A pH-responsive MRI probe targeting M2 macrophages and a preparation method and application thereof

Abstract

The present application relates to a kind of pH-responsive MRI probe targeting M2 macrophage and its preparation method and application, the pH-responsive MRI probe includes CD206 targeting group, pH-responsive group and Gd 3+ Imaging group.The pH-responsive MRI probe of the present application has a dual mechanism of "receptor-targeted enrichment + microenvironment signal activation", which ensures the delivery efficiency of the probe to the target cells and realizes the "precise switch" of the signal inside the target point, effectively solving the imaging interference problem caused by non-specific uptake in the traditional single targeting strategy.

Description

Technical Field

[0001] This invention belongs to the field of molecular imaging technology, and specifically relates to a pH-responsive MRI probe targeting M2 macrophages, its preparation method, and its application. Background Technology

[0002] Rotator cuff tear (RCT) is the most common type of tendon-bone interface (TBI) injury in clinical practice, and arthroscopic repair and reconstruction is currently the preferred treatment option for this condition. However, clinical experience has shown that the regeneration process of the natural fibrocartilage layer at the tendon-bone interface is easily hindered after surgery, and the injured site often forms fibrous scar tissue with weak mechanical properties, resulting in a re-tear rate as high as 20%-94%. This makes it difficult for patients to effectively restore shoulder joint function, becoming a core challenge in clinical treatment.

[0003] Tendon-bone healing is a complex, multi-stage physiological and pathological process, divided into the inflammatory phase, proliferative phase, matrix synthesis phase, and matrix remodeling phase. The regulation of the early inflammatory microenvironment is a core factor determining the final quality of tendon-bone healing. M2 macrophages, as key cells exerting anti-inflammatory and repair functions during the inflammatory phase, can secrete various tissue repair factors such as transforming growth factor-β and interleukin-10, effectively promoting inflammation resolution, local angiogenesis, and orderly collagen fiber synthesis. Their spatiotemporal distribution and enrichment at the tendon-bone interface can reflect the early healing potential after tendon-bone injury. Therefore, achieving non-invasive, highly specific, and visual detection of M2 macrophages in vivo is of significant clinical importance for predicting early efficacy after rotator cuff repair surgery, adjusting clinical intervention plans in a timely manner, and improving patient prognosis.

[0004] Currently, visualization imaging technology for macrophages has been applied in animal model studies of some diseases. However, for precise imaging of M2 macrophages during tendon and bone healing and early postoperative assessment, existing technologies still have three major shortcomings that make it difficult to meet the needs of clinical applications. These are as follows:

[0005] 1. Traditional imaging techniques and conventional contrast agents have functional limitations, making it impossible to achieve non-invasive, deep, and targeted visualization of M2 macrophages.

[0006] While optical imaging offers high sensitivity and low cost, its tissue penetration depth is limited (typically <1cm), making it suitable only for small animal experiments or intraoperative local navigation and unable to penetrate deep tendon and bone structures in the human body. Nuclear medicine imaging can achieve whole-body quantitative imaging, but it carries the risk of ionizing radiation and has a spatial resolution of only 1-2mm, making it unsuitable for long-term dynamic monitoring after surgery. Furthermore, commonly used traditional MRI contrast agents lack targeted recognition capabilities and rely solely on passive diffusion distribution, failing to achieve specific imaging of M2 macrophages. All three methods are ill-suited to the non-invasive, targeted visualization requirements of M2 macrophages in deep tendon and bone structures in the human body.

[0007] 2. Existing MRI probes targeting M2 macrophages lack specificity and stability, making it impossible to accurately quantify the actual number of M2 macrophages.

[0008] Current MRI imaging strategies targeting M2 macrophages largely rely on surface markers such as CD206 and CD163 for identification, achieving targeted binding by modifying probes with ligands such as mannose and antibody fragments. However, these probes have significant drawbacks: firstly, markers such as CD206 are not specifically expressed in M2 macrophages, but are also expressed at low levels in non-target cells such as endothelial cells and immature dendritic cells, easily leading to non-specific uptake of the probe and false positives in imaging; secondly, the expression level of markers is easily affected by microenvironmental factors such as the degree of inflammation and nutritional status, resulting in insufficient imaging signal stability and an inability to accurately quantify the actual number of M2 macrophages at the tendon-bone interface.

[0009] 3. Clinical practice lacks early molecular-level assessment tools for tendon-bone healing, and there is no correlation system between imaging indicators and healing outcomes, resulting in a delayed intervention window.

[0010] Current clinical assessment methods for tendon-bone healing after rotator cuff repair surgery mostly focus on anatomical observation in the mid-to-late postoperative period. These methods can only reflect the morphological repair status of the tendon-bone interface and lack molecular-level assessment tools that can reflect the repair microenvironment during the critical inflammatory period (about 2 weeks postoperatively). At the same time, existing technologies have not established a correlation system between imaging indicators and the final outcome of tendon-bone healing, making it impossible to predict the healing prognosis through early detection. This results in the inability to provide timely and effective intervention within the optimal intervention window, further reducing the quality of tendon-bone healing. Summary of the Invention

[0011] This invention provides a pH-responsive MRI probe targeting M2 macrophages, its preparation method, and its application. The technical problems to be solved include:

[0012] (1) Break through the functional limitations of traditional imaging technology and conventional contrast agents, overcome the problems of insufficient tissue penetration depth of optical imaging, low spatial resolution of nuclear medicine imaging with ionizing radiation, and lack of targeting of traditional MRI contrast agents, and achieve non-invasive, deep tissue penetration, and highly specific visualization imaging of M2 macrophages in vivo, adapting to the detection needs of deep tendon and bone structures in the human body.

