Janus structure based on TEMPO 1 H / 19 F dual-mode MRI contrast agent and a preparation method thereof

Abstract

The application belongs to the technical field of magnetic resonance contrast agents, and particularly relates to a TEMPO-based Janus-structure 1 H / 19 F bimodal MRI contrast agent and a preparation method thereof.A TEMPO-based 1 H / 19 F bimodal MRI contrast agent preparation method is disclosed, which uses sodium p-styrenesulfonate, potassium persulfate, styrene monomer, 2-(diisopropylamino)ethyl methacrylate and the like as raw materials, and adopts seed emulsion polymerization phase separation technology to synthesize a TEMPO-based MRI contrast agent.Under the condition of the presence of hydrophobic monomer TMPMA, the bimodal MRI contrast agent can form nanoparticles with Janus structure, and can effectively enhance the MRI imaging effect of the lesion site, has efficient 1 H / 19 F bimodal imaging performance, and shows excellent application prospects in the field of magnetic resonance imaging.

Description

Technical Field

[0001] This invention belongs to the field of magnetic resonance imaging (MRI) contrast agents, specifically relating to TEMPO with a Janus structure. 1 H / 19 F-modal MRI contrast agent and its preparation method. Background Technology

[0002] To date, many imaging techniques have been used for precise localization of lesions. However, single-modal imaging often fails to meet the needs of clinical practice, especially in the early diagnosis of cancer. Therefore, multimodal imaging, as a more promising imaging modality, has attracted widespread attention from researchers. Multimodal imaging utilizes appropriate imaging contrast agents to complement the advantages of different imaging techniques, thereby obtaining more accurate and reliable results. Current research has explored the combination of different instruments or the development of multimodal imaging techniques using the same imaging instrument. Among these, MRI is one of the most popular imaging diagnostic techniques in clinical practice. It can non-invasively visualize the internal anatomical structures of the human body without ionizing radiation, has deep tissue penetration capabilities, and is particularly suitable for high-resolution imaging of soft tissues. 1 1H MRI can generate detailed anatomical images, and the use of contrast agents can further improve the contrast between lesions and normal tissues, thus improving diagnostic accuracy. 1 The high background signal of the H nucleus greatly limits its applications such as quantitative tracking. 19 fMRI, due to its low biological background signal, makes... 19 fMRI contrast agents exhibit "hotspot" properties, enabling hotspot imaging and visualization of lesion sites, but they cannot perform whole-body imaging. Therefore, developing high-performance contrast agents is crucial. 1 H / 19 f MRI dual-modal imaging contrast agents can achieve precise imaging of lesion sites.

[0003] Currently, nanomaterials are considered a popular choice for MRI contrast agents due to their unique nanoscale correlation properties and ease of surface functionalization. 1 H / 19 Further development of fMRI dual-modal imaging requires nanoparticles with more complex functional structures that can simultaneously act as contrast agents in different MRI techniques, thereby enhancing imaging resolution and sensitivity. Currently, commonly used dual-modal contrast agents are generally prepared by combining paramagnetic metal chelates with a fluorine-containing moiety. Because the close proximity of these two components causes the PRE effect, the fluorine signal is weakened to almost nothing. Therefore, bioactive substances are typically designed to respond by breaking the connection between the two components to enhance the fluorine signal, thus achieving... 1 H / 19fMRI dual-modal imaging. However, such contrast agents are generally too reliant on response mechanisms and have low controllability. Therefore, it is necessary to explore more stable, reliable, and biocompatible multifunctional nanomaterials to achieve [fMRI dual-modal imaging]. 1 H / 19 fMRI dual-modal imaging.

[0004] Janus nanoparticles, also known as anisotropic nanoparticles, possess two or more spatial regions with differing physicochemical properties, allowing for the combination of different or even incompatible functions. By spatially separating these regions on their two faces, multiple properties are aggregated into a single structural unit to achieve synergistic effects, while maximizing the stability of the properties of each region and preventing mutual interference—something impossible with homogeneous nanoparticles. Therefore, applying Janus nanoparticles to… 1 H / 19 fMRI dual-modal imaging holds promise for fundamentally solving the problem of... 19 The fluorine-containing portion of f MRI is used for enhancement. 1 The problem of excessive influence from paramagnetic substances in hMRI.

