A multimodal contrast agent, its preparation method and application

Abstract

This invention provides a multimodal contrast agent, its preparation method, and its application. The multimodal contrast agent comprises, by weight, 1 part fluorescent precursor, 10-15 parts contrast agent, 5-8 parts dipalmitoylphosphatidylcholine, and 1-3 parts cholesterol. The fluorescent precursor has a structure as shown in Formula 1, which improves the relaxation rate and overcomes the limitations of low MRI sensitivity and high background noise. By utilizing the complementarity of MRI and fluorescence imaging, dual-modal imaging is achieved. This multimodal contrast agent is used to monitor the biodistribution of cells introduced into the body. Based on its advantages of large tissue penetration depth, high spatial resolution, high signal-to-noise ratio, and high sensitivity, high-resolution imaging results can be obtained, achieving the requirement of immediate, non-invasive, and real-time detection of the presence, distribution, quantity, and activity of cells in vivo.

Description

Technical Field

[0001] This invention belongs to the field of diagnostic imaging technology, specifically relating to a multimodal contrast agent, its preparation method, and its application. Background Technology

[0002] Research shows that cancer develops through a multi-step carcinogenic process involving many cellular physiological systems, such as cell signaling and apoptosis, making it a highly difficult and complex disease to understand. Current cancer treatments include surgical interventions, radiation, and chemotherapy drugs, which often kill healthy cells and are toxic to patients.

[0003] Understanding the role and function of immune cells in combating cancer has facilitated the development of novel immunotherapies. Immune cells, such as T cells and natural killer cells (NK cells), are increasingly being used to study the pathogenesis of various diseases or for tumor treatment. In cell-based immunotherapy, dynamic cell imaging can provide crucial information on immune cell migration, expansion, and consumption, as well as optimal delivery routes and individual treatment doses, helping to monitor treatment progress. Effective cell tracking strategies can guide correct decision-making in preclinical and clinical studies, improving the efficacy and safety of immune cell therapy and promoting the development and implementation of novel immunotherapies. An ideal in vivo cell tracing method should detect the presence, distribution, quantity, and activity of cells in vivo in a timely, non-invasive, and real-time manner. Therefore, developing a non-invasive method to observe the biodistribution of cells after injection is an important requirement for the development of cell therapies.

[0004] Currently, non-invasive cell tracking methods can be broadly categorized into two types: direct labeling and indirect labeling. Direct labeling does not involve genetic modification of cells. It uses tracers to directly label immune cells, and then reinfuses these labeled immune cells into the patient for imaging. Its advantage is that it provides a direct view of the entire process of immune function reconstitution and the homing of immune cells to lymph nodes and tumor sites. Its disadvantage is that the markers are diluted or lost during mitosis, thus limiting the observation time to a few days, and it cannot observe cell proliferation, cell activation, or cell death. Indirect labeling uses genetic engineering techniques to transfect reporter genes into immune cells, and then reinfuses the genetically modified immune cells into the patient, enabling real-time in vivo monitoring of immune cells through imaging. This method achieves real-time, long-term dynamic, and reproducible monitoring of immune cells in vivo, especially in monitoring the functional and activation states of immune cells. However, it requires the development of corresponding probes for each reporter gene, resulting in higher costs, and the immunogenicity of exogenous reporter gene-related proteins can trigger a strong immune rejection response in vivo.

[0005] Recently, with the rapid development of molecular imaging technology, the advancements in related materials such as imaging agents, reporter structures, ligands, and probes have facilitated the development of direct labeling methods for dynamic cell tracking. Various molecular imaging techniques, including computed tomography (CT), magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence imaging (FLI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET), are actively applied to the tracking of immune cells and stem cells. MRI and CT are limited by low sensitivity and high instrument costs; bioluminescence imaging cannot be used in large animals or clinical settings due to the physical limitations of light penetration into deep tissues; cytotoxicity and patient safety concerns limit the use of radionuclide-based methods for cell tracking. Multimodal fusion molecular imaging is now widely used to overcome the limitations of single imaging modalities and is gradually being applied to in vivo cell imaging and tracking. This system integrates various combinations of optical, PET, SPECT, CT, and MRI imaging. These multimodal methods allow for the simultaneous acquisition of different imaging techniques, combining the best features and utilities of each modality.

