Application of horse radish peroxidase as exogenous activator of artemisinin and derivatives thereof

Abstract

The invention belongs to the technical field of biological medicine, provides application of horse radish peroxidase as an exogenous activator of artemisinin and derivatives thereof, and is based on the characteristic that the horse radish peroxidase can catalyze the artemisinin and the derivatives thereof to generate high-level ROS (reactive oxygen species). The invention also provides an application of the horse radish peroxidase in preparation of anti-tumor nano drug-loaded particles.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the use of horseradish peroxidase as an exogenous activator of artemisinin and its derivatives. Background Art

[0002] Horseradish peroxidase (HRP) is a monomeric, heme-containing plant enzyme belonging to the peroxidase family (Journal of the American Chemical Society, 2024, 146, 13247-13257). It is one of the most commonly used enzymes in biochemistry and molecular biology experiments, favored for its high catalytic efficiency, stability, and broad substrate adaptability. It plays an irreplaceable role in biomedical research, clinical diagnostics, and industrial testing. Its core value lies in converting invisible biomolecular interactions into detectable signals, serving as a bridge between biological and chemical analysis.

[0003] Artemisinin and its derivatives are a series of compounds derived from the natural product artemisinin (ART) through chemical modification. These compounds include artemisinin, artesunate, dihydroartemisinin, artemether, and arteether. They are primarily used to treat malaria, particularly drug-resistant falciparum malaria. Studies have shown that the peroxide bridge structure in artemisinin and its derivatives breaks down upon exposure to activators, generating highly reactive alkoxy free radicals that alkylate biomacromolecules such as proteins, thereby causing the death of malarial parasites or cancer cells (Medicinal Research Reviews. 2017.37:1492-1517). While the peroxide bridge structure in artemisinin and its derivatives is key to their efficacy, it also makes them susceptible to environmental degradation, limiting their potential for direct cancer treatment. Furthermore, artemisinin and its derivatives are prodrugs of ROS, resulting in poor water solubility and requiring metabolic activation in vivo to exert their pharmacological effects (Chemical Society Reviews. 1998.27:273-279). The insufficient amount of endogenous activators of artemisinin and its derivatives in the body significantly reduces the anti-tumor efficacy of directly delivering artemisinin and its derivatives. Therefore, the development of an exogenous activator that efficiently catalyzes the release of artemisinin and its derivatives is of great significance in improving their anti-tumor efficacy in the body.

[0004] As "prodrugs" of ROS, artemisinin and its derivatives must undergo metabolic activation in the body, with their peroxide bridge structures oxidized, before they can exert their pharmacological effects. When artemisinin and its derivatives are delivered alone for anti-tumor therapy, the lack of endogenous activators in the body limits their ROS generation efficiency to a certain extent, further reducing their pharmacological effects. Therefore, the development of exogenous activators for artemisinin and its derivatives is beneficial for enhancing their pharmacological activity in vivo. Summary of the Invention

[0005] The present inventors have discovered that horseradish peroxidase can be used to efficiently catalyze the production of high levels of ROS from artemisinin and its derivatives. This discovery is surprising because it is not predicted that enzymes known to catalyze hydrogen peroxide would be able to catalyze the production of high levels of ROS from artemisinin and its derivatives.

[0006] Therefore, according to the present invention, a first aspect provides the use of horseradish peroxidase as an exogenous activator of artemisinin and its derivatives.

[0007] The artemisinin and its derivatives described herein include compounds containing peroxide bridges, such as artemisinin, artesunate, dihydroartemisinin, artemether, and arteether. The applications described include: HRP can efficiently catalyze the release of ROS from artemisinin and its derivatives, further oxidizing TMB and HPPA. Further electron paramagnetic resonance experiments revealed that the ROS generated during this catalytic process originate from alkoxy free radicals generated by the cleavage of peroxide bridges in the structures of artemisinin and its derivatives.

[0008] Secondly, based on the fact that HRP can catalyze artemisinin and its derivatives to produce high levels of ROS, the present invention also provides the use of horseradish peroxidase in the preparation of anti-tumor drug-loaded nanoparticles. The application steps include: using artemisinin and its derivatives as ROS precursors and covalently linking an exogenous activator, HRP, via a nanocarrier to construct an anti-tumor drug-loaded nanoparticle with a ROS-responsive cascade amplification effect, which is used to enhance tumor immunotherapy.