[0005] In summary, in order to prepare more stable and efficient... 1 H / 19 Developing a dual-modal imaging contrast agent for fMRI and expanding its application in related biological fields such as lesion imaging and quantitative analysis of biological targets, as well as preparing a multifunctional nanoparticle with a Janus structure, is of great practical significance. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention provides an MRI contrast agent based on TEMPO with a Janus structure, achieving... 1 H / 19 f MRI dual-modal imaging can effectively enhance the MRI imaging effect of lesion sites and has important potential application value.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a Janus structure based on TEMPO. 1 H / 19 The preparation method of the F-mode MRI contrast agent includes the following steps:

[0009] S1. Sodium styrene sulfonate (NaVBs) and potassium persulfate (KPS) are dissolved in water respectively. The two solutions are combined and then styrene monomer (Sty), 2-(diisopropylamino)ethyl methacrylate (DIPAEMA) and crosslinking agent are added. Then, a polymerization reaction is carried out under an inert gas atmosphere to prepare seed nanoparticles (Ps-DIPAEMA).

[0010] S2. Seed nanoparticles (Ps-DIPAEMA) were dispersed in water to prepare solution A. After adjusting the pH to 8.5-9.6, deoxygenation treatment was carried out. Separately, 3-TSPM and TMPMA were dispersed in deoxygenated water to prepare solution B. Solution B was added to solution A, followed by deoxygenated KPS aqueous solution. After adjusting the pH to 9.0-10.0, polymerization reaction was carried out under an inert gas atmosphere to prepare Janus nanoparticles JNPs.

[0011] S3, Functional modification of nanoparticles:

[0012] S31. Disperse JNPs in an organic solvent, dilute epoxyphosphorus fluoride (POF) in the organic solvent, and add it dropwise to the JNPs solution. Then carry out a polymerization reaction under an inert gas atmosphere to prepare fluorine-containing JNPs, namely JNPs-F.

[0013] S32. Disperse JNPs-F in an organic solvent, first add 30-40% hydrogen peroxide and react at room temperature for a period of time, then add hydrogen peroxide in multiple batches to prepare oxidized fluorinated JNPs, i.e., JNPs-FO, which is a Janus structure based on TEMPO. 1 H / 19 F-modal MRI contrast agent.

[0014] The present invention 1 H / 19 The F-modal MRI contrast agent was prepared using a seed emulsion polymerization phase separation technique and functionalized at the nanoparticle scale. This was achieved through a two-step reaction involving the oxidation of a tertiary amine structure with hydrogen peroxide and the ring-opening linkage of a quaternary amine with epoxide phosphorus fluoride (POF). 1 H / 19 Modification of the properties of F-modal MRI contrast agents.

[0015] Preferably, the polymerization reaction in steps S1 and S2 is carried out at a temperature of 65-80°C for 20-36 hours.

[0016] Preferably, in step S1, the mass ratio of sodium p-styrene sulfonate (NaVBs) to potassium persulfate (KPS) is 27-35:10-15, and the concentration of sodium p-styrene sulfonate (NaVBs) in water is 1-2 mg / mL; the ratio of sodium p-styrene sulfonate (NaVBs), styrene, 2-(diisopropylamino)ethyl methacrylate (DIPAEMA), and crosslinking agent is 270-350 mg:5-7 mL:20-30 mL:250-300 μL.

[0017] Preferably, in step S1, the crosslinking agent includes, but is not limited to, p-styrene (DVB).

[0018] Preferably, in step S2, the concentration of solution A is 20-30 mg / 1-3 mL, the concentrations of 3-TSPM and TMPMA in solution B are 700-900 μL / 15-20 mL and 100-300 μL / 15-20 mL, respectively, and the volume ratio of solution A to solution B is 37-50:15-20.

[0019] Preferably, in step S2, the concentration of the KPS aqueous solution is 1-3%.

[0020] Preferably, in step S31, the polymerization reaction is carried out at a temperature of 65-80°C for 100-120 hours.

[0021] Preferably, in step S31, the concentration of JNPs in the organic solvent is 470-530 mg / 1-3 mL, the concentration of epoxyphosphorus fluoride in the organic solvent is 250-300 mg / 1-2 mL, and the volume ratio of the JNPs solution to the epoxyphosphorus fluoride solution is 2:1.

[0022] Preferably, in step S32, the hydrogen peroxide is added in multiple batches every 4-8 hours, and the reaction is carried out at room temperature for a total of 32-64 hours.

[0023] Preferably, the organic solvent in step S31 includes, but is not limited to, dimethyl sulfoxide (DMSO), and the organic solvent in step S32 includes, but is not limited to, ethanol.

