Construction method and application of radiolabeled magnetic T1-T2 synergetic MRI-PET bimodal image probe

Abstract

The invention belongs to the technical field of molecular imaging and biomedical materials, and particularly relates to a construction method and application of a radiolabeled magnetic T1-T2 synergistic MRI-PET bimodal image probe. The preparation method comprises the following steps: dissolving a polyphenol substance in methanol, adjusting the pH value, dispersing SPIO in tetrahydrofuran, and mixing the two substances to obtain polyphenol-modified water-phase SPIO. The preparation method comprises the following steps: firstly, preparing a target MRI (Magnetic Resonance Imaging) probe, then dissolving streptavidin in water, forming a polydopa wrapping layer through dopa in-situ polymerization, coupling the polydopa wrapping layer with the streptavidin, combining with a biotin-modified target ligand to obtain the target MRI probe, and finally, efficiently chelating radioactive metal elements such as 64Cu and 68Ga to obtain the T1-T2 synergistic MRI-PET bimodal probe. The probe is strong in stability, high in hydrophilicity, good in biocompatibility, high in relaxation rate and good in tumor targeting property, and has the advantages of PET high-sensitivity whole-body quantification, MRI T1-T2 high soft tissue contrast and high spatial resolution.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular imaging and biomedical materials, and particularly relates to a method for constructing a radioactively labeled magnetic T1-T2 synergistic MRI-PET bimodal imaging probe and application thereof. BACKGROUND

[0002] Magnetic resonance imaging (MRI) occupies a core position in clinical applications due to its excellent soft tissue contrast, three-dimensional high spatial resolution and tissue penetration, but its detection sensitivity is restricted by factors such as molecular thermal motion and limited number of excitable atoms, and its recognition ability for biological markers and small lesions is insufficient. Meanwhile, in the imaging process, it faces the limitations of local focused radio frequency pulses and single organ diagnosis and treatment orientation of signal acquisition coils, and cannot realize the synchronous evaluation of multi-organ functions, which has a blind area for the detection of early tumors and metastatic tumors. Compared with MRI, positron emission tomography (PET) can capture molecular information in deep tissues with ultra-high sensitivity and quickly obtain whole-body functional imaging; however, its spatial resolution is limited by the freedom of positron decay of the probe, and it cannot fully and accurately reflect the physiological and structural characteristics of the lesion. Combining two or more imaging methods can overcome the limitations of single modality technology, fully utilize the inherent advantages of different imaging principles, and obtain multiple signals of the same biological event to meet clinical needs. The first PET-MRI system was born in 2010, which significantly improved the recognition accuracy and comprehensiveness of complex lesions. However, existing clinical PET-MRI detection can only rely 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, causing the signal source not to be the same molecular target or the same lesion area, and the spatial and temporal information of imaging cannot be accurately corresponded. Therefore, to fully utilize the advantages of PET-MRI, the key is to develop efficient, safe and stable bimodal probes, and the development process needs to consider the half-life of the nuclide and the structural stability, the performance of the magnetic unit, the surface modification and the biological safety of the system. The rationality of the related preparation strategy determines the PET-MRI imaging effect, molecular recognition targeting and clinical transformation potential.

