A reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, its preparation method and application

Abstract

This invention belongs to the field of biomedical nanomaterials technology, specifically relating to a reactive oxygen species (ROS)-responsive iron oxide nanocluster probe targeting type I collagen, its preparation method, and its application in early diagnostic agents for liver fibrosis. The probe consists of nanoclusters with an average particle size >50 nm, formed by cross-linking multiple ultra-small iron oxide nanoparticles coated with polyacrylic acid via an ROS-responsive cross-linking agent containing chalcogenide groups. In the highly reactive oxygen species (ROS) microenvironment of early liver fibrosis, the chalcogenide groups undergo oxidative breakage, leading to cluster dissociation and a shift in T2-weighted MRI signal from low to high. Simultaneously, the dissociated ultra-small iron oxide nanoparticles coated with polyacrylic acid target type I collagen. This probe possesses both active targeting and signal activation mechanisms, enabling non-invasive, highly specific diagnostic agents for early liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology, specifically relating to a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, its preparation method, and its application in early diagnostic agents for liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological process in the progression of various chronic liver diseases to cirrhosis and liver cancer. Early diagnosis is crucial for halting disease progression and improving prognosis. Currently, the gold standard for diagnosing liver fibrosis is liver biopsy, but this method is invasive, carries risks of sampling error and complications, limiting its clinical application. Therefore, developing a non-invasive, highly sensitive, and specific early diagnostic agent for liver fibrosis is of great significance.

[0003] Magnetic resonance imaging (MRI), with its core advantages such as no ionizing radiation, excellent soft tissue resolution, and multi-parameter, multi-sequence imaging, has become the preferred imaging method for non-invasive diagnosis of liver diseases. Targeted MRI contrast agents can specifically identify key pathological markers in the process of liver fibrosis, achieving precise imaging at the molecular level and effectively compensating for the lack of specificity in conventional MRI imaging. In recent years, the development of targeted MRI imaging probes for liver fibrosis has become a research hotspot in the field, with various functional probes being continuously developed and undergoing preclinical validation.

[0004] Most of the MRI probes reported for liver fibrosis at present utilize modified carriers such as hyaluronic acid, targeting peptides, and small-molecule specific ligands to actively target fibrosis markers such as activated hepatic stellate cells and type I collagen. However, these probes generally suffer from a core deficiency: their imaging contrast relies solely on the target-mediated passive accumulation effect, lacking a controllable signal activation mechanism. The enriched signal at the lesion site is difficult to distinguish from the non-specific uptake signal caused by the destruction of hepatic sinusoidal structure and local inflammatory response, which easily leads to false positive results and severely reduces the specificity and reliability of liver fibrosis diagnosis.

[0005] To address the issue of signal nonspecificity, some studies have shifted to developing enzyme-responsive or pathological microenvironment-responsive MRI probes. These probes utilize pathologically relevant factors such as fibroblast activator protein α, glutathione, and myeloperoxidase as signal triggers to "activate" or regulate the switching of imaging signals. However, the signal activation of these probes is highly dependent on the concentration threshold of the triggering factors in the microenvironment. Furthermore, the low expression levels and strong spatial heterogeneity of markers in the early stages of liver fibrosis make it difficult to effectively trigger probe signal responses, resulting in weak imaging signal changes and low signal-to-noise ratios, failing to meet the reliable detection requirements for early fibrosis.

[0006] Another type of nano-assembled smart probes based on disulfide bond crosslinking or enzyme-responsive aggregation can achieve a certain degree of signal switching regulation, but the metabolic pathways and clearance pathways of these probes after response in vivo are still unclear. Long-term retention of probe aggregates in the liver, or the release of free metal ions after dissociation, both pose potential biosafety risks, becoming a core bottleneck restricting their clinical translation.

[0007] In addition, liver fibrosis MRI probes constructed from reactive oxygen species-responsive materials still face two challenges in early diagnosis: first, most probes lack efficient active targeting capabilities, making it difficult to achieve efficient enrichment at early fibrotic lesion sites; second, the probe imaging mechanism is not well adapted to the pathophysiological characteristics of early liver fibrosis, and the specific recognition capability needs further verification, making it impossible to accurately identify early lesions.

[0008] In addition to shortcomings in imaging performance and biosafety, existing probes also face industrialization process barriers. The synthetic routes of some probes are lengthy and the molecular structures are complex, requiring multiple steps of chemical modification and ordered assembly and purification. This not only leads to large batch-to-batch variations and low synthesis yields, but also significantly increases the preparation cost, making it difficult to achieve standardized production and quality control, further hindering the clinical translation process.

[0009] In summary, current technologies lack an MRI probe that can precisely target key pathological markers of liver fibrosis while simultaneously achieving highly sensitive and specific signal activation, especially in the early stages of liver fibrosis. Therefore, developing a novel MRI contrast agent that combines active targeting with microenvironment-responsive activation is of significant clinical importance for the early and accurate diagnosis of liver fibrosis. Summary of the Invention

[0010] The purpose of this invention is to address the aforementioned problems in the existing technology by proposing a reactive oxygen species-responsive iron oxide nanocluster probe that targets type I collagen. This probe can actively target type I collagen overexpressed in liver fibrosis tissue and undergo structural dissociation in a highly reactive oxygen species microenvironment, thereby achieving a change in MRI signal from "dark" to "bright", which can be used for the early and specific diagnosis of liver fibrosis.

