Pathological environment response functional nano-enzyme material preparation method and diagnosis and treatment application thereof

Abstract

The invention discloses a preparation method and diagnosis and treatment application of a pathological environment response functional nano-enzyme material. The pathological environment response functional nano enzyme can be used for diagnosing, treating, relieving and / or preventing arterial diseases. The pathological environment response functional nano-enzyme can target a disease microenvironment, and responsively releases the ultra-small nano-enzyme with reactive oxygen species (ROS) elimination capability, on one hand, the ultra-small nano-enzyme is metabolized into urine, and the disease course can be reflected by detecting the enzyme-like catalytic activity of the nano-enzyme in the urine; on the other hand, due to the excellent antioxidant activity of the ultra-small nano enzyme, the inflammation condition of the disease part can be relieved, and the effect of preventing and / or treating arterial diseases is achieved. Through the efficient design, the kit not only has the ability to diagnose diseases, but also has treatment characteristics in multiple aspects, and can treat, relieve and / or prevent arterial diseases which are mainly represented by inflammation due to the rise of ROS level.

Description

Technical Field

[0001] The present invention relates to a method for preparing a pathological environment responsive functional nanoenzyme material and its diagnostic and therapeutic application, and belongs to the field of biomedicine and material engineering. Background Art

[0002] Aneurysm refers to a type of vascular dilatation disease in which the arterial wall locally expands more than 1.5 times its normal diameter and presents a tumor-like morphology. Unlike the invasive growth and metastasis of true tumors, its main hazard is that with the continuous impact of blood flow, the aneurysm wall becomes weaker and eventually ruptures, leading to fatal massive bleeding. Aneurysm is a potentially fatal disease that causes about 170,000 deaths worldwide each year. Aneurysms can affect arterial vessels throughout the body, such as the aorta, intracranial arteries, peripheral arteries, and visceral arteries. Aneurysms grow rapidly and have a high risk of rupture. Currently, surgical repair, whether open surgery or endovascular aortic repair, is still the only effective treatment for aneurysms. Guidelines recommend surgical intervention for large asymptomatic (≥5.0 mm for women; ≥5.5 mm for men), symptomatic, and ruptured aneurysms. However, surgical procedures such as open surgery and endovascular aneurysm repair are invasive and traumatic, and are accompanied by serious complications such as infection, kidney damage, spinal cord damage, and endoleak (about 16-30%). Patients who do not meet the criteria for surgical treatment remain at high risk because sudden rupture of aneurysms is often unpredictable and fatal. There are currently no effective surgical or medical therapies available for small asymptomatic aneurysms or patients with anatomical limitations. Despite the risk of sudden rupture, patients with smaller aneurysms are often not treated and are monitored. This "close observation" approach not only causes considerable psychological stress, but also poses risks because the progression of aneurysm rupture is not linear but unpredictable. Therefore, effective pharmacological methods are urgently needed in clinical practice to mitigate the progression of aneurysm pathology and reduce the risk of rupture as a supplement and alternative to surgical intervention. More importantly, new methods should be developed to manage the course of aneurysms non-invasively.

[0003] Preclinical studies have shown that antihypertensive drugs, statins, matrix metalloproteinase (MMP) inhibitors, and mast cell stabilizers have significant efficacy in inhibiting aneurysms. However, to date, no drug has been proven effective in clinical trials, prompting the search for new potential therapies to inhibit the progression of this deadly disease. The lack of drug-based aneurysm therapy can be attributed to two main reasons. One is that the multifactorial etiology behind it is not yet fully understood. The contribution of hemodynamic forces has long been considered critical to the pathogenesis of aneurysms. However, many drugs that regulate hemodynamics, such as angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, have failed to alleviate the progression of aneurysms in clinical trials. These drugs only target traditional risk factors (such as hypertension) but cannot specifically inhibit the intrinsic pathogenic pathways within the aortic wall, ultimately resulting in poor efficacy. In order to optimize the treatment of aneurysms, a comprehensive assessment of the key pathological cell types involved in their pathogenesis must be performed, and then a rational treatment strategy must be developed.

[0004] The second reason for the lack of drug treatment for aneurysms is the lack of targeting affinity for the disease. Aneurysms are primarily characterized by structural dilation changes, which result in limited in situ drug accumulation. In addition, rapid blood flow in the aorta complicates the enrichment of drugs on the aortic wall, resulting in poor therapeutic efficacy. Therefore, achieving effective drug concentrations may result in increased side effects. To ensure effectiveness and safety, precise lesion targeting is essential for the advancement of aneurysm treatment.

[0005] With the rapid aging of the population, there is an increasing demand for non-invasive diagnosis and treatment of elderly patients with aortic aneurysms. It is difficult to accurately diagnose aneurysms through imaging methods in the early stages of the disease, so that when they are diagnosed, there is often a risk of rupture, so the mortality rate caused by aneurysms remains high. How to diagnose and intervene in the early stages of the disease is the key to reducing the mortality rate of aneurysms. At present, the clinical diagnosis of aneurysms mainly relies on medical imaging examinations, such as ultrasound, computed tomography, and magnetic resonance imaging. These methods require the use of sophisticated and complex medical imaging equipment, are expensive and relatively time-consuming. Although medical imaging can diagnose aneurysms with a larger degree of dilation, it lacks the accuracy to detect small, asymptomatic aneurysms, so surgical intervention is not the best strategy for these cases. Taking into account the limitations of drug treatment and diagnosis methods for aneurysms, it is necessary to develop a new method / material to achieve integrated diagnosis and treatment of aneurysms. It is expected to reduce invasive operations for patients and improve the efficiency of diagnosis and treatment, which has important scientific research significance and clinical practical value.

