A method for constructing and applying a radiolabeled magnetic T1-T2 co-modal MRI-PET imaging probe.

Abstract

The application belongs to the technical field of molecular imaging and biomedical materials, and particularly relates to a construction method of a radioactively labeled magnetic T1-T2 synergistic MRI-PET bimodal imaging probe and application thereof. The polyphenol substance is dissolved in methanol, the pH is adjusted, SPIO is dispersed in tetrahydrofuran, after mixing, the water-phase SPIO modified by the polyphenol is obtained. Then, the streptavidin is dissolved in water, the polydopa coating layer is formed by in-situ polymerization of the polydopa and is coupled with the streptavidin, the biotin-modified targeting ligand is combined, the targeted MRI probe is obtained, and finally the radioactive metal element such as Cu, 64 Cu, 68 Ga is efficiently chelated to obtain the T1-T2 synergistic MRI-PET bimodal probe, the probe has the advantages of strong stability, high hydrophilicity, good biocompatibility, high relaxivity, good tumor targeting, high sensitivity of PET for whole body quantification, high soft tissue contrast of MRI T1-T2 and high spatial resolution.

Description

Technical Field

[0001] This invention belongs to the field of molecular imaging and biomedical materials technology, specifically relating to a method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) holds a central position in clinical applications due to its excellent soft tissue contrast, high three-dimensional spatial resolution, and tissue penetration. However, its detection sensitivity is limited by factors such as molecular thermal motion and the limited number of excitable atoms, resulting in insufficient ability to identify biomarkers and small lesions. Furthermore, the imaging process faces limitations such as localized focused radiofrequency pulses and single-organ diagnostic guidance of signal acquisition coils, preventing simultaneous assessment of multi-organ functions and creating blind spots in the detection of early tumors and metastases. In contrast, positron emission tomography (PET) can capture molecular information in deep tissues with ultra-high sensitivity, rapidly acquiring whole-body functional imaging; however, its spatial resolution is limited by the degree of freedom of positron decay of the probe, failing to comprehensively and accurately reflect the physiological and structural characteristics of lesions. Combining two or more imaging methods can overcome the limitations of single-modal techniques, fully leveraging the inherent advantages of different imaging principles to acquire multiple signals of the same biological event to meet clinical needs. The first PET-MRI system was developed in 2010, significantly improving the accuracy and comprehensiveness of identifying complex lesions. However, current clinical PET-MRI detection relies on the separate use of two types of imaging probes, resulting in a lack of consistency in the in vivo distribution, pharmacokinetics, and metabolic pathways of the two imaging signals. This means that the signals do not originate from the same molecular target or the same lesion region, making it impossible to achieve precise spatiotemporal correspondence in imaging. Therefore, to fully leverage the advantages of PET-MRI, the core lies in developing efficient, safe, and stable dual-modality probes. The development process must consider the half-life and structural stability of the radionuclide, the performance of the magnetic unit, the surface modification, and the biocompatibility of the system. The rationality of the related preparation strategies determines the PET-MRI imaging effect, molecular recognition targeting, and clinical translation potential.

[0003] Current PET probes are mainly divided into two categories: one is covalently labeled systems, such as common radioactive... 18 F and 11 The combination of C and other radioactive elements with a support structure, especially those with short half-lives, requires highly selective and rapid reaction preparation conditions, along with complex purification processes and in-situ cyclotrons, which is not conducive to the preparation of dual-mode probes. In contrast, metal chelate system PET probes, such as... 64 Cu、 68Ga is linked to chelating molecules, the preparation process is convenient, and radioactive metal elements generally have long half-lives, making them widely used for antibody and nanoscale labeling. MRI probes are mainly paramagnetic Gd. 3+ The chelates and superparamagnetic ferrite nanoparticle SPIO system, when combined with PET functional components, require careful consideration of structural stability and magnetic relaxation properties. However, traditional Gd... 3+ While the paramagnetic core construction strategy offers advantages such as intuitive MRI-T1 imaging and clear contrast, it inevitably competes with radioactive metal nuclides in ligand binding reactions, leading to decreased labeling efficiency and imaging performance loss. Furthermore, bimetallic chelate products can alter key parameters such as rotation time and water exchange constant, causing uncontrollable changes in relaxation efficiency. Superparamagnetic iron oxide nanoparticles (SPIO), on the other hand, possess a stable crystal structure, and nuclide modification is unlikely to affect magnetic properties. Moreover, the high specific surface area of ​​the nanosystem facilitates stable radionuclide labeling, resulting in minimal mutual interference between dual-modal imaging functions. However, its clinical application faces challenges due to the "negative enhancement" effect generated by shortening the transverse relaxation time (T2) through magnetic field inhomogeneity, making image interpretation relatively difficult and susceptible to background signal interference. Functionalizing the SPIO interface molecular structure can alter imaging effects, obtaining synergistic MRI-T1 and T2 signals, potentially improving imaging accuracy.