[0011] The first objective of this invention is achieved through the following technical solution: A reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, wherein the nanoclusters have an average particle size > 50 nm. The nanoclusters are formed by cross-linking multiple ultra-small iron oxide nanoparticles with an average particle size of <10 nm coated with polyacrylic acid through an active oxygen-responsive cross-linking agent containing chalcogenide groups. The sulfide groups in the linking groups formed after cross-linking undergo oxidative breakage in the reactive oxygen microenvironment.

[0012] Preferably, the chalcogenide group includes at least one of a ketethiolated group, a thioether group, or a selenide group.

[0013] Preferably, the reactive oxygen species responsive crosslinking agent is 2,2′-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine (TK-NH2).

[0014] Preferably, the chalcogenide groups in the linking groups formed after crosslinking undergo oxidative breakage in the active oxygen microenvironment, causing the nanoclusters to dissociate into ultra-small iron oxide nanoparticles coated with polyacrylic acid.

[0015] Preferably, the reactive oxygen microenvironment includes an environment containing superoxide anions and / or hydrogen peroxide.

[0016] Further preferably, the reactive oxygen species microenvironment includes superoxide anions and / or hydrogen peroxide generated by leakage from the mitochondrial electron transport chain, as well as superoxide anions and / or hydrogen peroxide generated by activation of NADPH oxidase.

[0017] Preferably, the nanoclusters are formed by crosslinking at least six ultra-small iron oxide nanoparticles with an average particle size of <10 nm coated with polyacrylic acid using a reactive oxygen species responsive crosslinking agent.

[0018] Preferably, the ultra-small iron oxide nanoparticles coated with polyacrylic acid have a core-shell structure, with the core being ultra-small iron oxide nanoparticles IONP and the shell being a polyacrylic acid coating layer PAA.

[0019] Further preferably, the average particle size of the ultrasmall iron oxide nanoparticles is <10 nm.

[0020] More preferably, the average particle size of the ultrasmall iron oxide nanoparticles is <5nm.

[0021] Further preferred, the ultrasmall iron oxide nanoparticles exhibit superparamagnetism.

[0022] Further preferably, the mass percentage of PAA in the ultrafine iron oxide nanoparticles coated with polyacrylic acid is 65-80 wt%.

[0023] In this invention, the PAA content of PAA@IONP in the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen is too low, resulting in insufficient colloidal stability of IONP and easy aggregation, which in turn affects the uniformity of subsequent cross-linking reactions and the controllability of cluster structure. If the PAA content is too high, the excessively long or dense PAA segments may cause spatial shielding of surface carboxyl groups, reducing the accessibility to the activator and cross-linking agent, leading to decreased cross-linking efficiency and uneven nanocluster particle size distribution. At the same time, an excessively high proportion of non-magnetic components will dilute the saturation magnetization per unit mass, affecting the sensitivity of magnetic resonance imaging, and excessively strong surface negative charge may induce non-specific protein adsorption, interfering with in vivo targeting efficiency and biodistribution.

[0024] Preferably, the polyacrylic acid coating layer is tightly coated on the surface of the ultra-small iron oxide nanoparticles through electrostatic adsorption and coordination.

[0025] In a further preferred embodiment, the polyacrylic acid coating layer binds to the surface of the positively charged iron oxide nanoparticles through electrostatic adsorption and coordination, thereby further tightly coating the surface of the ultra-small iron oxide nanoparticles (IONP).

[0026] Preferably, the preparation method of the ultra-small iron oxide nanoparticles PAA@IONP coated with polyacrylic acid includes the following steps: Under an inert atmosphere, Fe 3+ and Fe 2+ The iron precursor solution was added to a preheated polyacrylic acid aqueous solution at 60-80°C. Under constant temperature conditions, alkali solution was slowly added dropwise until the pH reached 8.2-8.9. After the reaction was complete, the solution was purified by dialysis.

[0027] This invention employs a one-step co-precipitation method combined with in-situ surface modification to obtain ultrafine iron oxide nanoparticles PAA@IONP coated with polyacrylic acid. If the preheating temperature of the polyacrylic acid aqueous solution is low (i.e., the reaction temperature is low), it leads to slower iron ion hydrolysis and crystal nucleus growth, resulting in insufficient nucleation, poor crystallinity, and uneven particle size. If the iron precursor solution contains only Fe... 3+ Or contains only Fe 2+ This results in the inability to generate magnetic iron(III) oxide, leading to products with extremely weak magnetic responsiveness or complete lack of magnetism.

[0028] Further preferably, the Fe in the iron precursor solution 3+ and Fe 2+ The molar ratio is (1.5~5):1.

[0029] More preferably, the Fe in the iron precursor solution 3+ and Fe 2+ The molar ratio is (3.1~3.6):1.

[0030] Further preferably, the Fe in the iron precursor solution 3+ and Fe 2+ The ratio of the total molar amount to the molar amount of polyacrylic acid in the aqueous polyacrylic acid solution is (3~9):1.