[0006] The pathological features of aneurysms include inflammatory cell infiltration, aortic wall dilation and calcification, oxidative damage, increased MMP expression and activity, vascular smooth muscle cell (VSMC) apoptosis, and degradation of the arterial elastic matrix. These features indicate that the pathogenesis of aneurysms is complex. Among these mechanisms, inflammation plays a central role and is closely related to the overproduction of reactive oxygen species (ROS) and oxidative stress. Elevated levels of ROS, including superoxide (O 2 ·- ) and hydrogen peroxide (H 2 O 2 ), which triggers an inflammatory response and leads to aneurysm formation. These oxidative stress factors lead to extracellular matrix (ECM) degradation and induce VSMC apoptosis, resulting in vascular damage and ultimately promoting aneurysm formation. Antioxidant enzymes, including catalase (CAT) and superoxide dismutase (SOD), play a key role in counteracting these processes, but their activity is significantly reduced in patients with aneurysms. Studies have shown that restoring the activity of these enzymes in the aortic wall can inhibit ECM degradation by MMP, thereby preventing aneurysm progression. However, the therapeutic use of natural enzymes or antioxidants is limited because they are unstable and inactivated after use. But their single enzyme activity limits the elimination of multiple ROS at the same time. At the same time, natural enzymes and antioxidants do not have targeting capabilities, and high-dose use can lead to toxic side effects. Therefore, it is necessary to develop new drugs that are targeted and have sustainable catalytic capabilities.

[0007] Nanozymes are functional nanomaterials with enzyme properties that can be given ideal functions through rational design. They show good application prospects in biomedicine and have been used for in vitro diagnosis and in vivo bioanalysis. The sustainable catalytic ability of nanozymes can greatly exert antioxidant activity, thereby eliminating excessive ROS in the disease process and alleviating the damage caused by excessive inflammatory response to the body. Among them, nanozymes with good antioxidant activity, such as Prussian blue PB, cerium oxide CeO 2 , Mn 3 O 4 etc. have successfully achieved the treatment of cardiovascular diseases. However, these nanozymes only have therapeutic functions and cannot achieve the desired function of non-invasive disease detection.

[0008] In summary, in view of the shortcomings of current aneurysm treatment drugs and the limitations of diagnostic methods, the use of nanozymes as the main body is a method that can simultaneously diagnose and treat aneurysms. Summary of the invention

[0009] Purpose of the invention: In view of the shortcomings of the prior art, the present invention provides a method for preparing a pathological environment responsive functional nanozyme material and its diagnostic and therapeutic application, and prepares aneurysm-targeting nanozyme by giving ultra-small nanozymes the ability to respond to pathological microenvironments.

[0010] Technical solution: In order to solve the above technical problems, the present invention provides a method for synthesizing a pathological environment responsive functional nanozyme based on surface modification, comprising the following steps:

[0011] (1) dissolving a group having oxidation response ability and / or a high molecular polymer in a polar solvent A liquid;

[0012] (2) dissolving and dispersing one or more nanozymes in liquid B with good biocompatibility;

[0013] (3) The solutions obtained in step (1) and step (2) are mixed under ultrasound and an emulsifier or stabilizer is added to stabilize the emulsion and form a polymer matrix of the nanoparticles, thereby obtaining the pathological environment responsive functional nanozyme based on surface modification.

[0014] The synthesis method further comprises collecting the product in step (3) and performing one or more treatments such as centrifugation, dialysis, and drying.

[0015] Among them, the groups and / or high molecular polymers with oxidation response ability in step (1) include one or more of N,N'-carbonyldiimidazole (CDI), 4-hydroxyphenylboronic acid pinacol ester (PBAP), tricyanuric chloride (TCT), 4-hydroxy-2,2,6,6-tetramethylpiperidinyl nitroxide free radical (Tpl), oxidation-sensitive dextran (Oxidizable dextran, Oxi-DEX), etc.

[0016] Preferably, the group and / or high molecular polymer having oxidation responsiveness is oxidation-sensitive dextran (dextran having a molecular weight of less than 10 kDa).

[0017] Wherein, the liquid A in step (1) comprises one or more combinations of isooctane, trifluoroacetic acid, trimethylpentane, cyclopentane, heptane, trichloroethylene, isobutanol, chloroform, butanol, n-propanol, tetrahydrofuran, ethanol, ethyl acetate, petroleum ether, cyclohexane, chloroform, benzene, toluene, dichloromethane, ether, ethyl acetate, isopropanol, pyridine, acetone, acetic acid, acetonitrile, aniline, dimethylformamide, methanol, ethylene glycol, dimethyl sulfoxide or water.

[0018] Preferably, the liquid A is a combination of chloroform and methanol.

