Inflammation-targeted diagnosis and treatment integrated nano system as well as preparation method and application thereof

Abstract

The invention provides an inflammation-targeted diagnosis and treatment integrated nano system as well as a preparation method and application thereof, and relates to the technical field of molecular imaging. The nano system comprises a magnetic resonance imaging nano enzyme core and a targeting and anti-inflammatory functional molecule, the core of the magnetic resonance imaging nano-enzyme is polydopamine particles doped with gadolinium element and arginine, the targeting and anti-inflammatory functional molecule is microRNA-146a, and the targeting and anti-inflammatory functional molecule is connected with a plurality of functional keys on the surface of the core of the nano-enzyme through a pi-pi bond, a hydrogen bond and an electrostatic interaction mode. After intravenous injection of the nano system, a scavenger receptor SR-A related to the inflammation degree on targeted macrophages is enriched in the atherosclerotic inflammatory plaque, the gadolinium element in the nano system enhances imaging of a magnetic resonance T1 signal, the inflammation process of the inflammatory plaque is accurately and visually displayed, and the treatment accuracy is clinically improved; active oxygen substances in a plaque area can be catalytically removed, local inflammation is relieved, lipid excretion is promoted, the intercellular effect of macrophages is recovered, plaque loads are reduced, plaque inflammation is relieved, and the plaque stability is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular imaging, and specifically relates to an inflammation-targeted integrated diagnosis and treatment nanosystem, a preparation method and application thereof, and in particular to an inflammation-targeted integrated diagnosis and treatment nanosystem for atherosclerotic inflammatory plaques, a preparation method and application thereof. Background Art

[0002] Atherosclerosis is the root cause of common cardiovascular and cerebrovascular diseases such as ischemic stroke and ischemic heart disease, which are the leading causes of death and disability worldwide. Lipid-lowering therapy is the standard treatment for atherosclerosis in clinical practice, but due to poor compliance and other reasons, more than 50% of patients do not fully benefit from it. Therefore, the clinical diagnosis and treatment of atherosclerotic cardiovascular and cerebrovascular diseases remains one of the most critical challenges in the medical field.

[0003] Essentially, atherosclerosis is a chronic inflammatory process and vascular wall remodeling caused by hyperlipidemia. As plaque inflammation progresses, oxidative stress and inflammation in the plaque promote each other, lipid deposition increases, macrophage efferocytosis is impaired, and cell apoptosis increases, resulting in a gradual increase in the area of ​​necrotic core, forming a "fragile plaque". Fragile plaques are prone to rupture, triggering platelet activation, leading to thrombus formation and arterial occlusion, triggering a series of life-threatening acute cardiovascular and cerebrovascular events. Therefore, regulating inflammation in atherosclerotic plaques and monitoring their inflammatory process are key strategies for the diagnosis and treatment of atherosclerotic plaques.

[0004] The Chinese invention patent with the announcement number CN117797273A provides a method for preparing a mesoporous polydopamine nanodiagnostic agent for MRI-visualized cancer treatment, using gadolinium ions (Gd 3+ ) and arginine (Arg) doped mesoporous polydopamine (MPDA) nanoparticles are used as carriers to load the anti-inflammatory drug meloxicam (MX) to prepare the nanoformulation MX@Arg-Gd-MPDA (MAGM), which is used for T1-T2 dual-mode MRI-guided cancer photothermal and anti-inflammatory treatment, improving the efficiency of cancer treatment and reducing the risk of recurrence. The Chinese invention patent with announcement number CN202010367277.3 provides an inflammation-targeted and microenvironment-responsive nanosystem and its preparation method and application. The nanosystem includes a targeting functional molecule, a nanocarrier, and a core crystal; the targeting functional molecule is a CD163 targeting binding short peptide, which is covalently linked to the polyethylene glycol on the surface of the nanoparticle; the core crystal is encapsulated in the nanocarrier in a biomineralized manner. After intravenous injection, the nanosystem targets CD163-positive macrophages enriched in epileptic foci and binds to H +It reacts with H2O2 to enhance the magnetic resonance T1 signal imaging of the epileptic focus, accurately and intuitively displaying the idiopathic / cryptogenic epileptic focus, which helps to improve the accuracy and safety of clinical treatment; it can also catalyze the removal of reactive oxygen species in the epileptic focus to reduce oxidative stress, generate oxygen to improve the local hypoxic microenvironment, and protect the neuronal vitality in the epileptic area.

[0005] Anti-inflammatory therapy is an important treatment strategy for atherosclerotic plaques. However, most of the anti-inflammatory drugs for atherosclerosis currently in clinical trials face the problem of ineffective efficacy, including no significant decrease in the incidence of anti-inflammatory factors and cardiovascular events. In addition, most anti-inflammatory drugs cause a series of adverse reactions due to the lack of targeted design. In the diagnosis of atherosclerotic plaques, routine clinical imaging methods fail to diagnose and monitor the inflammatory process of plaques. Therefore, there is an urgent need for a new targeted diagnosis and treatment integrated anti-inflammatory strategy to achieve the diagnosis and treatment of atherosclerosis. Summary of the invention

[0006] The present invention provides an integrated nanosystem for diagnosis and treatment of inflammation targeting, and a preparation method and application thereof. The gadolinium-containing nanozyme core of the present invention can be used as a magnetic resonance contrast agent to enrich in atherosclerotic plaques by targeting the inflammation-related scavenger receptor SR-A on macrophages through microRNA-146a. The magnetic resonance imaging nanozyme core enhances the magnetic resonance T1 signal for imaging, reflecting the degree of inflammation of the plaque. At the same time, the nanozyme core removes reactive oxygen in the plaque, and microRNA-146a alleviates the inflammatory response in the plaque, thereby reducing the plaque load and improving the stability of the plaque.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: an integrated nanosystem for diagnosis and treatment of targeted inflammation, the nanosystem comprising the following components in parts by weight:

[0008]

[0009] Furthermore, the nanozyme is polydopamine nanoparticles; the active groups of the polydopamine nanoparticles include carboxyl, phenolic hydroxyl and amino groups.