[0003] Current PET probes are mainly divided into two categories. One is a covalent bond labeling system, such as the combination of common radioactive elements such as F and C with carrier structures. Most of these radioactive elements have short half-lives, require high selectivity and rapid reaction preparation conditions, and need to be combined with complex purification processes and on-site cyclotrons, which is not conducive to the preparation of bimodal probes. In contrast, metal chelate system PET probes, such as Cu, Ga and In, have long half-lives, and can be prepared under mild conditions, which is conducive to the preparation of bimodal probes. 18 F and 11 C, most of which have short half-lives, require high selectivity and rapid reaction preparation conditions, and need to be combined with complex purification processes and on-site cyclotrons, which is not conducive to the preparation of bimodal probes. In contrast, metal chelate system PET probes, such as 64 Cu, 68Ga is linked to the chelating molecule, the preparation steps are convenient, and the radio metal element has a long general half-life, which has been widely used for antibody and nano system labeling. MRI probes are mainly paramagnetic Gd 3+ chelates and superparamagnetic iron oxide nanoparticles SPIO systems, which need to consider the structural stability and magnetic relaxation performance when combined with PET functional components. However, the traditional construction strategy with Gd 3+ as a paramagnetic core can have the advantages of intuitive MRI-T1 imaging and clear contrast, but will inevitably compete with the radio metal element in the ligand binding reaction, resulting in decreased labeling efficiency and lost imaging performance; in addition, the bimetallic chelate product will also change the key parameters such as rotation time and water exchange constant, causing uncontrollable changes in relaxation efficiency. Superparamagnetic iron oxide nanoparticles (SPIO) have a stable crystal structure, and the modification of the nuclide is difficult to affect the magnetic performance, and the high specific surface area characteristics of the nano system are easy to stably label the radio nuclide, so the influence of the dual-mode imaging function on each other is small, but the clinical application dilemma is that the image "negative enhancement" effect caused by the inhomogeneity of the magnetic field shortens the transverse relaxation time (T2), making the image interpretation relatively difficult and easy to be interfered by the background signal; and the functionalization of the SPIO interface molecular structure can change the imaging effect and obtain the dual synergistic signal of MRI-T1 and T2, which is expected to improve the imaging accuracy.

[0004] Therefore, how to construct a SPIO ligand interface with a determined structure to ensure the preparation of high magnetic crystal, provide excellent dispersibility and magnetic performance of SPIO to meet the needs of MRI-T1 and T2 cooperative imaging, and realize stable and efficient radio nuclide chelating ability has become a technical bottleneck. In addition, giving the PET-MRI dual-mode probe specific targeting function can significantly improve the signal-to-noise ratio of imaging, enhance the diagnostic sensitivity and accuracy at the molecular level. The most commonly used strategy is to covalently couple the targeting ligand (such as polypeptide, antibody fragment, etc.) to the particle surface. However, such chemical coupling reaction usually has the disadvantages of low coupling efficiency, complicated operation steps and loss of biological activity caused by chemical modification, which also reduces the batch consistency and clinical convertibility of the product. SUMMARY

[0005] In view of the problems existing in the prior art, the present application provides a method for constructing a radioactively labeled magnetic T1-T2 cooperative MRI-PET dual-mode image probe and its application.

[0006] The present application realizes the rapid phase inversion of nanoparticles from organic phase to aqueous phase by utilizing the high-efficiency coordination of polyphenol molecules to the Fe element of superparamagnetic ferroferric oxide SPIO, and simultaneously provides 64 Cu, 68Ga and other radioactive metals provide high-efficiency chelation sites to meet the needs of PET labeling; further introducing streptavidin (SA), through its high affinity with biotin, biotinylated antibodies are efficiently coupled to the surface of SPIO under mild conditions, constructing a modular connection platform for universal and replaceable targeted ligands, meeting the needs of specific targeting of probes in PET-MRI dual-mode imaging. The probe has strong stability, high hydrophilicity, good biocompatibility, high relaxation rate and good tumor targeting, and has the advantages of high sensitivity of PET whole body quantification and high soft tissue contrast and high spatial resolution of MRI T1-T2, which can realize the imaging path of "whole body quantitative screening-local fine positioning", improve the reliability of cross-individual, cross-time point comparison and pharmacokinetic / efficacy evaluation, and has good biocompatibility, targeting and clinical transformation potential.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is: The first aspect of the present application provides a method for constructing a metal-coordinated targeted T1-T2 MRI / PET dual-mode probe by ultrasonic self-assembly, comprising the following steps: Step S1: dissolve the polyphenolic substance in methanol to obtain solution A, and adjust the pH of solution A; Step S2: dissolve the ferroferric oxide in tetrahydrofuran to obtain solution B; Step S3: after mixing solution A and solution B, ultrasonic precipitation is generated, the supernatant is removed, deionized water is added for redissolution, and a polyphenolic substance modified SPIO, i.e. solution C, is obtained; Step S4: dissolve dopamine hydrochloride in deionized water, then mix with solution C to obtain a 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, ultrasonically add solution D to solution E to obtain solution F at room temperature, and the reaction is immediately ended; Step S7: dialysis of the reaction ended solution F at room temperature to obtain a solution F containing a coordinated Fe-based nanoparticle; Step S8: mix the dialyzed solution F with biotinylated targeted polypeptide, 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 volume, then incubate to obtain a radioactive labeled product solution J; Step S11: purify and separate the radioactive labeled product solution J by centrifugation to obtain a radioactive labeled magnetic T1-T2 synergistic MRI-PET dual-mode imaging probe.

