Renal tubule epithelial cell membrane wrapped nano-particles and preparation method and application thereof

Abstract

The invention particularly discloses nanoparticles wrapped by renal tubular epithelial cell membranes as well as a preparation method and application of the nanoparticles, and relates to the technical field of biological medicines. The invention provides nano-particles (FNMs) wrapped by a renal tubular epithelial cell membrane. The nano-particles comprise the renal tubular cell membrane, a contrast agent and a dye. The FNMs can accurately target carotid artery plaques in an APOE <- / -> mouse AS model to realize fluorescence / magnetic resonance bimodal imaging, and lesion information of the plaques can be comprehensively reflected from multiple angles. The defects of low bioavailability of traditional medicine treatment and high trauma risk of traditional surgical treatment are overcome. The PDT effect can be generated under the illumination effect when the concentration is 40 mu g / mL, and the treatment effect of relieving the AS plaque lesion is achieved. No matter in a cell experiment or an animal experiment, the FNMs show high biological safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to nanoparticles wrapped by renal tubular epithelial cell membranes, and a preparation method and application thereof. Background Art

[0002] A 2021 report in the Lancet Neurology stated that stroke is the third leading cause of death worldwide, second only to ischemic heart disease and COVID-19. Stroke is characterized by high morbidity, high disability, high mortality, high recurrence rate, and high economic burden. It is not only one of the leading causes of death worldwide, but also a significant factor affecting the quality of human life, resulting in a heavy disease burden worldwide. Among all new stroke cases, ischemic stroke (IS) accounts for the largest proportion, at 65.3%. AS is an immune-mediated chronic and progressive inflammatory pathological process characterized by the formation of subendothelial atherosclerotic plaques. Vulnerable arterial plaques are a hallmark of AS's high-risk state, characterized by rapid progression of vascular stenosis and plaque rupture leading to thrombosis. Acute ischemic stroke (AIS) caused by vulnerable plaque rupture and subsequent thrombosis is associated with expansion of the lipid-rich necrotic core, inflammatory cell infiltration, thinning of the fibrous cap, and neovascularization. However, there is currently a lack of effective, prospective methods for identifying and accurately assessing plaque vulnerability and for targeted intervention. This misses the golden opportunity for early treatment of vulnerable carotid plaques, ultimately leading to the development of AIS. Therefore, developing effective methods to accurately assess the vulnerability of carotid atherosclerotic plaques for risk stratification and early intervention is urgent to reduce major adverse cardiovascular and cerebrovascular events.

[0003] Macrophages are the most abundant leukocyte subset in AS lesions and play a key role in the formation of vulnerable carotid artery plaques. Under different environmental and inflammatory stimulation, they can form two subtypes: pro-inflammatory and anti-inflammatory. These subtypes can also transform into each other. Transformation into a pro-inflammatory type promotes plaque inflammation and destabilization, while transformation into an anti-inflammatory type promotes plaque repair and stability. The central role of macrophages in the development of AS makes them an attractive therapeutic target for preventing and stabilizing AS progression. β2M is a non-glycosylated protein composed of 119 amino acid residues, with a secreted form consisting of 99 amino acids and a molecular weight of 11.8 kDa. It is synthesized in all nucleated cells and non-covalently binds to the heavy chain of the major histocompatibility complex class I antigen on the cell surface. It is a key structural protein regulating host immune recognition and immunoglobulin transport, and is a core regulatory protein in the immune response. Current research has found that platelets are the primary source of plasma β2M. β2M mediates the proinflammatory transformation of monocytes, independent of its major histocompatibility complex I (MHC I) transport function and its function as a chaperone protein. Studies have shown that β2M and transforming growth factor-β (TGF-β) have opposing effects on monocytes by inducing and inhibiting inflammation, respectively. They both bind and signal through the same transforming growth factor β receptor (TGFβR). Numerous studies have shown that TGF-β can stabilize vulnerable carotid artery plaques by promoting the anti-inflammatory transformation of macrophages.