[0010] Furthermore, the arginine and gadolinium are doped with the nanozyme to form a nanozyme core with magnetic resonance imaging function. Arginine is doped in the nanozyme to improve enzyme activity and increase amino structure, and gadolinium is a magnetic resonance contrast agent.

[0011] Furthermore, the targeting and anti-inflammatory functional molecule is microRNA-146a, the sequence of which is 5'-UGAGAACUGAAUUCCAUGGGUU-3', which is bound to the surface of the nanozyme core through II-II bonds, hydrogen bonds and electrostatic interactions.

[0012] The present invention discloses a method for preparing the above-mentioned nanosystem, comprising the following steps:

[0013] A. dissolving gadolinium nitrate hexahydrate in ultrapure water to obtain a gadolinium nitrate solution and adjusting the solution to neutrality, dissolving dopamine hydrochloride in ultrapure water, and mixing the solution with the gadolinium nitrate solution to obtain a gadolinium-dopamine chelate solution;

[0014] B. Dissolve arginine in ultrapure water, add the gadolinium-dopamine chelate solution obtained in step A and stir to obtain the arginine-gadolinium-doped magnetic resonance imaging polydopamine nanozyme core AGPDA;

[0015] C. Add microRNA-146a to the AGPDA solution and mix and stir to obtain the nanosystem AGPDAR-146a.

[0016] Furthermore, the step A specifically comprises the following steps:

[0017] Gadolinium nitrate hexahydrate is dissolved in ultrapure water to obtain a gadolinium nitrate solution, and the gadolinium nitrate solution is adjusted to neutrality using ammonia water with a concentration of 25% to 28% under vigorous stirring at a speed of 100 to 200 rad / min; dopamine hydrochloride is uniformly dissolved in ultrapure water by blowing and ultrasonic dispersion to obtain a dopamine solution; the dopamine solution is slowly added to the gadolinium nitrate solution under vigorous stirring to obtain a gadolinium-dopamine chelate solution; wherein the molar ratio of the gadolinium nitrate solution to the dopamine solution is 1:3, and the stirring time is 1 hour.

[0018] Furthermore, the concentration of the gadolinium nitrate solution is 0.36-0.54 mg / mL, and the concentration of the dopamine solution is 0.45-0.67 mg / mL.

[0019] Furthermore, the step B specifically includes the following steps: taking arginine and dispersing and dissolving it in ultrapure water through ultrasonic treatment to obtain an arginine solution, the concentration of the arginine solution is 2.2-3.4 mg / mL, and the arginine solution is quickly injected into the gadolinium-dopamine chelate solution at a speed of 200-300 rad / min and continued to stir; wherein the mass ratio of arginine to dopamine is 1:1, the stirring time is 3 hours, and the arginine and gadolinium-doped magnetic resonance imaging polydopamine nanozyme core AGPDA is obtained.

[0020] Furthermore, the step C specifically comprises the following steps:

[0021] The AGPDA solution was centrifuged in an ultra-high speed centrifuge, and ultrapure water was used for repeated dispersion and centrifugal washing for a total of 3 times, the centrifugal speed was 13000 rpm / min, the time was 30 min, the targeting and anti-inflammatory functional molecule microRNA-146a solution was added to the AGPDA solution, and gently stirred at 60-100 rad / min and 4°C for 12 h to obtain the nanosystem AGPDAR-146a.

[0022] The present invention also discloses the application of the above-mentioned nanosystem, in which the inflammatory regulator microRNA-146a with inflammation-related receptor targeting is connected to the surface of a nanozyme carrier with magnetic resonance imaging function, and is used to prepare a preparation that uses inflammatory macrophages as inflammatory targets to achieve precise imaging of atherosclerotic plaques and targeted delivery of anti-inflammatory drugs.

[0023] Lipid-lowering therapy is the standard treatment for atherosclerosis in clinical practice, but more than 50% of patients do not fully benefit from it. Recent studies have shown that atherosclerosis is a chronic inflammatory process and vascular wall remodeling caused by hyperlipidemia, and is essentially a chronic inflammatory disease. Internalized low-density lipoprotein is oxidized in the arterial wall, inducing inflammation and immune responses. Locally infiltrated macrophages polarize to pro-inflammatory types, increase lipid endocytosis, and produce reactive oxygen species (ROS) to induce cell apoptosis. In the late stage of the inflammatory response, due to the impairment of the function of macrophages to phagocytize apoptotic cells (efferocytosis), apoptotic foam cells escape clearance, promote the expansion of the necrotic area in atherosclerotic plaques, lead to plaque instability and rupture, thrombus formation and shedding, and induce cardiovascular and cerebrovascular occlusion. Therefore, anti-inflammatory therapy is a key strategy for the root cause of atherosclerosis.

[0024] In the past decade, microRNA has played a vital role in gene therapy for inflammatory diseases. Among them, microRNA-146a is considered to be an inhibitor of several key proteins in the classic NF-κB inflammatory signaling pathway, including interferon regulatory factor 5 (IRF5). Recently, studies have found that IRF5 is not only related to macrophage polarization and lipid transport, but also impairs the efferocytosis function of macrophages. In addition, microRNA has been found to target the inflammation-related scavenger receptor SR-A on the macrophage membrane. Therefore, microRNA-146a may be an ideal therapeutic factor that can target inflammatory macrophages and regulate inflammation within the cell to play an anti-inflammatory role and restore the efferocytosis function of macrophages.