[0031] More preferably, the Fe in the iron precursor solution 3+ and Fe 2+ The ratio of the total molar amount to the molar amount of polyacrylic acid in the aqueous polyacrylic acid solution is (7.6~8.1):1.

[0032] Preferably, the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen is paramagnetic.

[0033] The second objective of this invention is achieved through the following technical solution: A method for preparing a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen includes the following steps: (1) Under the protection of an inert atmosphere, Fe is contained in 3+ and Fe 2+ The iron precursor solution was added to a polyacrylic acid aqueous solution preheated to 60-80℃. Under constant temperature conditions, alkali solution was slowly added dropwise until the pH reached 8.2-8.9. After the reaction was complete, the solution was purified by dialysis to obtain a dispersion of ultra-small iron oxide nanoparticles coated with polyacrylic acid. Fe in the iron precursor solution 3+ and Fe 2+ The molar ratio is (1.5~5):1; Fe in the iron precursor solution 3+ and Fe 2+ The ratio of the total molar amount to the molar amount of polyacrylic acid in the aqueous polyacrylic acid solution is (3~9):1; (2) The dispersion of ultra-small iron oxide nanoparticles coated with polyacrylic acid was freeze-dried to obtain PAA@IONP solid powder; (3) Disperse PAA@IONP solid powder in buffer solution, add activator to partially activate the carboxyl groups on the surface of polyacrylic acid, and obtain an activated system; (4) Add reactive oxygen species responsive crosslinking agent to the activation system and react under weak alkaline conditions to crosslink multiple PAA@IONP through amide bonds to form nanoclusters; after dialysis purification, TK-Fe, a reactive oxygen species responsive iron oxide nanocluster probe targeting type I collagen, is obtained.

[0034] In this invention, EDC / NHS only activates some of the carboxyl groups on the PAA surface, thus reserving a reaction window for the subsequent controllable growth of clusters and avoiding excessive cross-linking.

[0035] Preferably, in step (1), the iron precursor solution is added in two parts, with the first addition being 60-80% and the remaining amount added after an interval of 1-30 minutes.

[0036] Preferably, in step (1), the iron precursor solution contains a trivalent iron source and a divalent iron source, wherein the trivalent iron source is selected from at least one of ferric chloride, ferric nitrate or ferric sulfate, and the divalent iron source is selected from at least one of ferrous chloride, ferrous sulfate or ferrous nitrate.

[0037] Preferably, in step (2), the freeze-drying temperature is -80℃ and the time is 1~6h.

[0038] Preferably, in step (3), the activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0039] Further preferably, in step (3), the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC added is 70-80 wt% of the mass of PAA@IONP solid powder; The amount of N-hydroxysuccinimide (NHS) added is 50-70 wt% of the mass of PAA@IONP solid powder.

[0040] Preferably, in step (4), the reactive oxygen species responsive crosslinking agent is 2,2′-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine.

[0041] Preferably, in step (4), the amount of the reactive oxygen responsive crosslinking agent added is 55-70 wt% of the mass of the PAA@IONP solid powder added to the activation system.

[0042] Further preferred, in step (4), the amount of the reactive oxygen responsive crosslinking agent added is 60 wt% of the mass of the PAA@IONP solid powder added to the activation system.

[0043] The third objective of this invention is achieved through the following technical solution: An agent for the early diagnosis of liver fibrosis, comprising 0.1 to 100 wt% of the above-mentioned reactive oxygen species-responsive iron oxide nanoclusters probe targeting type I collagen.

[0044] Preferably, when the formulation is used for magnetic resonance imaging diagnosis, under imaging parameters of TR = 2210 ms and TE = 89 ms, within a time window of 2 to 3 hours after injection, in the highly reactive oxygen microenvironment of liver fibrosis tissue, the T2-weighted MRI signal of the target area shows a transition from low signal to high signal.

[0045] Further preferred, the highly reactive oxygen species microenvironment is an environment conducive to early liver fibrosis.

[0046] Compared with the prior art, the present invention has the following beneficial effects: 1. The reactive oxygen species (ROS) responsive iron oxide nanocluster probe targeting type I collagen of the present invention uses polyacrylic acid-coated ultrasmall iron oxide nanoparticles (PAA@IONP) as the basic unit. Multiple ultrasmall iron oxide particles are cross-linked by a ROS-responsive cross-linking agent with a ketethiolated structure to form nanoclusters with an average particle size >50 nm. The clusters are structurally stable under physiological conditions. In the high reactive oxygen species microenvironment of early liver fibrosis, the ketethiolated bonds break, and the nanoclusters dissociate into ultrasmall iron oxide particles with an average particle size <10 nm, realizing the transformation of T2-weighted imaging signals from low signal to high signal. At the same time, the dissociated small particles can pass through the windows of hepatic sinusoidal endothelial cells and further target type I collagen overexpressed in hepatic stellate cells, thereby achieving high sensitivity and high specificity magnetic resonance imaging diagnosis of liver fibrosis.