[0004] Therefore, constructing a SPIO ligand interface with a defined structure to ensure the preparation of high-magnetic crystals, providing excellent SPIO dispersibility and magnetic properties to meet the requirements of MRI-T1 and T2 co-imaging, and simultaneously achieving stable and efficient radionuclide chelation capabilities has become a technical bottleneck. Furthermore, endowing PET-MRI dual-modal probes with specific targeting capabilities can significantly improve the imaging signal-to-noise ratio and enhance the sensitivity and accuracy of molecular-level diagnosis. Currently, the most common strategy is to connect targeting ligands (such as peptides, antibody fragments, etc.) to the particle surface through covalent coupling. However, this type of chemical coupling reaction typically suffers from low coupling efficiency, cumbersome operation steps, and loss of biological activity due to chemical modification, which also reduces batch consistency and clinical translatability of the product. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe and its application.

[0006] This invention utilizes the efficient coordination of polyphenol molecules with the Fe element in superparamagnetic iron(III) oxide (SPIO) to achieve a rapid phase transformation of nanoparticles from the organic phase to the aqueous phase, while simultaneously providing... 64 Cu、 68Radioactive metals such as Ga provide efficient chelation sites to meet the labeling requirements of PET. Furthermore, streptavidin (SA) is introduced, leveraging its high affinity for biotin to efficiently conjugate biotinylated antibodies to the SPIO surface under mild conditions. This constructs a modular connection platform with universal and replaceable targeting ligands, satisfying the need for probe-specific targeting in PET-MRI dual-modal imaging. The probe of this invention exhibits strong stability, high hydrophilicity, good biocompatibility, high relaxation rate, and good tumor targeting. It combines the advantages of high-sensitivity whole-body quantification in PET with high soft tissue contrast and high spatial resolution in MRI T1-T2 imaging, enabling a "whole-body quantitative screening—local fine-tuning" imaging pathway. This improves the reliability of cross-individual and cross-time point comparisons and pharmacokinetic / efficacy assessments, demonstrating excellent biocompatibility, targeting capabilities, and clinical translational potential.

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

[0008] The first aspect of this invention provides a method for constructing a metal-coordinated targeted T1-T2 MRI / PET dual-modal probe via ultrasound self-assembly, comprising the following steps:

[0009] Step S1: Dissolve the polyphenols in methanol to obtain solution A, and adjust the pH of solution A;

[0010] Step S2: Dissolve iron(III) oxide in tetrahydrofuran to obtain solution B;

[0011] Step S3: Mix solution A and solution B, sonicate to produce a precipitate, remove the supernatant, add deionized water to redissolve, and obtain polyphenol-modified SPIO, i.e., solution C;

[0012] Step S4: Dissolve dopamine hydrochloride in deionized water, then mix it with solution C to obtain mixed solution D;

[0013] Step S5: Dissolve streptavidin in deionized water to obtain solution E, and adjust the pH of solution E;

[0014] Step S6: Using the ultrasonic self-assembly method, solution D is ultrasonically added to solution E to obtain solution F at room temperature, and the reaction is immediately terminated.

[0015] Step S7: Dialyze the solution F after the reaction has ended at room temperature to obtain solution F containing coordinated Fe-based nanoparticles;

[0016] Step S8: Mix the dialyzed solution F with the biotinylated targeting peptide and incubate to obtain solution G;

[0017] Step S9: Mix the radioactive metal element with sodium acetate buffer solution to obtain solution H;

[0018] Step S10: Mix solution G and solution H in equal volumes, then incubate to obtain radiolabeled product solution J;

[0019] Step S11: The radiolabeled product solution J was purified and separated by centrifugation to obtain the radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe.