[0004] Currently, clinical imaging techniques for AS primarily include intravascular ultrasound, magnetic resonance imaging (MRI), computed tomography (CT), and digital subtraction angiography (DSA). However, these imaging techniques primarily focus on the morphological characteristics of carotid plaques, such as plaque morphology and the degree of vascular stenosis they cause, and their predictive value for plaque stability is limited. In particular, there is a lack of effective means to characterize the core mechanism of plaque progression: the inflammatory state of the carotid intimal plaque. Regarding intervention, the main treatments for AS currently include lipid-lowering drugs, stent implantation, and endarterectomy surgery. However, traditional oral medications are associated with significant adverse reactions, low bioavailability, and high surgical risk. Therefore, effective assessment and precise intervention of the level and stability of carotid intimal plaques based on the pathological characteristics of vulnerable plaques, as well as the development of clinically needed, highly specific, early-stage, and precise assessment and treatment agents, are crucial for the early prevention and intervention of AIS. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In view of this, one of the main purposes of the present invention is to provide a renal tubular epithelial cell membrane-coated nanoparticles (FNMs). The nanoparticles provided by the present invention are composed of indocyanine green (ICG) and gadolinium ( 157 Nanoparticle cores (NMs) constructed by coupling FNMs with Gd) are coated with renal tubular epithelial cell membranes and can precisely target proinflammatory macrophages in carotid artery plaques through β2-microglobulin (β2M). FNMs possess dual-modality fluorescence / nuclear magnetic resonance imaging capabilities, enabling comprehensive molecular characterization of plaques and quantitative analysis of their inflammatory state. Furthermore, FNMs generate large amounts of reactive oxygen species (ROS) under 808nm laser irradiation, inducing apoptosis in proinflammatory macrophages, thereby stabilizing plaques and alleviating atherosclerotic lesions. These FNMs possess high biosafety and excellent therapeutic efficacy, making them suitable for the early and precise assessment and intervention of carotid atherosclerotic plaques.

[0007] (2) Technical solution

[0008] In order to achieve the above object, the present invention provides a nanoparticle coated with a renal tubular epithelial cell membrane, wherein the nanoparticle comprises a renal tubular epithelial cell membrane, a contrast agent and a dye.

[0009] In one embodiment, the contrast agent includes one or a combination of a gadolinium (Gd)-based contrast agent, a dysprosium (Dy)-based contrast agent, and a tantalum oxide-based contrast agent.

[0010] In one embodiment, the contrast agent is a gadolinium (Gd) based contrast agent.

[0011] In one embodiment, the gadolinium (Gd)-based contrast agent includes one or a combination of gadolinium chloride, gadoveline, gadopentetate dimeglumine, gadonate meglumine, gadoxetate disodium, gadoteridol, and gadobutrol.

[0012] In one embodiment, the gadolinium (Gd)-based contrast agent is gadolinium chloride.

[0013] In one embodiment, the dye comprises one or a combination of indocyanine green (ICG), IR-780, IR-783, MHI-148 and DZ-1.

[0014] In one embodiment, the dye is indocyanine green (ICG).

[0015] In one embodiment, the nanoparticles further comprise metformin.

[0016] In one embodiment, the renal tubular epithelial cell membrane comprises renal tubular epithelial cell membrane vesicles.

[0017] In one embodiment, the molar ratio of gadolinium chloride:indocyanine green:metformin is 1:(1-10):(1-5).

[0018] In one embodiment, the molar ratio of gadolinium chloride:indocyanine green:metformin is 1:3:(1-2).

[0019] In one embodiment, the molar ratio of gadolinium chloride:indocyanine green:metformin is 1:3:1.5.

[0020] In another aspect, the present invention provides a method for preparing the above-mentioned nanoparticles, comprising:

[0021] S1: preparing nanoparticle cores by mixing gadolinium chloride solution, indocyanine green solution, and metformin solution;

[0022] S2: The nanoparticle core is mixed with the renal tubular epithelial cell membrane to prepare the nanoparticle.

[0023] In one embodiment, the gadolinium chloride solution is prepared by dissolving 0.0023 g of ICG in 9 mL of pure water to prepare an ICG aqueous solution.

[0024] In one embodiment, the preparation method of the metformin solution is: dissolving 0.0025 g of metformin in 5 mL of ethanol to prepare the metformin solution.

[0025] In one embodiment, the gadolinium chloride solution is prepared by dissolving 0.0037 g of gadolinium chloride (GdCl 3 ·6H 2 O) in 10 mL of pure water to prepare the gadolinium chloride solution.

[0026] In one embodiment, the S1 further comprises: adding 0.5 mL of metformin solution to the ICG solution, mixing, and then adding 1 mL of gadolinium chloride solution to obtain a mixed solution.

[0027] In one embodiment, the S1 further comprises: freeze-drying the mixed solution to obtain a dark green solid powder, which is the nanoparticle core (NMs).

[0028] In one embodiment, the S2 further comprises: mixing the NMs solution with renal tubular epithelial cell membrane vesicles to prepare nanoparticles.

[0029] In one embodiment, the volume ratio of the NMs solution to the renal tubular epithelial cell membrane vesicles is 1:(0.1-10).

[0030] In one embodiment, the volume ratio of the NMs solution to the renal tubular epithelial cell membrane vesicles is 1:1.

[0031] In one embodiment, the NMs solution is prepared by dissolving NMs in H2O to prepare a NMs solution with a final concentration of 200 μg / mL.

[0032] In one embodiment, the preparation method specifically comprises:

[0033] S1: Dissolve 0.0023 g ICG in 9 mL pure water to prepare an ICG aqueous solution; dissolve 0.0025 g metformin in 5 mL ethanol to prepare a metformin solution; dissolve 0.0037 g gadolinium chloride (GdCl3·6H2O) in 10 mL pure water to prepare a gadolinium chloride solution;

[0034] 0.5 mL of metformin solution was added to the ICG solution, and after mixing, 1 mL of gadolinium chloride solution was added to obtain a mixed solution;

[0035] The mixed solution is freeze-dried to obtain a dark green solid powder, which is the core of the nanoparticles.