[0006] Existing technical solution: Silane-added near-infrared fluorophores (CF-MPTMS) are radiolabeled with 89Zr to obtain bimodal PET / NIRF nanoparticles. Then, protamine and heparin are coated onto the nanoparticles to obtain a bimodal imaging agent that promotes chimeric antigen receptor T-cell immunotherapy (CAR T-cell) labeling for direct labeling of human CAR-T cells. PET / NIRF nanoparticles do not affect cell viability or function. In an ovarian cancer peritoneal carcinoma model, PET and NIRF were used to achieve one week of whole-body CAR-T cell tracking ("A biomimetic model of 3D fluid extracellular macromolecular crowding microenvironment fine-tunes ovarian cancer cells dissemination phenotype", Rümeyza Bascetin, et al., Biomaterials, 2021, 269:120630.).

[0007] Existing MRI contrast agents are limited by low sensitivity and high background noise, and may suffer from low labeling efficiency and cytotoxicity; optical imaging is prone to photobleaching, and has limited penetration depth and spatial resolution. Therefore, developing a multimodal contrast agent with high relaxation rate and aggregation-induced emission properties is an urgent problem to be solved in this field. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a multimodal contrast agent, its preparation method, and its application. This improves the relaxation rate of existing contrast agents, overcomes the limitations of low MRI sensitivity and high background noise, and is used to monitor the biodistribution of cells introduced into the body. It also boasts advantages such as large tissue penetration depth, high spatial resolution, high signal-to-noise ratio, and high sensitivity.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a multimodal contrast agent, the multimodal contrast agent comprising, by weight: 1 part of a fluorescent precursor, 10-15 parts of a contrast agent, 5-8 parts of dipalmitoylphosphatidylcholine and 1-3 parts of cholesterol, wherein the fluorescent precursor has a structure as shown in Formula 1.

[0011]

[0012] The contrast agent is in the form of 10-15 parts by weight, for example, 10, 11, 12, 13, 14, or 15 parts, as well as specific point values ​​between the above-mentioned point values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0013] The dipalmitoylphosphatidylcholine is present in 5-8 parts by weight, for example, 5 parts, 6 parts, 7 parts, 8 parts, and specific values ​​between the above-mentioned values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0014] The cholesterol is in parts by weight of 1-3 parts, for example, 1 part, 2 parts, 3 parts, and specific values ​​between the above-mentioned points. Due to space limitations and for the sake of brevity, this invention will not exhaustively list the specific values ​​included in the range.

[0015] The multimodal contrast agent provided by this invention can achieve MRI imaging and near-infrared two-zone imaging, overcoming the shortcomings of existing methods such as low safety, low sensitivity, and poor spatial resolution. By co-incubating this multimodal imaging contrast agent with CAR-NK cells, it is possible to observe the series of movement and physiological activities of CAR-NK cells carrying the contrast agent after entering the tumor site, enabling visual tracking of immune cells and promoting the development of immunotherapy.

[0016] The multimodal contrast agent prepared by this invention exhibits a significantly improved relaxation rate compared to commercially available gadolinium phosphate. The multimodal contrast agent provided by this invention has controllable and stable dimensions. The multimodal contrast agent prepared by this invention can be used for direct cell labeling, providing clear imaging and enabling real-time, non-invasive cell tracking. The T1 value and relaxation rate of the multimodal contrast agent prepared by this invention are superior to currently available commercial contrast agents, with significant contrast differences and long durations, facilitating long-term cell tracking in vivo. The multimodal contrast agent possesses aggregation-induced emission properties, emitting strong fluorescence in the aggregated state, thus avoiding the influence of the physiological environment. The multimodal imaging contrast agent of this invention has good biocompatibility, reducing the side effects of single-image imaging.

