6):1.

[0010] The non-ROS-dependent photoenzyme provided by this invention is prepared through the self-assembly and reduction reaction of a ligand (6-aza-2-thiothymidine, abbreviated as ATT) and a metal precursor (chloroauric acid, HAuCl4). This preparation method is simple and efficient; the ligand only requires a one-step reduction process to synthesize nanomaterials, eliminating the need for complex subsequent separation and purification procedures. Furthermore, this photoenzyme exhibits ultra-high photocatalytic activity, a non-ROS-dependent catalytic mechanism, and excellent biocompatibility, enabling photocontrolled activation of Nrf2 within cells. This solves the problems of ROS-dependent photoenzymes leading to increased oxidative damage, uncontrollable Nrf2 activation, and overactivation of signaling pathways.

[0011] Furthermore, the inventors discovered during their research that the catalytic performance of the nano-photoenzyme mainly depends on its size, structure, and surface chemical properties. Reaction temperature and molar ratio influence the catalytic activity of the final product by regulating these intrinsic properties. Specifically, a suitable temperature (25-30℃) ensures that ATT molecules have sufficient kinetic energy for self-assembly, forming an ordered, stable ligand layer with a specific conformation on the gold core surface. Nano-photoenzymes synthesized within this temperature range exhibit suitable size, high uniformity, and a stable surface active interface, thus demonstrating the highest catalytic activity. The molar ratio of 6-aza-2-thiothymidine to chloroauric acid directly determines the stoichiometry of the reaction system, affecting the degree of reduction of the gold core, the surface ligand density, and the overall structural stability. When ATT:HAuCl4 = (2.1-2.6):1, such as ATT:HAuCl4 = 2.2:1, 2.4:1, or 2.6:1, ATT provides sufficient electrons to convert Au... 3+ Fully restored to Au 0 Or Au + This forms the core of gold nanoclusters. Excessive ATT helps form tiny, precisely numbered nanoclusters, whose specific electronic energy levels are key to generating enzyme-like activity. Meanwhile, temperature and molar ratio do not act in isolation. Only at the optimal molar ratio and suitable temperature can the most ideal non-ROS-dependent photoenzyme be formed.

[0012] In some other embodiments, the concentration of sodium hydroxide in the mixed solution is 0.15-0.25 mol / L;

[0013] The stirring reaction time is 0.5-2 h.

[0014] Specifically, the concentration of sodium hydroxide in the mixed solution is 0.15, 0.2, or 0.25 mol / L. The alkali concentration directly affects the reduction potential and the surface charge (Zeta potential) of the particles. At this optimized concentration, it works synergistically with ATT to create an ideal electrostatic stability and susceptibility environment. This ensures that the nanoparticles do not struggle to form stable nuclei due to excessive electrostatic repulsion during growth, while also preventing excessive aggregation due to insufficient repulsion.

[0015] Specifically, the stirring reaction time is 0.5, 1, 1.5, or 2 hours. Within a sufficient reaction time of 0.5-2 hours, the entire system, under the combined maintenance of temperature and alkalinity, completes atomic-level structural rearrangement and stabilization, ultimately preparing a nano-photoenzyme with excellent catalytic performance.

[0016] In some other embodiments, the molar ratio of 6-aza-2-thiothymidine to chloroauric acid is 2.2:1;

[0017] The stirring reaction was carried out at a temperature of 30°C for 1 hour.

[0018] The concentration of sodium hydroxide in the mixed solution is 0.2 mol / L.

[0019] Reaction temperature, molar ratio, sodium hydroxide concentration, and reaction time are crucial parameters in the synthesis of nano-photoenzymes. Studies have found that within a specific range, these parameters collectively constitute an optimized preparation system, and their synergistic regulation leads to the best final performance of the prepared nano-photoenzymes.

[0020] In some other embodiments, the average size of the non-ROS-dependent photoenzyme is 2-3 nm. Specifically, the average size of the non-ROS-dependent photoenzyme is 2, 2.36, 2.5, or 3 nm. Non-ROS-dependent photoenzymes within this range exhibit a molecular-like state, ensuring cell membrane permeability and biocompatibility.

