CN118340740A - A multifunctional nanozyme with endoplasmic reticulum targeting and its preparation method and application - Google Patents

Abstract

The present invention discloses a multifunctional nanozyme with endoplasmic reticulum targeting, and a preparation method and application thereof. The nanozyme uses ruthenium nanoparticles as carriers, and the endoplasmic reticulum targeting functional group p-dodecylbenzenesulfonamide is coated on the surface of the ruthenium nanoparticles. The multifunctional nanozyme thus formed can effectively eliminate the accumulation of reactive oxygen, inhibit the activation of ROS and MMP2/9 in the damaged site after cerebral hemorrhage, reduce the expression level of ROS and MMP2/9 in the damaged site after cerebral hemorrhage, and play a role in alleviating pain hypersensitivity. In addition, the present invention can achieve precise regulation of oxidative stress through the combination of endoplasmic reticulum precise antioxidant nanozymes.

Description

A multifunctional nanozyme with endoplasmic reticulum targeting and its preparation method and application

Technical Field

The present invention belongs to the field of biopharmaceuticals, and relates to the technical field of new drugs with endoplasmic reticulum targeting, and more specifically to a multifunctional nanozyme with endoplasmic reticulum targeting, and a preparation method and application thereof.

Background technique

Central poststroke pain (CPSP) occurs after stroke with an estimated prevalence of 56%, and is higher when there is thalamic hemorrhage or ischemia. Generalized hyperalgesia, allodynia, and/or various forms of pain (neck pain, shoulder pain, hand pain, and foot pain) are the most common symptoms, and available drug treatments are often ineffective. Therefore, new therapeutic targets are urgently needed to develop more effective analgesics. After intracerebral hemorrhage (ICH), an inflammatory cascade of neuroinflammation is unleashed. In response, activated microglia release factors such as reactive oxygen species (ROS, e.g., superoxide), chemokines, and cytokines, as well as matrix metalloproteinases (MMPs). Emerging evidence suggests that ROS and MMPs play an important role in pain transduction. Reactive oxygen species include hydrogen peroxide (H2O2), superoxide anion (·O ₂ ^- ), and hydroxyl radical (·OH), etc., whose excessive production causes secondary damage and scavenges O ^2·- and H ₂ O ₂ (not ·OH) to simulate superoxide dismutase (SOD) and catalase. However, their low bioavailability, short half-life, low efficiency in penetrating the blood-brain barrier, and side effects on kidney and liver function limit their clinical application. Therefore, there is an urgent need to design and develop drugs with strong reactive oxygen scavenging activity and ideal physicochemical properties for the treatment of cerebral hemorrhage.

The endoplasmic reticulum (ER) is the base for the synthesis of a series of important biomacromolecules in cells other than nucleic acids, such as proteins, lipids (such as triglycerides) and carbohydrates. The smooth endoplasmic reticulum also has a detoxification function. For example, the smooth endoplasmic reticulum in hepatocytes contains some enzymes to remove fat-soluble waste and harmful substances produced by metabolism. Studies have shown that [Yingying Shi, YuLiu, et al. Endoplasmic reticulum-targeted inhibition of CYP2E1 with vitamin Enanoemulsions alleviates hepatocyte oxidative stress and reverses alcoholic liver disease, Biomaterials, 2022] After constructing a nanoemulsion of vitamin E, the nanoemulsion containing vitamin E is loaded and delivered to the endoplasmic reticulum of hepatocytes by dodecylbenzenesulfonamide, which can enhance the therapeutic effect and safety compared with free drugs by providing good intracellular distribution and pharmacological effects.

Natural antioxidants such as catalase and superoxide dismutase are widely used in the field of inflammation treatment and tissue engineering materials, and enzyme immobilization technology has attracted more and more attention. There are still problems in the natural antioxidant and anti-inflammatory developed in recent years, such as: 1. Expensive; 2. Easy to degrade in vivo; 3. Poor tolerance of enzyme catalysts to alcohol. Compared with natural enzymes, nanozymes have lower R&D costs, stronger stability, and excellent recyclability. At the same time, they can also catalyze some non-natural biological processes and have been applied to disease diagnosis and treatment, biosensing, environmental governance, and antibacterial and antifouling fields. Patent 202110706467.8 discloses a transition metal single-atom nanozyme and its preparation method and use. The patent achieves high catalytic performance by imitating the activity of POD, and is used for antibacterial, wastewater treatment and immunoblotting; however, in the prior art, there is no patent on the combination of endoplasmic reticulum targeting groups and nanozymes for relieving central post-stroke pain.

Therefore, how to provide a new type of nanozyme with endoplasmic reticulum targeting and use it to relieve central post-stroke pain is a problem that technicians in this field urgently need to solve.