[0017] Preferably, the fluorescent precursor is prepared by the following method, the method comprising:

[0018] 4,8-Bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole was reacted with triphenylamine 4-boronic acid in the presence of a palladium catalyst to obtain the fluorescent precursor.

[0019] Preferably, the 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole has the structure shown in Formula 2.

[0020]

[0021] Preferably, the molar ratio of 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole to triphenylamine 4-boronic acid is 1:(2-3), for example, it can be 1:2, 1:2.1, 1:2.3, 1:2.5, 1:2.8, 1:3, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0022] Preferably, the palladium catalyst comprises tetra(triphenylphosphine)palladium.

[0023] Preferably, the molar ratio of 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole to tetra(triphenylphosphine)palladium is 1:(0.1-0.3), for example, 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0024] Preferably, the reaction is carried out in the presence of an alkaline substance.

[0025] Preferably, the alkaline substance includes potassium carbonate.

[0026] Preferably, the molar ratio of 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole to potassium carbonate is 1:(2.5-3.5), for example, it can be 1:2.5, 1:2.7, 1:2.9, 1:3, 1:3.2, 1:3.5, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0027] Preferably, the reaction is carried out in the presence of a solvent.

[0028] Preferably, based on 1 mol of 4,8-bis(5-bromo-(2-ethylhexyl)-2-thiophene)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole, the volume of the solvent is 30-50 mL, for example, 30 mL, 32 mL, 35 mL, 38 mL, 40 mL, 45 mL, 50 mL, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0029] Preferably, the solvent includes a combination of toluene, ethanol and water.

[0030] Preferably, the volume ratio of toluene to ethanol is (15-20):(0.5-1.5), for example, it can be 15:0.5, 15:1, 15:1.5, 18:0.5, 18:1, 18:1.5, 20:0.5, 20:1, 20:1.5, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0031] Preferably, the volume ratio of toluene to water is (15-20):(0.5-1.5), for example, it can be 15:0.5, 15:1, 15:1.5, 18:0.5, 18:1, 18:1.5, 20:0.5, 20:1, 20:1.5, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0032] Preferably, the reaction temperature is 90-110℃, for example, it can be 90℃, 92℃, 95℃, 100℃, 105℃, 110℃, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0033] Preferably, the reaction time is 10-12 hours, for example, 10 hours, 11 hours, 12 hours, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific values ​​included in the range.

[0034] Preferably, the reaction further includes post-processing.

[0035] Preferably, the post-processing includes concentration and column chromatography separation.

[0036] Preferably, the developing solvent for column chromatography is a combination of n-hexane and ethyl acetate.

[0037] Preferably, the volume ratio of n-hexane to ethyl acetate is (250-300):1, for example, it can be 250:1, 260:1, 270:1, 280:1, 290:1, 300:1, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0038] Preferably, the contrast agent comprises distearylphosphatidylethanolamine-polyethylene glycol-gadolinium contrast agent.

[0039] In a second aspect, the present invention provides a method for preparing a multimodal contrast agent as described in the first aspect, characterized in that the preparation method comprises:

[0040] A fluorescent precursor, contrast agent, dipalmitoylphosphatidylcholine, and cholesterol are mixed and dispersed with a first solvent to obtain a first solution. The solvent in the first solution is removed to form a liposome membrane. The liposome membrane is mixed with a second solvent and hydrated to obtain a liposome solution. The liposome solution is repeatedly extruded and purified to obtain the multimodal contrast agent.

[0041] Preferably, the first solvent is an organic solvent.

[0042] Preferably, the first solvent includes chloroform.

[0043] Preferably, the dispersion is performed under ultrasonic conditions.

[0044] Preferably, the ultrasound duration is 5-10 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific values ​​included in the range.

[0045] Preferably, the temperature of the ultrasound is 25-35℃, for example, it can be 25℃, 26℃, 28℃, 30℃, 33℃, 35℃, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0046] Preferably, the method for removing the solvent from the first solution includes rotary evaporation under reduced pressure.