[0024] The present invention also provides a TEMPO-based Janus structure prepared by the above preparation method. 1 H / 19 F-modal MRI contrast agent.

[0025] The MRI contrast agent prepared by this invention achieves... 1 H / 19 fMRI dual-modal imaging can effectively enhance the MRI imaging effect of lesion sites, and has high efficiency. 1 H / 19 F-mode imaging performance has broad application prospects.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] This invention discloses a TEMPO-based 1 H / 19 A method for preparing a dual-modal MRI contrast agent was developed. Using sodium p-styrene sulfonate, potassium persulfate, styrene monomer, and 2-(diisopropylamino)ethyl methacrylate as raw materials, seed nanoparticles (Ps-DIPAEMA) were synthesized via soap-free emulsion polymerization. Janus nanoparticles (JNPs) were then synthesized via a seed emulsion polymerization phase separation method using 3-TSPM and TMPMA. Functional modification at the nanoparticle scale was achieved through two steps: oxidation of the tertiary amine structure with hydrogen peroxide and ring-opening linkage of a quaternary amine with epoxide phosphorus fluoride (POF). 1 H / 19 Modification of the properties of the F-modal MRI contrast agent. This bimodal MRI contrast agent, in the presence of the hydrophobic monomer TMPMA, can form nanoparticles with a Janus structure, effectively enhancing MRI imaging of lesions and exhibiting high efficiency. 1 H / 19 The F-mode imaging performance shows excellent application prospects in the field of magnetic resonance imaging. Attached Figure Description

[0028] Figure 1 for 1 H / 19 F-mode MRI contrast agent synthesis technology roadmap;

[0029] Figure 2 Scanning electron microscopy and hydrated particle size data for seed nanoparticles;

[0030] Figure 3 Scanning electron microscopy and hydrated particle size data for Janus nanoparticles JNPs-FO;

[0031] Figure 4 The results validate the in vitro imaging performance of Janus nanoparticles JNPs-FO;

[0032] Figure 5 Cell compatibility of Janus nanoparticles JNPs-FO;

[0033] Figure 6 A schematic diagram illustrating the application of Janus nanoparticles JNPs-FO in in vivo MRI imaging;

[0034] Figure 7 The experimental results of in vivo tail vein injection imaging of JNPs-FO in animals;

[0035] Figure 8 The results are from an imaging experiment on intratumoral injection of JNPs-FO in animals. Detailed Implementation

[0036] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0038] Example 1: Synthesis of Janus-structured nanoparticles based on TEMPO

[0039] like Figure 1 As shown, the synthesis of this nanoparticle includes the following steps:

[0040] 1. Synthesis of Seed Nanoparticles

[0041] Seed nanoparticles (Ps-DIPAEMA) were synthesized via soap-free emulsion polymerization: 300 mg of sodium p-styrene sulfonate (NaVBs) and 135 mg of potassium persulfate (KPS) were dissolved in 300 mL of water, respectively. The two solutions were combined, and then 5.4 mL of styrene monomer (Sty), 21.6 mL of 2-(diisopropylamino)ethyl methacrylate (DIPAEMA), and 270 μL of crosslinked p-styrene (DVB) were added. Argon gas was then introduced for 20 minutes to remove oxygen, and the polymerization reaction was carried out for 24 h under argon protection at a temperature maintained at 70 °C to obtain Ps-DIPAEMA. Finally, the nanoparticles were washed sequentially with anhydrous ethanol and ultrapure water by centrifugation.

[0042] 2. Synthesis of Janus nanoparticles

[0043] Janus nanoparticles (JNPs) were synthesized using a seed emulsion polymerization phase separation method.

[0044] (1) Preparation of solution A: 500 mg of Ps-DIPAEMA was dispersed in 40 mL of ultrapure water, the pH of the solution was adjusted to 9.4 with NH3·H2O, and argon gas was introduced for 30 minutes to remove oxygen.

[0045] (2) Preparation of solution B: 800 μL of 3-TSPM (3-(trimethoxysilyl)propyl acrylate) and 200 μL of TMPMA (trimethylolpropane trimethacrylate) were dispersed in 17 mL of deoxygenated ultrapure water and ultrasonically emulsified for 2 minutes under ice bath conditions.

[0046] (3) Solution B was slowly added dropwise to solution A, and finally 2 mL of deoxygenated 1% KPS aqueous solution was added. The pH was adjusted to 9.4, and the polymerization reaction was carried out for 24 h under argon protection, with the reaction temperature maintained at 70 °C. JNPs nanoparticles were prepared and finally washed sequentially with anhydrous ethanol and ultrapure water by centrifugation. Figure 2 As shown in the scanning electron microscope and hydrated particle size, it can be seen that it has a Janus structure, a clear particle morphology, and no agglomeration.