Summary of the invention

In view of this, the present invention provides a multifunctional nanozyme with endoplasmic reticulum targeting, and a preparation method and application thereof.

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

A multifunctional nanozyme with endoplasmic reticulum targeting, wherein the nanozyme uses ruthenium nanoparticles as carriers and the surface of the ruthenium nanoparticles is coated with p-dodecylbenzenesulfonamide.

As the above technical solution, the present invention also claims a method for preparing the above multifunctional nanozyme with endoplasmic reticulum targeting, comprising the following steps:

(1) Weigh 2-methylimidazole and dissolve hexadecyltrimethylammonium bromide in deionized water, stir at 400 rpm to mix, then drop deionized water solution containing 1 mg/mL bovine serum albumin and deionized water solution containing 29.06 mg/mL Zn(NO ₃ ) ₂ ·6H ₂ O into the mixed solution in sequence, and stir continuously at room temperature for 20 min. After the reaction is completed, centrifuge the reaction solution at 5000 rpm for 5 min, collect the precipitate, resuspend the precipitate in deionized water containing RuCl ₃ , and then perform ultrasonic dispersion. Stir continuously for 12 h, add sodium borohydride to react for 10 min, collect the precipitate again, wash the precipitate twice, freeze, and dry it to obtain RuMOF;

(2) p-Dodecylbenzenesulfonamide, lecithin, and cholesterol were dissolved in chloroform/methanol and then dried under a rotary evaporator. The dried lipid film was then hydrated with Ru MOF solution (1 mg/mL) and then probe sonicated for 5 min to obtain ER-RuMOF.

Furthermore, in step (1), the mass ratio of the 2-methylimidazole, hexadecyltrimethylammonium bromide, bovine serum albumin, Zn(NO ₃ ) ₂ ·6H ₂ O, RuCl ₃ and sodium borohydride is 100-400:1:1:10-30.

Furthermore, in step (1), the mass ratio of RuCl ₃ , precipitate and sodium borohydride is 1:100:1.

Furthermore, in step (2), the mass ratio of p-dodecylbenzenesulfonamide lecithin to cholesterol is 1:1:5.

Furthermore, in step (2), the volume ratio of chloroform/methanol is 2:1.

As the above technical solution, the present invention also requests protection for the use of the above multifunctional nanozyme with endoplasmic reticulum targeting in the preparation of antioxidant drugs.

Furthermore, the multifunctional nanozyme can enhance the activity of superoxide dismutase and scavenge free radicals.

As the above technical solution, the present invention also requests to protect the use of the above multifunctional nanozyme with endoplasmic reticulum targeting in the preparation of drugs for inhibiting the activation of ROS and MMP2/9 in the damaged site after cerebral hemorrhage.

Furthermore, the multifunctional nanozyme reduces the expression levels of ROS and MMP2/9 in the damaged area after cerebral hemorrhage.

As the above technical solution, the present invention also requests protection for the use of the above multifunctional nanozyme with endoplasmic reticulum targeting in the preparation of drugs for treating central post-stroke pain.

It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a multifunctional nanozyme with endoplasmic reticulum targeting and a preparation method and application thereof, and its beneficial effects are:

(1) The multifunctional nanozyme in the present invention uses ruthenium (Ru) nanoparticles as the core carrier, and the endoplasmic reticulum targeting functional group p-dodecylbenzenesulfonamide is coated on the surface of the ruthenium nanoparticles. The new nanozyme formed can effectively eliminate the accumulation of reactive oxygen, inhibit the activation of ROS and MMP2/9 in the injured part after cerebral hemorrhage, and reduce the expression level of ROS and MMP2/9 in the injured part after cerebral hemorrhage, thereby playing a role in alleviating pain hypersensitivity. In addition, the present invention uses nanozymes as the core, which has stronger modifiability than natural enzymes.

(2) The experiment proved that the nanozyme of the present invention appeared on the first day after microinjection, reached a peak on the 3rd to 7th day after microinjection, and lasted for at least 28 days after contralateral microinjection. Systemic nano-administration eliminated the accumulation of reactive oxygen species, rapidly and effectively inhibited the activation of MMP-2 and MMP-9 in CPSP mice, and alleviated pain hypersensitivity; and the use of nano-injection within the appropriate time window can not only reduce further ICH damage, but also produce analgesic effects.