[0020] The highly efficient metal-coordinated targeted PET / MRI bimodal probe constructed in this invention is simple, rapid, and under mild conditions. In this reaction system, polyphenols (catechins, quercetin, pyrogallol, etc.) are first dissolved in methanol and the pH is adjusted to 8-10. Simultaneously, superparamagnetic iron oxide nanoparticles (SPIO) are dispersed in tetrahydrofuran. The two are then rapidly mixed, utilizing the efficient coordination and anchoring effect between the phenolic hydroxyl groups in the polyphenol molecules and the Fe sites on the SPIO surface to drive a rapid phase transformation of the nanoparticles from the organic phase to the aqueous phase, obtaining polyphenol-modified aqueous SPIO. Then, amino acids (SA) are dissolved in the aqueous system, and an in-situ polymerization process of dopamine (DA) is introduced. Under mild conditions, a polydopamine (PDA) coating layer is formed, and SA is simultaneously immobilized / coupled, thus obtaining a targeted MRI probe with a bio-coupling interface. Finally, direct radiolabeling technology is used to target the polyphenol coordination sites. 64 Cu、 68 Highly efficient chelation and stable labeling of radioactive metal elements such as Ga were used to obtain a single-probe PET / MRI dual-modal probe. During the construction of this probe, polyphenol molecules achieved rapid aqueous phase conversion and interfacial stabilization of nanoparticles through strong coordination with Fe on the SPIO surface, while also... 64 Cu、 68 Radioactive metals such as Ga provide high-affinity chelating sites to meet the needs of PET labeling; further, by introducing SA, its ultra-high affinity with biotin can be used to efficiently couple biotinylated antibodies / targeting ligands to the SPIO surface under mild conditions, thereby constructing a universal, modular, and target-ligand-replaceable connection platform, which facilitates rapid expansion and application for different targets.

[0021] Further, in step S1, the polyphenolic substance is at least one of quercetin, emodin, glycyrrhizic acid ammonium salt, catechin, and pyrogallol, and adjusting the pH of solution A means adjusting the pH value to 8-10.

[0022] Further, the mass ratio of the polyphenolic substance to iron oxide is (40~80):(20~40), the mass ratio of the polyphenolic substance to dopamine hydrochloride is (1.5~5):1, and the mass ratio of dopamine hydrochloride to streptavidin is (1~2):(1.5~3).

[0023] Furthermore, the concentration of streptavidin in solution E is (0.5-1) mg / mL, and the concentration of dopamine hydrochloride in solution D is (0.5-1) mg / mL.

[0024] Further, in step S8, if the biotinylated targeting peptide is a lyophilized powder, the lyophilized powder is dissolved in deionized water at a concentration of (0.5-1) mg / mL; the biotinylated targeting peptide is at least one of Biotin-cRGD, Biotin-cNGR, and Biotin-BBN; the molar ratio of streptavidin to the biotinylated targeting peptide is (4-10):(1-2).

[0025] Further, in step S9, the radioactive metallic element is 64 Cu、 68 Ga、 89 At least one of Zr; the sodium acetate buffer solution has a concentration of 0.1 M and a pH of 5.5; the radioactive metal element activity in the solution H is (1 ~ 10) mCi.

[0026] Furthermore, in step S7, the dialysis time is 24-78 hours.

[0027] Further, in step S8, the incubation refers to incubation at 4 °C overnight, and the solution G obtained from the incubation is used with an ultrafiltration tube of (10-100) kDa to remove unbound biotinylated target peptides.

[0028] Furthermore, in step S10, the incubation refers to incubation at 60 ℃ for 30 min.

[0029] A second aspect of the present invention provides a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe prepared by the above method, wherein the relaxation rate R1 of the probe is greater than 10 [Fe] mM -1 s -1 R² > 200 [Fe] mM -1 s -1 .

[0030] The third aspect of this invention provides an application of the above-mentioned radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe in magnetic resonance imaging (MRI), positron emission tomography (PET), in vivo tracing and pharmacokinetics / biodistribution studies, and targeted molecular imaging platforms.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] This invention achieves rapid phase transformation of nanoparticles from the organic phase to the aqueous phase through the strong coordination and anchoring effect between polyphenol molecules and Fe-based SPIO surface sites. Furthermore, it utilizes the in-situ self-polymerization of DA under mild conditions to form a PDA coating layer and immobilize SA, thereby constructing a magnetic nanoplatform with a universal coupling interface. Simultaneously, relying on the high affinity of polyphenol / phenolic hydroxyl sites for radioactive metal ions, direct radiolabeling is employed to achieve... 64 Cu、 68 The efficient chelation and stable loading of radionuclides such as Ga ultimately yields a single-probe form of a targeted PET / MRI dual-modality probe. Specifically, this invention has the following advantages:

[0033] (1) Based on polyphenol coordination-driven interface modification and phase transformation (steps S1~S2), this invention forms a PDA coating layer and fixes SA through DA in situ polymerization (steps S4~S7), thereby achieving tight integration and structural stabilization among multiple components, reducing the complex operation and risk of bioactivity damage caused by traditional multi-step covalent modification, and improving the stability and biosafety of the system.

