A basic phosphatase-induced t1 and t2 signal-switching responsive magnetic resonance imaging contrast agent, and a preparation method and application thereof

Abstract

The application provides a kind of basic phosphatase induced T1 and T2 signal switching response type magnetic resonance imaging contrast agent and its preparation method and application, the response type magnetic resonance imaging contrast agent includes superparamagnetic nanoparticle, and polyethylene glycol phosphoric acid monoester modified to the surface of superparamagnetic nanoparticle;The superparamagnetic nanoparticle includes ferroferric oxide nanoparticle.The contrast agent of the application can realize T1 and T2 signal switching, is used for stem cell osteogenic differentiation monitoring, not only improves the detection sensitivity of magnetic resonance imaging to microstructure and function, also provides new tools and methods for stem cell treatment in biomedical research and clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a responsive magnetic resonance imaging contrast agent that induces alkaline phosphatase-induced T1 and T2 signal switching, its preparation method, and its application. Background Technology

[0002] Stem cell therapy has shown great potential in the field of bone tissue regeneration and repair. However, there is currently a lack of effective real-time, non-invasive, in vivo monitoring methods for the survival, distribution, and directed differentiation of transplanted stem cells into osteoblasts. Traditional histological analysis methods are destructive and cannot perform longitudinal tracking; optical imaging is limited by the depth of tissue penetration and is difficult to use in deep tissues or large animal models.

[0003] Magnetic resonance imaging (MRI) is considered an ideal in vivo tracing tool due to its advantages such as being radiation-free, having high soft tissue resolution, and unlimited penetration depth. Ultrasmall superparamagnetic iron oxide nanoparticles, as classic T2-negative (low signal) MRI contrast agents, have been widely used for cell labeling. However, simple T2 signal attenuation can easily be confused with low-signal areas such as hemorrhage and calcification, resulting in insufficient specificity. More importantly, traditional "always-on" contrast agents can only provide cell location information and cannot reflect the functional state of cells (such as differentiation state).

[0004] Alkaline phosphatase (ALP) is a key biomarker in the early osteogenic differentiation of stem cells and in bone-related diseases such as osteosarcoma. Currently, several ALP-responsive optical or magnetic resonance probes have been reported. However, most of these probes are complex in design or only produce a single signal change (e.g., enhancement only on T1 or decrement only on T2), with limited signal-to-noise ratio and sensitivity, and are difficult to achieve high-specificity, high-contrast imaging of ALP activity in the complex biological environment. In particular, intelligent probes capable of switching drastically different MRI signal modes based on ALP activity (e.g., from high signal on T1 to low signal on T2) have not yet been reported. This "signal switching" capability is crucial for clearly distinguishing undifferentiated from differentiated cells and improving diagnostic confidence.

[0005] Therefore, there is an urgent need to develop an MRI tracer probe that can be specifically activated by ALP and generate significant and easily identifiable signal pattern shifts, thereby enabling highly sensitive, highly specific, and non-invasive in vivo dynamic monitoring of biological processes such as osteogenic differentiation of stem cells. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an alkaline phosphatase-induced T1 and T2 signal switching responsive magnetic resonance imaging contrast agent, its preparation method, and its application. This invention provides an ALP-responsive MRI contrast agent that is rapid, sensitive, specific, and versatile, enabling T1 and T2 signal switching for monitoring osteogenic differentiation of stem cells. This not only promises to improve the sensitivity of MRI in detecting microstructure and function but also provides new tools and methods for stem cell therapy in biomedical research and clinical practice.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a responsive magnetic resonance imaging contrast agent that induces alkaline phosphatase-induced T1 and T2 signal switching, the responsive magnetic resonance imaging contrast agent comprising superparamagnetic nanoparticles and polyethylene glycol monophosphate modified on the surface of the superparamagnetic nanoparticles; the superparamagnetic nanoparticles comprising ferric oxide nanoparticles.

[0008] This invention modifies the surface of superparamagnetic nanoparticles with polyethylene glycol phosphate monoester, achieving a change from a monodisperse state to a clustered state through ALP response, thereby realizing the switching of T1 and T2 signals. It has good liquid phase dispersibility, storage stability and biocompatibility, and can be effectively applied to MRI imaging and monitoring of osteogenic differentiation of stem cells. It is expected to not only improve the sensitivity of MRI in detecting microstructure and function, but also provide new tools and methods for stem cell therapy in biomedical research and clinical practice.

