Monatomic iron nano-enzyme with targeting effect as well as preparation method and application of monatomic iron nano-enzyme

Abstract

The invention discloses a monatomic iron nano-enzyme with a targeting effect as well as a preparation method and application thereof. The preparation method specifically comprises the following steps: (1) mixing and stirring water, absolute ethyl alcohol and stronger ammonia water, adding a polyphenol substance aqueous solution, stirring again, adding a ferric salt ethanol solution, continuously stirring, centrifuging, washing and freeze-drying; (2) calcining and grinding; and (3) ultrasonically dispersing in water, stirring, adding an NH2-PEG-iRGD solution, continuously stirring, centrifuging, washing, and freeze-drying, thereby obtaining the product. The monatomic iron nano-enzyme with the targeting effect prepared by the invention has very good peroxidase-like activity and photothermal conversion property, can simultaneously play the therapeutic effects of CDT and PTT, and can greatly improve the therapeutic effect of tumors through a combined therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of functional materials, and more specifically to a single-atom iron nanozyme with targeting effect and a preparation method and application thereof. Background Art

[0002] Malignant tumors are a major global public health problem. Their intractability stems from the high heterogeneity of the tumor microenvironment, immunosuppression, and the nonspecific killing mechanism of traditional therapies. At present, mainstream clinical treatments such as surgical resection, radiotherapy, chemotherapy, and targeted drugs have common limitations: surgery is difficult to eradicate micrometastatic lesions, radiotherapy and chemotherapy have systemic toxic side effects, and targeted drugs have drug resistance. The efficacy bottleneck of a single therapy can easily lead to poor tumor treatment effects or tumor recurrence and metastasis. Therefore, there is an urgent need to develop new synergistic treatment strategies with high selectivity and low toxicity.

[0003] In recent years, nanozymes have provided new treatment methods for tumor treatment due to their unique enzyme-like catalytic activity and nanomaterial properties. Among them, single-atom nanozymes have shown catalytic activity far exceeding that of traditional nanozymes due to their atomically dispersed active centers. They can efficiently degrade overexpressed H in the tumor microenvironment through chemodynamic therapy (CDT). 2 O 2 It is catalyzed into highly toxic ·OH, thereby specifically inducing apoptosis in cancer cells. However, single CDT is limited by the endogenous H 2 O 2 Insufficient concentration and limited diffusion distance of free radicals make it difficult to maximize the therapeutic effect. Therefore, CDT can be combined with other treatment methods to enhance the synergistic effect.

[0004] Photothermal therapy (PTT) directly kills tumor cells by irradiating near-infrared lasers with near-infrared responsive materials to generate local high temperatures, while accelerating the generation of H in the tumor microenvironment. 2 O 2 Generating and enhancing the catalytic efficiency of nanozymes can achieve the synergistic effect of PTT and CDT. However, existing photothermal nanozymes generally have problems such as low catalytic activity, insufficient photothermal conversion efficiency, and lack of targeting, which limits the effectiveness of tumor treatment.

[0005] Therefore, how to develop a photothermal nanozyme with targeted effect is an urgent problem that technicians in this field need to solve. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a single-atom iron nanozyme with targeted effect and its preparation method and application, so as to solve the shortcomings of the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing a single-atom iron nanozyme with a targeted effect, specifically comprising the following steps:

[0009] (1) mixing water, anhydrous ethanol and concentrated ammonia water, stirring, adding the polyphenol aqueous solution, stirring again, adding the iron salt ethanol solution, continuing to stir, centrifuging, washing, and freeze-drying to obtain a single-atom iron nanozyme precursor;

[0010] (2) calcining and grinding the single-atom iron nanozyme precursor in sequence to obtain the single-atom iron nanozyme;

[0011] (3) Ultrasonic dispersion of single-atom iron nanozymes into water, stirring, and adding NH 2 -PEG-iRGD solution, continue stirring, centrifuging, washing, and freeze-drying to obtain single-atom iron nanozyme with targeted effect.