[0025] Nanocarriers are an advantageous strategy for targeted nanoprobe and drug delivery. Key issues limiting the application of microRNA, including inherent instability, suboptimal delivery efficiency, and rapid blood clearance, have been reported to be improved by nanosystem delivery. Bionanomaterials have shown particular potential and advantages in the diagnosis and treatment of atherosclerosis.

[0026] Polydopamine nanozymes have high biosafety and excellent reactive oxygen scavenging ability, inherent biocompatibility and biodegradability, and good biological application prospects. They have been widely used to construct therapeutic nanoparticles for various diseases. The rich functional groups of polydopamine also give it modifiability, cargo loading and metal chelation capabilities. Among them, the ability of polydopamine to scavenge free radicals can be further enhanced by introducing amino acids, such as arginine. Therefore, the construction of diagnostic and therapeutic nanosystems based on polydopamine nanozymes to deliver anti-inflammatory drugs to improve the oxidative stress and inflammatory microenvironment of atherosclerosis is a promising strategy.

[0027] Each of the seven orbits of the gadolinium atom has one electron, which is the largest number of unpaired electrons among rare earth elements. It can generate a large magnetic moment and is therefore used as a conventional clinical magnetic resonance imaging agent. Through the ability of polydopamine to chelate metals, it is possible to give nanoparticles magnetic resonance imaging capabilities, shorten the magnetic resonance T1 relaxation time, and amplify the magnetic resonance imaging signal.

[0028] Based on the above background, the inventors of the present application intend to construct polydopamine nanoparticles doped with arginine and gadolinium as the core of magnetic resonance imaging nanozymes to obtain better antioxidant activity and impart magnetic resonance imaging capabilities, and bind microRNA-146a to the surface of the nanozyme core to construct a nucleic acid-gadolinium-nanozyme diagnosis and treatment integrated nanosystem, targeting the SR-A receptor on the surface of inflammatory plaque macrophages, depositing in the inflammatory plaque, and using magnetic resonance to intuitively display the inflammatory process in the plaque. At the same time, ROS are cleared in the plaque and an anti-inflammatory effect is exerted, so as to reduce the plaque load, relieve plaque inflammation, and improve plaque stability, providing new technologies and methods for the integrated diagnosis and treatment of atherosclerotic plaques.

[0029] Compared with the prior art, the at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects:

[0030] (1) The present invention discloses an integrated diagnosis and treatment nanosystem for targeted inflammation, as well as a preparation method and application thereof, which is used for integrated diagnosis and treatment of magnetic resonance imaging and anti-inflammatory and antioxidant effects on atherosclerotic plaques. The nanosystem comprises a nanozyme core with magnetic resonance imaging function, and a targeting and anti-inflammatory functional molecule; the nanozyme core with magnetic resonance imaging function is a polydopamine particle doped with gadolinium and arginine; the targeting and anti-inflammatory functional molecule is microRNA-146a, which is connected to the functional bond on the surface of the nanozyme core by II-II bonds, hydrogen bonds and electrostatic interactions;

[0031] (2) After intravenous injection, the nanosystem of the present invention uses macrophages as inflammatory targets to facilitate accurate imaging and targeted drug delivery of inflammatory plaques. By targeting the SR-A receptors on macrophages and enriching them in inflammatory plaques, gadolinium enhances the magnetic resonance T1 signal for imaging, which helps to improve the accuracy of clinical diagnosis and treatment.

[0032] (3) The nanosystem of the present invention binds to the SR-A receptor on macrophages that is related to the degree of inflammation, and indirectly quantifies the number of SR-A receptors, which helps to evaluate the inflammatory process in the plaque, serves as one of the reference indicators for evaluating the vulnerability of the plaque, and helps to evaluate the changes in the degree of inflammation before and after treatment;

[0033] (4) The nanosystem of the present invention solves the problem that the prior art cannot evaluate the progression of plaque inflammation from the perspective of inflammation imaging, and can provide reference information for the formulation of treatment plans and evaluate the treatment effects;

[0034] (5) The nanosystem of the present invention can regulate the inflammatory response in macrophages through microRNA-146a, thereby achieving an anti-inflammatory effect, reducing lipid deposition, and restoring damaged efferocytosis to reduce plaque inflammation and improve plaque stability;

[0035] (6) The nanosystem of the present invention can catalyze the removal of reactive oxygen species within the plaque, reduce oxidative stress, reduce cell apoptosis, promote inflammation relief, and reduce the area of ​​the necrotic core, thereby reducing and stabilizing the plaque. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without paying creative work.

[0037] Figure 1 Schematic diagram of the preparation method of the nanosystem of the present invention;

[0038] Figure 2 Characterization diagram of the nanosystem prepared in Example 1;

[0039] Figure 3 This is the MR-T1 weighted imaging image of the nanosystem prepared in Example 1;

[0040] Figure 4 The UV-visible light spectrum and two active oxygen scavenging rate bar graphs obtained from the free radical (ABTS, DPPH and PTIO) scavenging experiment of the nanosystem prepared in Example 1;

[0041] Figure 5 In Example 2, an in vitro macrophage inflammation model was established in the application of an inflammation-targeted integrated diagnosis and treatment nanosystem, and the fluorescence images of the internalization of the nanosystem in Raw 264.7 cells were compared with and without lipopolysaccharide (LPS)-induced inflammation;

[0042] Figure 6 This is a schematic diagram of establishing an animal model of atherosclerosis in the application of an integrated nanosystem for diagnosis and treatment of inflammation targeted in Example 3, and analyzing the deposition of targeted nanoparticles in aortic plaques;

[0043] Figure 7 In Example 4, an inflammation model of Raw 264.7 cells was established in the application of an integrated nanosystem for diagnosis and treatment of inflammation, and the fluorescence image of nanoparticles removing intracellular reactive oxygen species was analyzed;