[0009] Specifically, after contrast agents are applied to stem cells implanted in the body, stem cells can be traced. When stem cells differentiate into osteoblasts and produce a large amount of ALP, the contrast agent responds to it, switching from T1 to T2 signals, thereby tracing the osteoblast differentiation of stem cells and providing new tools and methods for stem cell therapy in biomedical research and clinical practice.

[0010] Preferably, the polyethylene glycol monophosphate has a structure as shown in Formula I, Formula II, or Formula III:

[0011] Formula I;

[0012] Formula II;

[0013] Formula III; Where n is an integer from 1 to 300, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 150, 200, 250 or 300, etc.

[0014] Preferably, the mass ratio of the superparamagnetic nanoparticles to polyethylene glycol monophosphate is 1:(5~10).

[0015] The specific point values ​​in (5~10) can be 5, 5.2, 5.5, 5.7, 6, 6.3, 6.5, 6.8, 7, 7.5, 8, 8.5, 9, 9.5 or 10, etc.

[0016] Preferably, the shape of the superparamagnetic nanoparticles includes any one or a combination of at least two of the following: spherical, disc-shaped, sheet-like, cubic, triangular, rod-shaped, or flower-shaped.

[0017] Preferably, the hydrated particle size of the responsive magnetic resonance imaging contrast agent before alkaline phosphatase response does not exceed 100 nm, for example, it can be 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm or 10 nm, etc.

[0018] The contrast agent of the present invention is in a monodisperse state before ALP response and belongs to the T1 type contrast agent.

[0019] Preferably, the hydrated particle size of the responsive magnetic resonance imaging contrast agent after alkaline phosphatase response is not less than 1000 nm, for example, it can be 1000 nm, 1200 nm, 1500 nm, 1700 nm, 2000 nm, 2300 nm, 2500 nm, 2800 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm or 5000 nm, etc.

[0020] The contrast agent of the present invention is in a clustered state after ALP response, with weakened T1 signal and enhanced T2 signal, belonging to the T2 type contrast agent.

[0021] Preferably, the superparamagnetic nanoparticles further include any one or a combination of at least two of gadolinium ions, manganese ions, or zinc ions.

[0022] Metal ions may be added to the contrast agent of the present invention to enhance imaging signals or to achieve diagnostic and therapeutic effects.

[0023] In a second aspect, the present invention provides a method for preparing a responsive magnetic resonance imaging contrast agent as described in the first aspect, the method comprising: Ferric chloride and oleate react 1 to generate ferric oleate; ferric oleate and oleic acid react 2 to generate superparamagnetic nanoparticles; the superparamagnetic nanoparticles and polyethylene glycol monophosphate react 3 in a dispersant, are mixed with a precipitant, and the precipitate is collected to obtain the responsive magnetic resonance imaging contrast agent.

[0024] In this invention, polyethylene glycol monophosphate undergoes ligand exchange with oleic acid on the surface of ferric oxide nanoparticles, and is linked to the surface of the nanoparticles through strong coordination.

[0025] Preferably, the molar ratio of ferric chloride to oleate is 1:(2~3).

[0026] The specific point values ​​in (2~3) can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3, etc.

[0027] Preferably, the molar ratio of ferric oleate to oleic acid is 1:(2~5).

[0028] The specific point values ​​in (2~5) can be 2, 2.2, 2.5, 2.7, 3, 3.3, 3.5, 3.8, 4, 4.5 or 5, etc.

[0029] Preferably, the temperature of reaction 1 is 65~75℃, for example, it can be 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃ or 75℃, etc.; the time is 2~5 h, for example, it can be 2 h, 2.2 h, 2.5 h, 2.7 h, 3 h, 3.3 h, 3.5 h, 3.8 h, 4 h, 4.5 h or 5 h, etc.

[0030] Preferably, the temperature of reaction 2 is 90~250℃, for example, 90℃, 100℃, 120℃, 150℃, 170℃, 200℃, 230℃ or 250℃; the time is 2~4 h, for example, 2 h, 2.2 h, 2.5 h, 2.7 h, 3 h, 3.3 h, 3.5 h, 3.8 h or 4 h.