[0019] Among them, the nanozyme described in step (2) includes oxygen compound nanozyme, nitrogen compound nanozyme, carbon-based nanozyme, metal nanozyme, metal compound-based nanozyme, covalent organic framework-based nanozyme, metal coordination nanozyme or composite material nanozyme.

[0020] The oxygen compound nanozymes include one or more combinations of oxide-based nanozymes, sulfide-based nanozymes, selenide-based nanozymes or telluride-based nanozymes, etc. The oxide-based nanozymes include one or more combinations of cerium oxide-based nanozymes, manganese oxide-based nanozymes, copper oxide-based nanozymes, iron oxide-based nanozymes, nickel oxide-based nanozymes, cobalt oxide-based nanozymes, zirconium oxide-based nanozymes, and hafnium oxide-based nanozymes.

[0021] Among them, the nitrogen compound nanozyme includes one or more combinations of nitride-based nanozymes or phosphide-based nanozymes.

[0022] Among them, the carbon family compound nanozymes include one or more combinations of fullerene-based nanozymes, carbon fiber-based nanozymes, carbon nanotube-based nanozymes, graphene-based nanozymes or other carbon-based nanozymes.

[0023] Among them, the metal nanozyme includes one or more combinations of gold nanozyme, copper nanozyme, silver nanozyme, platinum nanozyme, palladium nanozyme, rhodium nanozyme, ruthenium nanozyme or alloy nanozyme.

[0024] Among them, the metal compound-based nanozymes include one or more of iron oxide nanozymes, titanium dioxide-based nanozymes, manganese oxide nanozymes, vanadium oxide nanozymes, molybdenum sulfide-based nanozymes, iron sulfide-based nanozymes, copper sulfide-based nanozymes, metal selenide-based nanozymes, metal phosphide nanozymes, etc.

[0025] Among them, the organic metal framework-based nanozymes include iron-based organic metal framework nanozymes, manganese-based organic metal framework nanozymes, zinc-based organic metal framework nanozymes, copper-based organic metal framework nanozymes, gallium-based organic metal framework nanozymes, zirconium-based organic metal framework nanozymes, hafnium-based organic metal framework nanozymes, vanadium-based organic metal framework nanozymes, and one or more combinations of metal-doped organic metal framework nanozymes.

[0026] Preferably, the nanozyme is an organic metal framework-based nanozyme; the organic metal framework-based nanozyme is an iron-based organic metal framework nanozyme.

[0027] Among them, the iron-based organic metal framework nanozyme precursor includes one or more combinations of organic metal salts, metal chloride salts, metal bromide salts, metal nitrate salts, metal acetate salts, metal sulfate salts or metal cyanide complexes.

[0028] The metal cyanide complex includes one or more combinations of single metal cyanide complexes, double metal cyanide complexes, transition metal cyanide complexes, noble metal cyanide complexes and / or polynuclear metal cyanide complexes.

[0029] Preferably, the precursor of the iron-based organic metal framework nanozyme is a metal cyanide complex and / or a metal chloride salt and a combination thereof.

[0030] The metal cyanide complex includes one or more combinations of mononuclear metal cyanide complexes, mixed ligand metal cyanide complexes and multi-metal cyanide complexes.

[0031] Preferably, the metal cyanide complex is a mononuclear metal cyanide complex.

[0032] The mononuclear metal cyanide complex includes one or more combinations of ferrocyanide and ferrocyanide.

[0033] Preferably, the mononuclear metal cyanide complex is potassium ferrocyanide.

[0034] Among them, the precursor of the non-iron metal element contained in the iron-based organic metal framework nanozyme includes one or more combinations of organic metal salts, metal chloride salts, metal bromide salts, metal nitrate salts, metal acetate salts, metal sulfate salts or metal cyanide complexes.

[0035] Preferably, the precursor of the non-iron metal element contained in the iron-based organic metal framework nanozyme is a metal chloride salt.

[0036] Among them, the non-ferrous metal elements contained in the iron-based organic metal framework nanozyme include one or more combinations of lithium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, gallium, germanium, rubidium, strontium, iridium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, tin, antimony, cerium, hafnium, iridium, platinum, gold or bismuth.

[0037] Preferably, the non-ferrous metal element contained in the iron-based organic metal framework nanozyme is zinc.

[0038] The precursor of the non-ferrous metal element includes one or more combinations of organic metal salts, metal chloride salts, metal bromide salts, metal nitrate salts, metal acetate salts, metal sulfate salts or metal cyanide complexes.

[0039] Preferably, the precursor of the non-ferrous metal element is a chloride metal salt.

[0040] Wherein, the liquid B in step (2) is one or more combinations of phosphate buffer, physiological saline and ultrapure water.

[0041] Preferably, solution B is phosphate buffered saline.

[0042] Wherein, the emulsifier or stabilizer in step (3) is one or more combinations of polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, chitosan, gelatin, Tween 20, Tween 80, Span 60, Span 80, lecithin, gum, etc.

[0043] Preferably, the emulsifier or stabilizer is polyvinyl alcohol.

[0044] The present invention also includes the use of the nanoenzyme material based on pathological environment targeting and responsive release in the preparation of treatment, diagnosis, relief and / or prevention of arterial diseases, etc.