[0044] Figure 8 In Example 4, an inflammation model of Raw 264.7 cells was established in the application of an integrated nanosystem for diagnosis and treatment of inflammation, and the fluorescence image of the nanoparticles rescuing the cell mitochondrial membrane potential damaged by reactive oxygen species was analyzed;

[0045] Fig. 9 In Example 4, an inflammation model of Raw 264.7 cells was established in the application of an integrated nanosystem for diagnosis and treatment of inflammation, and the fluorescence image of the nanoparticles saving cell viability was analyzed;

[0046] Fig.10 In Example 4, an inflammation model of Raw 264.7 cells was established in the application of an integrated nanosystem for diagnosis and treatment of inflammation, and flow cytometry was used to analyze the effect of nanoparticles on reducing cell apoptosis;

[0047] Fig.11 In Example 4, an inflammation model of Raw 264.7 cells was established in the application of an integrated nanosystem for diagnosis and treatment of inflammation targeting, and the immunofluorescence image of the effect of nanoparticles on regulating the polarization of inflammatory cells was analyzed;

[0048] Fig.12For example 5, an atherosclerosis animal model was established in the application of an inflammation-targeted integrated diagnosis and treatment nanosystem, and MR-T1 weighted imaging images of nanoparticles deposited in aortic plaques;

[0049] Fig.13 This is a pathological section diagram of the effect of nanoparticles on reducing plaque load, relieving plaque inflammation, and improving plaque stability in establishing an animal model of atherosclerosis in the application of an inflammation-targeted integrated nanosystem for diagnosis and treatment in Example 6.

[0050] Fig.14 This is an immunofluorescence image of pathological sections showing the effects of nanoparticles on regulating macrophage polarization, increasing the expression of lipid transport proteins, and restoring macrophage efferocytosis in establishing an animal model of atherosclerosis in the application of an inflammation-targeted integrated nanosystem for diagnosis and treatment in Example 6. DETAILED DESCRIPTION

[0051] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0052] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0053] Example 1

[0054] An integrated nanosystem for diagnosis and treatment of inflammation targeting includes the following components in parts by weight: 100 parts of nanozymes; 100 parts of arginine; 80 parts of gadolinium; and 4 parts of targeting and anti-inflammatory functional molecules. The nanozymes are polydopamine nanoparticles, and the active groups of the polydopamine nanoparticles include carboxyl, phenolic hydroxyl and amino groups; arginine and gadolinium are doped with nanozymes to form a nanozyme core with magnetic resonance imaging function, arginine is doped in the nanozyme to improve enzyme activity and increase amino structure, and gadolinium is a magnetic resonance contrast agent; the targeting and anti-inflammatory functional molecules are microRNA-146a, and the sequence of microRNA-146a is 5'-UGAGA ACUGAAUUCCAUGGGUU-3', which is bound to the surface of the nanozyme core by II-II bonds, hydrogen bonds and electrostatic effects; the particle size of the nanosystem is 60nm.

[0055] like Figure 1 As shown; its preparation method comprises the following steps:

[0056] A. Preparation of gadolinium-dopamine chelate solution: dissolving gadolinium nitrate hexahydrate in ultrapure water to obtain a gadolinium nitrate solution with a concentration of 0.452 mg / mL; adjusting the gadolinium nitrate solution to neutrality with 25% to 28% ammonia water under vigorous stirring at a speed of 120 rad / min; uniformly dissolving dopamine hydrochloride in ultrapure water by blowing and ultrasonic dispersion to obtain a dopamine solution with a concentration of 0.56 mg / mL; slowly adding the dopamine solution to the gadolinium nitrate solution under vigorous stirring to obtain a gadolinium-dopamine chelate solution; wherein the molar ratio of the gadolinium nitrate solution to the dopamine solution is 1:3, and the stirring time is 1 hour.

[0057] B. Preparation of arginine-gadolinium-polydopamine (AGPDA) nanozyme core: Arginine was dispersed and dissolved in ultrapure water by ultrasonic treatment to obtain an arginine solution with a concentration of 2.8 mg / mL; the gadolinium-dopamine chelate solution was rapidly injected into the arginine solution under stirring at 240 rad / min and continued to stir for 3 hours; wherein, the mass ratio of arginine to dopamine was 1:1.

[0058] C. Preparation of AGPDAR-146a nanosystem: The AGPDA solution obtained in step B was centrifuged in an ultrahigh-speed centrifuge, and ultrapure water was used for repeated dispersion and centrifugal washing for a total of 3 times; the centrifugal speed was 13000 rpm / min, and the centrifugal time was 30 min; 4 portions of the targeting and anti-inflammatory functional molecule microRNA-146a solution were added to the AGPDA solution (the weight ratio of the AGPDA nanozyme core to microRNA-146a was 500:7), and the mixture was gently stirred at a speed of 80 rad / min and 4°C for 12 h to obtain the nanosystem AGPDAR-146a (i.e., AGPDAR-146a nanoparticles).

[0059] The morphology of the nanoparticles was observed and the diameter was measured using a transmission electron microscope, and the hydrated particle size of the nanoparticles was determined using a particle size and Zeta potential analyzer. Figure 2 As shown: A is a projection electron microscope image of AGPDAR-146a nanoparticles and a bar graph of the diameter measurement results; B is a statistical curve of the measured hydrated particle size. The AGPDAR-146a nanoparticles are spherical with a rough surface and uniform size. The average diameter is about 60nm and the hydrated particle size is about 210nm. MRI was used to analyze the magnetic sensitivity characteristics and T1 signal intensity of the nanoparticles. The results are as follows Figure 3 As shown: The r1 value of the nanoparticles is 6.07mM -1 s -1, which is 1.25 times that of the gadodiamide solution. The effect of the nanoparticles in scavenging DPPH, ABTS, PITO free radicals and ·OH was evaluated using an ultraviolet spectrophotometer, and the effect of the nanoparticles in scavenging ·O2- was evaluated using an enzyme marker. The results are as follows Figure 4 As shown: The nanoparticles exhibited efficient free radical and reactive oxygen scavenging activity in DPPH, ABTS, PITO free radical tests, ·OH and ·O2- scavenging experiments, and this activity was positively correlated with its concentration.