[0036] S2: NMs were dissolved in H2O to prepare a NMs solution with a final concentration of 200 μg / mL;

[0037] The NMs solution was mixed with renal tubular epithelial cell membrane vesicles in a volume ratio of 1:1 to prepare nanoparticles.

[0038] In another aspect, the present invention further provides nanoparticles obtained by the above preparation method.

[0039] In another aspect, the present invention also provides the use of the above-mentioned nanoparticles in preparing an atherosclerotic plaque diagnostic agent.

[0040] In another aspect, the present invention further provides a pharmaceutical composition comprising:

[0041] (1) a therapeutically effective amount of the above-mentioned nanoparticles;

[0042] (2) A pharmaceutically or immunologically acceptable carrier or excipient.

[0043] In another aspect, the present invention further provides a pharmaceutical preparation comprising the above-mentioned pharmaceutical composition.

[0044] In another aspect, the present invention further provides a product comprising the above-mentioned pharmaceutical preparation.

[0045] In another aspect, the present invention further provides the use of the above-mentioned nanoparticles, pharmaceutical compositions or pharmaceutical preparations in the preparation of drugs for preventing and / or treating atherosclerotic plaques.

[0046] Beneficial effects

[0047] The present invention provides a nanoparticle coated with renal tubular epithelial cell membrane. Compared with the existing technology, it has the following advantages:

[0048] 1. FNMs in APOE - / - In the mouse AS model, fluorescence / magnetic resonance dual-modal imaging can be achieved by precisely targeting carotid artery plaques, comprehensively reflecting plaque pathological information from multiple angles.

[0049] 2. FNMs overcome the low bioavailability of traditional drug treatments and the high risk of trauma from traditional surgical treatments. A concentration of 40 μg / mL can produce a PDT effect under light, achieving a therapeutic effect that reduces AS plaque lesions.

[0050] 3. FNMs have demonstrated high biosafety in both cell and animal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0052] Figure 1 β2M exacerbates atherosclerosis; (A) The lesion area on the surface of the aorta of the four groups of mice was determined by the macroscopic Oil Red O staining method; (B) The statistical histogram quantitatively calculated the positive area of ​​the aorta of the four groups of mice.

[0053] Figure 2 β2M promotes the transformation of macrophages into pro-inflammatory types; (A) Immunofluorescence staining results, blue is DAPI, marking the cell nucleus, green is CD68, specifically marking macrophages, red is Arg-1, specifically marking anti-inflammatory macrophages, pink is INOS, specifically marking pro-inflammatory macrophages; (B) Quantitative analysis of INO S-positive areas in macrophages detected by immunofluorescence staining; (C) Quantitative analysis of Arg-1-positive areas in macrophages detected by immunofluorescence staining.

[0054] Figure 3Characterization of NMs and FNMs; (A) TEM image of NMs; (B) TEM image of FNMs; (C) Particle size distribution of NMs and FNMs; (D) Fourier transform infrared spectrum of NMs; (E) UV absorption spectrum of FNMs; (F) Changes in the singlet oxygen generation curve per minute of NMs under 808 nm laser irradiation.

[0055] Figure 4 In vitro fluorescence / magnetic resonance imaging performance of NMs; (A) In vitro fluorescence imaging performance of NMs; (B) In vitro magnetic resonance imaging performance of NMs.

[0056] Figure 5 The in vitro photodynamic effect of FNMs; (A) Cellular reactive oxygen species production as shown by fluorescence microscopy (scale bar = 100 μm); (B) Effect of FNMs on the survival rate of pro-inflammatory macrophages under 808 nm laser irradiation; (C) Cell live-dead staining results (scale bar = 100 μm).

[0057] Figure 6 In vivo fluorescence / magnetic resonance imaging performance of FNMs; (A) Fluorescence imaging of carotid artery plaques in mice before and 24 hours after FNMs injection; (B) Magnetic resonance imaging of carotid artery plaques in mice before and 24 hours after FNMs injection.

[0058] Figure 7 Figure 3 is the in vivo PDT effect of FNMs; (A) Oil Red O staining of blood vessels in mice after treatment; (B) Quantitative analysis of Oil Red O staining (n=3).

[0059] Figure 8 Biosafety of FNMs; (A) HE staining of major organs; (B) Effect of FNMs on the survival rate of pro-inflammatory macrophages; (C) Hemolysis rate of FNMs. DETAILED DESCRIPTION

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0061] Terms and Definitions

[0062] As used herein, the term "pharmaceutical composition" refers to a composition comprising nanoparticles formulated together with one or more pharmaceutically acceptable carriers.