[0009] As a preferred embodiment of the second aspect of the present invention, the application comprises the steps of: Figure 1As shown, artemisinin and its derivatives are loaded in the hydrophobic cavity of the nano delivery carrier, and the exogenous activator HRP is connected to the nano delivery carrier by a click reaction to construct the anti-tumor nano drug-carrying particles, which are used to achieve cascade amplification of endogenous reactive oxygen species response in the tumor microenvironment; wherein the nano delivery carrier is a ROS-responsive organic silicon oxide cross-linked micelle RSCLMs, which is self-assembled from a silicon source and an amphiphilic polymer in a hydrochloric acid solution, and cyclohexane is used as a pore-expanding agent and dimethyldimethoxysilane is used as a silicon source hydrolysis terminator, and the hydrated particle size is 5 to 100 nm; wherein the mass ratio of the silicon source to the amphiphilic polymer is (1 to 3):1; and the concentration of the hydrochloric acid solution is 0.05 to 3.5 mol·L -1 ; The volume fraction of cyclohexane is 0-3.5%, and the volume fraction of dimethyldimethoxysilane is 0.4-3.0%; it should be noted that the volume fraction here refers to the ratio of the solute to the volume of the solution; wherein, the mass ratio of RSCLMs, HRP, artemisinin and its derivatives is 100:(5-10):(0.5-5); it should be noted that the click reaction of the present invention is a reaction between a thiol group and a maleimide, or a reaction between an amino group and an N-hydroxysuccinimide ester; for example, by thiolating the exogenous activator HRP and modifying the maleimide on the amphiphilic polymer in the nanodelivery carrier, the click reaction can be achieved; for example, by modifying the N-hydroxysuccinimide ester group on the amphiphilic polymer in the nanodelivery carrier, a click reaction occurs with the amino group on the HRP. As a better embodiment of the second aspect of the present invention, artemisinin and its derivatives and tumor treating drugs are simultaneously loaded in the hydrophobic cavity of the nanodelivery carrier, and the tumor treating drug is one of 7-ethyl-10-hydroxycamptothecin SN38, doxorubicin hydrochloride, cisplatin, gemcitabine, paclitaxel, and cyclophosphamide; the mass ratio of the RSCLMs, HRP, artemisinin and its derivatives and the tumor treating drug is 100: (5~10): (0.5~5): (0.1~2).

[0010] Furthermore, the silicon source is composed of two types: tetraalkyl orthosilicate and a bridging silane containing a thioacetal bond (TK chain), wherein the tetraalkyl orthosilicate is any one of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate, and the full name of the bridging silane containing a thioacetal bond (TK chain) is 3,3'-(propane-2,2-diylbis(thio))bis(N-(3-triethoxysilylpropyl)propionamide and its abbreviation is APTES-TK-APTES; wherein the raw material configuration volume ratio of the tetraalkyl orthosilicate and APTES-TK-APTES is (1 to 4):1.

[0011] Furthermore, the amphiphilic polymer is composed of a nonionic surfactant and a modified nonionic surfactant; wherein the raw material configuration mass ratio of the nonionic surfactant and the modified nonionic surfactant is (5-50):1; the nonionic surfactant is selected from any one of Pluronic F108, Pluronic F127, and Pluronic P123, and the modified nonionic surfactant is a nonionic surfactant modified with maleimide or N-hydroxysuccinimide ester.

[0012] Based on the present invention, HRP can be used as an exogenous activator for artemisinin and its derivatives, efficiently catalyzing the oxidation of TMB and HPPA by high levels of ROS produced by artemisinin and its derivatives. This overcomes the dilemma of low catalytic efficiency when artemisinin and its derivatives are delivered as ROS prodrugs to exert their pharmacological activity. The present invention demonstrates that HRP can efficiently catalyze the production of high levels of ROS by artemisinin and its derivatives through oxidation experiments with TMB and HPPA. Electron paramagnetic resonance experiments also confirm that the high levels of ROS produced by HRP-catalyzed artemisinin and its derivatives are alkoxy free radicals.