[0034] (2) Compared with the common approach of "grafting chelating agents such as DOTA / NOTA first and then labeling" in the prior art, the present invention utilizes the multi-site coordination environment provided by polyphenols (steps S1~S3) to directly label the polyphenols. 64 Cu、 68 Coordination chelation with radioactive metals such as Ga (steps S9-S10) reduces the chemical modification steps and intermediate purification processes of the material, significantly improving the convenience and operability of labeling.

[0035] (3) The polyphenol-metal coordination sites of the present invention have high density and strong binding force, which can promote the rapid capture and stable binding of radioactive metals; combined with centrifugation purification / separation (step S11), it is beneficial to obtain higher radiochemical purity and reduce the proportion of free nuclides, thereby improving the signal-to-noise ratio and specificity of in vivo imaging.

[0036] (4) In this invention, SA is introduced as a bridging module (steps S5~S7). With the help of the high affinity of SA-Biotin, Biotin-cRGD can be efficiently coupled to the surface of nanoparticles under mild conditions (step S8). At the same time, this strategy can be extended to other biotinylated antibodies / peptides / small molecule ligands to achieve rapid adaptation and expansion of "the same nanoplatform - different targets".

[0037] (5) Polyphenol modification and PDA coating can significantly improve the hydrophilicity and interfacial stability of particles (steps S3-S7), reduce the occurrence of SPIO agglomeration and sedimentation in the aqueous phase, which is conducive to obtaining a stable and repeatable particle size distribution and performance, and facilitates subsequent dialysis to remove small molecule impurities to improve the purity of the system (step S7).

[0038] As can be seen, this invention is based on polyphenol coordination-driven SPIO aqueous phase conversion and direct radioactive metal labeling, combined with PDA in-situ coating and immobilization of SA to construct a modular targeting connection platform, thereby obtaining a targeted PET / MRI dual-modal probe that is easy to prepare, has mild conditions, strong stability, and scalable targeting ligands. It can be used for MRI imaging, PET imaging, PET / MRI fusion imaging, in vivo tracking and quantitative analysis, as well as related biomedical research and diagnostic applications, and has good application prospects. Attached Figure Description

[0039] Figure 1 In Example 1, [ 68 Flowchart of Ga-labeled SPIO@QDS-cRGD preparation.

[0040] Figure 2 In Example 1, [ 68 Transmission electron microscopy image of Ga-labeled SPIO@QDS-cRGD.

[0041] Figure 3 In Example 1, [ 68 Particle size stability diagram of Ga-labeled SPIO@QDS-cRGD.

[0042] Figure 4 In Example 1, [ 68 Comparison of particle size before and after freeze-drying of Ga-labeled SPIO@QDS-cRGD.

[0043] Figure 5 In Example 1, [ 68 Relaxation rate plot of Ga-labeled SPIO@QDS-cRGD.

[0044] Figure 6 In Example 1, [ 68 PET images of Ga-labeled SPIO@QDS-cRGD.

[0045] Figure 7 The particle size distribution is shown for the assembled particles in Examples 2-7.

[0046] Figure 8 For test case 1, [ 68 In vitro radiochemical stability testing of Ga-labeled SPIO@QDS-cRGD.

[0047] Figure 9 For test example 2 [ 68 MRI imaging of Ga-labeled SPIO@QDS-cRGD in a mouse model of liver metastases.

[0048] Figure 10 For test example 3 [ 68 Biocompatibility test results of Ga-labeled SPIO@QDS-cRGD in vivo. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] Example 1: MRI-PET Dual-Modal Imaging Probe [ 68 Preparation of Ga-labeled SPIO@QDS-cRGD

[0052] [ 68 The fabrication process of Ga-labeled SPIO@QDS-cRGD is shown in the figure below. Figure 1 As shown, the specific steps include:

[0053] Step S1: Dissolve quercetin in methanol to obtain solution A, and adjust the pH of solution A to 10;

[0054] Step S2: Dissolve iron(III) oxide in tetrahydrofuran to obtain solution B;

[0055] Step S3: Mix solution A and solution B (the mass ratio of quercetin to iron oxide is 2:1), sonicate to produce a precipitate, remove the supernatant, add deionized water to redissolve, and obtain polyphenol-modified SPIO, i.e., solution C;

[0056] Step S4: Dissolve dopamine hydrochloride in deionized water, then mix it with solution C (the mass ratio of quercetin to dopamine hydrochloride is 1.5:1) to obtain mixed solution D. The concentration of dopamine hydrochloride in mixed solution D is 0.5 mg / ml.