(3) The present invention uses ruthenium (Ru) nanoparticles as the core carrier and coats the endoplasmic reticulum targeting functional group dodecylbenzenesulfonamide on the surface of the ruthenium nanoparticles. It not only provides a new method for in situ synthesis of synergistic nanotherapy, but also reveals the application mechanism of CPSP in combating oxidative stress-induced hemorrhagic stroke damage, which can provide a theoretical basis for achieving precise regulation of oxidative stress.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

FIG1 is a transmission electron microscope image of the RuMOF and ER-Ru MOF synthesized in Example 1;

FIG. 2 is a diagram showing the results of the assay of the activity of the ER-Ru MOF-based SOD/CAT enzyme synthesized in Example 1;

FIG3 is a diagram showing the endoplasmic reticulum targeting verification result of the ER-Ru MOF synthesized in Example 1;

Figure 4 shows that CPSP mice develop pain hypersensitivity and increased expression of ROS and inflammatory factors in the injured area; wherein: A is the design plan for the animal model preparation and behavioral test timeline; B is a schematic diagram of cerebral hemorrhage in CPSP group mice on the third day after type VII collagenase injection; C is a diagram of the nervous system score of CPSP group mice; D is a schematic diagram of pain in the contralateral limbs of CPSP group mice after thalamic hemorrhage; E is a schematic diagram of pain in the ipsilateral limbs of two groups of mice after thalamic hemorrhage; Figures F, G, and H are diagrams of the expression levels of ROS (8-OHdG as a marker of oxygen free radicals) (F) and inflammatory factors TNFa (G) and IL-6 (H) after thalamic hemorrhage; I is a diagram of the changes in the number of Nissl bodies in the thalamic injury area of CPSP group mice.

Figure 5 is a graph showing the results of different assay methods for detecting the expression level of MMP2/9, a key pain protein, in the thalamus injury region of CPSP mice; wherein: A is a graph showing the results of QT-PCR detection of the expression level of MMP2/9, a key pain protein, in the thalamus injury region of CPSP mice; B is a graph showing the results of West Blotting detection of the expression level of MMP2/9, a key pain protein, in the thalamus injury region of CPSP mice; C is a graph showing the results of immunofluorescence detection of the expression level of MMP2/9, a key pain protein, in the thalamus injury region of CPSP mice;

Figure 6 shows the test results of CPSP and toxicity after administration of nanozyme; wherein: AC is a schematic diagram of the pain sensation in the contralateral limbs after thalamic hemorrhage in the CPSP group of mice administered with different doses of nanozyme; DF is a diagram showing the sensation of the ipsilateral limbs of mice administered with different doses of nanozyme; GH is a diagram showing the effect of 8 mg/kg nanozyme on the liver and kidney functions of the two groups of mice; I is a diagram showing the weight changes of mice in the nanozyme groups with different doses during the experiment; JL are diagrams showing the effect of 8 mg/kg nanozyme on the heart, lung, and spleen tissues of the two groups of mice.

Figure 7 shows the changes in ROS, inflammatory factors, cerebral hemorrhage and the number of nylon bodies in the thalamic lesion area of mice treated with nanozyme; wherein: A is a graph showing the changes in ROS in the thalamic lesion area of mice treated with nanozyme; B is a graph showing the changes in the inflammatory factor TNF-α in the thalamic lesion area of mice treated with nanozyme; C is a graph showing the changes in the inflammatory factor IL-6 in the thalamic lesion area of mice treated with nanozyme; D is a graph showing the changes in the amount of cerebral hemorrhage in the thalamic lesion area of mice treated with nanozyme; E is a graph showing the changes in the number of Nissl bodies in the thalamic lesion area of mice treated with nanozyme.

Figure 8 shows the changes in the expression levels of MMP2/9 in the damaged area after nanozyme administration measured by different methods; wherein: A is a graph showing the changes in pain behavior of CPSP model mice after nanozyme administration; B is a graph showing the changes in the expression levels of MMP2/9 in the damaged area after nanozyme administration measured by QT-PCR; C is a graph showing the changes in the expression levels of MMP2/9 in the damaged area after nanozyme administration measured by West Blotting.

Detailed ways

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

The experimental animals used in the examples are as follows:

C57 male mice (7-8 weeks, 20-25 g) were purchased and raised in the Animal Experiment Center of Zhengzhou University, with 5 mice per standardized cage, and were raised in a standard 12-h light/dark cycle with free access to clean water and food. The animal experiment was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Zhengzhou University (Ethics Approval Number: 2021056). In order to minimize individual differences in behavioral tests, animals were acclimated for 1-2 days before behavioral testing. During the behavioral test, in order to ensure objectivity and authenticity, the experimenter was unaware of the treatment conditions of the mice.