[0013] (2) To solve the technical defects of existing MRI probes targeting M2 macrophages that lack specificity and stability, and to overcome the problems of existing target probes that rely on surface markers being easily taken up by non-targeted cells, the imaging signal being interfered with by microenvironmental factors such as the degree of inflammation and nutritional status, and the inability to accurately quantify the actual number of M2 macrophages at the tendon-bone interface, so as to achieve accurate identification and quantitative imaging of M2 macrophages.

[0014] (3) Establish a molecular imaging system for early assessment and prognosis prediction of tendon and bone healing, address the pain point of lacking early molecular assessment tools for tendon and bone healing in clinical practice, realize the direct correlation between probe imaging indicators and the final outcome of tendon and bone healing, provide objective and quantitative judgment basis for timely clinical intervention in the critical period of inflammation after rotator cuff repair, and fill the technical gap in early clinical prognosis prediction.

[0015] This invention provides a pH-responsive MRI probe targeting M2 macrophages, wherein the pH-responsive MRI probe comprises a CD206 targeting group, a pH-responsive group, and Gd covalently linked by chemical bonds. 3+ Imaging group.

[0016] The pH-responsive MRI probe has a "ternary composite spherical nanoparticle" structure with a diameter of 80-90 nm. The three functional groups work together to achieve a closed-loop function of "targeted enrichment → microenvironment response → signal amplification".

[0017] Preferably, the CD206 targeting group is derived from NH2-PEG. 5k -Man polymer, molecular weight 5000 Da, utilizes mannose to specifically bind to the CD206 receptor highly expressed on the surface of M2 macrophages (binding constant Kd = 1.2 × 10⁻⁶). -6 (mol / L) to achieve active targeted delivery of probes, while the PEG segment enhances colloidal stability and biocompatibility, avoiding rapid in vivo clearance.

[0018] Preferably, the pH-responsive group is derived from N4C (C 14 H 27 NO2) monomer or N5C (C 16 H 31The NO2 monomer is protonated in the acidic environment (pH=4.4-4.9) of M2 macrophage lysosomes, triggering a reversal of the hydrophilicity and hydrophobicity of polymer segments, leading to probe dissociation; in the physiological neutral environment (pH=7.2-7.4), it maintains a hydrophobic core, encapsulating imaging groups in a "signal off" state.

[0019] Preferably, the Gd 3+ The imaging group is derived from DOTA-Gd, and this stable coordination compound avoids Gd. 3+ Leakage toxicity, utilizing Gd 3+ The seven unpaired electrons in the outer layer shorten the T1 relaxation time of water protons, serving as a source of MRI-T1 weighted signal.

[0020] This invention also provides a method for preparing a pH-responsive MRI probe targeting M2 macrophages, comprising the following steps:

[0021] (1) Gd was prepared by organic synthesis. 3+ Imaging groups, targeting polymers, and pH-responsive amphiphilic polymers are used as functional components;

[0022] (2) The above functional components are mixed by the self-assembly method of "organic phase dissolution-aqueous phase dispersion-dialysis to remove impurities" to form a spherical probe with uniform structure, which is a pH-responsive MRI probe targeting M2 macrophages.

[0023] Preferably, the targeting polymer in step (1) is PEG-PNXC-Man.

[0024] Preferably, the pH-responsive amphiphilic polymer in step (1) is mPEG-PNXC.

[0025] Preferably, the functional components in step (2) are mixed in an equimolar ratio.

[0026] This invention also provides the application of a pH-responsive MRI probe targeting M2 macrophages in the preparation of visualization detection products.

[0027] The pH-responsive MRI probe of this invention is named MR-CAs (Macrophage-targeted pH-Responsive contrast agents). Its mechanism of action is through… Figure 1 Clearly visible: The probe consists of pH-responsive groups, Gd... 3+ The imaging group, CD206 targeting group, and other components work together to assemble the product. After local injection into the rat shoulder joint, it accumulates in M2 macrophages at the tendon-bone interface via CD206 receptor-mediated targeting, and subsequently enters the lysosome via endocytosis. The lysosomal acidic microenvironment (pH...) L~4.9) Triggering protonation of pH-responsive groups leads to probe structure dissociation. Gd 3+ This probe fully exposes and enhances the interaction of water molecules, significantly amplifying the T1-weighted signal and enabling specific visualization of M2 macrophages. It innovatively employs a dual mechanism of "CD206 receptor targeting + lysosomal pH response," forming a closed-loop regulation from targeted enrichment to signal activation. Figure 2 A).

[0028] To ensure the reliability of the probes in vivo, this invention establishes a three-tiered cell validation system encompassing phenotypic specificity, biocompatibility, and functional effectiveness.

[0029] (1) Macrophage phenotype induction and identification: Primary bone marrow-derived macrophages (BMDM) were extracted from the bone marrow of SD rats. The M1 phenotype was induced by IFN-γ+LPS and the M2 phenotype was induced by IL-4+IL-13. The phenotype purity (M1:CD86) was verified by flow cytometry. + ≥75%, M2: CD206 + (≥85%), providing a standardized cell model for subsequent validation;

[0030] (2) Biocompatibility verification: The CCK-8 method was used to detect different Gd 3+ Survival rate of M1 and M2 macrophages after co-incubation with probes at concentrations (0-0.1mM) for 24 hours, ensuring cell survival rate ≥90% within the effective concentration range;

[0031] (3) Target specificity verification: M1 and M2 macrophages were co-incubated with fluorescently labeled probes, and the uptake efficiency of M2 macrophages by probes was verified to be significantly higher than that of M1 macrophages by immunofluorescence imaging and quantitative analysis.