[0057] Step S5: Dissolve streptavidin in deionized water to obtain solution E with a concentration of 1 mg / ml, and adjust the pH of solution E to 9;

[0058] Step S6: Using the ultrasonic self-assembly method, solution D is ultrasonically added to solution E (mass ratio of dopamine hydrochloride to streptavidin is 2:3), and solution F is obtained at room temperature, and the reaction is immediately terminated.

[0059] Step S7: Dialyze the solution F after the reaction has ended at room temperature for 24 hours to obtain solution F containing coordinated Fe-based nanoparticles;

[0060] Step S8: Mix the dialyzed solution F with Biotin-cRGD (a lyophilized Biotin-cRGD powder dissolved in deionized water to obtain a solution with a concentration of 1 mg / ml) (the molar ratio of streptavidin to Biotin-cRGD is 4:1), incubate overnight at 4 °C, and remove unbound Biotin-cRGD using a 100 kDa ultrafiltration tube to obtain solution G;

[0061] Step S9: Add radioactive metal elements 68 Ga is mixed with sodium acetate buffer (0.1 M, pH 5.5) to obtain solution H, in which radioactive metal elements are present. 68 The Ga activity is 5 mCi;

[0062] Step S10: Mix solution G and solution H in equal volumes and incubate at 60 °C for 30 min to obtain radiolabeled product solution J;

[0063] Step S11: The radiolabeled product solution J was purified and separated by centrifugation to obtain the radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe. 68 Ga]-labeled SPIO@QDS-cRGD.

[0064] For the prepared [ 68 The structure and physicochemical properties of Ga-labeled SPIO@QDS-cRGD were characterized. First, transmission electron microscopy (TEM) results showed that... Figure 2 The nanosystem exhibits a uniform spherical morphology with a core size of approximately 5 nm. Its colloidal properties were subsequently determined using a nanoparticle size / potential analyzer. 68 The hydrated particle size of Ga-labeled SPIO@QDS-cRGD was 18.86 ± 1.17 nm, and the zeta potential was -28.05 ± 2.66 mV, indicating that the particles have good dispersion stability and can maintain this particle size and zeta potential for a long time. Figure 3 Further lyophilization of the sample allows for rapid reconstitution, with the particle size remaining essentially consistent before and after reconstitution. Figure 4 This indicates that the freeze-drying process did not cause significant aggregation or structural damage. The above results suggest that... 68Ga-labeled SPIO@QDS-cRGD combines high hydrophilicity with good stability, making it suitable for long-term storage at low temperatures and easy to reconstitute before use.

[0065] The contrast agent prepared [ 68 Ga]-labeled SPIO@QDS-cRGD exhibits high MRI relaxation performance and PET imaging effects. Figure 5 and Figure 6 Its relaxation rate R1 is greater than 10 [Fe] mM -1 s -1 R² > 200 [Fe] mM -1 s -1 This is far superior to what has been reported in existing literature.

[0066] Example 2: MRI-PET Dual-Modal Imaging Probe 68 Preparation of Ga-labeled SPIO@CDS-cRGD

[0067] The polyphenols in Example 1 were replaced with catechins, while the rest remained the same as in Example 1, to prepare a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe. 68 Ga]-labeled SPIO@CDS-cRGD.

[0068] The nanoparticle size-potential detector was used to measure [ 68 The Ga-labeled SPIO@CDS-cRGD particles have a particle size of 22.90±1.02 nm, such as... Figure 7 As shown in (A).

[0069] Example 3: MRI-PET Dual-Modal Imaging Probe [ 68 Preparation of Ga-labeled SPIO@GDS-cRGD

[0070] The polyphenols in Example 1 were replaced with pyrogallic acid, while the rest remained the same as in Example 1, to prepare a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe. 68 Ga]-labeled SPIO@GDS-cRGD.

[0071] The nanoparticle size-potential detector was used to measure [ 68 The Ga-labeled SPIO@GDS-cRGD particles have a particle size of 21.16 ± 1.47 nm. Figure 7 As shown in (B).