[0047] 2. The reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen of the present invention has excellent biosafety and in vivo metabolic characteristics. It uses a high proportion of polyacrylic acid to coat ultra-small iron oxide nanoparticles, giving them good colloidal stability and dispersibility, avoiding non-specific aggregation of nanoparticles in the physiological environment. At the same time, the probe dissociates into ultra-small iron oxide particles (<10 nm) after responding to reactive oxygen species, which can be effectively cleared through the kidney or liver metabolic pathway, avoiding the potential biotoxicity risks caused by long-term retention of aggregates or release of metal ions.

[0048] 3. The formulation for early diagnosis of liver fibrosis prepared by the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen of the present invention has a clear imaging time window and excellent diagnostic specificity in the highly reactive oxygen microenvironment of liver fibrosis tissue. It can effectively distinguish fibrotic tissue from normal tissue and overcome the key technical problems in the prior art, such as poor signal specificity of simple targeting probes, insufficient early sensitivity of enzyme / microenvironment response probes, and unclear in vivo metabolism of smart probes. It provides a brand-new imaging tool for early, accurate and non-invasive diagnosis of liver fibrosis. Attached Figure Description

[0049] Figure 1 This is a TEM image of PAA@IONP in Embodiment 1 of the present invention.

[0050] Figure 2 This is a TEM image of PAA@IONP in Embodiment 2 of the present invention.

[0051] Figure 3 This is a thermogravimetric diagram of PAA@IONP in Embodiment 1 of the present invention.

[0052] Figure 4 These are TEM images of the reactive oxygen species-responsive iron oxide nanocluster probe TK-Fe targeting type I collagen in Example 1 of this invention at different magnifications.

[0053] Figure 5 This is a mapping diagram of the reactive oxygen species-responsive iron oxide nanocluster probe TK-Fe targeting type I collagen in Example 1 of the present invention.

[0054] Figure 6 This is a TEM image of the complete dissociation of the reactive oxygen species-responsive iron oxide nanocluster probe TK-Fe targeting type I collagen after the addition of H2O2 in Example 1 of the present invention.

[0055] Figure 7 This is a hysteresis regression curve of TK-Fe, a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, before and after the addition of H2O2 in Example 1 of the present invention.

[0056] Figure 8 This is an MRI image of TK-Fe, a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, as described in Example 1 of this invention.

[0057] Figure 9 The image shows the reactive oxygen species-responsive iron oxide nanocluster probe TK-Fe targeting type I collagen in Example 1 of this invention and its MRI relaxation maps after the addition of different concentrations of H2O2.

[0058] Figure 10 This is a cell survival rate graph of TK-Fe, a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, as shown in Example 1 of this invention.

[0059] Figure 11 These are in vivo imaging images of mice with liver fibrosis at different time points after injection of TK-Fe, a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, as described in Example 1 of this invention.

[0060] Figure 12 This is a diagram showing the distribution of iron in organs of mice with liver fibrosis after injection of TK-Fe, a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, in Example 1 of this invention.

[0061] Figure 13 This is a diagram showing the hemolysis results of TK-Fe, a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, in Example 1 of this invention.

[0062] Figure 14 This is a pathological staining result of TK-Fe, a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, as described in Example 1 of this invention.

[0063] Figure 15The images show MRI images of TK-Fe and SPIO, reactive oxygen species-responsive iron oxide nanoclusters targeting type I collagen, in mice in different groups, as described in Example 1 of this invention. Detailed Implementation

[0064] The technical solution of the present invention will be further described and illustrated below through specific embodiments. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the present invention.

[0065] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0066] In this article, the raw materials include: Polyacrylic acid, PAA, molecular weight ~2000 g / mol, purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Reactive oxygen species responsive crosslinking agent, TK-NH2, 2,2′-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine, CAS No.: 22907-30-8; purchased from Ningbo Gude Biotechnology Co., Ltd. SPIO was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0067] The equipment used in this paper includes: The Talos F200X transmission electron microscope from Thermo Fisher Scientific, Inc., USA; Netzsch TG 209F1 thermogravimetric analyzer (Germany); Testing of Siemens 3.0 T magnetic resonance imaging system; The IVIS Lumina XRMS Series III small animal live imaging system.

[0068] Example 1 The preparation method of the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen in this embodiment includes the following steps: (1) Dissolve polyacrylic acid (PAA, 6.20 mmol) in 60 mL of deionized water and stir magnetically until completely clear to obtain a PAA solution. Transfer the PAA solution to a 150 mL three-necked flask and bubble under alternating nitrogen and trace amounts of argon for 40 min to remove dissolved oxygen. Under an inert atmosphere, slowly heat the PAA solution to 75 °C and maintain the temperature. In another beaker, dissolve 0.162 g FeCl3·6H2O and 0.036 g FeCl2·4H2O in 3 mL of 0.5 M dilute hydrochloric acid solution to form an iron precursor solution. Add the iron precursor solution to the heated PAA solution in two portions (70% first, and the remaining 30% added after a 10 min interval). Subsequently, under strong stirring, diluted ammonia (20 mL, 10 wt%) was slowly added dropwise to adjust the pH of the solution to 8.5 ± 0.1; then, the solution was dialyzed in deionized water for 48 h using a dialysis bag with a molecular weight cutoff of 8000 Da to obtain a stable dispersion of PAA-coated ultrafine iron oxide nanoparticles (PAA@IONP).