[0008] The high-efficiency metal-coordinated targeted PET / MRI dual-mode probe constructed by the application has a simple and quick construction process and mild conditions; in the reaction system, the polyphenol substance (catechin, quercetin, pyrogallic acid, etc.) is dissolved in methanol and adjusted to a pH of 8-10, while the superparamagnetic ferroferric oxide nanoparticles (SPIO) are dispersed in tetrahydrofuran, then the two are quickly mixed, the high-efficiency coordination anchoring effect between the phenolic hydroxyl groups in the polyphenol molecules and the Fe sites on the surface of SPIO is utilized to drive the nanoparticles to quickly phase transfer from the organic phase to the aqueous phase, and the polyphenol-modified aqueous SPIO is obtained; then the SA is dissolved in the aqueous system, and the dopamine (DA) in-situ polymerization process is introduced, and the polydopamine (PDA) coating layer is formed under mild conditions and the fixation / coupling of SA is simultaneously realized, so that the targeted MRI probe with a biological coupling interface is obtained, finally, the direct radio-labeling technology is adopted, and the radio-metal elements such as Cu, 64 Cu, 68 Ga, etc. are efficiently chelated and stably labeled to obtain the PET / MRI dual-mode probe in the form of a single probe. In the construction process of the probe, the polyphenol molecules realize the quick aqueous phase of the nanoparticles and the interface stability through the strong coordination effect with the Fe on the surface of SPIO, and on the other hand, provide high-affinity chelation sites for the radio-metal elements such as Cu, 64 Cu, 68 Ga, etc. to meet the PET labeling requirements; after the introduction of SA, the biotinylated antibody / targeting ligand can be efficiently coupled to the surface of SPIO under mild conditions by virtue of the ultra-high affinity between the SA and the biotin, so that a universal, modular and replaceable targeting ligand connection platform is constructed, which is convenient for the quick expansion and application for different target points.

[0009] Further, in step S1, the polyphenol substance is at least one of quercetin, emodin, ammonium glycyrrhizinate, catechin and pyrogallic acid, and the pH of the solution A is adjusted to 8-10.

[0010] Further, the mass ratio of the polyphenol substance to the ferroferric oxide is (40-80):(20-40), the mass ratio of the polyphenol substance to dopamine hydrochloride is (1.5-5):1, and the mass ratio of the dopamine hydrochloride to the streptavidin is (1-2):(1.5-3).

[0011] Further, the concentration of the streptavidin in the solution E is (0.5-1) mg / mL, and the concentration of the dopamine hydrochloride in the solution D is (0.5-1) mg / mL.

[0012] Further, in step S8, if the biotinylated targeting polypeptide is a freeze-dried powder, the freeze-dried powder is dissolved in deionized water, and the concentration is (0.5-1) mg / mL; the biotinylated targeting polypeptide is at least one of Biotin-cRGD, Biotin-cNGR and Biotin-BBN; and the molar ratio of the streptavidin to the biotinylated targeting polypeptide is (4-10):(1-2).