[0072] Example 4: MRI-PET Dual-Modal Imaging Probe68 Preparation of Ga-labeled SPIO@ADS-cRGD

[0073] The polyphenolic substance in Example 1 was replaced with ammonium glycyrrhizate, while the rest remained the same as in Example 1, to prepare a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe. 68 Ga]-labeled SPIO@ADS-cRGD.

[0074] The nanoparticle size-potential detector was used to measure [ 68 The Ga-labeled SPIO@ADS-cRGD particles have a particle size of 27.13 ± 1.91 nm. Figure 7 As shown in (C).

[0075] Example 5: MRI-PET Dual-Modal Imaging Probe [ 64 Preparation of Cu-labeled SPIO@QDS-cRGD

[0076] The radioactive metal element in Example 1 was changed to... 64 Cu, and the rest remained consistent with Example 1, to prepare a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe. 64 Cu]-labeled SPIO@QDS-cRGD.

[0077] The nanoparticle size-potential detector was used to measure [ 64 The particle size of Cu-labeled SPIO@QDS-cRGD particles is 21.75±2.44 nm. Figure 7 As shown in (D).

[0078] Example 6: MRI-PET Dual-Modal Imaging Probe [ 68 Preparation of Ga-labeled SPIO@QDS-cNGR

[0079] The biotinylated targeting peptide in Example 1 was replaced with Biotin-cNGR to prepare a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modality imaging probe. 68 Ga]-labeled SPIO@QDS-cNGR.

[0080] The nanoparticle size-potential detector was used to measure [ 68 The Ga-labeled SPIO@QDS-cNGR particles have a particle size of 23.15 ± 5.73 nm. Figure 7 As shown in (E).

[0081] Example 7: MRI-PET Dual-Modal Imaging Probe [ 68 Preparation of Ga-labeled SPIO@QDS-BBN

[0082] The biotinylated targeting peptide in Example 1 was replaced with Biotin-BBN to prepare a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modality imaging probe. 68 Ga]-labeled SPIO@QDS-BBN.

[0083] The nanoparticle size-potential detector was used to measure [ 68 The Ga-labeled SPIO@QDS-BBN particles have a particle size of 18.63 ± 2.20 nm. Figure 7 As shown in (F).

[0084] Test Example 1: In Vitro Radiochemical Stability Test

[0085] To test the radiochemical stability of the obtained radiolabeled nanomaterials under different physiologically relevant conditions, they were incubated in different media, and the changes in radiochemical purity (RCP) before and after incubation were monitored by radio-iTLC to evaluate whether the radionuclides dissociated from the nanomaterials.

[0086] The [obtained in Example 1] 68 Ga-labeled SPIO@QDS-cRGD was diluted in the corresponding media to prepare test solutions for stability testing.

[0087] The experimental method is as follows:

[0088] Experimental group 1 ([ 68 Ga]-labeled SPIO@QDS-cRGD + physiological saline): Take a certain volume of [ 68 Ga-labeled SPIO@QDS-cRGD solution was added to 0.9% NaCl solution, mixed well, and incubated; samples were taken at preset time points (0, 30, 60, 120 min) for radio-iTLC detection;

[0089] Experimental group 2 ([ 68 Ga]-labeled SPIO@QDS-cRGD + PBS): Will [ 68Ga-labeled SPIO@QDS-cRGD solution was added to PBS buffer (pH 7.4), mixed well, and incubated under the same conditions. Samples were taken at the same time points for radio-iTLC detection.

[0090] Experimental group 3 ([ 68 Ga]-labeled SPIO@QDS-cRGD + serum environment): will [ 68 Ga-labeled SPIO@QDS-cRGD solution was added to 10% fetal bovine serum (FBS), mixed well, and incubated under the same conditions. Samples were taken at the same time points for radio-iTLC detection.

[0091] After sampling at each time point, radio-iTLC was used for analysis: the sample was spotted on the iTLC stationary phase and developed using a set mobile phase. After development, a radioactivity scan was performed to obtain the radioactivity distribution curve. The radiochemical purity (RCP) was calculated based on the migration difference between the radiolabeled product and the free nuclide on the stationary phase to characterize whether the nuclide dissociated during the incubation process.

[0092] Test results are as follows Figure 8 As shown, [ 68 Ga-labeled SPIO@QDS-cRGD maintained high radiochemical purity after incubation in physiological saline, PBS, and serum environments, indicating that the radiolabeled nanoprobe has good in vitro radiochemical stability in different physiologically relevant media.