[0069] (2) Transfer all of the PAA@IONP nanoparticle dispersion obtained in step (1) into centrifuge tubes and freeze them at -80°C for 3 hours. Then, place the centrifuge tubes into a low-temperature freeze dryer, evacuate and cool them down to freeze dry them to obtain loose PAA@IONP solid powder.

[0070] (3) Weigh 10 mg of PAA@IONP solid powder obtained in step (2), disperse it in 6 mL of MES buffer (0.05 M, pH=6.0), and sonicate for 5 min to obtain uniform dispersion. Then add 7.5 mg of EDC and 5.4 mg of NHS, and stir magnetically for 45 min at room temperature to obtain the activated system.

[0071] (4) Dissolve 6.0 mg of reactive oxygen species responsive crosslinking agent TK-NH2 in 1 mL of deionized water and slowly add it dropwise to the activation system in step (3). Under weakly alkaline conditions (pH=7.4), the reaction is carried out at room temperature with magnetic stirring for 8 h to induce crosslinking on the surfaces of multiple PAA@IONP, forming nanocluster structures. The system is transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in deionized water for 72 h, with the dialysate being replaced every 8 h to fully remove free crosslinking agent and byproducts; finally, a dispersion of reactive oxygen species responsive iron oxide nanocluster probes targeting type I collagen is obtained, wherein the reactive oxygen species responsive iron oxide nanocluster probe is denoted as TK-Fe.

[0072] Example 2 The preparation method of the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen in this embodiment includes the following steps: (1) The procedure was carried out according to step (1) of Example 1, except that the amount of polyacrylic acid added was 3.10 mmol; a dispersion of PAA-coated ultrafine iron oxide nanoparticles was obtained.

[0073] (2~4) Follow the steps (2~4) of Example 1 to obtain a dispersion of reactive oxygen species responsive iron oxide nanoclusters targeting type I collagen, wherein the reactive oxygen species responsive iron oxide nanoclusters are denoted as TK-Fe.

[0074] The PAA-coated ultrasmall iron oxide nanoparticles PAA@IONP and the reactive oxygen species-responsive iron oxide nanocluster probe TK-Fe targeting type I collagen obtained in Examples 1 and 2 were characterized and their performance was tested.

[0075] (11) Characteristic 1: Transmission electron microscopy (TEM) results show that PAA@IONP ( Figure 1 The particles have a uniform and dense surface, are monodisperse, show no obvious agglomeration, and have a particle size of less than 10 nm. In Example 2, PAA@IONP (… Figure 2 There is a certain degree of aggregation among the particles, resulting in poor dispersibility.

[0076] (12) Characteristic 2: Thermogravimetric analysis (TGA) was conducted under a nitrogen atmosphere, with the temperature increased from room temperature to 800°C at a rate of 10°C / min. The main weight loss phase was observed in the range of 100–800°C. Figure 3 This indicates that the mass percentage of PAA in PAA@IONP of Example 1 is as high as ~70%, while the mass percentage of PAA in PAA@IONP of Example 2 is 40%.

[0077] (13) Characteristic 3: TEM image ( Figure 4 The results show that the average particle size of the reactive oxygen species-responsive iron oxide nanocluster probe TK-Fe targeting type I collagen in Example 1 is 124.2 nm. Elemental distribution diagram ( Figure 5 The results showed that Fe and S elements were highly co-located, confirming the successful introduction of the TK-NH2 crosslinking agent.

[0078] (21) Test 1: 1 mL of 80 mM H2O2 was added to the TK-Fe dispersion of the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen in Example 1. Figure 6 The results showed that after the addition of H2O2, the cluster structure of TK-Fe completely dissociated and redispersed into uniformly sized monodisperse ultrasmall iron oxide nanoparticles.

[0079] Magnetic properties were tested separately, with a temperature range of 2 K to 400 K and a magnetic field strength range of -9 T to 9 T. The results are as follows: Figure 7 As shown, TK-Fe has a high initial saturation magnetization of 19.9 emu / g; after complete dissociation by adding H2O2, the saturation magnetization decreases to 6.27 emu / g, which weakens its ability to generate a local magnetic field gradient.

[0080] The above results indicate that the TK-Fe undergoes structural dissociation under the influence of reactive oxygen species, accompanied by changes in its magnetic properties, providing a structural basis for the regulation of its reactive oxygen species-responsive magnetic resonance signal.

[0081] (22) Test 2: The reactive oxygen species (ROS) responsive iron oxide nanocluster probe TK-Fe targeting type I collagen prepared in Example 1 was diluted to concentrations of 0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, and 0.01 mM, respectively. Each concentration sample was divided into three groups, and 1 mL (based on iron concentration) of H₂O₂ at concentrations of 0 mM, 10 mM, and 50 mM was added to each group for ROS responsiveness testing.

[0082] The test was conducted using a 3T magnetic resonance imaging scanner with parameters set to TR = 2210 ms and TE = 89 ms.