[0032] (4) Intracellular function verification: Through lysosomal colocalization experiment and pH response experiment, it was confirmed that the probe can be enriched in the lysosomes of M2 macrophages and activate signals in an acidic environment.

[0033] This invention establishes a preclinical application process of "animal model construction - probe injection imaging - healing outcome verification":

[0034] (1) A rat rotator cuff injury repair model was constructed by grouping rats (sham surgery group, simple suture group, and PRP-assisted repair group) to simulate different clinical repair scenarios;

[0035] (2) Two weeks after the operation (critical period of M2 macrophage enrichment), the probe (50 μL, Gd concentration 0.05 mM / kg) was injected locally, and dynamic T1WI scanning was performed by 3.0T MRI (before injection and 30, 60 and 180 min after injection). The ΔCNR peak value was calculated as the quantitative index of M2 macrophage enrichment.

[0036] (3) Eight weeks after surgery, the healing quality was verified by histological staining (HE, Masson, SOFG) and biomechanical testing (maximum load, stiffness), and the correlation between the peak value of ΔCNR and the healing outcome index was analyzed to clarify the prognostic predictive value of the probe.

[0037] Beneficial effects

[0038] Compared with existing technologies, this invention has comprehensive technical advantages in terms of probe structural design, cellular performance, and in vivo application value. Each beneficial effect is supported by specific experimental data, as follows:

[0039] (1) Innovative structural design, dual mechanisms work together to improve imaging specificity, and clear data support;

[0040] (2) Excellent performance at the cellular level, and biocompatibility and functional stability meet quantitative standards;

[0041] (3) Comprehensive in vivo imaging function, combining non-invasive, precise quantification and dynamic monitoring;

[0042] (4) It has outstanding clinical application value, accurate early prognosis prediction and strong intervention guidance. Attached Figure Description

[0043] Figure 1 This is a schematic diagram illustrating the function of the pH-responsive MRI probe of the present invention.

[0044] Figure 2 AG represents the synthesis and characterization of the pH-responsive MRI probe of this invention.

[0045] Figure 3 AG is used to explore the imaging of the pH-responsive MRI probe of this invention at the cellular level.

[0046] Figure 4 AF is an imaging study of the pH-responsive MRI probe of this invention at the animal level.

[0047] Figure 5 Assessment of tendon-bone healing outcomes after rotator cuff repair in animals (AH). Detailed Implementation

[0048] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0049] Example 1

[0050] (I) Probe preparation process

[0051] (1) Synthesis of pH-responsive groups (N4C / N5C): Taking the synthesis of N4C as an example, ethanolamine (20 mmol), bromobutane (40 mmol) and sodium carbonate (50 mmol) were added to a 100 mL single-necked round-bottom flask, and 40 mL of anhydrous acetonitrile was added. The mixture was stirred overnight at room temperature. After filtration, the solvent was removed by vacuum distillation, and the fraction was collected to obtain the intermediate. The intermediate (10 mmol), triethylamine (10 mmol) and hydroquinone (0.1 mmol) were added to a 50 mL three-necked flask, and methacryloyl chloride (10 mmol) was added dropwise under ice bath conditions. The mixture was stirred overnight at room temperature, and after filtration, it was purified by vacuum distillation to obtain N4C (yield 63%). N5C was synthesized using a similar process, and the yield met the experimental requirements.

[0052] (2) Gd 3+ Synthesis of the imaging group (DOTA-Gd): Tetraazacyclododecanetetraacetic acid-succinimide ester (1 mmol) was added to a 50 mL two-necked flask, dissolved in 15 mL of dichloromethane, and a mixture of hexadecylamine (2.1 mmol) and triethylamine (5.25 mmol) was added dropwise. The mixture was stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the precipitate was dried under vacuum after precipitation with diethyl ether. The intermediate was dissolved in dichloromethane, deprotected with trifluoroacetic acid for 6 h, and chelated with GdCl3·6H2O (2 mmol) in a 1,4-dioxane / water system (pH=6.4). The mixture was stirred overnight at 45 °C. After dialyzing for 24 h, the mixture was freeze-dried to obtain DOTA-Gd (yield 70%).

[0053] (3) Synthesis of targeted polymer (PEG-PNXC-Man): 0.1 mmol of 4-cyano-4-(phenylthiocarbonylthio)valerate N-hydroxysuccinimide CPADB-NHS and NH2-PEG were added to a 50 mL single-necked round-bottom flask. 5k -Man (0.2 mmol) was added to 10 mL of dichloromethane and triethylamine (0.15 mmol), and stirred at room temperature for 12 h. After dialyzing (molecular weight cutoff of 3 kDa) for 24 h, it was freeze-dried to obtain CTA-Man. CTA-Man (0.04 mmol), N4C / N5C monomer (0.4 mmol) and AIBN (0.008 mmol) were added to the polymerization tube, dissolved in 1 mL of 1,4-dioxane, and deoxygenated by "freeze-pump-pump" three times. The mixture was then reacted in an oil bath at 85 °C for 12 h. After precipitation with ice-cold ether, the mixture was centrifuged, washed, and freeze-dried to obtain PEG-PNXC-Man (yield 57%).