[0012] Furthermore, in the above step (1), the usage ratio of water, anhydrous ethanol, concentrated ammonia water, aqueous solution of polyphenols and ethanol solution of iron salt is 90mL:40mL:3mL:10mL:5mL; the aqueous solution of polyphenols is an aqueous solution of dopamine hydrochloride with a concentration of 50mg / mL; and the ethanol solution of iron salt is an ethanol solution of ferric acetylacetonate with a concentration of 0.84mg / mL.

[0013] Furthermore, in the above step (1), the stirring temperature is room temperature, the speed is 600-800 r / min, and the time is 30 min; the stirring time again is 1-2 h; the stirring time is continued for 12-24 h; the centrifugal speed is 12000 r / min, and the time is 10-20 min; the washing reagent is water, and the number of times is 3 times; and the freeze-drying time is 24-36 h.

[0014] Furthermore, in the above step (2), the calcination equipment is a tubular furnace, the atmosphere is nitrogen, the temperature is 900° C., and the time is 2 hours; and the grinding equipment is an agate mortar.

[0015] Furthermore, in the above step (3), the single-atom iron nanozyme, water and NH 2 The dosage ratio of -PEG-iRGD solution was 10 mg: 20 mL: 4 mL; NH 2 The concentration of the PEG-iRGD solution was 5 mg / mL.

[0016] Furthermore, in the above step (3), the power of ultrasonic dispersion is 250W, and the time is 10-30min; the stirring equipment is a magnetic stirrer, the temperature is room temperature, and the speed is 800-1000r / min; the stirring time is continued for 10-20h; the centrifugal speed is 12000r / min, and the time is 10-20min; the washing reagent is water, and the number of times is 3; and the freeze-drying time is 24-36h.

[0017] The present invention also seeks to protect a single-atom iron nanozyme with targeting effect obtained by the above preparation method.

[0018] The present invention also seeks to protect the use of a single-atom iron nanozyme with targeting effect obtained by the above-mentioned preparation method in the preparation of drugs for treating tumors.

[0019] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The present invention firstly generates a spherical single-atom iron nanozyme precursor by polymerization reaction of iron salt (ferric acetylacetonate) and polyphenolic substances (dopamine hydrochloride) under alkaline conditions (concentrated ammonia water), and then calcines the precursor at high temperature to obtain a spherical single-atom iron nanozyme. Then, a layer of targeted polypeptide iRGD is modified on the surface of the nanozyme to improve the targeting and biocompatibility of the nanozyme. The POD-like enzyme activity of the nanozyme of the present invention is used to efficiently catalyze H in the tumor microenvironment through CDT therapy. 2 O 2 , generating highly toxic ·OH, which kills tumor cells. At the same time, 808nm near-infrared laser is used to irradiate tumor tissue, and the local temperature of the tumor is increased through PTT therapy to kill tumor cells, while synergistically improving the efficacy of CDT and jointly exerting anti-tumor therapeutic effects.

[0021] 2. The present invention first uses dopamine nanozyme as a carrier to generate a single-atom iron nanozyme precursor, then obtains the single-atom iron nanozyme by calcination, and then electrostatically adsorbs NH 2 -PEG-iRGD peptide is evenly wrapped on the surface of single-atom iron nanozyme. The nanozyme prepared in this way has efficient peroxidase-like activity and good photothermal conversion effect. It can quickly catalyze hydrogen peroxide to generate hydroxyl free radicals and can also quickly convert light energy into heat energy.

[0022] 3. The present invention uses iron salt (ferric acetylacetonate), polyphenols (dopamine hydrochloride) and NH 2-PEG-iRGD peptide was used as raw material, and a single-atom iron nanozyme with targeted effect was obtained through oxidative self-polymerization, high-temperature calcination and electrostatic adsorption. This nanozyme has excellent peroxidase-like activity and photothermal conversion performance, and can quickly and efficiently exert the therapeutic effects of CDT and PTT in the tumor microenvironment. This nanozyme has high biosafety and good stability, and can effectively accumulate in the tumor site. It has good application prospects in the biomedicine field, and it is expected to use this nanozyme to improve the efficacy of malignant tumors through combined therapy.