Example 1

A multifunctional nanozyme with endoplasmic reticulum targeting, comprising the following steps:

(1) Weigh 331.65 mg 2-methylimidazole and 1 mg hexadecyltrimethylammonium bromide and dissolve them in 5 mL deionized water. Stir at 400 rpm to mix. Then, add 1 mL of deionized water containing 1 mg bovine serum albumin and 1 mL of deionized water containing 29.06 mg Zn(NO ₃ ) ₂ ·6H ₂ O to the mixed solution. Stir for 20 min at room temperature. After the reaction, centrifuge the reaction solution at 5000 rpm for 5 min, collect the precipitate, resuspend the precipitate in deionized water containing 1 mg RuCl ₃ , and then perform ultrasonic dispersion. Stir for 12 h, add 1 mg sodium borohydride, and react for 10 min. Collect the precipitate again, wash it twice, freeze it, and dry it to obtain Ru MOF.

(2) 2 mg of p-dodecylbenzenesulfonamide, 2 mg of cholesterol, and 10 mg of lecithin were dissolved in chloroform/methanol (v/v = 2:1) and then dried under a rotary evaporator. The dried lipid film was then hydrated with a RuMOF solution (1 mg/mL, 2 mL), followed by probe sonication for 5 min to obtain ER-RuMOF.

The products Ru MOF and ER-Ru MOF were analyzed by transmission electron microscopy. The results are shown in FIG1 . ER-Ru MOF has a uniform particle size of about 100 nm, which indicates that the present invention has the potential to effectively penetrate the blood-brain barrier.

Example 2

A multifunctional nanozyme with endoplasmic reticulum targeting, comprising the following steps:

(1) Weigh 100 mg of 2-methylimidazole and 1 mg of hexadecyltrimethylammonium bromide and dissolve them in 5 mL of deionized water. Stir at 400 rpm to mix well. Then, add 1 mL of deionized water containing 1 mg of bovine serum albumin and 1 mL of deionized water containing 20 mg of Zn(NO ₃ ) ₂ ·6H ₂ O to the mixed solution in turn. Stir at room temperature for 20 min. After the reaction is completed, centrifuge the reaction solution at 5000 rpm for 5 min, collect the precipitate, resuspend the precipitate in deionized water containing 1 mg of RuCl ₃ , and then perform ultrasonic dispersion. Stir for 12 h, add 1 mg of sodium borohydride, and react for 10 min. Collect the precipitate again, wash it twice, freeze it, and dry it to obtain Ru MOF.

(2) 1 mg of p-dodecylbenzenesulfonamide, 2 mg of cholesterol, and 10 mg of lecithin were dissolved in chloroform/methanol (v/v = 2:1) and then dried under a rotary evaporator. The dried lipid film was then hydrated with a RuMOF solution (1 mg/mL, 2 mL), followed by probe sonication for 5 min to obtain ER-RuMOF.

Example 3

A multifunctional nanozyme with endoplasmic reticulum targeting, comprising the following steps:

(1) Weigh 400 mg of 2-methylimidazole and 1 mg of hexadecyltrimethylammonium bromide and dissolve them in 5 mL of deionized water. Stir at 400 rpm to mix well. Then, add 1 mL of deionized water containing 1 mg of bovine serum albumin and 1 mL of deionized water containing 10 mg of Zn(NO ₃ ) ₂ ·6H ₂ O to the mixed solution in turn. Stir at room temperature for 20 min. After the reaction is completed, centrifuge the reaction solution at 5000 rpm for 5 min, collect the precipitate, resuspend the precipitate in deionized water containing 1 mg of RuCl ₃ , and then perform ultrasonic dispersion. Stir for 12 h, add 1 mg of sodium borohydride, and react for 10 min. Collect the precipitate again, wash it twice, freeze it, and dry it to obtain RuMOF.

(2) 2 mg of p-dodecylbenzenesulfonamide, 2 mg of cholesterol and 10 mg of lecithin were dissolved in chloroform/methanol (v/v=2:1) and then dried on a rotary evaporator. The dried lipid film was then hydrated with a RuMOF solution (1 mg/mL, 4 mL) and then probe sonicated for 5 min to obtain ER-RuMOF.

Test Example 1 Enzyme Activity Assay

The RuMOF and ER-RuMOF products prepared in Example 1 were used to measure the enzyme activity. The specific detection method is as follows: The test conditions of the absorbance ΔA1 of the control group are:

The microplate reader was used to measure at 550 nm for 1 min, and the xanthine oxidase concentration was adjusted to stabilize ΔA1 at about 0.0225;

Test conditions for ΔA2 of absorbance of the experimental group:

The microplate reader was used to measure at 550 nm for 1 min, and the samples were graded for dilution, for example: 1, 1/5, 1/5 ^{^} 2 ^, 1/5 ^ 3 ^, 1/5 ^ 4, 1/5 ^ 5 ^, 1/5 ^{^} 6; the UV spectrophotometer was used to measure ΔA2 at 550 nm for 1 min.