[0054] (4) Synthesis of pH-responsive amphiphilic polymer (mPEG-PNXC): Methoxy polyethylene glycol functionalized chain transfer agent CTA-mPEG (0.04 mmol), N4C / N5C monomer (0.4 mmol) and azobisisobutyronitrile AIBN (0.008 mmol) were added to the polymerization tube, dissolved in 1 mL of 1,4-dioxane, and reacted in an oil bath at 85 °C for 12 h after deoxygenation; the precipitate was precipitated by ice-cold ether, centrifuged and washed, and freeze-dried to obtain mPEG-PNXC (yield 39%).

[0055] (5) Probe self-assembly: Add mPEG-PNXC (0.02 mmol), DOTA-Gd (0.02 mmol), and PEG-PNXC-Man (0.02 mmol) to a 100 mL three-necked round-bottom flask, then add 20 mL of methanol. Stir to fully mix and dissolve the components. Slowly add the dissolved organic phase dropwise to deionized water, and sonicate to ensure uniform dispersion of the system. After the addition is complete, transfer the system to a dialysis bag and dialyze at room temperature for 72 h using deionized water as the dialysis medium. Change the dialysis solution every 8 h during the dialysis. After removing the organic phase, the system obtained is the pH-responsive MRI probe targeting M2 macrophages.

[0056] (II) In vitro characterization of the probe

[0057] (1) Chemical structure characterization:

[0058] 1H NMR spectrum ( 1 ¹H-NMR: 10 mg each of N4C, N5C, mPEG-PN4C, DOTA-Gd, and the final probe sample were dissolved in 0.5 mL of deuterated chloroform and measured using a 400 MHz nuclear magnetic resonance spectrometer (Varian, VNMRS-400). Chemical shifts were measured using tetramethylsilane (TMS) as an internal standard. The characteristic ¹H-NMR peaks of N4C were: δ6.10 (s, 1H), 5.56 (s, 1H) (C=C double bond hydrogen), δ4.27–4.20 (m, 2H) (-O-CH2-), δ2.78 (t, 2H), 2.50 (t, J=7.5Hz, 4H) (-N-CH2-), consistent with the theoretical structure.

[0059] Electrospray ionization mass spectrometry (ESI-MS): 1 mg of each of the above samples was dissolved in methanol to prepare a 1 mg / mL solution. The solution was then analyzed using an electrospray ionization mass spectrometer (Thermo Fisher Scientific, Q Exactive). The [M+H] of N4C was measured. + Theoretical value 242.2115, measured value 242.2113, error -0.5ppm; [M+Na] of DOTA-Gd +The theoretical value was 833.3783, the measured value was 833.3790, and the error was 0.9 ppm, confirming that the chemical structures of each component are correct.

[0060] (2) Polymer buffering capacity test

[0061] The acidic pH titration curves of polymers with different N4C / N5C ratios show that ( Figure 2 (B) All three polymer samples exhibited typical pH-responsive titration characteristics. As the degree of protonation increased, the pH value of the solution gradually decreased, and a characteristic buffer zone appeared in the pH range of 4.0 to 6.0 (the core region of the acidic microenvironment of M2 macrophage lysosomes). This confirms that the tertiary amine groups in the polymer can achieve an acidic environment response through protonation-deprotonation equilibrium, providing a chemical basis for the specific activation of the probe in the M2 lysosomal microenvironment.

[0062] Focusing on the core target analysis at pH=4.9 (the characteristic pH of M2 macrophage lysosomes), the N4C:N5C=1:1.5 group exhibited the best targeting response characteristics: its titration curve equilibrium point precisely fell at pH=4.9, and the curve slope was the gentlest, indicating that this ratio of polymer can achieve a stable buffering response through efficient protonation at the characteristic pH of M2 lysosomes, accurately matching the pH triggering requirements of the target microenvironment. The N4C:N5C=1:1 group was second best, with a titration equilibrium point at pH=5.2, a deviation of 0.3 pH units from pH=4.9. The N4C:N5C=2:1 group had the weakest targeting response ability, with a titration equilibrium point at pH=5.6, a deviation of 0.7 pH units from pH=4.9.

[0063] The above results clearly confirm that the polymer exhibits the strongest pH titration response when the N5C ratio increases to N4C:N5C=1:1.5. Its titration equilibrium point precisely matches the characteristic pH of M2 macrophage lysosomes at 4.9, enabling efficient and specific protonation triggering in the target microenvironment. This provides a crucial pH response basis for the subsequent structural dissociation and signal activation of the probe within the cell. Conversely, an excessively high N4C ratio can cause the titration equilibrium point to deviate from the target pH, reducing the probe's targeted response specificity.

[0064] (3) Morphology and particle size characterization:

[0065] Transmission electron microscopy (TEM): The probe was dispersed in PBS (pH=7.4 or 4.9) at a concentration of 0.5 mg / mL. 5 μL was dropped onto a copper grid covered with an amorphous carbon film, allowed to air dry at room temperature, and then negatively stained with 2% (w / v) phosphotungstic acid solution for 30 s. Excess liquid was aspirated, and this process was repeated three times. Observation was performed using a transmission electron microscope (FEI, Tecnai G2 F20) at an accelerating voltage of 200 kV. In a physiologically neutral environment (pH=7.4), the probe maintained a complete, round micelle structure, appearing as uniformly shaped spherical particles without significant aggregation. However, in an acidic environment (pH=4.9, simulating the pH of M2 macrophage lysosomes), the micelles dispersed, appearing as scattered particles. Figure 2 C).