[0013] Further, in step S9, the radioactive metal element is at least one of Cu, 64 , 68 Ga, 89 Zr; the concentration of the sodium acetate buffer is 0.1 M, and the pH value is 5.5; and the activity of the radioactive metal element in the solution H is (1-10) mCi.

[0014] Further, in step S7, the dialysis time is 24-78 h.

[0015] Further, in step S8, the incubation is overnight incubation at 4 ℃, and the unbound biotinylated targeting polypeptide is removed from the obtained solution G by using an ultrafiltration tube with a molecular weight cutoff of (10-100) kDa.

[0016] Further, in step S10, the incubation is incubation at 60 ℃ for 30 min.

[0017] The second aspect of the present application provides a radioactive labeled magnetic T1-T2 synergistic MRI-PET bimodal imaging probe prepared by the above method, and the relaxivity R1 of the probe is greater than 10 [Fe] mM -1 s -1 , and the relaxivity R2 is greater than 200 [Fe] mM -1 s -1 .

[0018] The third aspect of the present application provides an application of the above radioactive labeled magnetic T1-T2 synergistic MRI-PET bimodal imaging probe in magnetic resonance imaging (MRI), positron emission tomography (PET), in vivo tracing and pharmacokinetics / biodistribution research, and a targeting molecular imaging platform.

[0019] Compared with the prior art, the present application has the following beneficial effects: The present application realizes the rapid phase inversion of nanoparticles from an organic phase to an aqueous phase through the strong coordination anchoring effect between the polyphenol molecules and the surface sites of Fe-based SPIO; further, the PDA coating layer is formed by in-situ self-polymerization of DA under mild conditions, and the SA is fixed, so as to construct a magnetic nanoparticle platform with a universal coupling interface; meanwhile, relying on the high affinity coordination ability of the polyphenol / phenolic hydroxyl sites to the radioactive metal ions, the direct radioactive labeling is adopted to realize the labeling of64 Cu, 68 High-efficiency chelation and stable loading of Ga, etc. radionuclides, and finally obtain a single probe form of targeted PET / MRI dual-mode probe. Specifically, the present application has the following advantages: (1) The present application is based on interface modification and phase inversion driven by polyphenol coordination (steps S1-S2), and then PDA coating layer is formed by in-situ polymerization of DA and SA is fixed (steps S4-S7), which realizes the close integration and structural stabilization of multi-components, reduces the complex operation and biological activity damage risk caused by traditional multi-step covalent modification, and improves the stability and biological safety of the system.

[0020] (2) Compared with the common route of "first grafting DOTA / NOTA chelator and then labeling" in the prior art, the present application utilizes the multi-site coordination environment provided by polyphenol (steps S1-S3) to directly coordinate and chelate radioactive metals such as Cu, 64 Cu, 68 Ga (steps S9-S10), which reduces the material chemical modification steps and intermediate purification links, and significantly improves the labeling convenience and operability.

[0021] (3) The polyphenol-metal coordination site density of the present application is high and the binding force is strong, which can promote the rapid capture and stable combination of radioactive metals; combined with centrifugal purification / separation (step S11), it is beneficial to obtain higher radiochemical purity and reduce the proportion of free radionuclides, thereby improving the signal-to-noise ratio and specificity of in vivo imaging.

[0022] (4) The present application introduces SA as a bridging module (steps S5-S7), which can efficiently couple Biotin-cRGD to the surface of nanoparticles under mild conditions (step S8) by means of the high affinity of SA-Biotin; at the same time, this strategy can be extended to other biotinylated antibodies / polypeptides / small molecule ligands, realizing the rapid adaptation and expansion of "the same nano platform-different targets".

[0023] (5) Polyphenol modification and PDA coating can significantly improve the hydrophilicity and interface stability of the particles (steps S3-S7), reduce the occurrence of SPIO aggregation and sedimentation in aqueous phase, which is beneficial to obtain stable and repeatable particle size distribution and use performance, and facilitate subsequent dialysis to remove small molecule impurities to improve the purity of the system (step S7).