[0013] The anti-tumor drug-loaded nanoparticles constructed in this invention serve as a ROS-responsive cascade amplification nano-drug delivery system, which can be used to enhance the immune efficacy of tumors. In vivo anti-tumor testing results show that the constructed ROS-responsive cascade nano-amplification system, utilizing HRP-catalyzed production of high levels of ROS from artemisinin and its derivatives, can kill tumors and effectively inhibit tumor cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a flow chart for constructing the tumor microenvironment endogenous reactive oxygen species self-responsive cascade amplification nano-drug delivery system of the present invention;

[0015] Figure 2a The color change of the ART+TMB solution before and after the addition of HRP in the TMB color reaction test in Example 1; Figure 2b This is the absorption spectrum of the ART+TMB solution before and after the addition of HRP in the TMB colorimetric reaction test in Example 1; Figure 2c This is a graph showing the change in absorbance at 652 nm over time of the ART+TMB solution after HRP was added to the TMB color reaction test in Example 1;

[0016] Figure 2d This is a graph showing the change in absorbance at 652 nm over time of the ART+TMB solution after adding different concentrations of HRP to the TMB color reaction test in Example 1;

[0017] Figure 3aThis is the HPPA fluorescence detection test in Example 1, after HRP reacted with different concentrations of ART at room temperature for 1 hour, the fluorescence spectrum of HPPA;

[0018] Figure 3b This is a graph showing the change in HPPA fluorescence detection test in Example 1, in which HRP catalyzes the oxidation of HPPA by ART at different concentrations, and the change in HPPA fluorescence over time;

[0019] Figure 4 This is a comparative test of the ROS production rate of ART, AS, and DHA catalyzed by HRP in Example 2, and a graph showing the change in the absorbance of the solution at 652 nm over time;

[0020] Figure 5 This is a graph showing the change of the electron paramagnetic resonance spectrum over time in the detection experiment of HRP-catalyzed ART producing ROS in Example 3;

[0021] Figure 6a is the dynamic light scattering image of RSCLMs;

[0022] Figure 6b Dynamic light scattering image of RSCLMs after responding to H2O2;

[0023] Figure 7a Transmission scanning electron microscopy images of RSCLMs;

[0024] Figure 7b Transmission scanning electron microscopy images of RSCLMs after responding to H2O2;

[0025] Figure 8a The results of inverted fluorescence imaging in Example 7 for detecting intracellular ROS in 4T1 cells after incubation of different materials (Control, RSCLMs-H, S@RSCLMs-H, A@RSCLMs, A@RSCLMs-H, and A&S@RSCLMs-H);

[0026] Figure 8b To use Image J software Figure 8a Semi-quantitative statistics of green fluorescence, n = 5, , ***p < 0.001;

[0027] Figure 9a This is an image of the size of the tumor in 4T1 tumor-bearing mice after the treatment was completed after the different materials in Example 8 were injected through the tail vein;

[0028] Figure 9b The weight of the tumor in 4T1 tumor-bearing mice after the treatment was completed after the different materials in Example 8 were injected through the tail vein;

[0029] Figure 9c Figure 1 is a graph showing the changes in tumor volume during treatment in 4T1 tumor-bearing mice after injection of different materials via tail vein in Example 8, n=5. *p<0.05, **p<0.01, ***p<0.001; Figure 9d Figure 1 is a graph showing changes in body weight of 4T1 tumor-bearing mice during treatment after injection of different materials via tail vein in Example 8, n=5. *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0030] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0031] The present invention will be described in detail below with reference to the embodiments.

[0032] Example 1: Detection of high levels of ROS produced by HRP-catalyzed artemisinin ART

[0033] The classic chromogenic substrate of HRP, 3,3',5,5'-tetramethylbenzidine (TMB), was selected as the oxidation indicator for this catalytic reaction. HRP can catalyze hydrogen peroxide (H2O2) to oxidize colorless TMB into blue oxidized TMB (oxTMB) with absorption at 652nm. In order to verify that HRP can catalyze artemisinin and its derivatives to produce ROS and further oxidize TMB, the specific steps for the detection of HRP-catalyzed ART oxidation of TMB are as follows: Measure 2mL of TMB solution (2mg·mL -1 ) in a 2 mL centrifuge tube, add 10 μL of ART (100 mM, dissolved in ethanol), observe and record the color of the solution and measure the UV absorption spectrum of the TMB solution. Then add 10 μL of HRP (2 μg mL -1 ), record the change curve of the ultraviolet absorption spectrum of the TMB solution over time. After the reaction time of 30 minutes, observe and record the color change of the TMB solution.