[0045] The arterial disease is a disease that widely involves arteries, including atherosclerotic diseases, arterial inflammatory diseases, hereditary or congenital arterial diseases, local or specific arterial lesions, blood disorder-related diseases, and arterial losses caused by external factors.

[0046] Wherein, the atherosclerotic diseases include coronary heart disease, myocardial infarction, and peripheral arteriosclerosis.

[0047] Wherein, the local or specific arterial lesions include aneurysms, aortic dissection or aneurysms.

[0048] Wherein, the blood disorder-related diseases include arterial embolism, arterial thrombosis or functional arterial disease.

[0049] Wherein, the arterial loss caused by external factors includes traumatic iatrogenic arterial disease, arterial compression or crush syndrome.

[0050] In one embodiment, the aneurysmal disease includes abdominal aortic aneurysm, thoracic aortic aneurysm, intracranial aneurysm, peripheral arterial aneurysm and visceral artery aneurysm.

[0051] Preferably, the disease is abdominal aortic aneurysm.

[0052] Among them, the integrated diagnosis and treatment nanozyme with pathological microenvironment responsiveness is composed of a polymer with oxidation responsiveness and an ultra-small nanozyme. The pathological microenvironment responsiveness is provided by a polymer with oxidation responsiveness. The ultra-small nanozyme refers to a nanozyme with a size less than 5.5nm. The integrated diagnosis and treatment nanozyme refers to a material formed by co-building a polymer with oxidation responsiveness and an ultra-small nanozyme.

[0053] The present invention utilizes the ROS responsiveness of nanozymes to responsively release ultrasmall nanozymes with antioxidant capacity in the pathological microenvironment of high ROS levels in aneurysms. On the one hand, the ultrasmall nanozymes remove ROS to achieve an antioxidant effect, thereby alleviating the excessive inflammation state of the pathological site of the disease; on the other hand, the ultrasmall nanozymes are metabolized through the kidneys to reach the urine, and the activity of the nanozymes in the urine is detected to diagnose the course of the disease.

[0054] The present invention proposes for the first time to utilize ultra-small nanozymes to regulate the oxidative stress and ROS levels of aneurysm pathological sites by utilizing the various antioxidant enzyme activities (SOD-like enzymes, CAT-like enzyme activities, etc.) of nanozymes under the condition of renal metabolism, and utilize the constructed pathological microenvironment response capability to achieve the targeting of nanozymes. Ultra-small nanozymes are released through the responsiveness of pathological sites. On the one hand, renal metabolism can be effectively performed, and the enzyme activity of the released nanozymes is detected by collecting urine to reflect the disease process; on the other hand, the excellent antioxidant activity of ultra-small nanozymes is utilized to regulate the ROS level of pathological sites, inhibit smooth muscle cell apoptosis and calcification, and inhibit inflammatory cell infiltration to achieve local vascular reconstruction of aneurysms and repair of pathological microenvironments, and to achieve the treatment, relief and / or prevention of aneurysm-related diseases. The disease diagnosis method provided by the present invention avoids the traumatic injury of traditional invasive medical imaging. The present invention provides a method for urine detection to reflect the course of the disease, which is an efficient and economical disease detection method. The ultra-small nanozymes of the present invention can effectively perform renal metabolism and avoid the potential toxicity problems of nanomaterials.

[0055] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0056] 1. Achieve the purpose of disease diagnosis and treatment at the same time: The present invention utilizes the constructed pathological environment responsive module and the anti-inflammatory properties of the nanozyme itself to diagnose and treat aneurysms. On the one hand, the glucan with oxidation responsiveness constructed by the present invention responsively releases ultra-small nanozymes in the pathological site with high ROS levels in the aneurysm, which can be metabolized by the kidneys to urine. By detecting the enzyme-like activity of the nanozyme in the urine, the disease can be diagnosed and the course of the disease can be monitored. On the other hand, the ultra-small nanozymes released by the pathological environment have excellent antioxidant activity and anti-inflammatory properties and can treat aneurysms.

[0057] 2. Reduce the dosage and types of drugs: Nanozymes with disease microenvironment targeting capabilities achieve low-dose medication and efficient therapeutic effects. On the one hand, the nanozymes developed by the present invention have the ability to respond to pathological microenvironments, can effectively target pathological sites and release nanozymes with therapeutic capabilities, and circumvent the problem of limited in-situ drug accumulation and the need for large amounts of medication for the treatment of broad-spectrum vascular diseases. On the other hand, the enzyme-like catalytic activity of nanozymes is used to regulate the higher oxidative stress levels in the pathological sites of aneurysms, ultimately playing a relevant therapeutic role. This targeted nanozyme material development can achieve long-term regulation of the disease in an auxiliary treatment mode without medication or with less medication (broad-spectrum anti-inflammatory drugs), reducing the complications and economic burden caused by long-term, high-dose medication in traditional treatments.

[0058] 3. Safe to use and meet clinical needs: The nanozyme used in the present invention is less than 5.5nm in size and can be quickly metabolized out of the body through the kidneys, which has high safety. Nanozymes with antioxidant activity reduce the local expansion of aneurysms and the inflammation of local tissues of aneurysms, meeting clinical treatment needs. More importantly, the present invention can collect urine from the subjects and detect the enzyme-like activity of nanozymes in urine to quickly diagnose the disease, reducing the time required for traditional medical imaging examinations and reducing the economic burden on patients.