[0031] Preferably, the temperature of the reaction 3 is 35~45℃, for example, it can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃, etc.; the time is 36~60 h, for example, it can be 36 h, 40 h, 42 h, 44 h, 46 h, 48 h, 50 h, 55 h or 60 h, etc.

[0032] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0033] Preferably, the oleate comprises sodium oleate and / or potassium oleate.

[0034] Preferably, the dispersant comprises any one or a combination of at least two of tetrahydrofuran, dichloromethane, toluene, or dimethylformamide.

[0035] Preferably, the precipitant comprises any one or a combination of at least two of diethyl ether, acetone, or methanol.

[0036] Preferably, the reaction 1 process further includes the addition of any one or a combination of at least two of gadolinium ions, manganese ions, or zinc ions.

[0037] Thirdly, the present invention provides the application of the responsive magnetic resonance imaging contrast agent as described in the first aspect in the preparation of stem cell osteogenic tracers.

[0038] The ALP-responsive magnetic resonance imaging contrast agent designed in this invention can achieve T1 and T2 signal switching, and can be effectively applied to the osteogenic differentiation tracking of stem cells. It can obtain the fate of stem cells after transplantation in vivo at the living animal level, which has extremely important scientific significance and research value, and has broad application prospects, such as understanding the role process of stem cells in vivo and basic research.

[0039] Compared with the prior art, the present invention has the following beneficial effects: This invention modifies the surface of superparamagnetic nanoparticles to achieve ALP response, enabling switching between T1 and T2 signals, thus improving the sensitivity and contrast of MRI imaging. It also exhibits good biocompatibility in vivo. Applicable to MRI imaging under various physiological conditions, it provides a new tool for clinical medicine, biomedical research, and drug delivery. It can monitor the location and osteogenic differentiation status of stem cells, and also offers new tools and methods for stem cell therapy in biomedical research and clinical practice. Attached Figure Description

[0040] Figure 1 High-resolution transmission electron microscopy image of the magnetic resonance imaging contrast agent prepared in Example 1; Figure 2 X-ray photoelectron spectrum of the magnetic resonance imaging contrast agent prepared in Example 1; Figure 3 Transmission electron microscopy images of the magnetic resonance imaging contrast agent prepared in Example 1 before and after ALP response, and the magnetic resonance imaging contrast agent prepared in Comparative Example 1 without ALP treatment. Figure 4 Hydration particle size diagrams of the magnetic resonance imaging contrast agents prepared in Example 1 and Comparative Example 1 before and after ALP response; Figure 5 A graph showing the change in longitudinal relaxation time over time during ALP incubation of the magnetic resonance imaging contrast agent prepared in Example 1. Figure 6 The graph shows the change in transverse relaxation time of the magnetic resonance imaging contrast agent prepared in Example 1 during ALP incubation. Figure 7Linear fitting diagram of longitudinal and transverse relaxation rates of the magnetic resonance imaging contrast agent prepared in Example 1 before ALP response; Figure 8 Linear fitting diagram of longitudinal and transverse relaxation rates of the magnetic resonance imaging contrast agent prepared in Example 1 after ALP response; Figure 9 T1-weighted solution imaging of the magnetic resonance imaging contrast agent prepared in Example 1 before and after ALP response; Figure 10 T2-weighted solution imaging images of the magnetic resonance imaging contrast agent prepared in Example 1 before and after ALP response; Figure 11 This is a cell viability assay image of bone marrow mesenchymal stem cells after co-incubation of the magnetic resonance imaging contrast agent prepared in Example 1 with bone marrow mesenchymal stem cells for 24 hours. Figure 12 T2-weighted images of undifferentiated stem cells and osteogenic differentiated stem cells labeled with magnetic resonance imaging contrast agent prepared in Example 1. Detailed Implementation

[0041] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] Compound 1: ; Compound 2: ; Compound 3: ; Compound 4: .

[0044] Example 1 This embodiment provides a magnetic resonance imaging contrast agent, the preparation method of which includes: 40 mmol of ferric chloride hexahydrate and 120 mmol of sodium oleate were placed in a 500 mL round-bottom flask, dissolved in 80 mL of ethanol, 60 mL of deionized water, and 140 mL of n-hexane, and the mixture was heated to 70 °C and reacted for 4 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel, the organic phase was collected, washed three times with deionized water, and excess liquid was removed by rotary evaporation to obtain ferric oleate.