As shown in FIG2 , RuMOF and ER-RuMOF have good superoxide dismutase (SOD) activity analysis, and endoplasmic reticulum-targeted liposome encapsulation does not affect the enzyme-like activity of the nanozyme, indicating that the present invention can effectively scavenge free radicals.

Experimental Example 2 Verification of the endoplasmic reticulum targeting of ER-RuMOF

The ER-RuMOF prepared in Example 1 was stained with FITC, and co-localization was performed using a commercial ER=Tracker.

The results are shown in FIG3 . The ER-RuMOF prepared in the present invention can better target the endoplasmic reticulum, thereby precisely regulating the oxidative stress of the endoplasmic reticulum.

Experimental Example 3 Establishment of CPSP model in mice

The method for establishing CPSP model mice was based on previous literature, and some modifications were made based on our preliminary experiments. As shown in Figure 4A, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (50 mg/kg) and fixed in a brain stereotaxic device. Under the guidance of the stereotaxic device, type VII collagenase (Coll VII, C0773, Sigma-Aldrich Co, MO; 0.025 U/0.25 ul, dissolved in saline) was extracted with a microinjection needle and injected into the VPM and VPL areas on the right side of the thalamus (0.82-2.3 mm in front of the anterior bregma, 1.30-1.95 mm lateral to the midline, and 3.01-4.25 mm deep to the skull surface). The sham operation group was injected with 0.25 ul of sterile saline. After administration, the microinjection needle was kept for 10 minutes to prevent reflux, and then the injection needle was slowly rotated out; after microinjection, the needle hole was sealed with paraffin, the skin was sutured, and the skin of the surgical site was wiped with iodine.

(1) Bederson score

The modified Bederson scoring method was used to evaluate the neurological function of mice in the Control group and Coll VII group. The mice were lifted 10 cm above the table by their tails, and their behavior was observed and scored according to the following scoring system: 0 points, no limb abnormality; 1 point, forelimb bending; 2 points, reduced resistance to lateral push of unilateral limbs; 3 points, unidirectional circling of the limbs; 4 points, longitudinal rotation of the limbs; 5 points, unilateral limb paralysis.

As shown in Figure 4C , there was no statistically significant difference in the neurological function scores between the two groups of mice at 1d, 3d, 5d, 7d, 14d, 21d, and 28d after surgery.

(2) Behavioral science

The frequency of mouse withdrawal under mechanical stimulation was measured according to the previous research method. The mouse was placed alone in a plexiglass chamber on a high-mesh screen and adapted to the environment for 1 hour. Two calibrated von frey wires (0.07g and 0.4g, Stoelting Co) were inserted into the mouse's hind paw for about 1 second, and repeated 10 times. The bilateral hind paw measurements were taken 20 minutes apart. The rapid withdrawal of the mouse's paw is a positive response. The withdrawal frequency for every 10 stimulations was calculated as a percentage: [(number of withdrawals/10 trials) × 100% = response frequency].

Cold stimulation: Same as the mechanical pain static method for mice mentioned above. When the mice are quiet and adapt to the environment, a 0.3ml syringe is used to draw an appropriate amount of acetone (12.5uL) and sprayed on the palm of the mouse's back. The behavior of the mouse is observed and scored according to the following criteria: 0 points: no reaction; 1 point: rapid lifting and shaking of the foot; 2 points: long-term, repeated lifting and shaking of the foot, licking the instep; 3 points: repeated, alternating licking of the sole of the foot. The test was performed three times in a row, with an interval of 5 minutes each time, and the average score of the mice in each treatment group was recorded. The interval between the measurement of the bilateral hind paws was 20 minutes.

Nanozymes (3, 5, or 8 mg/kg) and vehicle (PBS) were administered via the tail vein 30 min after microinjection of type VII collagenase or saline, and behavioral tests were performed 30 min after nano/vehicle administration, 1 d before CollIV/saline microinjection and on d1 and d3 after CollIV/saline microinjection.

As shown in Figure 4C, unilateral microinjection of Coll IV resulted in mechanical allodynia to 0.07g and 0.4g compared with the unilateral saline injection group. These hyperalgesias occurred one day after the lesion, and the mechanical threshold and cold pain response latency (acetone test) of the contralateral hindlimb were significantly increased, which lasted for at least 28 days compared with the sham control group (Figure 4D). There were no obvious pain-related changes in the ipsilateral limb (Figure 4E).