[0083] MRI results as follows Figure 8 As shown, with increasing H2O2 concentration, the T2-weighted MRI signal exhibits a trend of changing from dark to bright. Further analysis of the relaxation rate (…) Figure 9 It was found that the transverse relaxation rate r2 of the probe decreased with increasing H2O2 concentration, dropping to 110.88 mM. -1 s -1 and 85.88 mM -1 s -1 .

[0084] The above results indicate that the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen has a significant response to H2O2 concentration in the in vitro environment, and can realize the transition of T2 signal from low signal to high signal, and the response intensity is positively correlated with reactive oxygen species concentration.

[0085] (23) Test 3: The cytocompatibility of the reactive oxygen species-responsive iron oxide nanoclusters targeting type I collagen in Example 1 was evaluated using the CCK-8 assay. Two cell groups were set up: a Hepg-2 cell group and a RAW 264.7 cell group.

[0086] The two cell types were respectively fed with 3×10 5Cells were seeded at a density of 1 cell / well in 12-well plates and incubated for 24 hours. The old culture medium was discarded, and 1 mL of TK-Fe medium with iron concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL were added to each well. Incubation continued for another 12 hours. After incubation, 100 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 40 minutes. After shaking to mix, 100 μL of supernatant was transferred from each well to a 96-well plate. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0087] The results are as follows Figure 10 As shown, the survival rates of Hepg-2 cells and RAW 264.7 cells were both greater than 80%, indicating that the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen has good cell compatibility.

[0088] (24) Tests 4-8: Establishment of a mouse model of liver fibrosis: Early and intermediate-stage mouse models of liver fibrosis were established using the carbon tetrachloride (CCl4) induction method. CCl4 and olive oil were mixed at a volume ratio of 1:3 to prepare a 25% (v / v) CCl4 solution, which was prepared fresh or stored at 4°C in a light-proof, sealed container. Administration was via intraperitoneal injection. Before injection, the lower abdomen was disinfected with an alcohol swab. The CCl4-ol-ol mixture was injected at a dose of 2 μL / g body weight (i.e., a CCl4 dose of 0.5 mL / kg body weight). Injections were administered twice weekly, taking care to avoid the midline of the abdomen and the bladder area before slowly pushing the injection.

[0089] (241) The TK-Fe dispersion of Example 1 at a concentration of 6 mM was injected into mice with liver fibrosis via the tail vein. Fluorescence imaging was performed using an IVIS Lumina XRMS Series III small animal in vivo imaging system before injection and at 10 min, 1 h, 2 h, 3 h, 4 h and 6 h after injection.

[0090] The results are as follows Figure 11 As shown, the fluorescence signal in the mouse liver region significantly increased within 2–3 hours after injection, reaching a peak intensity. This result indicates that the probe achieves efficient enrichment in the liver region within 2–3 hours after injection.

[0091] The aforementioned time-dependent enrichment features provide an important basis for the selection of subsequent magnetic resonance imaging time points, namely, 2-3 hours after injection is the optimal imaging time window for this probe to perform T2-weighted imaging of the liver.

[0092] In Example 2, the cluster aggregation in the TK-Fe dispersion led to a significant decrease in its colloidal stability, incomplete reactive oxygen species response fragmentation, inability to achieve effective liver T2-weighted imaging, and weakened ability to target type I collagen.

[0093] (242) The TK-Fe dispersion of Example 1 at a concentration of 8 mM was injected into a mouse model of liver fibrosis via the tail vein. At 2 h and 5 days after the tail vein injection, heart, liver, spleen and kidney tissues were collected, washed three times with PBS buffer to remove residual blood on the surface of the organs, lyophilized, weighed and digested.

[0094] Iron content in various organs was determined by inductively coupled plasma mass spectrometry (ICP-MS), and the difference was calculated using the control group without probe injection as the baseline. The original content and difference results are as follows: Figure 12 As shown.

[0095] The results showed that 2 hours after injection, the iron content in the liver was significantly higher than that in the control group, with a difference of approximately 35.7 ppm, indicating that the probe achieved efficient enrichment in the liver. Five days after injection, the iron content in all organs decreased compared to the 2-hour time point, but was still slightly higher than that in the control group (especially the liver and spleen), indicating that the probe was gradually metabolized and cleared in vivo.

[0096] (243) Blood samples were collected from mice with liver fibrosis using the orbital vein sampling method and transferred to 1.5 mL anticoagulant tubes. The anticoagulant tubes were placed in a low-temperature centrifuge and centrifuged at 1000 rpm / min for 5 min to separate red blood cells and serum. After centrifugation, the supernatant serum was carefully removed, and the red blood cell precipitate was retained. The red blood cells were washed three times with physiological saline to remove residual serum and impurities. After washing, 1 mL of physiological saline was added to resuspend the red blood cells to prepare a 4% (volume fraction) red blood cell suspension. TK-Fe from Example 1 was added to the red blood cell suspension in sequence at concentrations of 0, 25, 50, 100, 200, 300, and 400 μg / mL as experimental groups. Control groups were set up at the same time: samples with added deionized water were used as positive control groups (H2O), and samples with added physiological saline were used as negative control groups. All samples were incubated at 37°C in the dark for 2 h. After incubation, the samples were centrifuged again at low temperature, and the supernatant was collected and photographed. The absorbance of the supernatant in each group was measured at a wavelength of 540 nm using an ELISA reader to assess the hemolytic effect of the probe on red blood cells.