[0059] 4. Efficient ROS elimination ability: Since the ROS elimination effect of nanozymes is different from that of traditional antioxidant drug molecules and natural enzymes, antioxidants need to consume a large amount of drug molecules to eliminate ROS, and the ROS elimination ability can only be maintained after taking enough drugs. The ROS elimination nanozymes used in the present invention are a class of enzyme mimics with SOD and CAT activities, which can continuously catalyze in vivo. This cascade catalysis method can efficiently remove ROS, thereby treating aneurysm-related diseases, with high efficiency and low side effects.

[0060] 5. The preparation process is simple and the cost of raw materials is low, which facilitates industrial production and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 XRD of ultrasmall Prussian blue SPB and its analog nanozyme SPBZn;

[0062] Figure 2 XPS of ultrasmall Prussian blue SPB (a) and its analog nanozyme SPBZn (b);

[0063] Figure 3 TEM images of ultrasmall Prussian blue SPB and its analog nanozyme SPBZn;

[0064] Figure 4The SOD-like enzyme activity (a) and CAT-like enzyme activity (b) of ultrasmall Prussian blue and its analog nanozymes;

[0065] Figure 5 FT-IR of nanozymes with pathological microenvironment response;

[0066] Figure 6 Fluorescent image showing that nanozymes responding to pathological microenvironment slow down ROS production in macrophages;

[0067] Figure 7 Flow cytometry quantitative detection results of nanozymes that respond to pathological microenvironment to slow down ROS production in macrophages;

[0068] Figure 8 The biodistribution of nanozymes responsive to pathological microenvironment in rats with abdominal aortic aneurysm;

[0069] Fig. 9 The results of nanozyme urine metabolism validation for pathological microenvironment response;

[0070] Fig.10 Results of the main elements in urine of rats with aneurysms: (a) Fe content; (b) Zn content;

[0071] Fig.11 The results of CAT enzyme activity detection in urine of rats with abdominal aortic aneurysm;

[0072] Fig.12 The results of the pathological microenvironment-responsive nanozymes in reducing vascular relative calcium deposition in an abdominal aortic aneurysm rat model;

[0073] Fig.13 The results showed that the nanozymes that responded to the pathological microenvironment increased the proportion of elastin in the middle layer of blood vessels in the abdominal aortic aneurysm rat model;

[0074] Fig.14 The results showed that the pathological microenvironment-responsive nanozymes reduced the level of vascular inflammation in the abdominal aortic aneurysm rat model. DETAILED DESCRIPTION

[0075] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0076] Example 1 Preparation of ultra-small nanozymes

[0077] 1. Prepare 13.6 mg mL -1 ZnCl 2 and 32.4 mg mL -1 FeCl 3 spare;

[0078] 2. Add 20 μL ZnCl to 20 mL deionized water.2 (13.6 mg mL -1 ) and 40 μL FeCl 3 (32.4 mg mL -1 ) solution, heated to 60°C;

[0079] 3. Prepare 20 mL of 0.02 mM K 4 [Fe(CN) 6 ]·3H 2 O and 0.5 mM citric acid solution for later use;

[0080] 4. The solution obtained in step 2 was quickly and dropwise added to the solution obtained in step 3 through a peristaltic pump, and the reaction was vigorously stirred at 60°C for 10 minutes.

[0081] 5. Centrifuge the liquid obtained in step 4 at 5000×g for 1 hour, repeat this step 3 times; and wash three times with deionized water.

[0082] 6. Freeze-dry the sample in step 5 for 24 hours and set aside.

[0083] Example 2 Evaluation of Nanozyme Synthesis and Antioxidant Activity in Example 1

[0084] In Example 1, by using K 4 [Fe(CN) 6 ]·3H 2 O and ZnCl 2 As a precursor, Zn-doped 2+ The ultra-small PB analogue SPBZn was successfully prepared to prepare ultra-small Prussian blue analog nanozyme ( Figure 1 and Figure 2 ), the particle size of the nanozyme was maintained at 5 nm and exhibited good dispersibility ( Figure 3 ).

[0085] Subsequently, the enzyme-like activity of the nanozyme prepared in Example 1 was investigated. 2 ·- It is a free radical with asymmetric electrons. Due to the asymmetry of the electronic configuration, O 2 ·- It causes extremely high damage to biological systems. 2 ·- The elimination rate can be determined by WST-1 and O 2 ·- The formazan dye formed by the interaction is used to detect when the nanozyme reacts with O 2 ·- When the elimination effect occurs, its absorbance decreases. Figure 4As shown in a, the ultra-small nanozyme constructed by the present invention has good SOD enzyme activity. The CAT-like enzyme activity was then tested. 2 O 2 The self-polymerization of dopamine triggered by the reaction is inhibited, and if CAT or nanozymes with CAT-like activity are present in the environment, they catalyze the H 2 O 2 O produced by decomposition 2 The second step of the antioxidant cascade, H 2 O 2 Decomposition of. Figure 4 As shown in b, for H 2 O 2 The decomposition showed obvious concentration dependence.