[0021] In some other embodiments, photocatalysis uses a non-ROS-dependent photoenzyme as a catalyst to catalyze the reaction of a fluorescent red dye (Amplex Red, abbreviated as AR, a fluorescent probe highly sensitive to hydrogen peroxide and peroxidase) under nitrogen or air irradiation. The catalytic ability of this photoenzyme is determined by the photoinduced catalytic intermediate and is not affected by ROS; its catalytic mechanism is similar to that of natural photoenzymes.

[0022] In some other embodiments, the concentration of the non-ROS-dependent photoenzyme in the photocatalytic reaction is 15-25 µg / mL; the concentration of the fluorescent red dye is 0.05-5 µM; and the light treatment is irradiation with a 5-15 W full-band white light lamp for 2-10 min.

[0023] Specifically, the concentration of non-ROS-dependent photoenzyme in the photocatalytic reaction is 15, 20, or 25 µg / mL; the concentration of fluorescent red dye is 0.05, 1, 2, 3, 4, or 5 µM; and the light treatment is irradiation with white light of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15W for 2, 3, 4, 5, 6, 7, 8, 9, or 10 min.

[0024] In some other embodiments, a non-ROS-dependent photoenzyme is used as the photo-controlled activator for Nrf2 activation. The non-ROS-dependent photoenzyme only activates Nrf2 under photoexcitation, and the spatiotemporally controllable catalytic process ensures the controllability and safety of Nrf2 regulation.

[0025] In some other embodiments, the cells are incubated with light using a non-ROS-dependent photoenzyme.

[0026] In some other embodiments, the concentration of the non-ROS-dependent photoenzyme is 1-50 µg / mL; the light treatment is performed by irradiation with 700-850 nm red light for 2-6 h.

[0027] Specifically, the concentration of the non-ROS-dependent photoenzyme is 1, 10, 20, 30, 40 or 50 µg / mL; the light treatment is irradiation with a 700, 800 or 850 nm red light lamp for 2, 3, 4, 5 or 6 h.

[0028] The beneficial effects of this invention are:

[0029] (1) The non-ROS-dependent nanophotocatalytic enzyme of this invention exhibits ultra-high photocatalytic activity and excellent kinetic parameters. Among them, K m It is 0.46 µM, V max It is 9.75×10 -4 M / s, superior to most nanozymes of similar substrates. At the same time, it has a non-ROS-dependent catalytic mechanism, with its catalytic ability determined by photoinduced catalytic intermediates and unaffected by ROS, and its catalytic mechanism is similar to that of natural photoenzymes.

[0030] (2) The non-ROS-dependent photoenzyme of this invention has excellent biocompatibility. Its size is 2-3 nm, exhibiting a molecular-like state, which ensures cell membrane permeability and biocompatibility. After cytotoxicity evaluation, the photoenzyme showed that even when treated with light at a concentration as high as 50 µg / mL, the cytotoxicity remained very low, proving that Au-A can ensure the safety of biological applications. At the same time, it has Nrf2 photo-activated properties. Western blotting results show that Au-A only activates Nrf2 under photoexcitation. The spatiotemporally controllable catalytic process ensures the controllability and safety of Nrf2 regulation.

[0031] (3) The method for synthesizing non-ROS-dependent nanophotonic enzymes of the present invention is simple and efficient. The ligands can be further reduced to synthesize nanomaterials without subsequent complex separation and purification processes. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 This is a schematic diagram illustrating the preparation of the non-ROS-dependent nanophotoenzyme Au-A in Example 1 of the present invention;

[0034] Figure 2 This is a diagram of the purified Au-A solution under fluorescent light in Example 1 of the present invention;

[0035] Figure 3 This is a low-magnification transmission electron microscope image of Au-A in Embodiment 1 of the present invention;

[0036] Figure 4 This is a size distribution diagram of Au-A in Embodiment 1 of the present invention;

[0037] Figure 5 This is a graph showing the catalytic performance of Au-A with fluorescent red dye (AR) as a substrate under both light and dark conditions in Example 1 of this invention.