As can be seen from Figure 6, different doses can alleviate the pain hypersensitivity of the contralateral limbs left over from thalamic hemorrhage in the CPSP group mice in a dose-dependent manner. The pain hypersensitivity of the CPSP mice in the group given 8 mg/kg of nanomaterials was significantly reduced. As shown in Figure 8A, after the administration of the nanozyme, the pain behavior test showed that the pain hypersensitivity of the mice in the nanozyme administration group was improved.

(3) Serum biochemical index detection

The mice were injected with PBS solution or Nano solution (dissolved in PBS, 8 mg/kg) through the tail vein. Three days later, the mice were deeply anesthetized, and blood was collected by puncturing the eye sockets. The blood was placed in a 1.5 ml centrifuge tube and kept in a refrigerator at 4 degrees overnight. The next day, the supernatant was extracted by centrifugation at 3000 rpm for 10 minutes. The corresponding serum indicators were detected according to the steps of the biochemical kit (Ledo, Shenzhen).

As shown in Figure 6C, there was no statistically significant difference in the liver function-related indicators ALT, AST, ALP and the kidney function-related indicators Crea and Urea between the mice in the nanozyme administration group and those in the control group. Nanozyme administration did not cause abnormal liver and kidney function in mice.

(4) Immunohistochemistry

Paraffin sections were placed in different concentration gradients of xylene and ethanol solutions for dehydration, and tissue sections were placed in a repair box filled with citric acid antigen repair buffer in a microwave oven for antigen repair. After natural cooling, the slides were placed in PBS (PH7.4) and washed by shaking on a decolorizing shaker. The sections were then placed in a 3% hydrogen peroxide solution, incubated at room temperature in the dark for 25 minutes, and washed again to block endogenous peroxidase. An immunohistochemical pen was used to circle the tissue around the tissue, and 3% BSA was added to the tissue circle to evenly cover the tissue, and blocked at room temperature for 30 minutes. Primary antibody dilution (TNF-a, 1:200, servicebio, GB11188; IL-6, 1:200, servicebio, GB11117) was added to the sections, and the sections were placed flat in a humidified box and incubated overnight at 4°C. The next day, the slides were placed in PBS (PH7.4) and washed by shaking on a decolorizing shaker. After the slices are slightly dried, add anti-rabbit secondary antibody dilution (HRP labeled) in the circle to cover the tissue and incubate at room temperature for 50 minutes. Place the slides in PBS (PH7.4) again and shake and wash on a decolorizing shaker. After the slices are slightly dried, add freshly prepared DAB colorimetric solution in the circle. Control the color development time under a microscope. The positive color is brown-yellow. Rinse the slices with pure water to stop the color development. Use hematoxylin to counterstain the nuclei for 3 minutes and rinse with pure water. Finally, put the slices in different concentrations of ethanol and xylene in turn to dehydrate and make them transparent. Take the slices out of xylene and dry them slightly, seal them with sealing glue, observe them under a microscope and take pictures.

As shown in Figures 4G-4H, the expression of ROS and inflammatory factors TNF-α and IL-6 in the damaged area increased; as shown in Figure 7, the expression of inflammatory factors TNFa (Figure 7C) and IL-6 (Figure 7D) in the brain damaged area was significantly reduced after nanozyme administration.

(5) Immunofluorescence

Paraffin sections were placed in different concentration gradients of xylene and ethanol solutions for dehydration, and tissue sections were placed in a repair box filled with EDTA antigen repair buffer in a microwave oven for antigen repair. After natural cooling, the slides were placed in PBS (PH7.4) and washed by shaking on a decolorizing shaker. After the sections were slightly dried, circles were drawn around the tissue with a tissue pen, and BSA was added and blocked for 30 minutes. Primary antibody dilution (EMMPRIN, 1:200, Santa Cruze, sc-46700/mmp9, 1:200, abcam, 1:200, ab228402/mmp2, 1:200, proteintech, 10373-2-AP/8-OHdG, 1:200, abcam, ab62623) was added to the sections, and the sections were placed flat in a humidified box and incubated overnight at 4°C. The next day, the slides were placed in PBS (PH7.4) and washed by shaking on a decolorizing shaker. After the slices are slightly dried, add the dilution of the secondary antibody (HRP labeled) of the corresponding species to the primary antibody in the circle to cover the tissue, and incubate at room temperature for 50 minutes in strict light-proof. Then place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature in the dark for 10 minutes. Wash again, dry the slices slightly, and seal them with anti-fluorescence quenching sealing agent, observe and collect images under a fluorescence microscope.

As shown in Figure 4F, the expression of ROS (using 8-OHdG as an oxygen free radical marker) around the brain injury site was significantly increased in mice injected with Coll VII in the thalamus, i.e., CPSP mice. As shown in Figure 5C, the expression of EMMPRIN, MMP9, and MMP2 around the brain injury site of CPSP mice was significantly increased, and there was a significant co-labeling phenomenon between EMMPRIN and MMP9 and MMP2. As can be seen from Figure 7, the expression of ROS (Figure 7A) in the brain injury area was significantly reduced after the administration of nanozymes, and the administration of nanozymes can reduce the oxidative stress response of tissues around cerebral hematomas.