[0024] It can be seen that the application is based on SPIO water phase and direct labeling of radioactive metal driven by polyphenol coordination, and a modular targeted connection platform is constructed by combining PDA in-situ coating and fixing SA, so as to obtain a targeted PET / MRI bimodal probe which is simple to prepare, mild in condition, strong in stability and scalable in targeted ligand, can be used for MRI imaging, PET imaging, PET / MRI fusion imaging, in-vivo tracing and quantitative analysis, and related biomedical research and diagnosis and treatment application, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 For example 1, 68 Ga]-labeled SPIO@QDS-cRGD preparation flow chart.

[0026] Figure 2 For example 1, 68 Ga]-labeled SPIO@QDS-cRGD transmission electron microscopy diagram.

[0027] Figure 3 For example 1, 68 Ga]-labeled SPIO@QDS-cRGD particle size stability diagram.

[0028] Figure 4 For example 1, 68 Ga]-labeled SPIO@QDS-cRGD particle size comparison diagram before and after freeze-drying.

[0029] Figure 5 For example 1, 68 Ga]-labeled SPIO@QDS-cRGD relaxation rate diagram.

[0030] Figure 6 For example 1, 68 Ga]-labeled SPIO@QDS-cRGD PET imaging diagram.

[0031] Figure 7 For example 2-7,

[0032] Figure 8 For test example 1, 68 Ga]-labeled SPIO@QDS-cRGD in-vitro radiochemical stability test.

[0033] Figure 9 For test example 2, 68 Ga]-labeled SPIO@QDS-cRGD MRI imaging in liver metastasis model mice.

[0034] Figure 10 For test example 3 [ 68 Biocompatibility test results of Ga-labeled SPIO@QDS-cRGD in vivo. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] Example 1: MRI-PET Dual-Modal Imaging Probe [ 68 Preparation of Ga-labeled SPIO@QDS-cRGD [ 68 The fabrication process of Ga-labeled SPIO@QDS-cRGD is shown in the figure below. Figure 1 As shown, the specific steps include: Step S1: Dissolve quercetin in methanol to obtain solution A, and adjust the pH of solution A to 10; Step S2: Dissolve iron(III) oxide in tetrahydrofuran to obtain solution B; 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; 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. 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; 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. 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; Step S8: The solution F after dialysis was mixed with Biotin-cRGD (a solution of Biotin-cRGD lyophilized powder in deionized water with a concentration of 1 mg / ml) (the molar ratio of streptavidin to Biotin-cRGD was 4:1), and incubated at 4°C overnight. Unbound Biotin-cRGD was removed using a 100 kDa ultrafiltration tube to obtain solution G; Step S9: The radioactive metal element 68 Ga was mixed with sodium acetate buffer (0.1 M, pH 5.5) to obtain solution H, and the radioactive metal element 68 Ga in solution H had an activity of 5 mCi; Step S10: After mixing solution G with solution H in equal amounts, incubation was carried out at 60°C for 30 min to obtain radioactive labeling product solution J; Step S11: Centrifugation was used to purify and separate the radioactive labeling product solution J to obtain the radioactive labeling magnetic T1-T2 synergistic MRI-PET bimodal imaging probe [ 68 Ga]-labeled SPIO@QDS-cRGD.

[0038] The structure and physicochemical properties of the prepared [ 68 Ga]-labeled SPIO@QDS-cRGD were characterized. First, transmission electron microscopy (TEM) results showed that the nano-system had a uniform spherical morphology, and the core size was about 5 nm. Figure 2 Subsequently, the colloidal properties were determined using a nanoparticle size / potential analyzer, and the hydrated particle size of the [ 68 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 had good dispersion stability and could maintain this particle size and zeta potential unchanged for a long time. Figure 3 Further, the sample was freeze-dried, and the particle size remained basically unchanged before and after reconstitution ( Figure 4 ), indicating that the freeze-drying process did not cause significant aggregation or structural damage. The above results show that the [ 68 Ga]-labeled SPIO@QDS-cRGD has high hydrophilicity and good stability, is suitable for long-term storage at low temperature, and can be conveniently reconstituted before use.