[0038] Figure 6 This is a comparison chart of the catalytic performance of ATT and HAuCl4 at different molar ratios when AR is used as the substrate in Example 2 of the present invention;

[0039] Figure 7 This is the Michaelis-Menten kinetic analysis diagram of Au-A with AR as the substrate in Embodiment 1 of the present invention;

[0040] Figure 8 This is a Lineweaver-Burk double reciprocal plot of the Au-A nanozyme with AR as the substrate in Example 1 of the present invention;

[0041] Figure 9 This is a graph showing the catalytic performance of Au-A with AR as a substrate under nitrogen and air conditions in Example 1 of this invention.

[0042] Figure 10 This is a cytotoxicity diagram of different Au-A concentrations applied under an 800 nm red light lamp in Example 1 of the present invention;

[0043] Figure 11 This is a Western blot image of Au-A used for Nrf2 activation in Embodiment 1 of the present invention, where "+" indicates the application. Detailed Implementation

[0044] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0045] Example 1

[0046] This embodiment provides a non-ROS-dependent nanophotoenzyme, its preparation method, and its application, specifically including the following steps:

[0047] Synthesis of ROS-independent nanophotoenzyme (labeled Au-A):

[0048] Figure 1The diagram shows a non-ROS-dependent nanophotoenzyme prepared from chloroauric acid (HAuCl4) and aza-2-thiothymidine (ATT). Figure 1 As shown, 80 mM aza-2-thiothymidine (ATT) solution (containing 0.2 mol / L NaOH, 0.44 mol ATT) was added dropwise to 1 mL of 10 mg / mL chloroauric acid (HAuCl4, 0.2 mol) solution. The mixture was placed in a brown, light-proof synthesis flask and stirred at 30 °C for 1 h. After ultrafiltration purification, pure nanophotoenzyme (labeled Au-A) was obtained.

[0049] Figure 2 The image shows the purified Au-A solution under fluorescent light. Figure 2 As shown, the aqueous solution of Au-A photoenzyme is a clear, transparent, bright yellow color under natural light, exhibiting excellent water solubility and dispersibility. Figure 3 This is a low-magnification transmission electron microscope image of Au-A. Figure 4 This is a size distribution diagram of Au-A. (From...) Figure 3 and Figure 4 As shown, the average size of Au-A photoenzyme is approximately 2.36 nm, and it exhibits a uniform monodisperse particle state.

[0050] Example 2

[0051] Unlike Example 1, in step (1) of the synthesis of nano-photoenzymes, the molar ratios of 6-aza-2-thiothymidine solution and chloroauric acid solution were 2.4:1, 2.6:1, 2.8:1 and 3.0:1, respectively. After stirring at room temperature for 1 h, the mixture was purified by ultrafiltration using an ultrafiltration tube to obtain a series of nano-photoenzymes with different molar ratios.

[0052] Performance testing

[0053] (1) The highly efficient photo-driven catalytic ability of Au-A:

[0054] Prepare 1 mL HAc-NaAc buffer (containing 20 µg / mA Au-A and different concentrations (0.05-5 µM) of Amplex Red (AR, a fluorescent probe highly sensitive to hydrogen peroxide and peroxidase). Illuminate the HAc-NaAc buffer with a full-spectrum household white light (9W) for 5 min, and record the fluorescence intensity at different time points. Finally, obtain the kinetic constants according to the Michaelis–Menten equation.

[0055] Figure 5 This chart shows the catalytic performance of Au-A with Amplex Red (AR) as a substrate under both light and dark conditions. Figure 5 It can be seen that Au-A photoenzyme exhibits catalytic oxidation ability of AR substrate when excited by light, but does not exhibit catalytic performance when there is no light, indicating that the catalytic activity of Au-A is controlled by light.

[0056] Figure 6 This is a comparison of the catalytic performance of ATT and HAuCl4 at different molar ratios when AR is used as the substrate; from Figure 6 It can be seen that the ATT:HAuCl4 molar ratio of 2.2:1 has the strongest catalytic performance, while other molar ratios have very low catalytic performance (such as ATT:HAuCl4 molar ratios of 2.4:1 and 2.6:1), or even no catalytic ability (such as ATT:HAuCl4 molar ratios of 2.8:1 and 3.0:1).