[0045] 2 mmol of ferric oleate and 6 mmol of oleic acid were dissolved in 58 mmol of diphenyl ether. The mixture was heated to 90 °C under argon protection, degassed under vacuum for 2 h, and then heated to 250 °C at a rate of 10 °C / min for 0.5 h. After the reaction was complete, the mixture was rapidly cooled to 25 °C, 50 mL of acetone was added, and the precipitate was collected by centrifugation. The precipitate was dissolved in n-hexane and precipitated again with acetone. This process was repeated three times to obtain superparamagnetic nanoparticles of oleic acid.

[0046] 50 mg of superparamagnetic nanoparticles (oleic acid) and 250 mg of compound 1 were dissolved in 20 mL of tetrahydrofuran and reacted at 40 °C for 48 h. After the reaction was complete, the reaction solution was slowly added dropwise to 100 mL of ice-cold diethyl ether, and stirred vigorously for 10 min. The precipitate was collected. The precipitate was dissolved in deionized water and dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 35,000. The dialyzed solution was ultrafiltered, concentrated, and lyophilized to obtain the magnetic resonance imaging contrast agent (abbreviated as ESIONPs-PO-PEG).

[0047] Example 2 This embodiment provides a magnetic resonance imaging contrast agent, the preparation method of which includes: 50 mmol of ferric chloride hexahydrate and 100 mmol of sodium oleate were placed in a 500 mL round-bottom flask, dissolved in 60 mL of ethanol, 80 mL of deionized water, and 120 mL of n-hexane, and the mixture was heated to 75 °C and reacted for 2 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel, the organic phase was collected, washed three times with deionized water, and excess liquid was removed by rotary evaporation to obtain ferric oleate.

[0048] 1.5 mmol of ferric oleate and 7 mmol of oleic acid were dissolved in 50 mmol of diphenyl ether. The mixture was heated to 90 °C under argon protection, degassed under vacuum for 2 h, and then heated to 250 °C at a rate of 10 °C / min for 0.5 h. After the reaction was complete, the mixture was rapidly cooled to 25 °C, 50 mL of acetone was added, and the precipitate was collected by centrifugation. The precipitate was dissolved in n-hexane and precipitated again with acetone. This process was repeated three times to obtain superparamagnetic nanoparticles of oleic acid.

[0049] 25 mg of superparamagnetic nanoparticles (oleic acid) and 175 mg of compound 2 were dissolved in 10 mL of tetrahydrofuran and reacted at 35 °C for 60 h. After the reaction was complete, the reaction solution was slowly added dropwise to 100 mL of ice-cold diethyl ether, and stirred vigorously for 10 min. The precipitate was collected. The precipitate was dissolved in deionized water and dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 35,000. The dialyzed solution was ultrafiltered, concentrated, and lyophilized to obtain the magnetic resonance imaging contrast agent.

[0050] Example 3 This embodiment provides a magnetic resonance imaging contrast agent, the preparation method of which includes: 40 mmol of ferric chloride hexahydrate and 100 mmol of sodium oleate were placed in a 500 mL round-bottom flask, dissolved in 100 mL of ethanol, 40 mL of deionized water, and 160 mL of n-hexane, and the mixture was heated to 65 °C and reacted for 5 h. After the reaction was complete, the reaction mixture was transferred to a separatory funnel, the organic phase was collected, washed three times with deionized water, and excess liquid was removed by rotary evaporation to obtain ferric oleate.

[0051] 2.5 mmol of ferric oleate and 5 mmol of oleic acid were dissolved in 70 mmol of diphenyl ether. The mixture was heated to 90 °C under argon protection, degassed under vacuum for 2 h, and then heated to 250 °C at a rate of 10 °C / min for 0.5 h. After the reaction was complete, the mixture was rapidly cooled to 25 °C, 50 mL of acetone was added, and the precipitate was collected by centrifugation. The precipitate was dissolved in n-hexane and precipitated again with acetone. This process was repeated three times to obtain superparamagnetic nanoparticles of oleic acid.