[0063] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0064] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is a vehicle for administering a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers that are not essential to the active ingredient and are not unduly toxic upon administration. Suitable carriers are well known to those of ordinary skill in the art, and a comprehensive discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0065] In the composition, pharmaceutically acceptable carriers include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch or talc, or liquid carriers, such as water, fatty oils or liquid paraffin. Other examples of the carrier include culture media, such as DMEM or RPMI; and low-temperature storage media, comprising components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates and ion concentrations that balance the intracellular state at low temperatures; and mixtures of organic solvents and water.

[0066] The pharmaceutical composition disclosed in the present invention can be in the form of granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets, liquid solutions, nasal drops, sprays, or metered sprays.

[0067] The pharmaceutical compositions of the present invention can be administered using any known method.The term "administering" or "administering" a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those skilled in the art.

[0068] For example, the compound or agent can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). The compound or agent can also be suitably introduced by a rechargeable or biodegradable polymeric device or other device (e.g., a patch and pump or formulation) that provides extended, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0069] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and effective dosage levels include the type and severity of the subject, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors well known in the medical art.

[0070] As used herein, the term "treating" a condition or patient means taking steps to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially improving or alleviating the clinical manifestations of a condition, substantially preventing the clinical manifestations of a disease, condition, or disorder, and avoiding harmful or unpleasant symptoms. Treating also refers to accomplishing one or more of the following: (a) lessening the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and / or (e) limiting the recurrence of symptoms in patients who previously had no symptoms of the condition.

[0071] The terms "prevent" and "prevent" refer to reducing the likelihood of onset or recurrence of a disease, disorder, condition, or associated symptoms.

[0072] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0073] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0074] Example 1

[0075] Preparation of FNMs:

[0076] (1) Weigh 0.0023 g of ICG using an electronic balance and dissolve it in 9 mL of pure water. Use an ultrasonic oscillator to fully dissolve it and prepare an ICG aqueous solution.

[0077] (2) Weigh 0.0025 g of metformin and dissolve it in 5 mL of ethanol to prepare a metformin solution. Dissolve 0.0037 g of gadolinium chloride (GdCl3·6H2O) in 10 mL of pure water to prepare a gadolinium solution. After sufficient dissolution, take 0.5 mL of metformin solution and slowly add it dropwise to the ICG solution while stirring. Take 1 mL of gadolinium solution and add it to the mixture of the two solutions and stir at room temperature.

[0078] (3) After 24 hours, the mixture was placed on a rotary evaporator and subjected to rotary evaporation for 20 minutes. The mixture was then freeze-dried to obtain a dark green solid powder, which was NMs.

[0079] (4) Culture of renal tubular epithelial cells (TCMK-1 cells) and extraction of cell membranes: TCMK-1 cells were cultured in DMEM medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2. The cells were passaged when they grew to 80%-90%.

[0080] Extraction of TCMK-1 cell membranes: When the cell density reaches 90%, completely remove the old medium and wash the cells three times with 2 mL of phosphate buffered saline (PBS). Then, add 1 mL of PBS to the culture flask and scrape the cells with a cell scraper. Transfer the resulting cell suspension to a 2 mL centrifuge tube and centrifuge at 1500 rpm for 10 min at 4°C. Discard the supernatant and resuspend the cells in 10 μL / mL phenylmethanesulfonyl fluoride (PMSF) in the centrifuge tube containing the cell pellet. Incubate on ice for 15 min. The cell suspension is then freeze-thawed four times in liquid nitrogen (freezing at -200°C for 3 min and thawing at room temperature at 25°C for 10 min). Centrifuge at 700 g for 10 min at 4°C. Remove the supernatant and transfer it to a new centrifuge tube. Centrifuge again at 14,000 g for 30 min at 4°C and discard the supernatant. The pellet is the TCMK-1 cell membrane. The cell membranes were resuspended in ultrapure water and stored in a -80°C refrigerator.

[0081] Preparation of TCMK-1 membrane vesicles: TCMK-1 cell membrane fragments were repeatedly extruded through a 200 nm polycarbonate porous membrane using a nanoliposome extruder 14 times to obtain TCM K-1 cell membrane vesicles.

[0082] (5) Preparation of FNMs: NMs were dissolved in H2O (concentration of 200 μg / mL), and the NM solution was mixed with TCMK-1 cell membrane vesicles at a volume ratio of 1:1. The mixture was then sonicated in a water bath at 42 kHz and 100 W for 8 min. The suspension of NMs and TCMK-1 cell membranes was then transferred to a liposome extruder and co-extruded 14 times with a pore size of 200 nm. The product was collected as FNMs.

[0083] Example 2

[0084] β2M induces aggravation of atherosclerosis:

[0085] (1) Grouping and intervention (n=6): control group (C57BL / 6J male mice, fed with normal diet for 12 weeks), WD Ctrl group (ApoE - / - Male mice, high-fat diet for 10 weeks), WD TGF-β1 group (ApoE - / - Male mice, high-fat diet for 10 weeks), WDβ2M group (ApoE - / - Male mice were fed a high-fat diet for 10 weeks and the above four groups were intraperitoneally injected with normal saline, normal saline, TGF-β1, and β2M at a dose of 50 μg / kg, once a day for 2 consecutive weeks.