[0034] like Figure 2aAs shown in the figure, when an appropriate amount of artemisinin and its derivatives are added to the TMB solution, the solution is colorless when only artemisinin derivatives are present, indicating that TMB will not be oxidized by artemisinin and its derivatives. After adding HRP, the TMB solution turns blue after 30 minutes of catalytic reaction at room temperature, indicating that HRP catalyzes the production of highly oxidative ROS by artemisinin derivatives, oxidizing TMB into blue oxTMB. The results of the ultraviolet absorption spectrum show that the maximum absorption value at 652nm is significantly enhanced, and as the catalytic time increases, the ultraviolet absorption value at 652nm gradually stabilizes, as shown in the figure. Figure 2b and Figure 2c When the concentration of HRP increases, the catalytic rate increases, which shortens the time for the absorbance value of the solution at 652nm to reach equilibrium, indicating that high concentration HRP catalyzes ART to quickly produce high levels of ROS to further oxidize TMB. Figure 2d , indicating that HRP exhibits obvious enzymatic reactivity in the catalysis of artemisinin and its derivatives.

[0035] The fluorescent colorimetric substrate of HRP, p-hydroxyphenylpropionic acid (HPPA), was selected to further verify the ability of HRP to catalyze artemisinin and its derivatives to produce ROS. A certain amount of HRP was added to the mixed solution system of HPPA and ART and reacted at room temperature for a period of time. The fluorescence spectrometer recorded the changes in the fluorescence spectrum of the solution. The specific steps for detecting the oxidation of HPPA by ART catalyzed by HRP are as follows: 500 μL of HPPA solution (2 mg mL -1 ) were added to a 2 mL centrifuge tube, 0, 5, 10, 20 μL of ART (100 mM) were added, and then 10 μL of HRP (2 μg mL -1 ), react at room temperature, and record the fluorescence spectrum of the HPPA solution over time.

[0036] like Figure 3a As shown, after HRP reacted with different concentrations of ART at room temperature for 1 hour, the fluorescence intensity value of the fluorescence spectrum of HPPA at 410 nm gradually increased with the increase of ART concentration. Figure 3bThe results showed that HRP catalyzed the production of ROS at varying concentrations of artemisinin, which further oxidized HPPA into fluorescent products. With increasing catalytic time, the fluorescence intensity at 410 nm gradually stabilized, demonstrating that HRP catalyzed artemisinin exhibits enzymatic properties. These results suggest that, under certain HRP concentrations, the oxidation activity of artemisinin and its derivatives toward HPPA is concentration-dependent. Specifically, increasing the concentration of artemisinin and its derivatives intensified the degree of HPPA oxidation, with a significant increase in the fluorescence intensity at 410 nm. With increasing catalytic time, the fluorescence intensity of the HPPA solution reached a peak and remained constant. Furthermore, the peak fluorescence intensity of HPPA significantly increased with increasing concentrations of artemisinin and its derivatives. To further investigate the catalytic activity of HRP on artemisinin and its derivatives, varying concentrations of HRP were added to a mixed solution of HPPA and artemisinin and its derivatives, and the fluorescence spectra of the HPPA solutions were recorded using a fluorescence spectrometer. The results showed that high HRP concentrations accelerated the catalytic activity of artemisinin and its derivatives, significantly shortening the time it took for HPPA's fluorescence to reach its maximum value. While HRP concentrations varied, the catalytic rate of artemisinin analogs varied. However, over time, the maximum fluorescence intensity of HPPA remained constant, indicating that HRP only affected the rate of HPPA oxidation by ART. The greater the amount of HRP, the faster ART oxidized HPPA. These results demonstrate that, given the same substrate concentration, the higher the enzyme concentration, the faster the reaction, indicating that the reaction rate is enzyme concentration-dependent.

[0037] The results of the above two experimental methods both indicate that HRP can serve as an exogenous activator of ART, efficiently catalyzing ART to produce high levels of ROS, which further oxidizes TMB and HPPA. HRP only affects the reaction rate but not the reaction equilibrium, and has the characteristics of an enzymatic reaction.

[0038] Example 2: Comparison of HRP-catalyzed ROS production efficiency of artemisinin, artesunate, and dihydroartemisinin

[0039] To further evaluate the efficiency of HRP artemisinin and its derivatives in generating ROS, the steps are as follows: 100 μL of HRP artemisinin and its derivatives were measured at a concentration of 0.2 mg mL -1 TMB solution was added to a 96-well plate, and 4 μL of ART, artesunate AS, and dihydroartemisinin DHA dissolved in ethanol at a concentration of 12.5 mM were added, and an equal volume of blank solvent ethanol was added as a control group. Then, 4 μL of 1 μg mL -1 The HRP of TMB solution was added to record the change curve of UV absorption spectrum over time. The number of samples in each group was n=5.