(6) Evaluation of mouse body weight changes

The mouse was placed on the electronic balance tray and left to rest for 5 minutes. The weight data on the display screen was read after the mouse was quiet.

As shown in Figure 6I, CPSP mice were given a placebo (i.e., Vehicle group) and 3 mg/kg, 5 mg/kg, and 8 mg/kg of nanozymes, respectively. There was no statistically significant difference in body weight among the four groups of mice, indicating that the administration of nanozymes would not cause abnormal growth status of mice.

(7) HE staining

Paraffin sections are placed in different concentration gradients of xylene and ethanol solutions for dehydration and dewaxing until water is reached. After drying, add hematoxylin to stain for 3-8 minutes and rinse with pure water. Use 1% hydrochloric acid alcohol to differentiate for 30 seconds and rinse with water again. Use 0.6% ammonia water to turn blue and rinse again. Then stain the sections in eosin stain for 1-3 minutes. After completion, dehydrate and seal with neutral gum. Use a microscope to inspect and collect images

As shown in Figures 6J-L, administration of 8 mg/kg nanozyme did not cause damage to the mouse myocardial tissue (Figure 6J), lung tissue (Figure 6K), and spleen tissue (Figure 6L).

(7) Assessment of cerebral hemorrhage volume

Mice were deeply anesthetized with isoflurane and perfused transcardially with 50-100 ml of 4% paraformaldehyde (0.1 M PBS, pH 7.4). The brain was removed and fixed at 4°C for 24 h. The mouse brain tissue was placed in the mouse brain trough and sliced from the frontal part to the occipital part in sequence with a thickness of 1 mm. The volume of mouse brain hematoma (mm ³ ) was analyzed using imagine-pro 5.0 software. The hematoma volume was calculated according to the formula V=tx(A1+A2+…+An) reported in the literature, where V represents the volume, t represents the slice thickness, and A represents the hematoma area.

As shown in Figure 4B, injection of CollVII into the VPM and VPL regions of the thalamus can cause hematoma formation in the thalamus of mice. As shown in Figure 7D, the amount of cerebral hemorrhage was improved after nanozyme administration.

(8) Nissl staining

After perfusion, the brain tissue of mice was taken, dehydrated, paraffin-fixed, embedded, and sliced to a thickness of 5 μm. Dewaxed with xylene, washed with different concentrations of anhydrous ethanol, stained with Nissl staining solution (Biyuntian Biotechnology, C0117) for 30 min, and sealed with neutral gum after dehydration and transparent treatment. The pathological section scanning machine was used to take pictures. Image J was used to analyze the number of Nissl bodies in the lesion area.

As shown in Figure 1, the number of Nissl bodies in the thalamic injury area of mice in the CPSP group was significantly reduced; as shown in Figure 7E, the number of Nissl bodies did not change after the administration of nanozyme.

(9) Western blotting

The mouse was deeply anesthetized and the brain was removed. Then the operation was performed on ice, the brain hematoma was removed, and the tissue around the brain hematoma was removed for protein extraction. The protein sample was mixed with the loading buffer and heated at 99 ° C for 5 minutes, loaded into a 7.5% polyacrylamide gel, electrophoresed, and then transferred to a PVDF membrane (Millipore, USA), placed in 3% skim milk, and blocked for two hours at room temperature. The PVDF membrane was then taken out and incubated overnight with the primary antibody at 4 degrees, including EMMPRIN (Santa Cruze, USA sc-46700), MMP9 (Wuhan Sanying 10375-2-AP), MMP2 (ABCAM, ab92536), GAPDH (Wuhan Sanying 10494-1-AP). The next day, the PVDF membrane was washed three times with TBST for 10 minutes each time, and incubated with secondary antibodies for 2 minutes, including goat anti-rabbit secondary antibody (Wuhan Tri-Tek, SA00001-2) and goat anti-mouse secondary antibody (ABCAM, ab6789), followed by TBST washing three times for 10 minutes each time. ECL luminescent liquid (Biyuntian, P0018AFT) and ChemiDoc XRS system were used for development. Image software was used to measure the density of the blot intensity.

As shown in Figure 5A, the expression of MMP2/9, a key protein for pain, was increased in the thalamic injury area of mice in the CPSP group; as shown in Figure 8C, the expression of MMP2/9, a key protein for pain, was decreased in the brain tissue of CPSP mice in the thalamic injury area after nanozyme administration.