[0086] (2) Oil Red O staining of the aorta: Remove the aorta, remove the fat and adhesion tissue on the outer wall of the aorta, and fix it in 4% paraformaldehyde for 24 hours. Weigh 0.1g of Oil Red O powder and add 200mL of isopropanol. Place it in a heating mantle and heat until the oil red is fully dissolved. After dissolution, take it out and filter it. The saturated solution of Oil Red O is obtained after filtration. Mix the saturated solution with double distilled water in a ratio of 3:2, let it stand for 10 minutes, and then filter it to obtain the Oil Red O working solution. Place the aorta in a 6cm cell dish and add the Oil Red working solution to completely immerse the aorta. Then place the cell dish in a 37℃ water bath and incubate for 30 minutes. Discard the Oil Red dye solution and add 60% isopropanol to wash away the excess dye solution. When part of the aorta appears translucent white, remove the isopropanol and wash it with PBS three times. Observe and photograph it under a stereo microscope.

[0087] The results are as follows Figure 1 As shown, the plaques in the WDβ2M group were most widely distributed and had the largest area compared with the other groups. Statistical analysis using Graphpad Prism software showed that the differences were statistically significant.

[0088] Example 3

[0089] β2M promotes the transformation of macrophages into pro-inflammatory types:

[0090] Immunofluorescence staining of aortic sections:

[0091] (1) Heat the paraffin sections in a 65°C oven for 30-60 minutes.

[0092] (2) The sections were sequentially immersed in environmentally friendly dewaxing solution I (G1128-500ML, Sevier Biotechnology) for 10 minutes, environmentally friendly dewaxing solution II (G1128-500ML, Wuhan Sevier Biotechnology Co., Ltd.) for 10 minutes, and environmentally friendly dewaxing solution III (G1128-500ML, Wuhan Sevier Biotechnology Co., Ltd.) for 10 minutes, and then treated with anhydrous ethanol I, II, and III (Sinopharm Chemical Reagent Co., Ltd.) for 5 minutes each, and finally washed with distilled water.

[0093] (3) Place the tissue sections in a container containing EDTA antigen retrieval buffer (PH 8.0), heat in a microwave oven at 70°C for 8 minutes, let it stand for 8 minutes, and then continue heating at 37°C for 7 minutes. During this process, prevent the buffer from evaporating excessively and do not let the sections dry out.

[0094] (4) After cooling naturally, wash the slices with PBS (pH 7.4) on a shaker three times for 5 minutes each time. Shake the slices gently to remove the liquid and draw a circle around the tissue with a histochemical pen. Add 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes to block endogenous peroxidase. Wash the slices with PBS (pH 7.4) three times for 5 minutes each time. Shake the slices slightly to dry, add goat serum to cover the tissue, and block for 30 minutes.

[0095] (5) Gently shake off the blocking solution, add diluted primary antibody (ARG-1, 16001-1-AP, Wuhan Sanying Biotechnology Co., Ltd.), and incubate overnight at 4°C in a wet box. Wash 3 times with PBS (PH7.4), 5 minutes each time. After gently shaking off the solution, add HRP-labeled secondary antibody (goat anti-rabbit, G1213-100UL, Wuhan Sevier Biotechnology Co., Ltd.) and incubate at room temperature for 50 minutes. Wash 3 times with PBS (PH7.4), 5 minutes each time. After gently shaking off the solution, add IF488-TSA, incubate at room temperature in the dark for 10 minutes, then place the slide in TBST and wash 3 times on a decolorization shaker, 5 minutes each time.

[0096] (6) Repeat the microwave repair process of step (3) to dissociate the bound antibody. Add the second diluted primary antibody (INOS, 80517-1-RR, Wuhan Tri-Taiwan Biotechnology Co., Ltd.) and incubate in a humidified chamber at 4°C overnight. Wash with PBS (PH7.4) three times, 5 minutes each time. After gently shaking, add HRP-labeled secondary antibody (INOS, 80517-1-RR, Wuhan Tri-Taiwan Biotechnology Co., Ltd.) and incubate at room temperature for 50 minutes. Wash with PBS (PH7.4) three times, 5 minutes each time. Add IF555-TSA, incubate at room temperature in the dark for 10 minutes, and then wash with TBST.

[0097] (7) Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 8.0) and heat them in a microwave oven. Heat at 70°C for 8 minutes, then stop heating for 8 minutes, and then heat at 37°C for 7 minutes to remove the primary and secondary antibodies that have bound to the tissue. During this process, excessive evaporation of the buffer should be prevented and the sections should not be dried.