[0066] Dynamic light scattering (DLS): The probe was dispersed in PBS (pH 7.4 or 4.9) at a concentration of 0.1 mg / mL and sonicated for 2 min. The hydrated particle size was measured using a dynamic light scattering instrument (Malvin, Zetasizer Nano ZS) at 25℃ and a scattering angle of 90°. The average particle size of the probe was 94.83 ± 3.84 nm at pH 7.4, and dropped sharply to 0.68 ± 0.05 nm at pH 4.9, confirming that the probe underwent structural dissociation under acidic conditions. Figure 2 D).

[0067] (4) Stability characterization:

[0068] Physiological environment stability: The probe was dispersed in physiological saline (simulating the physiological ion environment in vivo) at a concentration of 0.1 mg / mL and incubated at 37℃. Samples were taken on days 0, 1, and 3, and particle size changes were measured by DLS. The average particle size was 82.78±2.36 nm on day 0, 85.01±2.32 nm on day 1, and 152.65±2.97 nm on day 3. The main peak around 80 nm still accounted for more than 75%, and there was no large-scale aggregation, which meets the requirements for short-term in vivo imaging. Figure 2 E).

[0069] (5) pH responsiveness characterization:

[0070] Relaxation rate response: formulation of different Gd 3+ Probe solutions at concentrations (0.05, 0.10, 0.15, 0.20 mM) were dissolved in PBS at pH 4.9 and pH 7.4, respectively. T1 mapping scans were performed using a 3.0T MRI scanner (Siemens, MAGNETOM Prisma) with the following parameters: TR = 24.00 ms, TE = 2.95 ms, NEX = 3 times, slice thickness = 2.0 mm, FOV = 130 mm × (130 mm × 75.0%). The T1 relaxation time was calculated using workstation software, and the ratio of 1 / T1 to Gd... 3+Concentration was plotted, and the slope represents the T1 relaxation rate (r1). The results showed significant differences in probe signal at different pH levels: at pH 4.9, the T1-weighted signal increased with increasing Gd content. 3+ The signal increased significantly with increasing concentration, while at pH 7.4, the signal changed less with concentration. Figure 2 F). Quantitative analysis showed that the probe's relaxation rate increased from 8.17 mM at pH=7.4. -1 s -1 The concentration rose to 11.71 mM at pH 4.9. -1 s -1 The increase reached 43.3% ( Figure 2 G). The core mechanism of this change lies in the fact that, in a physiologically neutral environment, the probe micelle structure will transfer Gd. 3+ Encapsulation restricts its interaction with water molecules, creating a "signal-off" state; in acidic environments, protonation of pH-sensitive groups triggers micelle dissociation, Gd 3+ By fully exposing the image to water and allowing it to freely exchange with water molecules, the "signal is turned on," and this intelligent regulation effectively enhances the imaging specificity.

[0071] (III) Cell-level validation methods for probes

[0072] 1. Macrophage isolation, culture, and phenotype induction

[0073] (1) Extraction of primary bone marrow-derived macrophages (BMDM): Four-week-old SPF-grade male SD rats were selected, and after being anesthetized with an overdose of isoflurane, they were euthanized by dislocation and disinfected by soaking in 75% ethanol for 10-30 min. The femur and tibia of the rats were separated in a biosafety cabinet, and the muscle fascia tissue was removed. The bone cavity was rinsed three times with PBS containing 10% penicillin and antibiotics, and the cell fluid was collected. After filtering through a 70 μm filter, the cells were centrifuged, and red blood cell lysis buffer was added. The mixture was allowed to stand at room temperature for 5 min. The process was repeated twice, and the supernatant was discarded after centrifugation. The precipitated cells were seeded in a complete culture medium containing recombinant rat macrophage colony-stimulating factor (M-CSF) and cultured in a 37℃, 5% CO2 incubator. Half of the culture medium was added after 3 days, and adherent M0 macrophages were obtained after 7 days.

[0074] (2) M1 / M2 macrophage induction: M0 macrophages were digested and collected, and seeded at an appropriate density in 6-well plates or confocal dishes; medium containing 20 ng / mL interferon-gamma (IFN-γ) and 100 ng / mL lipopolysaccharide (LPS) was added, and the cells were cultured for 24 h to induce M1 macrophages; medium containing 20 ng / mL interleukin-4 (IL-4) and 20 ng / mL interleukin-13 (IL-13) was added, and the cells were cultured for 48 h to induce M2 macrophages. Figure 3 As shown in Figure A.

[0075] (3) Phenotypic identification: Macrophage phenotype was verified by flow cytometry. Induced M1 and M2 macrophages were digested and collected, washed twice with PBS, and incubated at room temperature for 10 min with Fc blocking agent. Antibodies against CD11b (a universal macrophage marker), CD86 (an M1-specific marker), and CD206 (an M2-specific marker) were added respectively, and incubated at 4°C for 30 min. After washing with PBS, flow cytometry was used to detect the phenotype of M1 macrophages (CD86). + The proportion of M2 macrophages (CD206) is ≥75%. + The proportion is ≥85%, which meets the experimental requirements.

[0076] 2. Cytotoxicity verification

[0077] (1) Cell seeding: M1 and M2 macrophages in logarithmic growth phase were digested with 0.25% trypsin and seeded at a concentration of 1×10⁻⁶ cells / cells. 4 Cells / well were seeded at a density of 96 wells and cultured at 37°C with 5% CO2 for 24 hours until the cells were fully adhered.