[0097] The results are as follows Figure 13 As shown, within the tested concentration range, the hemolysis rate of each sample in the experimental group was less than 5%, indicating that the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen has good blood compatibility and meets the biosafety requirements for intravenous injection in vivo.

[0098] (244) A 6 mM TK-Fe dispersion from Example 1 was injected via the tail vein into mice with a liver fibrosis model. Two hours after tail vein injection, the mice were sacrificed, and the liver tissue was immediately dissected and separated. The liver tissue was washed three times with pre-cooled PBS to remove residual blood. The liver tissue was fixed in 4% paraformaldehyde solution at room temperature for 24 hours. After dehydration with graded ethanol and clearing with xylene, the tissue was embedded in paraffin and serial sections with a thickness of 5 μm were prepared. The sections were stained sequentially with Prussian blue stain to visualize iron deposition and Sirius red stain to visualize collagen fiber distribution.

[0099] The results are as follows Figure 14 As shown in the figure, in the normal control group (CCl40 weeks), Sirius red staining (top image) showed only a small amount of collagen fibers in the liver tissue, and Prussian blue staining (bottom image) showed no obvious blue granules, indicating that there was no probe-specific enrichment in normal liver tissue. In the model group (CCl42 weeks), Sirius red staining showed a significant increase in collagen fibers in the liver tissue, which were distributed in red cords in the portal area and liver lobules, confirming that the early liver fibrosis model was successfully established after 2 weeks of CCl4 induction; Prussian blue staining showed obvious blue granule deposition, and the blue signal showed a high degree of spatial overlap with the collagen area stained by Sirius red.

[0100] The above results indicate that the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen can specifically recognize and bind to type I collagen in early liver fibrosis tissue, achieving targeted enrichment.

[0101] (245) Set up three groups of experiments: Experimental group (LF+TK-Fe): Mice with early liver fibrosis were injected via tail vein with a 6mM concentration of TK-Fe dispersion from Example 1. Normal control group (Normal+TK-Fe): Normal mice were injected via tail vein with a 6mM TK-Fe dispersion from Example 1; Negative control group (LF+SPIO): Mice with early liver fibrosis were injected via tail vein with a conventional T2-weighted imaging contrast agent (non-responsive iron oxide nanoparticles, SPIO).

[0102] Mice in each group underwent magnetic resonance imaging (MRI) scans before injection and at 10 min, 1 h, 2 h, 3 h, 4 h, and 6 h after injection. Imaging parameters were set to TR=2210 ms and TE=89 ms, using a T2-weighted imaging sequence.

[0103] The results are as follows Figure 15As shown in the figure, in the experimental group, mice with early liver fibrosis showed a significant shift in T2-weighted imaging signal from low to high signal within 2-3 hours after probe injection; in the normal control group, no significant signal changes were observed in normal mice at any time point after probe injection; and in the negative control group, no significant signal changes were observed in mice with early liver fibrosis at any time point after injection of conventional T2 contrast agent.

[0104] The above results indicate that the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen exhibits specific response imaging only in the early liver fibrosis microenvironment, highlighting its unique advantage as a smart responsive contrast agent in the early diagnosis of liver fibrosis.

[0105] In summary, this invention provides a reactive oxygen species (ROS) responsive iron oxide nanocluster probe targeting type I collagen. It uses ultra-small iron oxide nanoparticles coated with a high proportion of polyacrylic acid as the basic unit, cross-linked with an ROS responsive cross-linking agent containing a ketethiolated structure to form stable nanoclusters with a particle size greater than 50 nm. In the highly reactive oxygen species microenvironment of early-stage liver fibrosis, the ketethiolated bonds specifically break, and the nanoclusters dissociate into ultra-small particles with a particle size less than 10 nm. This achieves an "opening" transition of T2-weighted MRI signals from low to high signal. Simultaneously, the dissociated small particles can pass through the endothelial pores of the liver sinusoids and target overexpressed type I collagen, thereby achieving highly sensitive and specific diagnosis of liver fibrosis. This probe works synergistically through a dual mechanism of "active targeting" and "signal activation," effectively overcoming key issues in existing technologies such as insufficient specificity of simple targeting probes, low early sensitivity of enzyme-responsive probes, unclear in vivo metabolism of smart probes, and questionable biosafety. It also exhibits good cell and blood compatibility (cell survival rate >80%, hemolysis rate <5%), with the optimal imaging window being 2-3 hours after injection. This provides an ideal imaging tool for the early, accurate, and non-invasive diagnosis of liver fibrosis.

[0106] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0107] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0108] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen, characterized in that, It consists of nanoclusters with an average particle size > 50 nm; The nanoclusters are formed by cross-linking multiple ultra-small iron oxide nanoparticles with an average particle size of <10 nm coated with polyacrylic acid through an active oxygen-responsive cross-linking agent containing chalcogenide groups. The sulfide groups in the linking groups formed after cross-linking undergo oxidative breakage in the reactive oxygen microenvironment.