[0093] Test Example 2: MRI Imaging Test in an In Vivo Mouse Model of Liver Metastases

[0094] To verify the magnetic resonance imaging (MRI) enhancement effect and dynamic distribution characteristics of the probe of the present invention on liver metastatic lesions in vivo, a mouse liver metastatic lesion model was established, and MRI scans were performed at different time points after the probe was injected into the tail vein. The imaging performance was evaluated by comparing the signal changes in the lesion area before and after injection.

[0095] The probes from Example 1 and the comparative application were prepared into injectable solutions. All samples were administered at a uniform dosage based on the metal (Fe / Gd) ratio, set at 0.05 mM / kg (per mouse), and were administered via tail vein injection for in vivo T1-MRI imaging testing.

[0096] The experimental method is as follows:

[0097] Experimental group ([ 68Ga]-labeled SPIO@QDS-cRGD): Tumor-bearing mice with liver metastases were selected, and baseline T1-MRI scans were performed before drug administration; subsequently, [Ga] was injected via the tail vein. 68 Ga-labeled SPIO@QDS-cRGD (Fe 0.05mM / kg) was used, and MRI scans were repeated at set time points to obtain T1-weighted and T2-weighted images of the liver and metastatic lesions at 60 min.

[0098] Control group 2 (Primovist): The procedure was the same as the experimental group, except that the injected sample was replaced with Primovist and MRI scans were performed at the same time points to compare the imaging performance of the clinical T1 probe in liver metastases.

[0099] Imaging results as follows Figure 9 As shown, at 60 min, the experimental group [ 68 Ga-labeled SPIO@QDS-cRGD produced significant T1 signal enhancement in the liver metastasis area after injection, making the lesion boundaries easier to identify; while Primovist showed limited improvement in contrast between the lesion and normal liver tissue due to background signal elevation caused by uptake by normal liver tissue. These results indicate that the present invention […]. 68 Ga-labeled SPIO@QDS-cRGD exhibits superior MRI imaging and detection capabilities for small lesions in liver metastasis models.

[0100] Test Example 3: Biocompatibility Test (Hemolysis Test and Histological Assessment of Major Organs)

[0101] To verify the biocompatibility and in vivo safety of the probe of this invention, in vitro hemolysis experiments and in vivo histological and endoscopic (H&E) assessments of major organs were conducted. By comparing the hemolysis of erythrocytes in different treatment groups and the changes in the tissue structure of major organs after drug administration, the blood compatibility and systemic toxicity risk of the material were comprehensively evaluated.

[0102] The probe from Example 1 was prepared into a testable solution, with the clinical control preparation Primovist used as the control group. Physiological saline was used as a negative control, and deionized water (H2O) as a positive control. In the hemolysis experiment, sample solutions were prepared according to a metal (Fe) concentration gradient, with concentration ranges of 2, 4, 6, 8, 10, and 20 μg / mL, to investigate the effect of different concentrations on erythrocyte membrane integrity. In the in vivo safety assessment, tissue sections were prepared from major organs of animals in each group after administration and H&E staining was performed for observation.

[0103] The experimental method is as follows:

[0104] Experimental group ([ 68 Ga-labeled SPIO@QDS-cRGD): Red blood cell suspensions were mixed with different Fe concentrations. 68 The Ga-labeled SPIO@QDS-cRGD sample solution was incubated and treated. After treatment, the color change of the supernatant was observed and the hemolysis rate was calculated. At the same time, after the animals were given the drug, the main organs such as heart, liver, spleen, lung, and kidney were taken, paraffin sections were prepared and H&E staining was performed to evaluate histological toxicity.

[0105] Control group 1 (physiological saline): The same treatment procedure as the experimental group was used, except that the incubation solution was replaced with PBS. It was used as a negative control to assess the background of hemolysis and normal tissue morphology.

[0106] Control group 2 (water): The same treatment process as the experimental group was used, except that the incubation solution was replaced with deionized water (H2O) to serve as a positive control and to verify the effectiveness of the hemolysis model.

[0107] Control group 3 (Primovist): The same treatment process as the experimental group was used, except that the incubation solution was replaced with Primovist. This was used to compare the blood compatibility and histological safety of the clinical formulation under the same conditions.