(10) QT-PCR

After the mice were euthanized with isoflurane, the brain tissue around the hematoma was removed and placed in a 1.5 ml sterile enzyme-free tube, frozen in liquid nitrogen, and then stored in a -80°C refrigerator. RNA was extracted using an RNA column extraction kit (Acori), and reverse transcribed into cDNA. The primer sequences were EMMPRIN upstream 5‘-GGTCGGAAAGAAATCAGAGCAT-3‘, downstream 5‘-GCAGTGAGATGGTTTC CCGAG-3‘, MMP9 upstream 5‘-GCTGGCAGAGGCATACTTGTAC-3‘, downstream 5‘-GGTGTTCGAATGGCCTTTAGTG-3‘, MMP2 upstream 5‘-TGATAACCT GGATGCCGTCG-3‘, downstream 5‘-CCAGCCAGTCTGATTTGATGC-3‘, GAPD H upstream 5‘-CCTCGTCCCGTAGACAAAATG-3‘, downstream 5‘-TGAGGTCAAT GAAGGGGTCGT-3‘, followed by QT-PCR according to the manufacturer’s instructions.

As shown in Figure 5B , the expression of MMP2/9, a key protein for pain, was increased in the thalamic injury area of mice in the CPSP group; as shown in Figure 8B , the expression of MMP2/9, a key protein for pain, was decreased in the brain tissue of CPSP mice in the thalamic injury area after nanozyme administration.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

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 (10)

1. A multifunctional nanozyme with endoplasmic reticulum targeting, characterized in that the nanozyme uses ruthenium nanoparticles as carriers and p-dodecylbenzenesulfonamide is coated on the surface of the ruthenium nanoparticles. 2. A method for preparing a multifunctional nanozyme with endoplasmic reticulum targeting, characterized in that it comprises the following steps: (1) 2-Methylimidazole and hexadecyltrimethylammonium bromide were dissolved in deionized water, stirred at 400 rpm, and then a deionized water solution containing 1 mg/mL bovine serum albumin and a deionized water solution containing 29.06 mg/mL Zn(NO ₃ ) ₂ ·6H _2O were added dropwise in sequence. The mixture was stirred at room temperature for 20 min. After the reaction was completed, the reaction solution was centrifuged at 5000 rpm for 5 min, the precipitate was collected, and then the precipitate was resuspended in deionized water containing RuCl ₃ and ultrasonically dispersed. The mixture was stirred for 12 h, sodium borohydride was added for reaction for 10 min, the precipitate was collected again, washed, frozen, and dried to obtain Ru MOF; (2) dissolving p-dodecylbenzenesulfonamide, lecithin and cholesterol in a chloroform-methanol mixed solution, drying by rotary evaporation to obtain a lipid membrane, and then hydrating with a solution containing the RuMOF of step (1) and subjecting to probe ultrasound for 5 minutes to obtain ER-Ru MOF, i.e., an antioxidant functional nanozyme. 3. The method for preparing a multifunctional nanozyme with endoplasmic reticulum targeting according to claim 2, characterized in that in step (1), the mass ratio of 2-methylimidazole, hexadecyltrimethylammonium bromide, bovine serum albumin and Zn(NO ₃ ) ₂ ·6H ₂ O is 100-400:1:1:10-30. 4. The method for preparing a multifunctional nanozyme with endoplasmic reticulum targeting according to claim 2, characterized in that, in step (1), the mass ratio of RuCl ₃ , precipitate and sodium borohydride is 1:100:1. 5. The method for preparing a multifunctional nanozyme with endoplasmic reticulum targeting according to claim 2 is characterized in that, in step (2), the mass ratio of dodecylbenzenesulfonic acid, lecithin and cholesterol is 1:1:5; the volume ratio of chloroform and methanol in the chloroform-methanol mixed solution is 2:1. 6. Use of the multifunctional nanozyme with endoplasmic reticulum targeting according to any one of claims 1 to 5 in the preparation of antioxidant drugs. 7. The use according to claim 6, characterized in that the multifunctional nanozyme increases the activity of superoxide dismutase and scavenges free radicals. 8. Use of the endoplasmic reticulum-targeted multifunctional nanozyme according to any one of claims 1 to 5 in the preparation of a drug for inhibiting the activation of ROS and MMP2/9 at the damaged site after cerebral hemorrhage. 9. The use according to claim 7, characterized in that the multifunctional nanozyme reduces the expression levels of ROS and MMP2/9 in the damaged area after cerebral hemorrhage. 10. Use of the multifunctional nanozyme with endoplasmic reticulum targeting according to any one of claims 1 to 5 in the preparation of a drug for treating central post-stroke pain.