[0052] 20 mg of superparamagnetic nanoparticles (oleic acid) and 200 mg of compound 3 were dissolved in 15 mL of tetrahydrofuran and reacted at 45 °C for 36 h. After the reaction was complete, the reaction solution was slowly added dropwise to 100 mL of ice-cold diethyl ether, and stirred vigorously for 10 min. The precipitate was collected. The precipitate was dissolved in deionized water and dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 35,000. The dialyzed solution was then ultrafiltered, concentrated, and lyophilized to obtain the magnetic resonance imaging contrast agent.

[0053] Comparative Example 1 This comparative example provides a magnetic resonance imaging contrast agent, which differs from Example 1 only in that compound 1 is replaced with an equal amount of compound 4, while the other raw materials and steps remain unchanged.

[0054] Test Example 1 The ESIONPs-PO-PEG from Example 1 was mixed with anhydrous ethanol and dropped onto an ultrathin carbon support film. Excess liquid was absorbed with filter paper and the film was allowed to dry naturally at 25°C until the solvent was completely evaporated. The ultrathin carbon support film was then placed under a transmission electron microscope for morphological observation.

[0055] like Figure 1 As shown, the responsive magnetic resonance imaging contrast agent provided by this invention is a typical monodisperse small-sized spherical nanoparticle with uniform particle size and an average diameter of about 3 nm. The lattice fringes of a single nanoparticle are clearly visible, indicating that it has good crystallinity, and the lattice spacing is about 0.25 nm, corresponding to the (311) interplanar spacing of spinel iron oxide.

[0056] Test Example 2 The ESIONPs-PO-PEG from Example 1 was ground into a uniform powder. 2 mg of the sample was attached to a double-sided carbon conductive adhesive, and the diffraction pattern of the sample was obtained by using the diffraction phenomenon of X-rays in the crystal.

[0057] like Figure 2 As shown, the surface of the responsive magnetic resonance imaging contrast agent provided by the present invention contains iron, oxygen, carbon and phosphorus elements, wherein the characteristic peak of phosphorus element indicates the successful modification of phosphate ester-terminated polyethylene glycol.

[0058] Test Example 3 The magnetic resonance imaging contrast agents (iron concentration of 1 mM) prepared in Examples 1-3 were mixed with pH 9.8 Tris-HCl buffer and 100 U / mL ALP enzyme solution (prepared with pH 9.8 Tris-HCl buffer), respectively, and reacted at 37°C for 12 hours. After the reaction, 5 μL of the liquid was dropped onto an ultrathin carbon support membrane, excess liquid was absorbed with filter paper, and the membrane was allowed to dry naturally at 25°C until the solvent was completely evaporated. The ultrathin carbon support membrane was then observed under a transmission electron microscope for morphological observation. The results showed that the magnetic resonance imaging contrast agents prepared in Examples 1-3 were monodisperse small-sized spherical nanoparticles before the ALP response, and formed larger clusters after the ALP response, indicating that the contrast agents have good ALP response capability.

[0059] The magnetic resonance imaging contrast agent (iron concentration of 1 mM) prepared in Comparative Example 1 was mixed with pH 9.8 Tris-HCl buffer. 5 μL of the liquid was dropped onto an ultrathin carbon support membrane. Excess liquid was absorbed with filter paper, and the membrane was allowed to dry naturally at 25°C until the solvent completely evaporated. The ultrathin carbon support membrane was then observed under a transmission electron microscope for morphological analysis. Figure 3 As shown, the ESIONPs-PO-PEG of Example 1 was in a monodisperse state before ALP response and in a clustered state after ALP response; however, the contrast agent prepared in Comparative Example 1 was in a clustered state before ALP incubation and could not achieve ALP response.

[0060] Test Example 4 The ESIONPs-PO-PEG (iron concentration of 1 mM) from Example 1 and the contrast agent prepared in Comparative Example 1 were mixed with pH 9.8 Tris-HCl buffer and 100 U / mL ALP enzyme solution (prepared with pH 9.8 Tris-HCl buffer), respectively, and reacted at 37°C for 12 hours. After the reaction, the dynamic light scattering hydrated particle size was measured using a laser particle size analyzer.

[0061] like Figure 4As shown, the contrast agent provided by this invention has a particle size of approximately 20 nm before ALP response and a particle size exceeding 1000 nm after ALP response, indicating that it can achieve a change from a monodisperse state to a clustered state through ALP response. However, the contrast agent prepared in Comparative Example 1 has a particle size greater than 1000 nm before ALP incubation and cannot achieve ALP response.