[0086] Example 3 Construction of nanozymes responsive to pathological microenvironment

[0087] 1. Dissolve 4-hydroxyphenylboronic acid pinacol ester PBAP (1.10 g, 4.7 mmol) and N,N'-carbonyldiimidazole CDI (1.219 g, 7.5 mmol) in 15 mL of anhydrous CH 2 Cl 2 In N 2 The mixture was stirred at room temperature for 3 hours under protection.

[0088] 2. The product obtained in step 1 was extracted and washed with deionized water for 3 times and then washed with saturated brine once. 4 After drying, the product was concentrated by rotary evaporation to obtain a white PBAP-CDI solid for later use.

[0089] 3. Dissolve 90 mg of dextran (Mw = 10 kDa) in 1.575 mL of anhydrous DMSO, add 149.3 mg of 4-dimethylaminopyridine and 149.3 mg of PBAP-CDI. 2 Under protection, the reaction was stirred at room temperature overnight.

[0090] 4. Collect the product in step 3, centrifuge at 10250×g for 15 minutes, and wash three times with deionized water.

[0091] 5. Freeze-dry the sample obtained in step 4 and set aside.

[0092] 6. Using the double emulsification method, the nanozyme material OZn with pathological microenvironment response was constructed with the nanozyme obtained in Example 1 as the center.

[0093] (1) The nanozyme prepared in Example 1 was added at 4 mg mL -1Dissolve in PBS and set aside.

[0094] (2) Dissolve 26 mg of oxidation-sensitive dextran Oxi-DEX in 0.8 mL of CHCl 3 After 0.5 mL of MeOH, the nanozyme solution was added. Ultrasonic treatment was performed on ice (ultrasonication for 30 seconds, interval of 10 seconds, a total of 3 minutes, power output 80%). 1.5 mL of 3% w / w PVA solution (with PBS as solvent) was added and ultrasonicated on ice for 30 seconds under the same conditions.

[0095] 7. Add 0.3% w / w PVA solution (10 mL) to the double emulsion obtained in step 6 and stir at room temperature for two hours.

[0096] 8. Collect the product obtained in step 7, centrifuge at 10250×g for 15 minutes, and wash three times with deionized water. Freeze-dry the sample for later use. Figure 5 The FT-IR test results show that the nanozyme with pathological microenvironment response constructed in the present invention has a wavelength of 2070 cm -1 The C≡N stretching vibration of about 1.5 nm indicates that it retains the characteristic peak of the ultra-small nanozyme SPBZn itself, and also confirms the incorporation of the ultra-small nanozyme. In addition, its incorporation does not affect the structure of the oxidation-sensitive polymer itself, and the nanozyme with pathological microenvironment response is successfully constructed.

[0097] Example 4: Further verification of the antioxidant function of the nanozyme responsive to the pathological microenvironment prepared in Example 3 at the cellular level

[0098] 1. RAW 264.7 cells were plated at a density of 1×10 5 Seed in 6-well plates and incubate overnight.

[0099] 2. Add different concentrations (20, 50, 100 μg / mL) of nanozymes (SPB, SPBZn, OZn, OPB) to the cells in step 1 and let them act for 2 hours. Wash the cells 3 times with PBS and add 60 μM H 2 O 2 The preparation method of SPB is as follows: weigh 0.75g polyvinyl pyrrolidone PVP and 0.0275g K 3 [Fe(CN) 6 ], dispersed in 10 mL of 75% ethanol solution containing 0.01 M HCl. The obtained solution was reacted in an 80°C water bath for 3 hours, cooled to room temperature, and the obtained dark blue solution was washed three times with deionized water, and freeze-dried for 24 hours to collect the product for use. The preparation method of OPB is the same as that of Example 3, except that the nanozyme in step 6 is replaced with SPB to obtain OPB.

[0100] 3. After washing with PBS three times, DMEM containing 10 μM DCFH-DA (Biyuntian Biotechnology Co., Ltd.) was added to the cells in step 2 for 30 minutes. After washing with PBS, fluorescence images were obtained using a laser scanning confocal microscope, and the intracellular ROS level was semi-quantitatively analyzed using a flow cytometer. Figure 6 As shown, the nanozymes with pathological microenvironment response have good antioxidant activity and are concentration-dependent.

[0101] At the same time, the quantitative detection results of flow cytometry are as follows Figure 7 As shown, it is also shown that the nanoenzyme material constructed by the present invention has excellent active oxygen scavenging ability.

[0102] Example 5 Diagnostic ability of pathological microenvironment-responsive nanozymes in an abdominal aortic aneurysm model

[0103] 1. SD rats (Weitong Lihua Experimental Animal Technology Co., Ltd.) were divided into a healthy group (Healthy), a sham operation group (AAA - ) and modeling group (AAA + ). The healthy rats were not treated, and urine was collected as a blank control. Four hours before the experiment, the rats were fasted and each rat was housed individually. 2 To induce elastin damage in the abdominal aorta region of rats, an AAA model was established. The specific operation was as follows: After SD rats were anesthetized, the abdominal hair was shaved, and after disinfection with alcohol and iodine, the skin and muscles were carefully cut along the midline of the abdomen. The organs in the abdominal cavity were isolated with cotton balls containing physiological saline. The diaphragm was carefully separated with ophthalmic forceps to expose the infrarenal abdominal aorta. A piece of scalp tissue soaked in CaCl in advance was placed on the scalp. 2 Sterile gauze (0.5 cm × 0.5 cm) in a 0.50 M solution was wrapped around the aortic adventitia. After 15 minutes, the abdominal cavity was cleaned with saline, the abdominal organs were returned to their positions, and the muscles and skin were sutured to form the model group (AAA + ). Normal control group (sham operation, i.e. AAA - ) SD rats also underwent the same surgical procedure, but saline was used instead of CaCl 2 to be processed.