[0078] (2) Probe treatment: Prepare different Gd 3+ Probe solutions at concentrations (0, 0.0125, 0.025, 0.05, 0.10 mM) were filtered through a 0.22 μm filter membrane and then added to 96-well plates for co-incubation with cells for 24 h. Complete culture medium without probes was set up as a control group.

[0079] (3) Toxicity test: After incubation, the cells were washed twice with PBS, and fresh culture medium containing 10 μl of CCK-8 reagent was added to each well. Incubation was continued for 2 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Cell viability was calculated as (mean OD value of experimental group / mean OD value of control group) × 100%, verifying the biocompatibility of the probe within its effective concentration range. The results showed that Gd... 3+ At concentrations ranging from 0 to 0.05 mM, the viability of both M1 and M2 macrophages remained above 90%. When the concentration increased to 0.1 mM, the viability of both macrophage phenotypes decreased slightly, but remained above 90%. Figure 3 (BC). This confirms that the probe has no significant cytotoxicity within the commonly used concentration range (0-0.1 mM) in in vitro experiments. This meets the cytotoxicity requirements for subsequent experiments.

[0080] 3. Cell targeting validation

[0081] (1) Preparation of fluorescently labeled probes: Fluorescently labeled targeted probes MR-CAs and non-targeted probes NMR-CAs were prepared separately. 0.1 mg FITC was dissolved in 1 mL of deionized water and mixed with 1 mg of probe in the dark. The mixture was stirred magnetically at room temperature for 24 h. After centrifugation and washing 2-3 times, the mixture was resuspended in PBS and stored at 4 °C in the dark for later use.

[0082] (2) Cell incubation: M1 and M2 macrophages were seeded into confocal dishes with glass bottoms (2-3×10⁻⁶ cells / mL). 5 Cells (cells / plate) were induced to mature using the method described above and then divided into three groups. The M1, M2, and M2 cell groups were respectively replaced with culture media containing fluorescently labeled MR-CAs, MR-CAs, and NMR-CAs, and incubated at 37°C for 2 hours.

[0083] (3) Fluorescence observation: After incubation, cells were washed three times with PBS to remove unbound probes, fixed with 4% paraformaldehyde for 15 min, and incubated with DAPI staining solution at room temperature for 3 min. Intracellular fluorescence distribution was observed using a laser confocal microscope, and the fluorescence signal intensity was quantitatively analyzed using ImageJ software to compare the differences in probe uptake between M1 and M2 macrophages. Results showed that after incubation with the targeting probes (MR-CAs), the intracellular fluorescence signal intensity of M2 macrophages was significantly higher than that of the M1 macrophage group (…). Figure 3 D). ImageJ quantitative analysis showed that the fluorescence signal intensity of the M2 macrophage + MR-CAs group (10.61±0.36) was higher than that of the M1 macrophage + MR-CAs group (8.01±0.13), and the difference was statistically significant (P < 0.001). The fluorescence signal intensity of the M2 macrophage + MR-CAs group was higher than that of the M2 macrophage + NMR-CAs group (9.68±0.29), and the difference was statistically significant (P < 0.05). Figure 3 E).

[0084] 4. Verification of lysosomal colocalization

[0085] (1) Cell treatment: M2 macrophages were seeded in confocal dishes and induced to mature. Then, fluorescently labeled probes were added and incubated at 37°C for 2 h. After washing with PBS 3 times, 100 nM lysosome-specific labeling probe (Lysotrack-Red) was added and incubated for another 30 min. Hoechst 33342 staining solution was used to incubate in the dark for 5 min to label the cell nuclei.

[0086] (2) Confocal imaging: Laser confocal microscopy showed that the MR-CAs probe (green fluorescence) and the lysosomal labeling probe Lysotrack-Red (red fluorescence) exhibited significant overlap in distribution within M2 macrophages. Figure 3F). A linear region was plotted within the cell field of view, and co-localization analysis was performed on the red and green fluorescent signals along this line segment. Quantitative analysis using ImageJ software confirmed that the two fluorescent signals had a high degree of overlap, indicating that the MR-CAs probe and lysosomes have good co-localization characteristics, further demonstrating that after the probe is taken up by M2 macrophages, it is ultimately mainly enriched and localized in lysosomes. Figure 3 G).

[0087] (iv) Application of probes in early assessment of tendon-bone healing after rotator cuff repair surgery

[0088] 1. Animal Model Establishment: SPF-grade SD rats were selected, anesthetized with isoflurane, fixed in a supine position, and shaved and disinfected. An incision was made along the long axis of the humerus, and the tissue was separated to expose the supraspinatus tendon. The supraspinatus tendon was transected at its insertion point at the greater tubercle of the humerus and sutured and fixed using a modified Mason-Allen method to construct a rotator cuff injury repair model. According to the intervention method, the rats were divided into a sham surgery group (only the rotator cuff was exposed, without tendon transection and repair), a simple suture group (only tendon suture and fixation), and a suture + PRP gel group (PRP gel was applied to the tendon-bone interface after suturing).

[0089] 2. PRP gel preparation: Blood was collected from rats via intracardiac puncture and mixed with citrate phosphate buffer. The mixture was centrifuged at 500g, 4℃ for 10 min, and the supernatant plasma was collected. This supernatant was then centrifuged at 2200g, 4℃ for 10 min, and the precipitate was collected as PRP. The PRP (platelet concentration 1.5 × 10⁻⁶) was then... 9 Mix PRP solution (5000U / mL) with thrombin solution (5000U / mL) at a volume ratio of 1:1, and let stand at room temperature for 5 minutes until solidification to obtain PRP gel.