[0098] (8) Add a third primary antibody (CD68, 97778s, CST Antibody Company) prepared in a certain proportion in PBS to the slices, and place the slices flat in a humidified box and incubate overnight at 4°C. Place the slides in PBS (PH7.4) and shake on a decolorization shaker to wash 3 times, 5 minutes each time. After the slices are slightly dried, add HRP-labeled fluorescent secondary antibodies of the same species as the primary antibody to cover the tissue in the circle and incubate at room temperature in the dark for 50 minutes. After incubation, place the slides in PBS (PH7.4) and shake on a decolorization shaker to wash 3 times, 5 minutes each time. After incubation, place IF647-TSA in the circle and incubate at room temperature in the dark for 10 minutes. After incubation, place the slides in TBST and shake on a decolorization shaker to wash 3 times, 5 minutes each time. Add DAPI and incubate in the dark for 10 minutes. After washing with PBS (pH 7.4), an autofluorescence quencher (G1221-5 ml, Wuhan Sevier Biotechnology Co., Ltd.) was added dropwise for 5 minutes, and the sections were rinsed with running water for 10 minutes. The sections were washed again with PBS (pH 7.4) and gently shaken before mounting with an anti-fluorescence quenching mounting medium (G1407-25 ml, Wuhan Sevier Biotechnology Co., Ltd.).

[0099] GraphPad Prism software was used to analyze and plot the experimental data. Figure 2 As shown in the results, the performance of the WDβ2M group was opposite to that of the WD TGF-β1 group. The number of pro-inflammatory macrophages increased significantly, while the number of anti-inflammatory macrophages decreased significantly. The differences were statistically significant (P<0.05). The above results indicate that TGF-β1 may alleviate the inflammatory response of plaques by promoting the polarization of macrophages to anti-inflammatory macrophages, while β2M tends to induce macrophage polarization to pro-inflammatory type, thereby aggravating the progression of lesions.

[0100] Example 4

[0101] Characterization of FNMs and their ability to generate singlet oxygen in vitro:

[0102] (1) Observation of the particle size and morphology of NMs by transmission electron microscopy (TEM): The prepared NMs were diluted with pure water to an appropriate concentration and uniformly dispersed by ultrasonic vibration. 10 μL of the sample solution was taken with a pipette and carefully dropped onto the copper grid of the transmission electron microscope. The solution was allowed to stand and dry. 1% phosphotungstic acid negative staining solution was prepared with deionized water and dropped onto the copper grid.

[0103] After the copper mesh was dried, it was observed and photographed under a transmission electron microscope. It was found that the shape of NMs was a circular structure, and the shape of FNMs was a "core-shell" structure ( Figure 3 A, B).

[0104] (2) Dynamic light scattering (DLS) was used to detect the particle size of NMs and FNMs: the prepared nanoparticles were diluted with pure water to an appropriate concentration, 3 mL of the sample solution was placed in a cuvette, and the hydrated particle size of NMs was about 53.6 nm and that of FNMs was about 200.2 nm at room temperature. Figure 3 C).

[0105] (3) Analysis of Fourier transform infrared spectrum of NMs: A small amount of potassium bromide was added to an agate mortar and ground thoroughly as a blank control. Then NMs, ICG and gadolinium chloride samples were mixed with potassium bromide at a ratio of about 1:100 and ground thoroughly. The mixed sample was evenly spread on a tablet pressing mold and placed in a tablet press to be pressed into tablets with a pressure exceeding 10 MPa. After waiting for about 1 minute, the pressed tablets were taken out and placed in a Fourier transform infrared spectrometer for scanning with a scanning range of 4000 cm-400 cm-1. The results showed that NMs had the same characteristic absorption peaks as ICG and gadolinium chloride, proving that NMs combined the two raw materials and was successfully prepared ( Figure 3 D).

[0106] (4) UV-visible spectrophotometer to detect the UV absorption spectrum of FNMs: the prepared nanoparticles were diluted with pure water to an appropriate multiple, 3 mL of the sample solution was placed in a cuvette, and the cuvette was placed in a UV spectrophotometer to measure the UV absorption spectrum of FNMs between 200 and 600 nm ( Figure 3 E).

[0107] (5) Use singlet oxygen ( 1 The photodynamic properties of NMs under 808 nm laser irradiation were detected by using 1,3-diphenylisobenzofuran (DPBF), a photosensitive agent for O2 capture. Under laser excitation, nanomaterials containing photosensitizer ICG can generate singlet oxygen ( 1 O2), the fluorescence intensity will be weakened after DPBF combines with it. Therefore, this can be used to indirectly react with the material to produce 1A 100 μg / mL NMs solution was prepared, 1 mL was taken and placed in a transparent glass dish, 20 μL DPBF (1.5 μg / mL) was added, and 808 nm laser irradiation was used at 0.8 W / cm2. The absorbance change of the mixed solution was measured every 1 minute using a UV-visible spectrometer for a total of 6 minutes. The results showed that the absorbance gradually decreased with each minute, which fully demonstrated that the synthesized nanoparticles were able to produce reactive oxygen species under 808 nm laser irradiation ( Figure 3 F).