[0057] Figure 7 Michaelis-Menten kinetic analysis of Au-A with AR as the substrate; Figure 8 Lineweaver-Burk double reciprocal plot of Au-A nanozyme with AR as substrate. Figure 7 and 8 It can be seen that the dynamic parameter K of Au-A m Value and V max The values ​​were 0.46 µM and 9.75 × 10⁻⁶, respectively. -4 The M / s indicates that Au-A photoenzyme possesses excellent AR substrate affinity and ultra-high efficiency photocatalytic ability.

[0058] Figure 9 This is a graph showing the catalytic performance of Au-A with AR as a substrate under nitrogen and air conditions. Figure 9 It can be seen that,

[0059] Unlike traditional nanozymes, Au-A photoenzymes exhibit highly efficient catalytic performance even under anaerobic conditions, unaffected by reactive oxygen species (ROS). This indicates that Au-A can be photoexcited to become an independent catalyst participating in catalytic reactions, a catalytic mechanism similar to that of natural photoenzymes.

[0060] (2) Au-A photocontrolled activation of Nrf2 in cancer cells:

[0061] HepG2 cells were seeded into six-well plates (3.6 × 10⁶ cells per well). 5 Cells were cultured at 37°C for 24 h in a cell culture incubator. Then, the cells were subjected to different treatments: the control group received no drugs and no treatment; the experimental group underwent three treatments: Au-A incubation for 1 h + light treatment for 4 h, Au-A incubation for 1 h, and Au-A light treatment for 4 h. After treatment (incubation), cell proteins were extracted, and the expression level of Nrf2 in HepG2 cells was detected by Western blotting.

[0062] Figure 10 Cytotoxicity graphs of different Au-A concentrations under red light (wavelength 800 nm). Figure 10 It is known that Au-A photoenzyme (≤ 50 µg / mL) exhibits negligible cytotoxicity under light irradiation, demonstrating excellent biocompatibility and ensuring the safety of biological applications.

[0063] Figure 11 Western blot images showing the expression of Nrf2 protein in the nucleus of HepG2 cells in the control and experimental groups. Figure 11 It was found that, compared with the control group cells which remained in their original biological state without any treatment, the experimental group showed a significant increase in the expression level of Nrf2 in the cell nucleus after incubation with Au-A photoenzyme for 1 hour under photoexcitation. However, neither incubation with Au-A photoenzyme alone nor light exposure alone increased the expression level of Nrf2 in the cell nucleus. This indicates that Au-A photoenzyme can only induce nuclear aggregation of Nrf2 under photoexcitation, making it an excellent light-controlled Nrf2 activator.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of non-ROS-dependent photoenzymes in photocatalysis and Nrf2 activation, characterized in that, The preparation method of the non-ROS-dependent photoenzyme is as follows: chloroauric acid is added to a mixed solution of 6-aza-2-thiothymidine and sodium hydroxide, and the mixture is stirred at 25℃-30℃ to obtain nano-photoenzyme. The molar ratio of 6-aza-2-thiothymidine to chloroauric acid is (2.1-2.6):1; The average size of the non-ROS-dependent photoenzyme is 2-3 nm; Photocatalysis is the catalytic reaction of fluorescent red dyes by phototreatment under nitrogen or air conditions using non-ROS-dependent photoenzymes as catalysts. The concentration of the non-ROS-dependent photoenzyme in the photocatalytic reaction is 15-25 µg / mL; the concentration of the fluorescent red dye is 0.05-5 µM; and the light treatment is irradiation with a 5-15 W full-band white light lamp for 2-10 min. The Nrf2 activation uses a non-ROS-dependent photoenzyme as the Nrf2 photocontrolled activator; The concentration of the non-ROS-dependent photoenzyme is 1-50 µg / mL; the light treatment is irradiation with 700-850 nm red light for 2-6 h.

2. The application according to claim 1, characterized in that, The concentration of sodium hydroxide in the mixed solution is 0.15-0.25 mol / L.

3. The application according to claim 1, characterized in that, The stirring reaction time is 0.5-2 h.

4. The application according to claim 1, characterized in that, The molar ratio of 6-aza-2-thiothymidine to chloroauric acid is 2.2:

1.

5. The application according to claim 1, characterized in that, It also includes using non-ROS-dependent photoenzymes to incubate cells with light.

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