[0090] 3. Probe Injection and MRI Imaging: Two weeks post-surgery, rats were anesthetized with intraperitoneal injection of afodin, and 50 μL of the probe (Gd concentration 0.05 mM / kg) was injected locally into the shoulder joint. T1WI scans were performed using a 3.0T MRI scanner (MAGNETOM Prisma) at pre-injection and 30, 60, and 180 minutes post-injection. Scanning parameters were: TR = 950 ms, TE = 79.0 ms, NEX = 4.0 fractions, baseline resolution = 320, slice thickness = 0.60 mm, FOV = 87 mm × (87 mm × 58.8%), acquisition matrix = 188 × 320, and scan bandwidth = 372 Hz / pixel. Utilizing the sub-millimeter spatial resolution of 3.0T MRI, deep tendon-bone interface structures can be clearly displayed, overcoming the limitations of optical imaging (penetration depth < 1 cm) and nuclear medicine imaging (spatial resolution 1-2 mm), achieving simultaneous acquisition of anatomical and molecular-level information. MRI images and pseudo-color images are shown below. Figure 4 A and Figure 4 D.

[0091] 4. Imaging Data Analysis: Images were processed using MR Body diffusion (1.4.0) software. The signal intensity (SI) of the supraspinatus tendon, the SI of the supraspinatus muscle, and the standard deviation (SD) in air were measured. CNR was calculated as (SI supraspinatus tendon - SI muscle) / SD air. ΔCNR was the difference between the CNR after injection and the CNR before injection. The peak value of ΔCNR was used as a quantitative indicator of M2 macrophage enrichment. T1-weighted signal quantification results showed ( Figure 4 B), T1 MRI images showed that the signal values ​​of the supraspinatus tendon (yellow arrow) at the supraspinatus muscle interface (white box) of the humerus were all low before the injection of the three probes. The signal values ​​of each group reached their peak 30 minutes after probe injection and then gradually decreased. Compared with baseline, the PRP group showed the largest increase in peak signal, with a T1-weighted signal intensity of 307.43±9.88, which was higher than that of the Control group (288.6±7.37) and the Sham group (261.6±5.47), and the differences were statistically significant (P<0.01). The T1-weighted signal intensity of the Control group was higher than that of the Sham group, and the difference was statistically significant (P<0.001). To rigorously compare imaging results, at 30 min post-injection, the ΔCNR of the PRP group (21.84±4.25) was higher than that of the Control group (17.27±2.64) and the Sham group (11.34±1.70), with a statistically significant difference compared to the Sham group (P=0.001), but no statistically significant difference compared to the Control group (P>0.05). At 60 min post-injection, the ΔCNR of the PRP group (17.92±0.90) was higher than that of the Control group (12.69±5.26) and the Sham group (5.21±0.69), with statistically significant differences compared to both (P<0.05). At 30 min and 60 min post-injection, the ΔCNR of the Control group was higher than that of the Sham group, with statistically significant differences (P=0.022 and P=0.008). At 180 min post-injection, the overall differences among the three groups were not significant, and there were no statistically significant differences between the groups (P>0.05). Immunohistochemical staining results showed ( Figure 4 At 2 weeks post-surgery, the relative positive area of ​​CD206, a marker of M2 macrophages at the tendon-bone interface, in the PRP group was 2.55±0.54, which was higher than that in the Control group (1.48±0.40, P<0.05) and higher than that in the Sham group (0.16±0.08, P<0.01). (Control group: 1.48±0.40)

[0092] The ratio was higher than that in the Sham group (0.16±0.08, P<0.01). The ratio among the three groups was consistent with the inter-group trend of MRI imaging results.