[0062] Test Example 5 ESIONPs-PO-PEG (1 mM iron concentration) from Example 1 was mixed with pH 9.8 Tris-HCl buffer and 100 U / mL ALP enzyme solution (prepared with pH 9.8 Tris-HCl buffer), and reacted at 37°C for 12 hours. After the reaction, 150 μL of solution was taken at time points of 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and the longitudinal relaxation time and transverse relaxation time were measured on a 0.5 T pulsed magnetic resonance imaging (MRI) scanner. A curve was plotted with reaction time on the x-axis and longitudinal or transverse relaxation time on the y-axis to observe the trend of relaxation time change of ESIONPs-PO-PEG during incubation.

[0063] like Figure 5 As shown, during ALP incubation, the longitudinal relaxation time gradually increases with time, but remains almost unchanged in the buffer solution, indicating that the T1 signal of the contrast agent weakens after ALP response.

[0064] like Figure 6 As shown, during incubation with ALP, the transverse relaxation time gradually decreased over time, but remained almost unchanged in the buffer solution, indicating that the T2 signal of the contrast agent was enhanced after the ALP response.

[0065] Test Example 6 Longitudinal and transverse relaxation rate tests were performed before the ALP response: ESIONPs-PO-PEG from Example 1 was prepared into solutions of different concentrations (0.25 mM, 0.50 mM, 1.0 mM, 2.0 mM, 4.0 mM). 150 μL of each solution was added to a sample cell, and T1 and T2 tests were performed on a 0.5 T pulsed NMR analyzer. Based on the relaxation times obtained for different concentrations, a linear fit was performed with 1 / T1 or 1 / T2 as the ordinate and iron concentration as the abscissa to obtain r1 and r2 before the ESIONPs-PO-PEG response.

[0066] like Figure 7 As shown, the longitudinal relaxation rate of the contrast agent before the ALP response is 2.2 mM. -1 s -1 The lateral relaxation rate is 3.0 mM. -1 s-1 This indicates that the contrast agent used before the response was a T1 type contrast agent.

[0067] Longitudinal and transverse relaxation rate tests were performed after ALP response: The ESIONPs-PO-PEG solution from Example 1 was mixed with 100 U / mL ALP enzyme solution to prepare samples of different concentrations (0.25 mM, 0.50 mM, 1.0 mM, 2.0 mM, 4.0 mM), and reacted at 37℃ for 12 h. 150 μL of each sample was taken and tested for T1 and T2 on a 0.5 T pulsed NMR analyzer. Based on the relaxation times obtained for different concentrations, a linear fit was performed with 1 / T1 or 1 / T2 as the ordinate and iron concentration as the abscissa to obtain r1 and r2 after the ESIONPs-PO-PEG response.

[0068] like Figure 8 As shown, the longitudinal relaxation rate of the contrast agent after the ALP response was 1.3 mM. -1 s -1 The transverse relaxation rate is 25.6 mM. -1 s -1 This indicates that the contrast agent after the response is a T2 type contrast agent, realizing the switching of T1 to T2 signals.

[0069] Test Example 7 ESIONPs-PO-PEG from Example 1 was prepared into solutions of different concentrations (0.25 mM, 0.50 mM, 1.0 mM, 2.0 mM, and 4.0 mM) using pH 9.8 Tris-HCl buffer and 100 U / mL ALP enzyme solution (prepared with pH 9.8 Tris-HCl buffer). After incubation at 37°C for 12 h, 150 μL of the solution was transferred to the sample cell, and then T1-weighted and T2-weighted solution imaging was performed on a 0.5 T pulsed NMR analyzer.

[0070] like Figure 9 As shown, the contrast agent in the T1-weighted solution after ALP response is darker, the T1 signal is weakened, and the T2 signal is enhanced.

[0071] like Figure 10 As shown, the contrast agent in the T2-weighted solution after the ALP response is darker, the T1 signal is weakened, and the T2 signal is enhanced, indicating that the contrast agent can switch from T1 to T2 signals through the ALP response.

[0072] Test Example 8 The effect of ESIONPs-PO-PEG co-incubated with bone marrow mesenchymal stem cells for 24 h in Example 1 on cell viability was detected by the CCK8 assay.

[0073] like Figure 11 As shown, even at iron concentrations as high as 2 mM, cell viability remained above 95%, indicating that the contrast agent had no significant cytotoxicity to stem cells.