[0040] like Figure 4As shown in the figure, within the same catalytic time, the absorbance change rates of the solutions in different groups were roughly the same, indicating that HRP can efficiently catalyze ART, AS and DHA to produce high levels of ROS, and the catalytic rates are the same. This is attributed to the fact that artemisinin and its derivatives AS and DHA contain the same number of peroxide bridge bonds in their structures.

[0041] Example 3: Detection of ROS species generated by HRP-catalyzed ART

[0042] 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was used as a ROS scavenger in electron paramagnetic resonance (EPR) to verify the generation of ROS. 30 μL of 12.5 mM ART solution was added to 500 μL of PBS (pH = 7.4), followed by 20 μL of 1 μg mL -1 Prepare a reaction mixture by combining 50 μL of 1 M HRP solution and 50 μL of 1 M DMPO. Immediately transfer the mixture to a capillary tube for EPR analysis. Spectra were acquired at the designated time points of 5, 15, and 30 minutes to monitor the formation of DMPO-ROS adducts.

[0043] like Figure 5 As shown, the obtained EPR spectrum has broad and overlapping hyperfine splitting peaks, and the hyperfine coupling constant A N and A Hβ These spectra are consistent with the characteristics of alkoxyl radicals. Therefore, HRP-catalyzed ART can generate a large number of alkoxyl radicals.

[0044] Example 4: Preparation of ROS-responsive organosilica cross-linked micelles RSCLMs

[0045] Preparation of ROS-responsive organosilica cross-linked micelles RSCLMs, the steps are as follows:

[0046] Step 1. Preparation of bridged silane APTES-TK-APTES containing a thioacetal bond (TK chain): 28.30 mmol of trimercaptopropionic acid and 14.65 mmol of anhydrous acetone were placed in a dry container, 50 μL of trifluoroacetic acid was added, and the mixture was stirred overnight. The mixture was crystallized in an ice-water bath, filtered and washed several times, and dried in vacuo to obtain 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK-COOH). 10.00 mmol of the prepared TK-COOH was added to 100 mL of dichloromethane, stirred to dissolve, 30.00 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 30.00 mmol of N,N-diisopropylethylamine (DIPEA) were added, and finally 30.00 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 30.00 mmol of N,N-diisopropylethylamine (DIPEA) were quickly added. N-hydroxysuccinimide (NHS) was stirred at room temperature for overnight reaction, excess solvent was removed by rotary evaporation, column chromatography was performed for purification and separation, and rotary evaporation was performed to obtain a white solid powder of bis(2,5-dioxopyrrolidin-1-yl) 3,3'-(propane-2,2-diylbis(thio))dipropionate (NHS-TK-NHS); 10.00 mmol of NHS-TK-NHS was weighed and dissolved in 100 mL of ultra-dry dichloromethane, 22.00 mmol of triethylamine (Et3N) and 22.00 mmol of 3-aminopropyltriethoxysilane (APTES) were added, and stirred overnight. After the reaction was completed, the solution was dried by rotary evaporation, and ethyl acetate (EA) was added to precipitate a solid. The solid was filtered, the solid was repeatedly washed, and the filtrate was dried to obtain a yellow oily liquid containing a bridging silane containing a thioacetal bond, namely APTES-TK-APTES.

[0047] Step 2, Preparation of Maleimide-Modified F108: Tetrahydrofuran (THF) (20 mL), triphenylphosphine (PPh3) (392 mg), N-hydroxymaleimide (112.5 mg), and F108 (Mw, 14.6 KDa, 7.4 g) were added sequentially to a 100 mL flask and cooled to 0°C. Diisopropyl azodicarboxylate (DIAD) (73.6 μL) was dissolved in THF (2 mL) and added dropwise to the flask, and the reaction was continued at 0°C for 30 minutes. Subsequently, the mixture was heated to room temperature and stirred overnight. Afterwards, the solvent was removed by evaporation under reduced pressure, and the precipitate was resuspended in a mixed solution of n-hexane and ethyl acetate (n-hexane:ethyl acetate, 50:50, volume ratio). The crude product was filtered, washed three times with a mixture of n-hexane and ethyl acetate (n-hexane:ethyl acetate, 50:50, volume ratio), and finally dried to obtain the product maleimide-modified F108, which is abbreviated as MAL-F108.