[0108] Example 5

[0109] In vitro fluorescence / magnetic resonance imaging performance of FNMs:

[0110] (1) In order to demonstrate the fluorescence imaging performance of FNMs, H2O and different concentrations of FN Ms (10, 20, 40, 80 μg / mL) were placed in a small animal in vivo imaging system, with the excitation wavelength set to 780 nm and the emission wavelength set to 825 nm, to detect the fluorescence performance of the nanoparticles. The results showed that no red color was observed in H2O. However, as the concentration of FNMs increased, the red color became brighter, indicating that the fluorescence intensity was gradually increasing, proving that the synthesized FNMs had good fluorescence imaging performance ( Figure 4 A).

[0111] (2) In order to demonstrate the magnetic resonance imaging performance of nanoparticles, different concentrations of FNMs (0, 0.031, 0.063, 0.125, 0.25, 0.5, 1 mg / mL) were placed in a 9.4T small animal magnetic resonance imaging system, and the parameters were set to image the nanoparticles. The results showed that as the concentration of FNMs increased, the displayed color became brighter and the T1 image intensity gradually increased, proving that the nanomaterial has good magnetic resonance imaging performance ( Figure 4 B).

[0112] Example 6

[0113] In vitro photodynamic effects of FNMs:

[0114] (1) DCFH-DA is a commonly used fluorescent probe for detecting intracellular ROS. Six groups were set up: ①Control group ②Control+Laser group ③NMs group ④NMs+Laser group ⑤FNMs group ⑥FNMs+Laser group. RAW264.7 cells were seeded in a 24-well plate and cultured in a 37°C, 5% CO2 incubator. After the cell density reached approximately 50%, LPS (100 ng / mL) was added for 24 h to induce pro-inflammatory macrophages. The original culture medium was aspirated, and the cells were washed three times with PBS. Pre-diluted NMs and FNMs solutions (concentration of 40 μg / mL) were added and the cells were continued to be incubated in the incubator. After 24 h, the well plate was removed. The illumination group was irradiated with 808 nm laser (0.8 W / cm2, 3 min) and then continued to be cultured in the incubator for 4 h. The well plate was removed, the original culture medium in the well was aspirated, and the cells were washed three times with PBS. Pre-diluted DCFH-DA probe (1:1000 diluted in serum-free culture medium) was added to each well. After incubation in a 37°C incubator for 25 min, the well plate was removed, the original culture medium was aspirated to remove incompletely bound probes, and the cells were carefully washed 2-3 times with PBS before being photographed under a fluorescence microscope. Compared with other groups, the NMs+Laser group and the FNMs+Laser group showed a large area of ​​green fluorescence in the visual field, indicating that the synthesized nanoparticles can produce ROS ( Figure 5 A).

[0115] (2) In order to verify the cytotoxicity of FNMs, the MTT method was used to investigate. As the concentration of FNMs increased, the survival rate of macrophages gradually decreased. When the concentration reached 40 μg / mL, the cell survival rate dropped below 20%, indicating that after laser irradiation, NMs can induce apoptosis of pro-inflammatory macrophages ( Figure 5 B).

[0116] (3) In order to further verify the PDT effect on pro-inflammatory macrophages, AM / PI live-dead staining experiments were used to prove it. Calcein AM can penetrate the membrane of living cells, and after being cut by the esterase in the cell, it is activated and emits green fluorescence. PI usually cannot penetrate the intact cell membrane, but can enter dead or damaged cells. Therefore, it will bind to DNA in dead cells and emit red fluorescence. By combining these two dyes, it can be observed under a microscope that living cells and dead cells emit different colors of fluorescence signals. Figure 5 As shown in (C), in the NMs+Laser group and the FNMs+Laser group, after irradiation with 808 nm laser (0.8 W / cm2, 3 min), the field of view was almost entirely red fluorescent, indicating that the cells were dead, further demonstrating the PDT effect of FNMs.

[0117] Example 7

[0118] In vivo fluorescence / magnetic resonance imaging performance of FNMs:

[0119] ApoE mice fed a high-fat diet for 16 weeks - / - The mouse's head was placed in a small animal gas anesthesia system mask and anesthetized under isoflurane, and the neck and chest areas were depilated.

[0120] (1) FNMs at a concentration of 40 μg / mL were injected into the rat tail vein, and the fluorescence imaging signals of the carotid artery plaques were recorded before and 24 hours after injection. There was a clear fluorescence signal in the carotid artery 24 hours after injection ( Figure 6 A).

[0121] (2) FNMs (concentration of 40 μg / mL) were injected through the tail vein, and the imaging signals of the magnetic resonance T1-weighted images of the carotid artery plaques of mice were recorded before and 24 hours after injection. The imaging of the carotid artery plaques 24 hours after injection was clearer than that before injection ( Figure 6 B).