[0023] 4. The single-atom iron nanozyme with targeted effect prepared by the present invention has excellent peroxidase-like activity and photothermal conversion properties, can simultaneously exert the therapeutic effects of CDT and PTT, and can greatly enhance the therapeutic effect of tumors through combined therapy.

[0024] 5. The present invention greatly improves the accumulation of nano-drugs in tumor sites through the modification effect of iRGD, overcomes the problem of low efficiency of traditional drug targeted delivery, and has a good prospect for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A high-angle annular dark field scanning-transmission electron microscopy (HADDF-STEM) image and an X-ray energy spectrum analysis (EDS-mapping) image of the single-atom iron nanozyme with targeted effect prepared in Example 1 of the present invention;

[0026] Figure 2 This is a high-resolution transmission electron microscopy (HR-TEM) image of the single-atom iron nanozyme with targeting effect prepared in Example 1 of the present invention;

[0027] Figure 3 This is the EPR spectrum of hydroxyl radicals generated by the single-atom iron nanozyme with targeted effect prepared in Example 1 of the present invention;

[0028] Figure 4 This is the photothermal conversion efficiency of the single-atom iron nanozyme with targeted effect prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] The preparation method of the single-atom iron nanozyme with targeted effect specifically comprises the following steps:

[0032] (1) 90 mL of water, 40 mL of anhydrous ethanol and 3 mL of concentrated ammonia were mixed, stirred at 600 r / min for 30 min at room temperature, 10 mL of 50 mg / mL dopamine hydrochloride aqueous solution was added, stirred again for 2 h, 5 mL of 0.84 mg / mL acetylacetonate iron ethanol solution was added dropwise, stirring was continued for 24 h, centrifuged at 12000 r / min for 15 min, washed repeatedly with water, and freeze-dried for 24 h to obtain a single-atom iron nanozyme precursor;

[0033] (2) The single-atom iron nanozyme precursor was transferred to a tube furnace and calcined at 900 °C for 2 h in a nitrogen atmosphere. After being taken out, it was fully ground with an agate mortar to obtain the single-atom iron nanozyme;

[0034] (3) 10 mg of single-atom iron nanozyme was dispersed into 20 mL of water at a power of 250 W for 30 min, stirred with a magnetic stirrer at a speed of 800 r / min at room temperature, and 4 mL of 5 mg / mL NH 2 -PEG-iRGD solution was stirred for 10 h, centrifuged at 12000 r / min for 10 min, washed with water three times, and freeze-dried for 24 h to obtain single-atom iron nanozyme with targeted effect.

[0035] Example 2

[0036] The preparation method of the single-atom iron nanozyme with targeted effect specifically comprises the following steps:

[0037] (1) 90 mL of water, 40 mL of anhydrous ethanol and 3 mL of concentrated ammonia water were mixed, stirred at 700 r / min for 30 min at room temperature, 10 mL of 50 mg / mL dopamine hydrochloride aqueous solution was added, stirred again for 1 h, 5 mL of 0.84 mg / mL acetylacetonate iron ethanol solution was added dropwise, stirring was continued for 12 h, centrifuged at 12000 r / min for 10 min, washed repeatedly with water, and freeze-dried for 30 h to obtain a single-atom iron nanozyme precursor;

[0038] (2) The single-atom iron nanozyme precursor was transferred to a tube furnace and calcined at 900 °C for 2 h in a nitrogen atmosphere. After being taken out, it was fully ground with an agate mortar to obtain the single-atom iron nanozyme;

[0039] (3) 10 mg of single-atom iron nanozyme was dispersed into 20 mL of water at a power of 250 W for 10 min, stirred with a magnetic stirrer at room temperature at a speed of 900 r / min, and 4 mL of 5 mg / mL NH 2-PEG-iRGD solution was stirred for 15 h, centrifuged at 12000 r / min for 15 min, washed three times with water, and freeze-dried for 30 h to obtain single-atom iron nanozyme with targeted effect.