[0048] Step 3. Accurately weigh 243.75 mg of F108 (purchased from Sigma-Aldrich Reagent Company) and 6.25 mg of MAL-F108 prepared in step 2 into a clean glass bottle, add 7.5 mL of 2 M HCl, and stir to dissolve; add 240 μL of pore-expanding agent cyclohexane, sonicate for 1 minute to obtain a milky white mixed solution, and continue stirring for 30 minutes; mix 150 μL of APTES-TK-APTES prepared in step 1 and 150 μL of tetraethyl orthosilicate, then add to the mixed solution and stir at room temperature for 12 hours; add 120 μL of dimethyldimethoxysilane as a silicon source hydrolysis terminator and continue stirring for 24 hours; after the reaction is completed, transfer the solution to a dialysis bag (MWCO = 30,000 Da) and dialyze with 2 L of deionized water for 12 hours, changing the water every 2 hours. The dialyzed solution was collected and cyclohexane was removed by rotary evaporation (50°C, 15 minutes). Large particles were removed by centrifugation (8000 rpm, 10 minutes). The precipitate was discarded, and the supernatant was taken. The solution was diluted to 15 mL with deionized water and stored at 4°C until further use. Water was removed by freeze-drying, and the mass concentration of RSCLMs was determined by weight loss.

[0049] The RSCLMs prepared in Example 4 were characterized by dynamic light scattering (DLS). Figure 6a The results showed that the hydrated particle size of the synthesized RSCLMs was uniform.

[0050] The RSCLMs prepared in Example 4 were characterized using a transmission scanning electron microscope (TEM). Figure 7a The results showed that the morphology of RSCLMs was uniform and stable, and the dispersion was good, indicating that the active oxygen-responsive RSCLMs were successfully prepared and were an ideal nanocarrier.

[0051] Example 5: Verification of ROS responsiveness of ROS-responsive organosilica cross-linked micelles RSCLMs

[0052] The ROS responsiveness of ROS-responsive organosilica cross-linked micelles (RSCLMs) was verified by the following steps:

[0053] Step 1: Preparation of RSCLMs is the same as that in Example 4;

[0054] Step 2: Dilute the RSCLMs prepared in step 1 to 2 mg mL -1 , reacted with 1 mM hydrogen peroxide solution (H2O2) at room temperature for 12 hours. After the reaction, DLS and TEM were used to characterize the RSCLMs to evaluate the ability of responding to ROS.

[0055] like Figure 6bAs shown in Figure 2, H2O2 in the system can significantly destroy the TK chain in the silicon skeleton of RSCLMs, causing the destruction of the micelle structure and the hydrated particle size becoming no longer uniform. Figure 7b As shown in the figure, the morphology of RSCLMs changed significantly, indicating that the synthesized RSCLMs have good ROS response ability.

[0056] Example 6: Construction of anti-tumor drug-loaded nanoparticles

[0057] The steps for constructing the anti-tumor drug-loaded nanoparticles provided in this embodiment are as follows:

[0058] Step 1: Preparation of RSCLMs was the same as that in Example 4;

[0059] Step 2. Thiolation of horseradish peroxidase HRP: Use the reaction of 2-iminothiolane hydrochloride with primary amines to introduce thiol groups into proteins. Weigh 20 mg of HRP into 2 mL of PBS (containing 3 mM EDTA), add 130 μL of 2-iminothiolane hydrochloride solution, and stir at room temperature for 1 hour. After the reaction is completed, remove the unreacted 2-iminothiolane hydrochloride by dialysis (dialysis bag MWCO = 3000 Da). After the dialysis is completed, store at 4 ° C for use. The mass of HRP is obtained by freeze-drying to 18.5 mg;

[0060] Step 3: Prepare artemisinin ART and 7-ethyl-10-hydroxycamptothecin SN38 into 100mM and 25mM DMSO solutions (stock solutions), respectively. Accurately measure 4μL of ART stock solution and 4μL of SN38 stock solution, and add them separately or simultaneously to 1mL of the solution prepared in step 1 to a concentration of 10mg·mL according to different preparation groups. -1 The RSCLMs solution was sonicated vigorously for 30 seconds to allow ART or SN38 to be loaded into the hydrophobic core of the RSCLMs, and different preparation groups were prepared: A@RSCLMs, i.e., the preparation group loaded with ART alone; S@RSCLMs, i.e., the preparation group loaded with SN38 alone; and A&S@RSCLMs, i.e., the preparation group loaded with both ART and SN38.