[0060] Example 2

[0061] Application of an integrated nanosystem for diagnosis and treatment of inflammation:

[0062] Raw 264.7 macrophages were stimulated with 100 ng / mL lipopolysaccharide (LPS) for 24 h to construct an inflammatory macrophage model with upregulated SR-A. The SR-A receptor ligand Fucoidan (250 μg / mL) was used to treat the above inflammatory macrophage model cells to form a competitive inhibition model. Each group of cells was co-incubated with Cy5.5-labeled AGPDA and AGPDAR-146a nanoparticles, and the intracellular uptake was observed by laser confocal microscopy, and the internalization amount was quantified by cell flow cytometry. The results are shown in Figure 2. Figure 5 As shown: A is a fluorescence image taken by confocal microscope; B is the result of flow cytometry analysis, LPS-induced inflammatory macrophages internalized more AGPDAR-146a nanoparticles than the control group, and internalized more AGPDAR-146a nanoparticles than AGPDA; after Fucoidan treatment, the internalization of AGPDAR-146a nanoparticles by inflammatory macrophages was significantly reduced. This shows that the targeted functional molecule microRNA-146a can be targetedly enriched in inflammatory cells, which is helpful for the targeted diagnosis and treatment of inflammatory lesions.

[0063] Example 3

[0064] Application of an integrated nanosystem for diagnosis and treatment of inflammation:

[0065] ApoE- / - (male) mice aged 4 to 5 weeks were fed a high-fat diet for 16 weeks (0.75% cholesterol was selected for the high-fat diet) to establish an atherosclerosis animal model. The mice were divided into two groups according to the non-targeted AGPDA and targeted AGPDAR-146a nanoparticles. The corresponding nanoparticle solution labeled with Cy5.5 was intravenously injected into the atherosclerosis model mice at a dose of 30 mg AGPDA / kg. The mice were killed after 3, 6, 12 and 24 hours, and the heart was perfused with 4% paraformaldehyde. The aorta was taken and photographed using a small animal fluorescence imaging system; the results are shown in Figure 6As shown in the figure, A is the in vitro fluorescence image of the aorta; B is the quantitative bar graph of the average fluorescence signal. The nanoparticles can be seen to be enriched in the aortic plaque 3 hours after injection, and the highest enrichment can be observed at 6 hours; at all test time points, the enrichment of AGPDAR-146a nanoparticles in the aorta is about twice that of AGPDA. This shows that AGPDAR-146a can be targeted and enriched in inflammatory plaques for further diagnosis and treatment.

[0066] Example 4

[0067] Application of an integrated nanosystem for diagnosis and treatment of inflammation:

[0068] The Raw 264.7 macrophage inflammation model was established by LPS induction to increase the level of intracellular reactive oxygen species. The cells were co-incubated with non-targeted antioxidant nanoparticles AGPDA and targeted anti-inflammatory antioxidant nanoparticles AGPDAR-146a, and the effects of nanoparticles on intracellular reactive oxygen species were investigated using the DCFH-DA fluorescent probe. The results are as follows: Figure 7 As shown: Compared with the control group, the fluorescence of reactive oxygen species in LPS-induced Raw 264.7 macrophages was significantly increased. After co-incubation with AGPDA and AGPDAR-146a nanoparticles, the intracellular fluorescence was significantly decreased, indicating that the nanoparticles can effectively remove intracellular reactive oxygen species.

[0069] LPS induced Raw 264.7 macrophages to construct an inflammatory cell model. Oxidative stress will destroy the stability of mitochondrial membrane potential in cells. Macrophages were co-incubated with nanoparticles. The mitochondrial membrane potential was indicated by JC-1 staining agent to investigate the effect of nanoparticles on the mitochondrial membrane potential in cells. The results are as follows: Figure 8 As shown: Macrophages in normal state showed significant red fluorescence, LPS-induced Raw 264.7 macrophages showed strong green fluorescence and reduced red / green fluorescence intensity ratio, and after co-incubation with AGPDA and AGPDAR-146a nanoparticles, the intracellular red fluorescence was restored, indicating that nanoparticles can rescue the changes in mitochondrial membrane potential caused by oxidative stress.

[0070] LPS induced Raw 264.7 macrophages to construct an inflammatory cell model. Oxidative stress will reduce cell viability. Macrophages were co-incubated with nanoparticles. Calcian AM / PI double staining reagent was used to show cell survival and investigate the effect of nanoparticles on cell viability. The results are as follows: Fig. 9 As shown: The LPS-induced Raw 264.7 macrophage viability was significantly reduced, and after co-incubation with AGPDA and AGPDAR-146a nanoparticles, the macrophage viability was significantly increased, indicating that nanoparticles can rescue the reduced cell viability caused by oxidative stress.

[0071] LPS induced Raw 264.7 macrophages to construct an inflammatory cell model. Oxidative stress will increase cell apoptosis. Macrophages were co-incubated with nanoparticles. The effect of nanoparticles on cell apoptosis was investigated using Annexin V-FITC apoptosis detection reagent and flow cytometry. The results are as follows: Fig.10 As shown: the apoptosis rate of Raw 264.7 macrophages induced by LPSd was significantly increased. After co-incubation with AGPDA and AGPDAR-146a nanoparticles, the apoptosis rate of macrophages was significantly decreased, indicating that nanoparticles can reduce cell apoptosis caused by oxidative stress.