[0040] Example 3

[0041] The preparation method of the single-atom iron nanozyme with targeted effect specifically comprises the following steps:

[0042] (1) 90 mL of water, 40 mL of anhydrous ethanol and 3 mL of concentrated ammonia were mixed, stirred at 800 r / min for 30 min at room temperature, 10 mL of 50 mg / mL dopamine hydrochloride aqueous solution was added, stirred again for 1.5 h, 5 mL of 0.84 mg / mL acetylacetonate iron ethanol solution was added dropwise, stirring was continued for 18 h, centrifuged at 12000 r / min for 20 min, washed repeatedly with water, and freeze-dried for 36 h to obtain a single-atom iron nanozyme precursor;

[0043] (2) The single-atom iron nanozyme precursor was transferred to a tube furnace and calcined at 900 °C for 2 h in a nitrogen atmosphere. After being taken out, it was fully ground with an agate mortar to obtain the single-atom iron nanozyme;

[0044] (3) 10 mg of single-atom iron nanozyme was dispersed into 20 mL of water at a power of 250 W for 20 min, stirred with a magnetic stirrer at room temperature at a speed of 1000 r / min, and 4 mL of 5 mg / mL NH 2 -PEG-iRGD solution was stirred for 20 h, centrifuged at 12000 r / min for 20 min, washed with water three times, and freeze-dried for 36 h to obtain single-atom iron nanozyme with targeted effect.

[0045] Performance Testing

[0046] 1. HADDF-STEM and EDS-mapping characterization

[0047] The high-angle annular dark field scanning-transmission electron microscopy (HADDF-STEM) image of the single-atom iron nanozyme with targeted effect prepared in Example 1 is as follows: Figure 1 As shown in AB, the X-ray energy spectrum analysis (EDS-mapping) diagram is as follows Figure 1 Shown in CD.

[0048] Depend on Figure 1 It can be seen that the four elements of carbon, nitrogen, oxygen and iron are evenly distributed in this nanozyme, and the iron content is extremely low.

[0049] 2. HR-TEM characterization

[0050] The high-resolution transmission electron microscopy (HR-TEM) image of the single-atom iron nanozyme with targeted effect prepared in Example 1 is as follows: Figure 2 shown.

[0051] Depend on Figure 2 It can be seen that the size of the nanozyme is about 80nm, and iRGD is wrapped on the surface of the single-atom nanozyme.

[0052] 3. Study on peroxidase-like activity

[0053] The single-atom iron nanozyme with targeted effect prepared in Example 1 was used to study the peroxidase-like activity. The steps are as follows:

[0054] (1) dispersing the single-atom iron nanozyme in ultrapure water to a concentration of 1 mg / mL, and subjecting the mixture to ultrasonic treatment at an ultrasonic power of 250 W for 30 min to obtain a single-atom iron nanozyme aqueous solution;

[0055] (2) 10 μL of single-atom iron nanozyme aqueous solution, 20 μL of hydrogen peroxide solution, 20 μL of TMB solution, 2 mL of HAc-NaAc buffer (PH = 4.5) and 2 μL of 1 M DMPO solution were uniformly mixed, and the centrifuge tube was vortexed and then waited for 5 min to obtain a mixed solution;

[0056] (3) The mixed solution is pipetted into a quartz capillary for electron paramagnetic resonance spectroscopy (EPR) analysis to detect the peroxidase-like activity.

[0057] The results are as follows Figure 3 shown.

[0058] Depend on Figure 3 It can be seen that the single-atom iron nanozyme with targeted effect prepared in Example 1 has efficient peroxidase-like activity and can quickly catalyze hydrogen peroxide to generate hydroxyl radicals.