[0093] 5. Verification of healing outcome: Eight weeks post-surgery, rats were sacrificed and tissue samples were collected for HE staining, Masson staining, and Safranin-Fix-Green (SOFG) staining. Histological staining results are shown in the image below. Figure 5 AC. The healing quality was assessed using a tendon-bone healing histological scoring system, and the collagen area ratio and fibrocartilage area ratio were calculated using ImageJ software. Biomechanical testing was performed using an electronic universal testing machine to detect maximum load and stiffness. The correlation between the peak ΔCNR and histological and biomechanical parameters was analyzed to verify the prognostic predictive value of the probe. HE staining results showed ( Figure 5 (A) At 4 weeks post-surgery, the PRP group had more lacunar cells at the rotator cuff tendon-bone interface than the Control group; at 8 weeks post-surgery, the PRP group had a more compact and orderly arrangement of collagen fibers than the Control group. Histological scoring results showed ( Figure 5 D), at four weeks postoperatively, the histological score of PRP at the tendon-bone interface was 5.00±1.16, slightly higher than the Control group (3.75±0.98, P>0.05). At eight weeks postoperatively, the histological score of PRP at the tendon-bone interface was 8.00±0.82, higher than the Control group (6.75±0.96, P>0.05). At both four and eight weeks postoperatively, both groups were lower than the Sham group (four weeks postoperatively: 11.50±1.00, eight weeks postoperatively: 11.75±0.50). SOFG staining results showed ( Figure 5 B), four weeks post-surgery, the PRP group showed an increase in the amount of cartilage at the rotator cuff tendon-bone healing interface compared to the Control group, with an orderly arrangement, and approached normal tissue at eight weeks post-surgery. Quantitative analysis results showed ( Figure 5 At four weeks post-surgery, the relative cartilage area in the PRP group was 0.43±0.04, significantly higher than that in the Control group (0.31±0.06). At eight weeks post-surgery, the relative cartilage area in the PRP group was 0.64±0.03, significantly higher than that in the Control group (0.53±0.04). Both groups showed lower relative cartilage areas at four and eight weeks post-surgery compared to the Sham group (four weeks: 0.79±0.06, eight weeks: 0.84±0.03). Masson staining results showed ( Figure 5 C), four weeks post-surgery, the collagen fibers at the rotator cuff tendon-bone healing interface were more orderly arranged in the PRP group than in the Control group, and increased in number at eight weeks post-surgery. Quantitative analysis results showed ( Figure 5At four weeks post-surgery, the relative collagen area in the PRP group was 0.44±0.02, slightly higher than that in the Control group (0.42±0.03), but the difference was not statistically significant (P<0.05). At eight weeks post-surgery, the relative collagen area in the PRP group was 0.69±0.08, higher than that in the Control group (0.55±0.03), and the difference was statistically significant (P<0.05). At both four and eight weeks post-surgery, both groups were lower than the Sham group (four weeks post-surgery: 0.89±0.06, eight weeks post-surgery: 0.90±0.06). Biomechanical testing results showed ( Figure 5 At four weeks post-operation, the maximum load at the bone insertion point of the rotator cuff tendon in the PRP group was 9.97±3.03, which was higher than that in the Control group (7.42±1.45, P>0.05) and lower than that in the Sham group (17.13±1.55). At eight weeks post-operation, the maximum load at the bone insertion point of the rotator cuff tendon in the PRP group was (13.97±5.39, P>0.05), which was higher than that in the Control group (9.28±3.77, P>0.05) and lower than that in the Sham group (17.32±4.55).

[0094] At four weeks post-surgery, the stiffness of the rotator cuff tendon bone insertion point in the PRP group was 1.56±0.79, which was higher than that in the Control group (1.25±0.37, P>0.05) and lower than that in the Sham group (5.25±0.99). At eight weeks post-surgery, the maximum load at the rotator cuff tendon bone insertion point in the PRP group was 2.84±1.29, which was higher than that in the Control group (2.15±0.96, P>0.05) and lower than that in the Sham group (5.98±0.87).

[0095] Correlation analysis (Table 1) showed that the peak ΔCNR at 2 weeks post-operation was significantly positively correlated with the histological score at 8 weeks post-operation (r=0.951, P<0.05) and the fibrocartilage area ratio (r=0.978, P<0.05). The peak ΔCNR in the simple suture group was also significantly positively correlated with the histological score (r=0.961, P<0.05), allowing for early prediction of healing outcomes. Based on imaging results, the effectiveness of different repair approaches could be differentiated. The peak ΔCNR in the PRP-assisted repair group was significantly higher than that in the simple suture group, suggesting that treatment can be optimized by supplementing with PRP, avoiding blind intervention.

[0096] Table 1 Correlation Analysis

[0097]

[0098] In summary, this invention demonstrates that pH-responsive MRI probes (MR-CAs) targeting M2 macrophages can non-invasively and specifically visualize M2 macrophages at the tendon-bone interface. Furthermore, their imaging parameters show a high correlation with some healing outcome indicators of tendon-bone healing—histological score and relative cartilage area—indicating that MR-CA imaging can predict the healing quality of rotator cuff repair surgery at an early stage. This study provides a non-invasive and precise molecular imaging tool for early efficacy assessment after clinical rotator cuff injury repair surgery, possessing significant translational application value. Besides assessment after rotator cuff repair surgery, this invention can be extended to other tendon-bone healing models such as anterior cruciate ligament reconstruction and Achilles tendon injury repair, and can also be used for M2 macrophage imaging in inflammation-related diseases, demonstrating strong versatility.

Claims

1. A pH-responsive MRI probe targeting M2 macrophages, characterized in that, The pH-responsive MRI probe contains a CD206 targeting group, a pH-responsive group, and a Gd group covalently linked by chemical bonds. 3+ Imaging group.

2. The pH-responsive MRI probe according to claim 1, characterized in that, The CD206 targeting group is derived from NH2-PEG. 5k -Man polymer.

3. The pH-responsive MRI probe according to claim 1, characterized in that, The pH-responsive group is derived from N4C monomer or N5C monomer.

4. The pH-responsive MRI probe according to claim 1, characterized in that, The Gd 3+ The imaging group is derived from DOTA-Gd.

5. A method for preparing a pH-responsive MRI probe targeting M2 macrophages, characterized in that, Includes the following steps: (1) Gd was prepared by organic synthesis. 3+ Imaging groups, targeting polymers, and pH-responsive amphiphilic polymers are used as functional components; (2) The above functional components are mixed by the self-assembly method of "organic phase dissolution-aqueous phase dispersion-dialysis to remove impurities" to form a spherical probe with uniform structure, which is a pH-responsive MRI probe targeting M2 macrophages.

6. The preparation method according to claim 5, characterized in that: The targeted polymer in step (1) is PEG-PNXC-Man.

7. The preparation method according to claim 5, characterized in that: The pH-responsive amphiphilic polymer in step (1) is mPEG-PNXC.

8. The preparation method according to claim 5, characterized in that: The functional components in step (2) are mixed in an equimolar ratio.

9. The application of a pH-responsive MRI probe targeting M2 macrophages as described in claim 1 in the preparation of visualization detection products.

Images