[0122] Example 8

[0123] In vivo PDT effect of FNMs:

[0124] (1) ApoE - / - Construction and subsequent treatment of mouse atherosclerotic plaque model:

[0125] Select 9 male ApoE mice aged 6-8 weeks - / - Transgenic mice were housed in a specific pathogen-free (SPF) laminar flow chamber at a constant temperature (20°C-26°C) and humidity (50%-65%). They were fed a high-fat, high-cholesterol diet (HFD) containing 15% fat and 0.25% cholesterol. Feed, bedding, and cages were sterilized with autoclaved steam and replaced as needed under sterile conditions for 16 weeks.

[0126] Nine mice were randomly divided into a control group, a FNMs group, a NMs+Laser group, and a FNMs+Laser group (n=3). Every two days, normal saline, FNMs, NMs, or FNMs (at a concentration of 40 μg / mL) were injected into the tail vein of each group. Simultaneously, the light-exposed group was irradiated with 808 nm laser light (0.8 W / cm², 3 minutes) for 19 days. The mice maintained their original diet during this period. After treatment, the mice were dissected.

[0127] (2) Aorta gross oil red O staining: The same steps as in Example 1 were used for aorta gross oil red O staining. Statistical analysis showed that the lipid plaques in the FNMs+Laser group were significantly reduced compared to those in the other groups ( Figure 7 A, B).

[0128] Example 9

[0129] Biosafety of FNMs:

[0130] (1) HE staining of various organs of mice: After the last treatment, the mice were killed, and the tissue and organ specimens (heart, liver, spleen, lung, and kidney) were fixed, dehydrated, and then embedded in paraffin. They were then stained with hematoxylin-eosin (H&E). The results showed that no obvious histological abnormalities or inflammatory lesions were observed in the main organs after treatment ( Figure 8 A).

[0131] (2) Effect of FNMs on the survival rate of pro-inflammatory macrophages: Similar to Example 5 (2), FNMs were co-incubated with pro-inflammatory macrophages, and it was determined that FNMs had almost no effect on the survival rate of pro-inflammatory macrophages without laser irradiation ( Figure 8 B).

[0132] (3) Collect 500 μL of whole blood from healthy mice from the eye socket and place it in a centrifuge tube moistened with sodium heparin for anticoagulation. Centrifuge at 4°C and 3000 rpm for 10 minutes to separate plasma and red blood cells. Red blood cells are collected from the bottom of the tube to avoid white blood cells and platelets on the surface. Wash with pre-cooled PBS three times until the supernatant is completely colorless. Prepare different concentrations of nanomaterials (10, 20, 40, 80 μg / mL), take 0.5 mL of nanomaterial solution, 0.5 mL of pure water, and 0.5 mL of PBS and mix them with 10 μL of red blood cells. Incubate at 37°C for 4 hours, then centrifuge at 3000 rpm for 10 minutes, place the samples on the same horizontal line, and take pictures of their hemolysis phenomenon; use a pipette to draw the sample supernatant into a 96-well plate, and use an enzyme marker to measure the absorbance of the sample at 542 nm: [Set up 3 replicate wells for each group and calculate SD]. Hemolysis rate calculation: Hemolysis rate (%) = (OD experimental group - OD negative control) / (OD positive control - OD negative control) × 100%. The results show that the presence of FNMs almost does not cause hemolysis and has good biosafety ( Figure 8 C).

[0133] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0134] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A nanoparticle coated with a renal tubular epithelial cell membrane, characterized in that: The nanoparticles include renal tubular epithelial cell membranes, contrast agents and dyes.

2. The nanoparticles according to claim 1, characterized in that The contrast agent includes one or a combination of a gadolinium-based contrast agent, a dysprosium-based contrast agent, and a tantalum oxide-based contrast agent.

3. The nanoparticles according to claim 1, characterized in that The dye includes one or a combination of indocyanine green, IR-780, IR-783, MHI-148 and DZ-1.

4. The nanoparticles according to any one of claims 1 to 3, characterized in that The nanoparticles also include metformin.

5. The nanoparticles according to claim 4, characterized in that The molar ratio of the contrast agent: dye: metformin is 1: (1-10): (1-5).

6. The method for preparing nanoparticles according to any one of claims 1 to 5, characterized in that: The preparation method comprises: S1: preparing nanoparticle cores by mixing gadolinium chloride solution, indocyanine green solution, and metformin solution; S2: The nanoparticle core is mixed with the renal tubular epithelial cell membrane to prepare the nanoparticle.

7. The preparation method according to claim 6, characterized in that The volume ratio of the nanoparticle core to the renal tubular epithelial cell membrane is 1:(0.1-10).

8. A nanoparticle, characterized in that The nanoparticles are obtained by the preparation method according to claim 6 or 7.

9. Use of the nanoparticles according to any one of claims 1 to 5 or claim 8 in the preparation of an agent for diagnosing atherosclerotic plaques.

10. Use of the nanoparticles according to any one of claims 1 to 5 or claim 8 in the preparation of a drug for preventing and / or treating atherosclerotic plaques.

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