[0072] LPS induced Raw 264.7 macrophages to construct an inflammatory cell model. Oxidative stress will cause macrophages to differentiate into pro-inflammatory types. Macrophages were co-incubated with nanoparticles. The effect of nanoparticles on macrophage polarization was investigated by immunostaining of the pro-inflammatory macrophage marker iNOS and the anti-inflammatory macrophage marker CD206. The results are as follows: Fig.11 As shown: After co-incubation with AGPDA and AGPDAR-146a nanoparticles, the iNOS marker of LPS-induced Raw 264.7 macrophages was significantly reduced, and the CD206 marker was significantly increased, indicating that the nanoparticles can reduce the differentiation of macrophages into pro-inflammatory types and promote their differentiation into anti-inflammatory types, thus playing an anti-inflammatory role.

[0073] Example 5

[0074] Application of an integrated nanosystem for diagnosis and treatment of inflammation:

[0075] A mouse model of atherosclerosis was established. AGPDAR-146a nanoparticles were administered via the tail vein at a dose of 30 mg AGPDA / kg. 3.0 MRI brain T1WI scans were performed 6 hours later. Fig.12 As shown: A is the T1-weighted imaging of the transverse, sagittal and coronal planes; B is the fat-suppressed T1-weighted imaging of the transverse planes at different levels. Compared with before injection (upper row), after nanoparticle injection (lower row), the T1 signal intensity of atherosclerotic plaques in the aortic arch and brachiocephalic trunk, bilateral common carotid arteries, and the proximal wall of bilateral subclavian arteries increased.

[0076] Example 6

[0077] Application of an integrated nanosystem for diagnosis and treatment of inflammation:

[0078] Atherosclerotic mice were divided into three groups, namely, control group, non-targeted antioxidant AGPDA group and targeted anti-inflammatory antioxidant AGPDAR-146a group. The corresponding nanoparticle solution or an equal amount of PBS was injected into the tail vein at a dose of 30 mg AGPDA / kg, once every 5 days, for a total of 8 injections. All mice were sacrificed 3 days after the last injection, and the aorta and heart were separated to make frozen sections for Oil Red O staining, HE staining, Masson staining, and immunohistochemical staining (including CD68, MMP9 and α-SMA) to evaluate the plaque area, necrotic core area, collagen fibers, macrophage infiltration, matrix protein deposition and smooth muscle cell infiltration. Macrophage polarization, the expression of lipid transport proteins, and the expression of receptors and ligands related to burial function were evaluated by immunofluorescence staining of different markers (including CD86, CD206, ABCA1, ABCG1, Itgβ3 and Mfge8).

[0079] like Fig.13 As shown, after nanoparticle treatment, the area of ​​atherosclerotic plaques was reduced, the proportion of necrotic core area was reduced, macrophage (CD68) infiltration was reduced, MMP-9 deposition was reduced, and collagen deposition was increased; and the therapeutic effect of the targeted AGPDAR-146a group was more obvious than that of the non-targeted AGPDA group; indicating that nanoparticles can reduce plaques, relieve plaque inflammation, and improve plaque stability.

[0080] like Fig.14 As shown in the data, after nanoparticle treatment, the pro-inflammatory macrophages (marker CD86) in atherosclerotic plaques were significantly reduced, the anti-inflammatory macrophages (marker CD206) were increased, the expression of lipid transport proteins ABCA1 and ABCG1 increased, and the expression of efferocytosis-related regulatory factors Itgβ3 and Mfge8 increased; and the therapeutic effect of the targeted AGPDAR-146a group was more obvious than that of the non-targeted AGPDA group; this shows that nanoparticles can regulate and relieve inflammation, promote lipid efflux, restore macrophage efferocytosis, relieve plaque inflammation and improve plaque stability.

[0081] Example 7

[0082] An integrated nanosystem for diagnosis and treatment of targeted inflammation includes the following components in parts by weight: 80 parts of nanozymes; 80 parts of arginine; 64 parts of gadolinium; and 3 parts of targeting and anti-inflammatory functional molecules. The nanozymes are polydopamine nanoparticles, and the active groups of the polydopamine nanoparticles include carboxyl, phenolic hydroxyl and amino groups; the arginine and gadolinium are doped with the nanozymes to form a nanozyme core with magnetic resonance imaging function, and arginine is doped in the nanozymes to improve enzyme activity and increase amino structure, and gadolinium is a magnetic resonance contrast agent; the targeting and anti-inflammatory functional molecules are microRNA-146a, and the sequence of microRNA-146a is 5'-UGAG AACUGAAUUCCAUGGGUU-3', which is bound to the surface of the nanozyme core by II-II bonds, hydrogen bonds and electrostatic effects; the particle size of the nanosystem is 49nm.

[0083] like Figure 1 As shown; its preparation method comprises the following steps:

[0084] A. Preparation of gadolinium-dopamine chelate solution: dissolving gadolinium nitrate hexahydrate in ultrapure water to obtain a gadolinium nitrate solution with a concentration of 0.36 mg / mL; adjusting the gadolinium nitrate solution to neutrality with 25% to 28% ammonia water under vigorous stirring at a speed of 100 rad / min; uniformly dissolving dopamine hydrochloride in ultrapure water by blowing and ultrasonic dispersion to obtain a dopamine solution with a concentration of 0.45 mg / mL; slowly adding the dopamine solution to the gadolinium nitrate solution under vigorous stirring to obtain a gadolinium-dopamine chelate solution; wherein the molar ratio of the gadolinium nitrate solution to the dopamine solution is 1:3, and the stirring time is 1 hour.