[0047] 3. Synthesis of Janus nanoparticles with high imaging performance

[0048] Janus nanoparticles with high imaging performance were prepared through a two-step functional modification reaction:

[0049] (1) First step of modification reaction: Under anhydrous conditions, 500 mg of JNPs were dispersed in 2 mL of dimethyl sulfoxide (DMSO), and 280 mg of epoxyphosphorus fluoride (POF) was diluted in 1 mL of DMSO and added dropwise to the JNPs solution. After the addition was completed, a polymerization reaction was carried out for 110 h under argon protection, with the reaction temperature maintained at 70 °C, to prepare fluorinated JNPs (JNPs-F). Finally, the JNPs were washed by centrifugation with anhydrous ethanol and ultrapure water, respectively.

[0050] (2) Second step modification reaction: Disperse 500 mg of JNPs-F in 4 mL of anhydrous ethanol, add 4 mL of 30% hydrogen peroxide first, then add 4 mL of hydrogen peroxide every 6 h until the reaction is completed at room temperature for 48 h, to prepare oxidized fluorinated JNPs (JNPs-FO), and finally wash with ultrapure water by centrifugation. Figure 3 As shown in the scanning electron microscope and hydrated particle size, it can be seen that it exhibits a Janus structure with a clear particle morphology and no agglomeration.

[0051] Example 1: Characterization and Performance Testing of JNPs-FO

[0052] (1) JNPs-FO as 1 H / 19 Characterization of in vitro imaging performance of fMRI contrast agents

[0053] Using JNPs-FO 19F NMR was used to determine whether it was successfully linked to POF and whether it had a fluorine signal; JNPs-FO was used for EPR detection to determine whether it was successfully oxidized and whether it had TEMPO nitroxide radicals; JNPs-FO was prepared into solutions with concentrations of 1-40 mg / mL and then used as... 1 H / 19 fMRI in vitro imaging was used to verify its good imaging performance; at the same time, through 1 The relaxation rate r1 can be calculated from the in vitro H MRI images.

[0054] pass Figure 4 A is evident. 19 The presence of fluorine signal detected by 1F NMR proves that POF was successfully ring-opened and attached to the nanoparticles; Figure 4 The EPR spectrum of B indicates that the nanoparticles were successfully oxidized by hydrogen peroxide, and TEMPO nitrile radicals were present; T1-weighted MRI imaging of JNPs-FO was performed according to... Figure 4 C shows its 1 The h MRI signal exhibited concentration-dependent enhancement characteristics, and its longitudinal relaxation rate was calculated to be 0.31 mM based on the relaxation time at various concentrations. -1 s -1 ; Figure 4 C shows its 19 fMRI signals exhibit concentration-dependent enhancement characteristics and have good... 19 fMRI imaging performance, Figure 4 D shows that JNPs-FO has good performance. 1 H MRI imaging performance; by performing JNPs-FO 19 fMRI imaging.

[0055] (2) JNPs-FO as 1 H / 19 Cell compatibility of f MRI contrast agents

[0056] The cytotoxicity of JNPs-FO was detected using the MTT assay. Two cell types were selected: normal human umbilical vein endothelial cells (HUVECs) and tumor cells, mouse breast cancer cells (4T1). Cells were cultured in 96-well plates with JNPs-FO concentrations ranging from 100 μg / mL to 2 mg / mL. After 24 hours of culture, 20 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated at 37°C for 4 hours. Then, the liquid in each well was aspirated, the cells were washed once with PBS, and 150 μL of DMSO was added to each well. The plates were then incubated on a shaker for 5 minutes. Finally, the absorbance of the cells was measured at 490 nm using a multi-mode microplate detector.

[0057] Based on the results of the MTT test ( Figure 5 The survival rate of both cell types was around 80% under the condition of JNPs-FO concentration of 2 mg / mL, indicating that JNPs-FO has good cell compatibility.