[0059] 4. Study on photothermal conversion performance

[0060] The single-atom iron nanozyme with targeted effect prepared in Example 1 was used to study the photothermal conversion performance. The steps are as follows:

[0061] (1) preparing a nanozyme solution with a volume of 1 mL and a concentration of 100 μg / mL of single-atom iron nanozyme;

[0062] (2) The nanozyme solution was irradiated with an 808 nm near-infrared laser for 10 min and then stopped, and the solution was allowed to cool naturally, and the temperature change of the solution was recorded every minute until the solution temperature stopped changing;

[0063] (3) Calculate the photothermal conversion efficiency of the nanozyme based on the results.

[0064] The results are as follows Figure 4 shown.

[0065] Depend on Figure 4 It can be seen that the single-atom iron nanozyme with targeted effect prepared in Example 1 has a good photothermal conversion effect and can quickly convert light energy into thermal energy.

[0066] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a single-atom iron nanozyme with targeting effect, characterized in that: The specific steps include: (1) mixing water, anhydrous ethanol and concentrated ammonia water, stirring, adding the polyphenol aqueous solution, stirring again, adding the iron salt ethanol solution, continuing to stir, centrifuging, washing, and freeze-drying to obtain a single-atom iron nanozyme precursor; (2) calcining and grinding the single-atom iron nanozyme precursor in sequence to obtain the single-atom iron nanozyme; (3) Ultrasonic dispersion of the single-atom iron nanozyme in water, stirring, adding NH2-PEG-iRGD solution, continuing stirring, centrifuging, washing, and freeze-drying to obtain the single-atom iron nanozyme with targeting effect.

2. The method for preparing a single-atom iron nanozyme with targeting effect according to claim 1, characterized in that: In step (1), the amount ratio of water, anhydrous ethanol, concentrated ammonia water, polyphenol aqueous solution and iron salt ethanol solution is 90mL:40mL:3mL:10mL:5mL; the polyphenol aqueous solution is an aqueous solution of dopamine hydrochloride with a concentration of 50mg / mL; the iron salt ethanol solution is an ethanol solution of ferric acetylacetonate with a concentration of 0.84mg / mL.

3. The method for preparing a single-atom iron nanozyme with targeting effect according to claim 1, characterized in that: In step (1), the stirring temperature is room temperature, the speed is 600-800 r / min, and the time is 30 min; the time for the second stirring is 1-2 h; the time for the continued stirring is 12-24 h; the speed of the centrifugation is 12000 r / min, and the time is 10-20 min; the washing reagent is water, and the number of times is 3 times; the time for the freeze-drying is 24-36 h.

4. The method for preparing a single-atom iron nanozyme with targeting effect according to claim 1, characterized in that: In step (2), the calcination equipment is a tubular furnace, the atmosphere is nitrogen, the temperature is 900° C., and the time is 2 hours; the grinding equipment is an agate mortar.

5. The method for preparing a single-atom iron nanozyme with targeting effect according to claim 1, characterized in that: In step (3), the dosage ratio of the single-atom iron nanozyme, water and NH2-PEG-iRGD solution is 10 mg:20 mL:4 mL; the concentration of the NH2-PEG-iRGD solution is 5 mg / mL.

6. The method for preparing a single-atom iron nanozyme with targeting effect according to claim 1, characterized in that: In step (3), the power of the ultrasonic dispersion is 250W, and the time is 10-30min; the stirring equipment is a magnetic stirrer, the temperature is room temperature, and the speed is 800-1000r / min; the time of continued stirring is 10-20h; the speed of the centrifugation is 12000r / min, and the time is 10-20min; the washing reagent is water, and the number of times is 3 times; the time of freeze-drying is 24-36h.

7. A single-atom iron nanozyme with targeting effect obtained by the preparation method as described in any one of claims 1 to 6.

8. Use of a single-atom iron nanozyme with targeting effect obtained by the preparation method as described in any one of claims 1 to 6 in the preparation of drugs for treating tumors.

Images