[0061] Step 4: Next, horseradish peroxidase is covalently modified onto the PEG termini of RSCLMs, A@RSCLMs, S@RSCLMs, or A&S@RSCLMs via a click reaction between thiol and maleimide groups. To 5 mL of RSCMs, A@RSCLMs, S@RSCLMs, or A&S@RSCLMs solution, 4 mg of the thiol-modified horseradish peroxidase prepared in Step 2 is added. 50 μL of triethanolamine is then added to the solution, and the mixture is stirred at room temperature for 4 hours. After the reaction, unreacted horseradish peroxidase is removed by dialysis (dialysis bag, MWCO = 50,000 Da). After dialysis, the resulting RSCLMs-H, A@RSCLMs-H, S@RSCLMs-H, or A&S@RSCLMs-H solution is stored at 4°C.

[0062] The following tests were performed on the RSCLMs-H, A@RSCLMs, A@RSCLMs-H, S@RSCLMs-H, and A&S@RSCLMs-H prepared in this example:

[0063] Example 7: Verification of the effect of anti-tumor drug-loaded nanoparticles A&S@RSCLMs-H on increasing ROS levels in tumor cells

[0064] The following operations were performed using RSCLMs-H, A@RSCLMs, A@RSCLMs-H, S@RSCLMs-H, and A&S@RSCLMs-H prepared in Example 4, respectively:

[0065] Step 1: 4T1 cells were co-incubated with the materials prepared in Example 4. 2×10 5 4T1 cells were seeded into 6-well plates at a density of 10 cells / well and incubated in an incubator for 24 hours. When the cells reached 50% to 60% growth, the culture medium was discarded and the cells were washed three times with PBS. Six groups were set up: the first group was the control group, which received only culture medium; the second group received culture medium containing RSCLMs-H; the third group received culture medium containing S@RSCLMs-H; the fourth group received culture medium containing A@RSCLMs; the fifth group received culture medium containing A@RSCLMs-H; and the sixth group received culture medium containing both A and S@RSCLMs-H. The cells were incubated in an incubator for 4 hours, the culture medium was replaced with fresh medium, and the cells were cultured in the incubator for a further 24 hours.

[0066] Step 2: Detect intracellular ROS levels using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). After the cell culture in step 1, replace each well with fresh culture medium containing DCFH-DA and continue incubation for 30 minutes. Wash three times with PBS and observe under a fluorescence microscope (λex = 488 nm, λem = 525 nm).

[0067] like Figure 8a and Figure 8b As shown in the figure, compared with the other treatment groups, the green fluorescence in 4T1 cells treated with A@RSCLMs-H and A&S@RSCLMs-H increased significantly, indicating that A@RSCLMs-H and A&S@RSCLMs-H can respond to endogenous ROS in 4T1 tumor cells and release ART in the hydrophobic cavity, which can be efficiently catalyzed by peripheral HRP to produce high levels of ROS.

[0068] Example 8: Tumor growth inhibition test of anti-tumor nanoparticles A&S@RSCLMs-H on 4T1 tumor-bearing mice

[0069] The following operations were performed using RSCLMs-H, A@RSCLMs, A@RSCLMs-H, S@RSCLMs-H, and A&S@RSCLMs-H prepared in Example 6, respectively:

[0070] Step 1. Establishment of mouse tumor model: The animals used in this experiment were 4-6 week old Balb / c female mice weighing approximately 20 g, 30 in number, purchased from the Experimental Animal Center of Yangzhou University. After one week of adaptive feeding, the hair on the back of the mice was removed and the mouse tumor model was established. 0.1 mL of 4T1 cell suspension (4 × 10 6 After 4T1 cell inoculation, a rice-sized tumor mass will appear at the inoculation site about one week later. The size of the tumor mass will be measured every two days until the tumor mass grows to 100 mm. 3 , 4T1 tumor-bearing mice were used for subsequent experiments;

[0071] Step 2. The model mice from Step 1 were randomly divided into 6 groups, each with 5 mice: control group (normal saline), RSCLMs-H group, S@RSCLMs-H group, A@RSCLMs group, A@RSCLMs-H group, and A&S@RSCLMs-H group. Normal saline, RSCLMs-H, S@RSCLMs-H, A@RSCLMs, A@RSCLMs-H, and A&S@RSCLMs-H were injected through the tail vein at 0.2 mL / mouse (SN38 25 μM, ART 200 μM). The drugs were administered every 4 days, and the body weight and tumor volume of the mice were recorded daily. Mice were sacrificed on day 21, and the tumor masses of each mouse were collected, photographed, and weighed.