[0058] (3) JNPs-FO as 1 H / 19 In vivo imaging performance verification of fMRI contrast agents

[0059] like Figure 6 As shown, JNPs-FO PBS solution (concentration 10 mg / mL, injection volume 100 μL) was injected into different mice via tail vein injection and intratumoral injection, respectively. The mice were then subjected to systemic and tumor site treatments after injection. 1 H / 19 FR imaging was used. Mice were imaged at 30 min, 1 h, 2 h, and 4 h after tail vein injection to observe the imaging effect of nanoparticles on the tumor site and compare the results with those before injection. Intratumoral injection was performed immediately to observe the imaging effect of nanoparticles on the tumor site and compare the results with those before injection.

[0060] Based on the experimental results of in vivo tail vein injection imaging ( Figure 7 , Figure 8 Compared to pre-injection levels, JNPs-FO showed signal enhancement at the tumor site at 30 min, 1 h, and 2 h post-injection. Imaging experiments involving intratumoral injection also showed significant signal enhancement at the tumor site immediately after JNPs-FO injection, compared to pre-injection levels. These results demonstrate that JNPs-FO possesses [the following properties / functionality] as [a specific therapeutic effect]. 1 H / 19 The potential of fMRI dual-modal contrast agents for tail vein injection imaging.

[0061] In summary, the MRI contrast agent with Janus structure based on TEMPO constructed in this invention achieves... 1 H / 19 fMRI dual-modal imaging can effectively enhance the MRI imaging effect of lesion sites and has important potential application value.

[0062] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A Janus-based TEMPO structure 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, Includes the following steps: S1. Sodium p-styrene sulfonate and potassium persulfate were dissolved in water, the two solutions were combined, and then styrene monomer, 2-(diisopropylamino)ethyl methacrylate and crosslinking agent were added. Then, a polymerization reaction was carried out under an inert gas atmosphere to prepare seed nanoparticles. S2. Disperse seed nanoparticles in water to prepare solution A. After adjusting the pH to 8.5-9.6, deoxygenate the solution. Separately, disperse 3-TSPM and TMPMA in deoxygenated water to prepare solution B. Add solution B to solution A and then add deoxygenated KPS aqueous solution. After adjusting the pH to 9.0-10.0, carry out polymerization reaction under an inert gas atmosphere to prepare Janus nanoparticles JNPs. S3, Functional modification of nanoparticles: S31. Disperse JNPs in an organic solvent, dilute epoxy phosphorus fluoride in the organic solvent, and add it dropwise to the JNPs solution. Then carry out a polymerization reaction under an inert gas atmosphere to prepare fluorinated JNPs, namely JNPs-F. S32. Disperse JNPs-F in an organic solvent, first add 30-40% hydrogen peroxide and react at room temperature for a period of time, then add hydrogen peroxide in multiple batches to prepare oxidized fluorinated JNPs, i.e., JNPs-FO, which is a Janus structure based on TEMPO. 1 H / 19 F-modal MRI contrast agent.

2. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, The polymerization reaction described in steps S1 and S2 is carried out at a temperature of 65-80°C for 20-36 hours.

3. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, In step S1, the mass ratio of sodium p-styrene sulfonate to potassium persulfate is 27-35:10-15, and the concentration of sodium p-styrene sulfonate in water is 1-2 mg / mL; the ratio of sodium p-styrene sulfonate, styrene, 2-(diisopropylamino)ethyl methacrylate and crosslinking agent is 270-350 mg:5-7 mL:20-30 mL:250-300 μL.

4. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, In step S1, the crosslinking agent includes p-styrene.

5. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, In step S2, the concentration of solution A is 20-30 mg / 1-3 mL, and the concentrations of 3-TSPM and TMPMA in solution B are 700-900 uL / 15-20 mL and 100-300 uL / 15-20 mL, respectively. The volume ratio of solution A to solution B is 37-50:15-20.

6. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, In step S2, the concentration of the KPS aqueous solution is 1-3%.

7. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, In step S31, the polymerization reaction is carried out at a temperature of 65-80 °C for 100-120 h.

8. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, In step S31, the concentration of JNPs in the organic solvent is 470-530 mg / 1-3 mL, the concentration of epoxyphosphorus fluoride in the organic solvent is 250-300 mg / 1-2 mL, and the volume ratio of the JNPs solution to the epoxyphosphorus fluoride solution is 2:

1.

9. A Janus structure based on TEMPO according to claim 1 1 H / 19 The preparation method of the F-mode MRI contrast agent is characterized by, In step S32, the hydrogen peroxide is added in multiple batches every 6-10 hours, and the reaction is carried out at room temperature for a total of 32-64 hours.

10. A Janus-based TEMPO material prepared by the preparation method according to any one of claims 1-9. 1 H / 19 F-modal MRI contrast agent.

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