[0108] Test results as follows Figure 10 As shown, in the hemolysis experiment ( Figure 10 As shown in (A), the H2O group showed obvious hemolysis, with a significantly increased hemolysis rate; while the PBS group, Primovist group, and [ 68 No significant hemolysis was observed in the Ga-labeled SPIO@QDS-cRGD group within the range of 2-20 μg / mL, and the color change of the supernatant was not significant, with the hemolysis rate remaining at a low level. In vivo histological evaluation results ( Figure 10 As shown in (B), the overall structure of the heart, liver, spleen, lung, and kidney tissues in each group remained intact, with no obvious abnormal pathological changes such as inflammatory cell infiltration, necrosis, or hemorrhage. The [68Ga]-labeled SPIO@QDS-cRGD group did not show additional tissue damage compared to the PBS and Primovist control groups. These results demonstrate that the probe of this invention possesses good biocompatibility and in vivo safety, exhibiting significant clinical application potential as a next-generation PET / MRI dual-modal contrast agent.

[0109] 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 method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe via ultrasound self-assembly, characterized in that, Includes the following steps: Step S1: Dissolve the polyphenols in methanol to obtain solution A, and adjust the pH of solution A; Step S2: Dissolve iron(III) oxide in tetrahydrofuran to obtain solution B; Step S3: Mix solution A and solution B, sonicate to produce a precipitate, remove the supernatant, add deionized water to redissolve, and obtain polyphenol-modified SPIO, i.e., solution C; Step S4: Dissolve dopamine hydrochloride in deionized water, then mix it with solution C to obtain mixed solution D; Step S5: Dissolve streptavidin in deionized water to obtain solution E, and adjust the pH of solution E; Step S6: Using the ultrasonic self-assembly method, solution D is ultrasonically added to solution E to obtain solution F at room temperature, and the reaction is immediately terminated. Step S7: Dialyze the solution F after the reaction has ended at room temperature to obtain solution F containing coordinated Fe-based nanoparticles; Step S8: Mix the dialyzed solution F with the biotinylated targeting peptide and incubate to obtain solution G; Step S9: Mix the radioactive metal element with sodium acetate buffer solution to obtain solution H; Step S10: Mix solution G and solution H in equal volumes, then incubate to obtain radiolabeled product solution J; Step S11: The radiolabeled product solution J was purified and separated by centrifugation to obtain the radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe; In step S1, the polyphenolic substance is at least one of quercetin, emodin, glycyrrhizic acid ammonium salt, catechin, and pyrogallol, and adjusting the pH of solution A means adjusting the pH value to 8-10. In step S8, the biotinylated targeting peptide is at least one of Biotin-cRGD, Biotin-cNGR, and Biotin-BBN. In step S9, the radioactive metal element is 64 Cu、 68 Ga、 89 At least one of Zr; the sodium acetate buffer solution has a concentration of 0.1 M and a pH of 5.5; the radioactive metal element activity in the solution H is (1 ~ 10) mCi.

2. The method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe by ultrasound self-assembly according to claim 1, characterized in that, The mass ratio of the polyphenolic substance to iron oxide is (40~80):(20~40), the mass ratio of the polyphenolic substance to dopamine hydrochloride is (1.5~5):1, and the mass ratio of dopamine hydrochloride to streptavidin is (1~2):(1.5~3).

3. The method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe by ultrasound self-assembly according to claim 1, characterized in that, The concentration of streptavidin in solution E is (0.5-1) mg / mL, and the concentration of dopamine hydrochloride in solution D is (0.5-1) mg / mL.

4. The method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe by ultrasound self-assembly according to claim 1, characterized in that, In step S8, the biotinylated targeting peptide is a lyophilized powder. The lyophilized powder is dissolved in deionized water at a concentration of (0.5-1) mg / mL. The molar ratio of streptavidin to the biotinylated targeting peptide is (4-10):(1-2).

5. The method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe by ultrasound self-assembly according to claim 1, characterized in that, In step S7, the dialysis time is 24-78 hours.

6. The method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe by ultrasound self-assembly according to claim 1, characterized in that, In step S8, the incubation refers to incubation at 4 °C overnight, and the solution G obtained after incubation is used to remove unbound biotinylated target peptides using an ultrafiltration tube with a capacity of (10-100) kDa.

7. The method for constructing a radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe by ultrasound self-assembly according to claim 1, characterized in that, In step S10, the incubation refers to incubation at 60 ℃ for 30 min.

8. A radiolabeled magnetic T1-T2 synergistic MRI-PET dual-modal imaging probe prepared by the method according to any one of claims 1-7, characterized in that, The relaxation rate R1 of the probe is greater than 10 [Fe] mM -1 s -1 R2 is greater than 200 [Fe] mM -1 s -1 .

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