[0039] The prepared contrast agent [ 68 Ga]-labeled SPIO@QDS-cRGD has high MRI relaxation performance and PET imaging effect ( Figure 5 and Figure 6 ), and the relaxation rate R1 is greater than 10 [Fe] mM-1 s -1 , R2 is greater than 200 [Fe] mM -1 s -1 , far superior to the existing literature reported.

[0040] Example 2: MRI-PET dual-mode imaging probe 68 Ga]-labeled SPIO@CDS-cRGD preparation The polyphenols in Example 1 are replaced with catechin, and the rest is consistent with Example 1, to prepare a radioactive labeled magnetic T1-T2 synergistic MRI-PET dual-mode imaging probe 68 Ga]-labeled SPIO@CDS-cRGD.

[0041] The nano particle size-potential detector measures the particle size of 68 Ga]-labeled SPIO@CDS-cRGD particles to be 22.90±1.02nm, as shown in Figure 7 (A).

[0042] Example 3: MRI-PET dual-mode imaging probe 68 Ga]-labeled SPIO@GDS-cRGD preparation The polyphenols in Example 1 are replaced with pyrogallic acid, and the rest is consistent with Example 1, to prepare a radioactive labeled magnetic T1-T2 synergistic MRI-PET dual-mode imaging probe 68 Ga]-labeled SPIO@GDS-cRGD.

[0043] The nano particle size-potential detector measures the particle size of 68 Ga]-labeled SPIO@GDS-cRGD particles to be 21.16±1.47nm, as shown in Figure 7 (B).

[0044] Example 4: MRI-PET dual-mode imaging probe 68 Ga]-labeled SPIO@ADS-cRGD preparation The polyphenols in Example 1 are replaced with ammonium glycyrrhizinate, and the rest is consistent with Example 1, to prepare a radioactive labeled magnetic T1-T2 synergistic MRI-PET dual-mode imaging probe 68 Ga]-labeled SPIO@ADS-cRGD.

[0045] The nano particle size-potential detector measures the particle size of 68The Ga-labeled SPIO@ADS-cRGD particles have a particle size of 27.13 ± 1.91 nm. Figure 7 As shown in (C).

[0046] Example 5: MRI-PET Dual-Modal Imaging Probe [ 64 Preparation of Cu-labeled SPIO@QDS-cRGD 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.

[0047] 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).

[0048] Example 6: MRI-PET Dual-Modal Imaging Probe [ 68 Preparation of Ga-labeled SPIO@QDS-cNGR 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.

[0049] 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).

[0050] Example 7: MRI-PET Dual-Modal Imaging Probe [ 68 Preparation of Ga-labeled SPIO@QDS-BBN 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.

[0051] The nanoparticle size-potential detector was used to measure [ 68The Ga-labeled SPIO@QDS-BBN particles have a particle size of 18.63 ± 2.20 nm. Figure 7 As shown in (F).

[0052] Test Example 1: In Vitro Radiochemical Stability Test 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.

[0053] The [obtained in Example 1] 68 Ga-labeled SPIO@QDS-cRGD was diluted in the corresponding media to prepare test solutions for stability testing.

[0054] The experimental method is as follows: 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; Experimental group 2 ([ 68 Ga]-labeled SPIO@QDS-cRGD + PBS): Will [ 68 Ga-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. 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.

[0055] 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.

[0056] 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.

[0057] Test Example 2: MRI Imaging Test in an In Vivo Mouse Model of Liver Metastases 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.

[0058] 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.