[0072] The results are as follows Figure 9a 、 Figure 9b 、 Figure 9cAs shown in Figure 2, compared with the control group (normal saline), RSCLMs-H group, and S@RSCLMs-H group, the A@RSCLMs-H group can significantly inhibit tumor growth; as a more preferred embodiment of the present invention, the A&S@RSCLMs-H group can also significantly inhibit tumor growth compared with the control group (normal saline), RSCLMs-H group, and S@RSCLMs-H group, and the inhibitory effect is better than that of the A&S@RSCLMs-H group; and as shown in Figure 2 Figure 9d As shown, the body weight of 4T1 tumor-bearing mice did not change significantly during the treatment period, indicating that the nano drug delivery system constructed in the present invention has good biocompatibility and does not cause obvious systemic toxicity.

Claims

1. Use of horseradish peroxidase as an exogenous activator for artemisinin and its derivatives.

2. The use according to claim 1, characterized in that The artemisinin and its derivatives include compounds containing peroxy bridge bonds such as artemisinin, artesunate, dihydroartemisinin, artemether, and arteether; wherein the horseradish peroxidase catalyzes artemisinin and its derivatives to produce a large amount of reactive oxygen species, and the large amount of reactive oxygen species produced comes from alkoxy free radicals generated by the cleavage of the peroxy bridge structure in the structure of artemisinin and its derivatives.

3. Application of horseradish peroxidase in the preparation of anti-tumor drug-loaded nanoparticles.

4. The use according to claim 3, characterized in that The application comprises the steps of: using artemisinin and its derivatives as precursors of ROS and covalently linking an exogenous activator HRP through a nanocarrier to construct anti-tumor nano drug-carrying particles with a cascade amplification effect of ROS response.

5. The use according to claim 4, characterized in that Artemisinin and its derivatives are loaded into the hydrophobic cavity of a nano-delivery carrier, and an exogenous activator HRP is connected to the nano-delivery carrier by a click reaction to construct the anti-tumor drug-loaded nanoparticles, which are used to achieve cascade amplification of endogenous reactive oxygen species response in the tumor microenvironment. The nano-delivery carrier is a ROS-responsive organic silicon oxide cross-linked micelle RSCLMs, which is self-assembled from a silicon source and an amphiphilic polymer in a hydrochloric acid solution, and cyclohexane is used as a pore-expanding agent and dimethyldimethoxysilane is used as a silicon source hydrolysis terminator, and the hydrated particle size is 5 to 100 nm. The mass ratio of the silicon source to the amphiphilic polymer is (1 to 3):1; the concentration of the hydrochloric acid solution is 0.05 to 3.5 mol·L -1 ; The volume fraction of cyclohexane is 0-3.5%, and the volume fraction of dimethyldimethoxysilane is 0.4-3.0%; wherein the mass ratio of RSCLMs, HRP, artemisinin and its derivatives is 100:(5-10):(0.5-5).

6. The use according to claim 5, characterized in that The silicon source is composed of tetraalkyl orthosilicate and a bridging silane containing a thioacetal bond, wherein the tetraalkyl orthosilicate is any one of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, and tetrabutyl orthosilicate, and the bridging silane containing a thioacetal bond is abbreviated as APTES-TK-APTES.

7. The use according to claim 5, characterized in that The raw material configuration volume ratio of the tetraalkyl orthosilicate and APTES-TK-APTES is (1-4):

1.

8. The use according to claim 5, characterized in that The amphiphilic polymer consists of a nonionic surfactant and a modified nonionic surfactant.

9. The use according to claim 8, characterized in that The raw material configuration mass ratio of the nonionic surfactant and the modified nonionic surfactant is (5-50):1; the nonionic surfactant is selected from any one of Pluronic F108, Pluronic F127, and Pluronic P123, and the modified nonionic surfactant is a nonionic surfactant modified with maleimide or N-hydroxysuccinimide ester.

10. The use according to claim 5, characterized in that Artemisinin and its derivatives and a drug for treating tumors are simultaneously loaded in the hydrophobic cavity of the nanodelivery carrier, wherein the drug for treating tumors is one of 7-ethyl-10-hydroxycamptothecin SN38, doxorubicin hydrochloride, cisplatin, gemcitabine, paclitaxel, and cyclophosphamide; the mass ratio of the RSCLMs, HRP, artemisinin and its derivatives and the drug for treating tumors is 100:(5-10):(0.5-5):(0.1-2).

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