[0047] Preferably, the second solvent is water.

[0048] Preferably, the hydration method includes alternating ice baths and hot water baths.

[0049] Preferably, the ice bath can be replaced with a liquid nitrogen environment for the reaction.

[0050] Preferably, the alternation is repeated 4-6 times, for example, 4, 5, or 6 times.

[0051] Preferably, the ice bath time is 5-15 minutes, for example, 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 15 minutes, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list the specific values ​​included in the range.

[0052] Preferably, the hot water bath time is 5-15 minutes, for example, 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 15 minutes, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list the specific values ​​included in the range.

[0053] Preferably, the temperature of the hot water bath is 90-100℃, for example, it can be 90℃, 92℃, 94℃, 96℃, 98℃, 100℃, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0054] Preferably, the number of repeated extrusions is 50-60 times, for example, 50 times, 52 times, 55 times, 58 times, 60 times, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list the specific values ​​included in the range.

[0055] Preferably, the purification method includes dialysis.

[0056] Preferably, the dialysis time is 40-50 hours, for example, 40 hours, 42 hours, 45 hours, 48 ​​hours, 50 hours, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific values ​​included in the range.

[0057] Preferably, the molecular weight cutoff of the dialysis bag used for dialysis is 3000-4000, for example, it can be 3000, 3200, 3500, 3800, 4000, and specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0058] The raw materials used in the preparation of this invention are simple and readily available, the synthesis steps are few, the yield is high, and it is suitable for industrial production.

[0059] Thirdly, the present invention provides the application of the multimodal contrast agent as described in the first aspect in the preparation of cell imaging reagents.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The multimodal contrast agent provided by this invention improves the relaxation rate and overcomes the limitations of low sensitivity and high background noise in MRI. By combining the complementarity of MRI and fluorescence imaging, dual-modal imaging is achieved. This multimodal contrast agent is used to monitor the biodistribution of cells introduced into the body. Based on its advantages of large tissue penetration depth, high spatial resolution, high signal-to-noise ratio, and high sensitivity, high-resolution imaging results can be obtained, achieving the requirement of immediate, non-invasive, and real-time detection of the presence, distribution, quantity, and activity of cells in vivo. Attached Figure Description

[0062] Figure 1 The proton spectrum of the fluorescent precursor obtained in Example 1;

[0063] Figure 2 The carbon spectrum of the fluorescent precursor obtained in Example 1;

[0064] Figure 3 The mass spectrum of the fluorescent precursor obtained in Example 1;

[0065] Figure 4 The absorption spectrum of the fluorescent precursor obtained in Example 1 is shown.

[0066] Figure 5 The emission spectrum of the fluorescent precursor obtained in Example 1 is shown.

[0067] Figure 6The image shows a transmission electron microscope (TEM) image of the multimodal contrast agent obtained in Example 1.

[0068] Figure 7 This is a particle size distribution diagram of the multimodal contrast agent obtained in Example 1;

[0069] Figure 8 The absorption spectrum of the multimodal contrast agent obtained in Example 1;

[0070] Figure 9 The emission spectrum of the multimodal contrast agent obtained in Example 1;

[0071] Figure 10 This is a blood routine measurement image of the multimodal contrast agent obtained in Example 1;

[0072] Figure 11 The graph shows the liver and kidney function measurements of the multimodal contrast agent obtained in Example 1.

[0073] Figure 12 The image shows the H&E staining of the multimodal contrast agent obtained in Example 1.

[0074] Figure 13 This is a fluorescence imaging measurement of the multimodal contrast agent obtained in Example 1;

[0075] Figure 14 The T1-weighted plot of the multimodal contrast agent and gadoterol obtained in Example 1;

[0076] Figure 15 The T1 curves for the multimodal contrast agent and gadoterol obtained in Example 1 are shown.