2. The reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen according to claim 1, characterized in that, The reactive oxygen microenvironment includes an environment containing superoxide anions and / or hydrogen peroxide.

3. The reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen according to claim 1, characterized in that, The chalcogenide group includes at least one of a ketethioyl group, a thioether group, or a selenide group. The reactive oxygen species responsive crosslinking agent is 2,2′-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine; The chalcogenide groups in the linking groups formed after cross-linking undergo oxidative breakage in the active oxygen microenvironment, causing the nanoclusters to dissociate into ultra-small iron oxide nanoparticles coated with polyacrylic acid.

4. The reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen according to claim 1, characterized in that, The ultra-small iron oxide nanoparticles coated with polyacrylic acid have a core-shell structure, with the core being ultra-small iron oxide nanoparticles and the shell being a polyacrylic acid coating layer.

5. The reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen according to claim 4, characterized in that, The mass percentage of PAA in the ultrafine iron oxide nanoparticles coated with polyacrylic acid is 65-80 wt%. And / or, the ultrasmall iron oxide nanoparticles are superparamagnetic; And / or, the polyacrylic acid coating layer is tightly coated on the surface of the ultra-small iron oxide nanoparticles through electrostatic adsorption and coordination.

6. The reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen according to claim 1, characterized in that, The ultrafine iron oxide nanoparticles PAA@IONP coated with polyacrylic acid were prepared by the following method: Under an inert atmosphere, Fe 3+ and Fe 2+ The iron precursor solution was added to a polyacrylic acid aqueous solution preheated to 60-80℃. Under constant temperature conditions, alkali solution was slowly added dropwise until the pH reached 8.2-8.

9. After the reaction was complete, the solution was purified by dialysis. Fe in the iron precursor solution 3+ and Fe 2+ The molar ratio is (1.5~5):1; Fe in the iron precursor solution 3+ and Fe 2+ The ratio of the total molar amount to the molar amount of polyacrylic acid in the aqueous polyacrylic acid solution is (3~9):

1.

7. A method for preparing a reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen as described in claim 1, characterized in that, Includes the following steps: (1) Under the protection of an inert atmosphere, Fe is contained in 3+ and Fe 2+ The iron precursor solution was added to a preheated polyacrylic acid aqueous solution at 60-80°C. Under constant temperature conditions, alkali solution was slowly added dropwise until the pH reached 8.2-8.

9. After the reaction was complete, the solution was purified by dialysis to obtain a dispersion of ultra-small iron oxide nanoparticles coated with polyacrylic acid. Fe in the iron precursor solution 3+ and Fe 2+ The molar ratio is (1.5~5):1; Fe in the iron precursor solution 3+ and Fe 2+ The ratio of the total molar amount to the molar amount of polyacrylic acid in the aqueous polyacrylic acid solution is (3~9):1; (2) The dispersion of ultra-small iron oxide nanoparticles coated with polyacrylic acid was freeze-dried to obtain PAA@IONP solid powder; (3) Disperse PAA@IONP solid powder in buffer solution, add activator to partially activate the carboxyl groups on the surface of polyacrylic acid, and obtain an activated system; (4) Add reactive oxygen species responsive crosslinking agent to the activation system and react under weak alkaline conditions to crosslink multiple PAA@IONP through amide bonds to form nanoclusters; after dialysis purification, TK-Fe, a reactive oxygen species responsive iron oxide nanocluster probe targeting type I collagen, is obtained.

8. The method for preparing the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen according to claim 7, characterized in that, In step (1), the iron precursor solution contains a trivalent iron source and a divalent iron source. The trivalent iron source is selected from at least one of ferric chloride, ferric nitrate, or ferric sulfate, and the divalent iron source is selected from at least one of ferrous chloride, ferrous sulfate, or ferrous nitrate.

9. The method for preparing the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen according to claim 7, characterized in that, In step (1), the iron precursor solution is added in two parts. The first addition is 60-80% of the solution, and the remaining amount is added after an interval of 1-30 minutes. And / or, in step (2), the freeze-drying temperature is -80°C and the time is 1~6h; And / or, in step (3), the activator comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS; the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC added is 70-80 wt% of the mass of PAA@IONP solid powder; the amount of N-hydroxysuccinimide NHS added is 50-70 wt% of the mass of PAA@IONP solid powder; And / or, in step (4), the reactive oxygen species responsive crosslinking agent is 2,2′-(propane-2,2-dimethylbis(sulfonamide diel))diethylamine; And / or, in step (4), the amount of the reactive oxygen responsive crosslinking agent added is 55~70wt% of the mass of PAA@IONP solid powder added to the activation system.

10. A preparation for the early diagnosis of liver fibrosis, characterized in that, It includes 0.1 to 100 wt% of the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen as described in any one of claims 1 to 6, or the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen prepared by the preparation method of the reactive oxygen species-responsive iron oxide nanocluster probe targeting type I collagen as described in any one of claims 7 to 9. When the formulation is used for magnetic resonance imaging diagnosis, under imaging parameters of TR=2210 ms and TE=89 ms, within a time window of 2-3 hours after injection, in the highly reactive oxygen microenvironment of liver fibrosis tissue, the T2-weighted MRI signal of the target area shows a transition from low signal to high signal.

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