[0104] 2. Replace the AAA in step 1 - and AAA + Rats in group A were injected with 10 mg / kg of fluorescently labeled nanozymes.

[0105] 3. Collect urine from the two groups of rats in step 2 every hour and store in a dark place.

[0106] After 4 and 8 h, the rats were euthanized, and the heart, liver, spleen, lungs, kidneys and other major organs were collected and the residual blood was washed with PBS.

[0107] 5. Use a small animal imaging system to obtain fluorescent images of the main organs of each group of rats obtained in step 4 to evaluate the distribution of OZn in vivo.

[0108] 6. Simultaneously observe the fluorescence intensity of OZn in the urine collected in step 3 to evaluate the metabolism of OZn.

[0109] 7. Perform a dopamine-based CAT enzyme activity test on the urine collected in step 3.

[0110] 8. The urine collected in step 3 was acid-hydrolyzed, and the content of Fe and Zn elements in the nanozyme was further detected by ICP-OES.

[0111] 9. The corresponding test results were analyzed and the results showed that the nanozymes injected via tail vein mainly accumulated in the kidneys of the abdominal aortic aneurysm rat model ( Figure 8 ) and can be metabolized into urine ( Fig. 9 ). Further ICP-OES test results confirmed this conclusion ( Fig.10 ).

[0112] 10. The results of urine CAT enzyme activity test showed that ( Fig.11 ), the urine of healthy rats, and the urine collected from the sham-operated rats after nanozyme injection had no CAT enzyme activity. On the contrary, the urine of the aneurysm model rats showed obvious CAT enzyme activity signals after nanozyme injection, indicating that the nanozyme of the present invention has good aneurysm diagnostic ability.

[0113] Example 6 Physiological performance of nanozymes responsive to pathological microenvironment in abdominal aortic aneurysm model

[0114] 1. SD rats (Weitong Lihua Experimental Animal Technology Co., Ltd.) were divided into control group, model group, oxidation-sensitive glucan group, ultra-small nanozyme group and pathological microenvironment-responsive nanozyme group. The control group was a sham operation group (Normal) and a model group (Model) injected with the same volume of saline (the construction method of the Normal group was the same as that of the AAA group). - The construction method of group and model is the same as AAA + The oxidation-sensitive dextran group (Oxi-DEX), the ultrasmall nanozyme group (SPBZn), and the pathological microenvironment-responsive nanozyme group (OZn) were injected with the same dose of the corresponding different drugs (5 mg / mL). The treatment was carried out twice a week (three days between each treatment) for three weeks. The experiment was terminated on the 26th day, and the rats were euthanized by carbon dioxide asphyxiation. The complete aorta and major organs were removed by laparotomy.

[0115] 2. Detect aortic lesion tissue, including alizarin red staining, EVG staining and CD68 staining of abdominal aorta;

[0116] 3. Perform statistical analysis on the test results, including relative calcium deposition ( Fig.12 ), the proportion of elastin in the middle layer of blood vessels ( Fig.13 ) and relative CD68 positive expression ( Fig.14 Compared with the rats in the modeling group and other drug treatment groups, the relative calcium deposition in the rat aorta was reduced after the nanozyme treatment in response to the pathological microenvironment ( Fig.12 ) decreased significantly, and the proportion of elastin in the middle layer of blood vessels ( Fig.13 ) increased significantly, indicating that the therapeutic effect of targeted nanozymes is better than that of other drug groups. From the statistical results of inflammatory cell CD68 immunohistochemistry ( Fig.14 ) It can be found that after targeted nanozyme treatment, rats showed the lowest CD68 expression and their inflammation levels were lower than those in the control group and other drug groups, indicating that targeted delivery of nanozymes can effectively alleviate inflammation in the pathological site of aneurysms.

[0117] In summary, the targeted nanozymes prepared by the present invention can achieve non-invasive diagnosis of abdominal aortic aneurysm and solve the problem of high inflammation levels in the pathological site of the aneurysm, thereby reducing calcium deposition in the aneurysm lesion site and repairing the integrity of vascular elastin. It has a certain universality and the possibility of clinical transformation.

Claims

1. A method for synthesizing a pathological environment responsive functional nanozyme based on surface modification, characterized in that: The following steps are involved: (1) dissolving a group having oxidation response ability and / or a high molecular polymer in a polar solvent A liquid; (2) dissolving and dispersing one or more nanozymes in liquid B with good biocompatibility; (3) The solutions obtained in step (1) and step (2) are mixed under ultrasound and an emulsifier or stabilizer is added to stabilize the emulsion and form a polymer matrix of the nanoparticles, thereby obtaining the pathological environment responsive functional nanozyme based on surface modification.