[0085] B. Preparation of arginine-gadolinium-polydopamine (AGPDA) nanozyme core: Arginine was dispersed and dissolved in ultrapure water by ultrasonic treatment to obtain an arginine solution with a concentration of 2.2 mg / mL; the gadolinium-dopamine chelate solution was rapidly injected into the arginine solution under stirring at a speed of 200 rad / min and continued to stir for 3 hours; wherein, the mass ratio of arginine to dopamine was 1:1.

[0086] C. Preparation of AGPDAR-146a nanosystem: The AGPDA solution obtained in step B was centrifuged in an ultra-high speed centrifuge, and ultrapure water was used for repeated dispersion and centrifugal washing for a total of 3 times; the centrifugal speed was 13000 rpm / min, and the centrifugal time was 30 min; 3 portions of aqueous solutions of the targeting and anti-inflammatory functional molecule microRNA-146a were added to the AGPDA solution, and gently stirred at a speed of 80 rad / min and 4°C for 12 h to obtain the nanosystem AGPDAR-146a (i.e., AGPDAR-146a nanoparticles).

[0087] After intravenous injection, the nanosystem targets inflammatory macrophages to facilitate accurate imaging and targeted drug delivery of atherosclerotic plaques. MicroRNA-146a targets the SR-A receptor on the surface of inflammatory macrophages and is enriched in inflammatory macrophages in atherosclerotic plaques. Gadolinium enhances the magnetic resonance T1 signal of the plaque for imaging, which can intuitively display the macrophage infiltration and inflammation level of the plaque, thereby improving the accuracy of diagnosis and treatment. Arginine-doped polydopamine can catalyze the removal of reactive oxygen species in the plaque to reduce oxidative stress response, reduce cell apoptosis, save cell vitality, and help relieve local inflammation. This establishes a microenvironmental study based on arginine-doped polydopamine nanozymes in improving plaque oxidative stress. Application in the environment; microRNA-146a regulates the NF-κB inflammatory pathway, promotes the differentiation of macrophages into anti-inflammatory macrophages, regulates the secretion of inflammatory factors, promotes lipid excretion, restores the macrophage efferocytosis, alleviates the local inflammatory environment of the plaque, reduces lipid deposition and the area of ​​necrotic core, helps to reduce the plaque load and improve the stability of the plaque; from the perspective of inflammation imaging, it solves the problem that the existing technology cannot judge the progress of plaque inflammation, and can intuitively display the degree of plaque inflammation; the targeted binding of magnetic resonance imaging nanoparticles to the inflammatory-related SR-A receptors indirectly quantifies the number of SR-A receptors and then reflects the degree of inflammation, which helps to judge the inflammatory process of the plaque and help to infer the progression trend and vulnerability of the plaque.

[0088] Example 8

[0089] An integrated nanosystem for diagnosis and treatment of inflammation targeting includes the following components in parts by weight: 120 parts of nanozymes; 120 parts of arginine; 96 parts of gadolinium; and 5 parts of targeting and anti-inflammatory functional molecules. The nanozymes are polydopamine nanoparticles, and the active groups of the polydopamine nanoparticles include carboxyl, phenolic hydroxyl and amino groups; the arginine and gadolinium are doped with the nanozymes to form a nanozyme core with magnetic resonance imaging function, and arginine is doped in the nanozymes to improve enzyme activity and increase amino structure, and gadolinium is a magnetic resonance contrast agent; the targeting and anti-inflammatory functional molecules are microRNA-146a, and the sequence of microRNA-146a is 5'-UGAGAACUGAAUUCCAUGGGUU-3', which is bound to the surface of the nanozyme core by II-II bonds, hydrogen bonds and electrostatic effects; the particle size of the nanosystem is 70nm.

[0090] like Figure 1 As shown; its preparation method comprises the following steps:

[0091] A. Preparation of gadolinium-dopamine chelate solution: dissolving gadolinium nitrate hexahydrate in ultrapure water to obtain a gadolinium nitrate aqueous solution with a concentration of 0.54 mg / mL; adjusting the gadolinium nitrate solution to neutrality with 25% to 28% ammonia water under vigorous stirring at a speed of 200 rad / min; uniformly dissolving dopamine hydrochloride in ultrapure water by blowing and ultrasonic dispersion to obtain a dopamine solution with a concentration of 0.67 mg / mL; slowly adding the dopamine solution to the gadolinium nitrate solution under vigorous stirring to obtain a gadolinium-dopamine chelate solution; wherein the molar ratio of the gadolinium nitrate solution to the dopamine solution is 1:3, and the stirring time is 1 hour.

[0092] B. Preparation of arginine-gadolinium-polydopamine (AGPDA) nanozyme core: Arginine was dispersed and dissolved in ultrapure water by ultrasonic treatment to obtain an arginine solution with a concentration of 3.4 mg / mL; gadolinium-dopamine chelate solution was rapidly injected into the arginine solution under stirring at 300 rad / min and continued to stir for 3 h; wherein, the mass ratio of arginine to dopamine was 1:1.

[0093] C. Preparation of AGPDAR-146a nanosystem: The AGPDA solution obtained in step B was centrifuged in an ultra-high speed centrifuge, and ultrapure water was used for repeated dispersion and centrifugal washing for a total of 3 times; the centrifugal speed was 13000 rpm / min, and the centrifugal time was 30 min; 5 portions of aqueous solution of the targeting and anti-inflammatory functional molecule microRNA-146a were added to the AGPDA solution, and gently stirred at a speed of 100 rad / min and 4°C for 12 h to obtain the nanosystem AGPDAR-146a (i.e., AGPDAR-146a nanoparticles).