[0074] Test Example 9 T2-weighted imaging of bone marrow mesenchymal stem cells was performed using a 0.5 T pulsed magnetic resonance imaging (MRI) scanner. (1) Blank group: undifferentiated bone marrow mesenchymal stem cells without any treatment; (2) Control group: undifferentiated bone marrow mesenchymal stem cells were co-cultured with culture medium containing 2 mM ESIONPs-PO-PEG from Example 1 at 37°C for 24 h; (3) OS group: bone marrow mesenchymal stem cells were induced to undergo osteogenic differentiation using osteogenic culture medium, and osteogenic differentiated bone marrow mesenchymal stem cells were co-cultured with culture medium containing 2 mM ESIONPs-PO-PEG from Example 1 at 37°C for 24 h.

[0075] like Figure 12 As shown, the image brightness of osteogenic differentiated stem cells labeled with contrast agent is much lower than that of undifferentiated cells, indicating that ALP produced by osteogenic differentiation of stem cells can trigger a T1 to T2 signal switch in the contrast agent, thereby causing the image to darken and enabling the monitoring of the osteogenic differentiation status of stem cells.

[0076] This invention illustrates, through the above embodiments, a responsive magnetic resonance imaging contrast agent for alkaline phosphatase-induced T1 and T2 signal switching, its preparation method, and its application. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials in the product, addition of auxiliary components, and selection of specific methods, all fall within the protection and disclosure scope of this invention.

[0077] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0078] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A responsive magnetic resonance imaging contrast agent that induces alkaline phosphatase-induced T1 and T2 signal switching, characterized in that, The responsive magnetic resonance imaging contrast agent includes superparamagnetic nanoparticles and polyethylene glycol monophosphate modified on the surface of the superparamagnetic nanoparticles; the superparamagnetic nanoparticles are ferric oxide nanoparticles. The polyethylene glycol monophosphate has a structure as shown in Formula I, Formula II or Formula III: Where n is an integer from 1 to 300.

2. The responsive magnetic resonance imaging contrast agent according to claim 1, characterized in that, The mass ratio of the superparamagnetic nanoparticles to polyethylene glycol monophosphate is 1:(5~10).

3. The responsive magnetic resonance imaging contrast agent according to claim 1, characterized in that, The hydrated particle size of the responsive magnetic resonance imaging contrast agent before alkaline phosphatase response does not exceed 100 nm. The responsive magnetic resonance imaging contrast agent has a hydrated particle size of not less than 1000 nm after alkaline phosphatase response.

4. The responsive magnetic resonance imaging contrast agent according to claim 1, characterized in that, The superparamagnetic nanoparticles also include any one or a combination of at least two of gadolinium ions, manganese ions, or zinc ions.

5. The method for preparing a responsive magnetic resonance imaging contrast agent according to any one of claims 1 to 4, characterized in that, The preparation method includes: Ferric chloride and oleate react 1 to generate ferric oleate; ferric oleate and oleic acid react 2 to generate superparamagnetic nanoparticles; the superparamagnetic nanoparticles and polyethylene glycol monophosphate react 3 in a dispersant, are mixed with a precipitant, and the precipitate is collected to obtain the responsive magnetic resonance imaging contrast agent.

6. The preparation method according to claim 5, characterized in that, The molar ratio of ferric chloride to oleate is 1:(2~3); The molar ratio of ferric oleate to oleic acid is 1:(2~5); The temperature of reaction 1 is 65~75℃, and the time is 2~5 h; The temperature of reaction 2 is 90~250℃, and the time is 2~4 h; The temperature of reaction 3 is 35~45℃ and the time is 36~60 h.

7. The preparation method according to claim 5, characterized in that, The oleate includes sodium oleate and / or potassium oleate; The dispersant includes any one or a combination of at least two of tetrahydrofuran, dichloromethane, toluene, or dimethylformamide; The precipitant includes any one or a combination of at least two of diethyl ether, acetone, or methanol.

8. The preparation method according to claim 5, characterized in that, The reaction 1 process also includes the addition of any one or a combination of at least two of gadolinium ions, manganese ions, or zinc ions.

9. The use of the responsive magnetic resonance imaging contrast agent according to any one of claims 1 to 4 in the preparation of stem cell osteogenic tracers.

Images