2. The synthesis method according to claim 1, characterized in that The group and / or high molecular polymer having oxidation responsiveness in step (1) includes one or more of N,N'-carbonyldiimidazole, 4-hydroxyphenylboronic acid pinacol ester, cyanuric chloride, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl nitroxide free radical, and oxidation-sensitive dextran.

3. The synthesis method according to claim 1, characterized in that The liquid A in step (1) includes one or more combinations of isooctane, trifluoroacetic acid, trimethylpentane, cyclopentane, heptane, trichloroethylene, isobutanol, chloroform, butanol, n-propanol, tetrahydrofuran, ethanol, ethyl acetate, petroleum ether, cyclohexane, chloroform, benzene, toluene, dichloromethane, ether, ethyl acetate, isopropanol, pyridine, acetone, acetic acid, acetonitrile, aniline, dimethylformamide, methanol, ethylene glycol, dimethyl sulfoxide or water.

4. The synthesis method according to claim 1, characterized in that The nanozyme described in step (2) includes oxide-based nanozymes, sulfide-based nanozymes, selenide-based nanozymes, telluride-based nanozymes, cerium oxide-based nanozymes, manganese oxide-based nanozymes, copper oxide-based nanozymes, iron oxide-based nanozymes, nickel oxide-based nanozymes, cobalt oxide-based nanozymes, zirconium oxide-based nanozymes, hafnium oxide-based nanozymes, nitride-based nanozymes, phosphide-based nanozymes, fullerene-based nanozymes, carbon fiber-based nanozymes, carbon nanotube-based nanozymes, graphene-based nanozymes, gold nanozymes, copper nanozymes, silver nanozymes, platinum nanozymes, palladium nanozymes, rhodium nanozymes, ruthenium nanozymes, alloy nanozymes, One or more combinations of iron oxide nanozymes, titanium dioxide-based nanozymes, manganese oxide nanozymes, vanadium oxide nanozymes, molybdenum sulfide-based nanozymes, iron sulfide-based nanozymes, copper sulfide-based nanozymes, metal selenide-based nanozymes, metal phosphide nanozymes, iron-based organic metal framework nanozymes, manganese-based organic metal framework nanozymes, zinc-based organic metal framework nanozymes, copper-based organic metal framework nanozymes, gallium-based organic metal framework nanozymes, zirconium-based organic metal framework nanozymes, hafnium-based organic metal framework nanozymes, vanadium-based organic metal framework nanozymes or metal-doped organic metal framework nanozymes.

5. The synthesis method according to claim 4, characterized in that The precursor of the iron-based organic metal framework nanozyme includes one or more combinations of organic metal salts, metal chloride salts, metal bromide salts, metal nitrate salts, metal acetate salts, metal sulfate salts or metal cyanide complexes; the metal cyanide complex includes one or more combinations of single metal cyanide complexes, double metal cyanide complexes, transition metal cyanide complexes, noble metal cyanide complexes or polynuclear metal cyanide complexes.

6. The synthesis method according to claim 4, characterized in that The precursor of the iron-based organic metal framework nanozyme includes a metal cyanide complex and / or a chloride metal salt and a combination thereof; the metal cyanide complex includes a combination of one or more of a mononuclear metal cyanide complex, a mixed ligand metal cyanide complex and a multi-metal cyanide complex; the mononuclear metal cyanide complex includes a combination of one or more of ferrocyanide and ferrocyanide, etc.; the precursor of the non-ferrous metal element contained in the iron-based organic metal framework nanozyme includes a combination of one or more of an organic metal salt, a chloride metal salt, a bromide metal salt, a nitrate metal salt, an acetate metal salt, a sulfate metal salt or a metal cyanide complex; the precursor of the non-ferrous metal element contained in the iron-based organic metal framework nanozyme is a chloride metal salt; The non-ferrous metal elements contained in the iron-based organic metal framework nanozyme include one or more combinations of lithium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, gallium, germanium, rubidium, strontium, iridium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, tin, antimony, cerium, hafnium, iridium, platinum, gold or bismuth; the precursor of the non-ferrous metal element includes one or more combinations of organic metal salts, metal chlorides, metal bromides, metal nitrates, metal acetates, metal sulfates or metal cyanide complexes.

7. The synthesis method according to claim 1, characterized in that The liquid B in step (2) includes one or more combinations of phosphate buffer, physiological saline or ultrapure water.

8. The synthesis method according to claim 1, characterized in that The emulsifier or stabilizer in step (3) includes one or more combinations of polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, chitosan, gelatin, Tween 20, Tween 80, Span 60, Span 80, lecithin or gum.

9. A nanozyme with pathological environment response function prepared by the synthesis method described in any one of claims 1 to 8.

10. Use of the nanozyme with pathological environment response function according to claim 9 in the preparation of a drug for diagnosing, treating, alleviating and / or preventing arterial diseases, characterized in that: The arterial diseases include atherosclerotic diseases, arterial inflammatory diseases, hereditary or congenital arterial diseases, local or specific site arterial lesions, blood disorder-related diseases or arterial losses caused by external factors.

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