[0094] After intravenous injection, the nanosystem targets inflammatory macrophages to facilitate accurate imaging and targeted drug delivery of atherosclerotic plaques. MicroRNA-146a targets the SR-A receptor on the surface of inflammatory macrophages and is enriched in inflammatory macrophages in atherosclerotic plaques. Gadolinium enhances the magnetic resonance T1 signal of the plaque for imaging, which can intuitively display the macrophage infiltration and inflammation level of the plaque, thereby improving the accuracy of diagnosis and treatment. Arginine-doped polydopamine can catalyze the removal of reactive oxygen species in the plaque to reduce oxidative stress response, reduce cell apoptosis, save cell vitality, and help relieve local inflammation. This establishes a microenvironmental study based on arginine-doped polydopamine nanozymes in improving plaque oxidative stress. Application in the environment; microRNA-146a regulates the NF-κB inflammatory pathway, promotes the differentiation of macrophages into anti-inflammatory macrophages, regulates the secretion of inflammatory factors, promotes lipid excretion, restores the macrophage efferocytosis, alleviates the local inflammatory environment of the plaque, reduces lipid deposition and the area of ​​necrotic core, helps to reduce the plaque load and improve the stability of the plaque; from the perspective of inflammation imaging, it solves the problem that the existing technology cannot judge the progress of plaque inflammation, and can intuitively display the degree of plaque inflammation; the targeted binding of magnetic resonance imaging nanoparticles to the inflammatory-related SR-A receptors indirectly quantifies the number of SR-A receptors and then reflects the degree of inflammation, which helps to judge the inflammatory process of the plaque and help to infer the progression trend and vulnerability of the plaque.

[0095] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. An integrated nanosystem for diagnosis and treatment of inflammation, characterized in that: The nanosystem comprises the following components in parts by weight: Nanozyme 80-120 parts; Arginine 80-120 parts; Gadolinium 64-96 parts; 3 to 5 targeting and anti-inflammatory functional molecules.

2. The nanosystem according to claim 1, characterized in that The nanozyme is polydopamine nanoparticles; the active groups of the polydopamine nanoparticles include carboxyl, phenolic hydroxyl and amino groups.

3. The nanosystem according to claim 1, characterized in that The arginine and gadolinium are doped with the nanozyme to form a nanozyme core with magnetic resonance imaging function. Arginine is doped in the nanozyme to improve enzyme activity and increase amino structure, and gadolinium is a magnetic resonance contrast agent.

4. The nanosystem according to claim 1, characterized in that The targeting and anti-inflammatory functional molecule is microRNA-146a, and the sequence of microRNA-146a is 5'-UGAGAACUGAAUUCCAUGGGUU-3', which is bound to the surface of the nanozyme core through II-II bonds, hydrogen bonds and electrostatic interactions.

5. A method for preparing a nanosystem according to any one of claims 1 to 4, characterized in that: The steps include: A. dissolving gadolinium nitrate hexahydrate in ultrapure water to obtain a gadolinium nitrate solution and adjusting the solution to neutrality, dissolving dopamine hydrochloride in ultrapure water, and mixing the solution with the gadolinium nitrate solution to obtain a gadolinium-dopamine chelate solution; B. Dissolve arginine in ultrapure water, add the gadolinium-dopamine chelate solution obtained in step A and stir to obtain the arginine-gadolinium-doped magnetic resonance imaging polydopamine nanozyme core AGPDA; C. Add microRNA-146a to the AGPDA solution and mix and stir to obtain the nanosystem, i.e., AGPDAR-146a nanoparticles.

6. The preparation method according to claim 5, characterized in that: The step A specifically comprises the following steps: Gadolinium nitrate hexahydrate is dissolved in ultrapure water to obtain a gadolinium nitrate solution, and the gadolinium nitrate solution is adjusted to neutrality using ammonia water with a concentration of 25% to 28% under vigorous stirring at a speed of 100 to 200 rad / min; dopamine hydrochloride is uniformly dissolved in ultrapure water by blowing and ultrasonic dispersion to obtain a dopamine solution; the dopamine solution is slowly added to the gadolinium nitrate solution under vigorous stirring to obtain a gadolinium-dopamine chelate solution; wherein the molar ratio of the gadolinium nitrate solution to the dopamine solution is 1:3, and the stirring time is 1 hour.

7. The preparation method according to claim 6, characterized in that: The concentration of the gadolinium nitrate solution is 0.36-0.54 mg / mL, and the concentration of the dopamine solution is 0.45-0.67 mg / mL.

8. The preparation method according to claim 5, characterized in that: The step B specifically comprises the following steps: Arginine is dispersed and dissolved in ultrapure water through ultrasonic treatment to obtain an arginine solution, wherein the concentration of the arginine solution is 2.2-3.4 mg / mL, and the arginine solution is quickly injected into the gadolinium-dopamine chelate solution at a speed of 200-300 rad / min and continued to be stirred; wherein the mass ratio of arginine to dopamine is 1:1, and the stirring time is 3 hours, to obtain the arginine and gadolinium-doped magnetic resonance imaging polydopamine nanozyme core AGPDA.

9. The preparation method according to claim 5, characterized in that: The step C specifically comprises the following steps: The AGPDA solution was centrifuged in an ultra-high speed centrifuge, and ultrapure water was used for repeated dispersion and centrifugal washing for a total of 3 times, the centrifugal speed was 13000 rpm / min, the time was 30 min, the targeting and anti-inflammatory functional molecule microRNA-146a solution was added to the AGPDA solution, and gently stirred at 60-100 rad / min and 4°C for 12 h to obtain the nanosystem AGPDAR-146a.

10. A use of the nanosystem according to any one of claims 1 to 4, characterized in that: The inflammatory regulator microRNA-146a with inflammation-related receptor targeting is connected to the surface of a nanozyme carrier with magnetic resonance imaging function, and is used to prepare a preparation that uses inflammatory macrophages as inflammatory targets to achieve precise imaging of atherosclerotic plaques and targeted delivery of anti-inflammatory drugs.

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