[0059] The experimental method is as follows: Experimental group ([ 68 Ga]-labeled SPIO@QDS-cRGD): Tumor-bearing mice with liver metastases were selected, and baseline T1-MRI scans were performed before 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. 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.

[0060] Imaging results as follows Figure 9 As shown, at 60 min, the experimental group [ 68Ga]-labeled SPIO@QDS-cRGD produced obvious T1 signal enhancement in the region of liver metastasis after injection, and the lesion boundary was easier to identify; while Primovist had limited improvement in the contrast between the lesion and normal liver tissue due to the increased background signal caused by normal liver tissue uptake. The above results show that the probe of the present application has better MRI imaging effect and micro-lesion detection ability in the liver metastasis model. 68 Ga]-labeled SPIO@QDS-cRGD produced obvious T1 signal enhancement in the region of liver metastasis after injection, and the lesion boundary was easier to identify; while Primovist had limited improvement in the contrast between the lesion and normal liver tissue due to the increased background signal caused by normal liver tissue uptake. The above results show that the probe of the present application has better MRI imaging effect and micro-lesion detection ability in the liver metastasis model.

[0061] Test Example 3: Biocompatibility test (hemolysis experiment and main organ histological evaluation) To verify the biocompatibility and in vivo safety of the probe of the present application, in vitro hemolysis experiment and in vivo main organ histology (H&E) evaluation were carried out. By comparing the hemolysis of red blood cells in different treatment groups and the changes in the tissue structure of the main organs after administration, the blood compatibility and systemic toxicity risk of the material were comprehensively evaluated.

[0062] The probe of Example 1 was prepared into a testable solution, and the clinical control preparation Primovist was used as a control group, and normal saline was used as a negative control, and deionized water (H2O) was used as a positive control. In the hemolysis experiment, sample solutions were prepared according to the metal (Fe) concentration gradient, and the concentration range was set to 2, 4, 6, 8, 10, 20 μg / mL, to investigate the influence on the integrity of red blood cell membranes at different concentrations; in the in vivo safety evaluation, the main organs of the animals after administration were taken to prepare tissue sections and observe H&E staining.

[0063] The experimental method is as follows: Experimental group ([ 68 Ga]-labeled SPIO@QDS-cRGD): The red blood cell suspension was incubated with different Fe concentrations of [ 68 Ga]-labeled SPIO@QDS-cRGD sample solution, and after the treatment, the supernatant color change was observed and the hemolysis rate was calculated; at the same time, the main organs such as heart, liver, spleen, lung and kidney of the animals after administration were taken to prepare paraffin sections and perform H&E staining for histological toxicity evaluation.

[0064] Control group 1 (normal saline): the same processing procedure as the experimental group was adopted, only the incubation solution was replaced with PBS, which was used as a negative control to evaluate the basic hemolysis background and normal tissue morphology.

[0065] Control group 2 (water): the same processing procedure as the experimental group was adopted, only the incubation solution was replaced with deionized water (H2O), which was used as a positive control to verify the effectiveness of the hemolysis model.

[0066] Control group 3 (Primovist): the same processing procedure as the experimental group was adopted, only the incubation solution was replaced by Primovist, which was used to compare the blood compatibility and histological safety performance of the clinical preparation under the same conditions.

[0067] The detection results are shown in Figure 10 As shown in (A) of Figure 10 in the hemolysis experiment, the H2O group showed obvious hemolysis, and the corresponding hemolysis rate was significantly increased; while the PBS group, the Primovist group and the [68Ga]-labeled SPIO@QDS-cRGD group did not show obvious hemolysis in the range of 2-20 μg / mL, the color change of the supernatant was not significant, and the hemolysis rate remained at a low level. 68 The results of in vivo histological evaluation (B) of Figure 10 showed that the heart, liver, spleen, lung and kidney tissue structures of each group were overall intact, and no obvious inflammatory cell infiltration, necrosis or hemorrhage and other abnormal pathological changes were observed; among them, the [68Ga]-labeled SPIO@QDS-cRGD group did not show additional tissue damage compared with the PBS and Primovist control groups. The above results show that the probe has good biocompatibility and in vivo safety, and shows significant clinical application potential as a new generation of PET / MRI dual-mode contrast agent.

[0068] The above describes the embodiments of the present application in detail, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of these embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A method for constructing a metal-coordinated targeted T1-T2 MRI / PET dual- modality probe by ultrasonic self-assembly, characterized in that, The method comprises the following steps: Step S1: dissolving polyphenols in methanol to obtain solution A, and adjusting the pH of solution A; Step S2: dissolving ferroferric oxide in tetrahydrofuran to obtain solution B; Step S3: mixing solution A and solution B, and then generating a precipitate by ultrasonic, removing supernatant, and then adding deionized water to dissolve to obtain polyphenol modified SPIO, i.e. solution C; Step S4: dissolving dopamine hydrochloride in deionized water, and then mixing with solution C to obtain mixed solution D; Step S5: dissolving streptavidin in deionized water to obtain solution E, and adjusting the pH of solution E; Step S6: using an ultrasonic self-assembly method, ultrasonically adding solution D to solution E to obtain solution F at room temperature, and then ending the reaction; Step S7: dialyzing the solution F after ending the reaction at room temperature to obtain solution F containing coordinated Fe-based nanoparticles; Step S8: mixing the dialyzed solution F with biotinylated targeting polypeptide, and incubating to obtain solution G; Step S9: mixing a radioactive metal element with sodium acetate buffer to obtain solution H; Step S10: mixing solution G and solution H in equal volume, and then incubating to obtain a radioactive labeling product solution J; Step S11: purifying and separating the radioactive labeling product solution J by centrifugation to obtain a radioactive labeling magnetic T1-T2 synergistic MRI-PET bimodal imaging probe.

2. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasonic self-assembly. In step S1, the polyphenols are at least one of quercetin, emodin, ammonium glycyrrhizinate, catechin, pyrogallol, and the pH of solution A is adjusted to 8-10.

3. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasonic self-assembly. The mass ratio of the polyphenols to ferroferric oxide is (40-80):(20-40), the mass ratio of the polyphenols to dopamine hydrochloride is (1.5-5):1, and the mass ratio of dopamine hydrochloride to streptavidin is (1-2):(1.5-3).

4. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasonic self-assembly. 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.

5. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasound self-assembly. In step S8, if the biotinylated targeting polypeptide is a freeze-dried powder, the freeze-dried powder is dissolved in deionized water to a concentration of (0.5-1) mg / mL, and the biotinylated targeting polypeptide is at least one of Biotin-cRGD, Biotin-cNGR and Biotin-BBN; the molar ratio of streptavidin to biotinylated targeting polypeptide is (4-10):(1-2).

6. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasound self-assembly. In step S9, the radioactive metal element is at least one of 64 Cu, 68 Ga, 89 Zr; the concentration of the sodium acetate buffer is 0.1 M, the pH value is 5.5; and the activity of the radioactive metal element in the solution H is (1 ~ 10) mCi.

7. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasound self-assembly. In step S7, the dialysis time is 24-78 h.

8. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasound self-assembly. In step S8, the incubation is overnight incubation at 4 ℃, and the obtained solution G is subjected to ultrafiltration with a (10-100) kDa ultrafiltration tube to remove unbound biotinylated targeting polypeptide.

9. The method of claim 1, wherein the metal-coordinated targeted T1-T2 MRI / PET dual- modality probe is constructed by ultrasound self-assembly. In step S10, the incubation is incubation at 60 ℃ for 30 min.

10. The radiolabeled magnetic Tl-T2 synergistic MRI-PET bimodal imaging probe prepared according to the method of any one of claims 1 to 9, characterized in that, The probe has a relaxation rate R1 greater than 10 [Fe] mM -1 s -1 R2 greater than 200 [Fe] mM -1 s -1 .

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