[0077] Figure 16 The image shows the r1 chromatograms of the multimodal contrast agent and gadoterol obtained in Example 1. Detailed Implementation

[0078] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0079] The experimental materials used in the embodiments of this invention are as follows:

[0080] (1) DSPE-PEG-DOTA contrast agent (Gd), brand name CP_20220909019, manufacturer Ruixi Biotechnology;

[0081] Preparation Example 1

[0082] This preparation example provides a fluorescent precursor, the preparation method of which includes:

[0083]

[0084] Under nitrogen protection, 4,8-bis(5-bromo-(2-ethylhexyl)-2-thiophene)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole, triphenylamine 4-borate, tetra(triphenylphosphine)palladium, and potassium carbonate in a molar ratio of 1:2.5:0.2:3 were accurately weighed and dissolved in a solution prepared with 36 mL toluene, 2 mL ethanol, and 2 mL pure water. The solution was refluxed and stirred overnight at 100 °C. The reaction was stopped when TLC monitoring showed no further reaction. The solvent was removed by vacuum distillation, and the solution was purified by column chromatography and vacuum dried to obtain 650 mg of a blue solid, which was the fluorescent precursor, with a yield of 81.25%. The 1H NMR spectrum was obtained using a Bruker AVANCE 400 NMR spectrometer. Figure 1 As shown, 1 ¹H NMR (400MHz, CDCl₃) δ 7.71–7.62 (m, 4H), 7.43 (s, 2H), 7.36–7.16 (m, 20H), 7.10 (dd, J = 11.3, 4.0Hz, 4H), 1.37 (d, J = 2.2Hz, 2H), 1.27–0.88 (m, 20H), 0.75 (d, J = 6.5Hz, 6H), 0.59 (d, J = 7.1Hz, 6H), [NMR spectrum shown in figure]. Figure 2 As shown, 13 C NMR (101MHz, CDCl3) δ153.28 (s), 147.61 (d, J = 11.6Hz), 146.71 (s), 144.73 (s), 129. 46(s),129.06(s),128.32(s),126.89(s),125.63(s),124.74(s),124.05(d,J=32.5 The following mass spectra were obtained using an Orbitrap Fusion Tribrid mass spectrometer: 123.44 (d, J = 26.3 Hz), 116.49 (s), 40.69 (s), 34.66 (s), 32.63 (s), 31.70 (s), 28.71 (s), 25.78 (s), 22.85 (d, J = 13.6 Hz), 14.24 (d, J = 12.6 Hz), 10.80 (s). Figure 3 As shown, the calculated HRMS m / z value C 66 H 64 N6S4:[[M + [Na] = 1069.52000; Experimental value: 1069.41647 indicates that the fluorescent precursor was successfully prepared. The absorption spectrum was obtained using a UV-Vis spectrophotometer. Figure 4As shown, the maximum absorption peak of the fluorescent precursor is around 700 nm. Fluorescence spectroscopy was used to measure the emission spectrum, as shown below. Figure 5 As shown, the maximum emission peak of the fluorescent precursor is around 950 nm.

[0085] Example 1

[0086] This embodiment provides a multimodal contrast agent and its preparation method. The multimodal contrast agent comprises, by weight, 1 part precursor, 10 parts DSPE-PEG-DOTA contrast agent (Gd), 5 parts dipalmitoylphosphatidylcholine, and 1 part cholesterol.

[0087] The specific preparation method of the multimodal contrast agent is as follows:

[0088] Accurately weigh DSPE-PEG-DOTA contrast agent (Gd), dipalmitoylphosphatidylcholine, cholesterol, and the fluorescent precursor, dissolve them in chloroform, disperse by sonication, remove the organic solvent by rotary evaporation under reduced pressure at 30-40℃, then add 10 mL of pure water for homogeneous hydration, and cycle through ice bath and hot water bath five times, 10 min each time, to obtain a blue liposome solution. Then, extrude the liposome solution 50-60 times using an extruder, and dialyze the extruded liquid in 1200 mL of pure water for 48 h using a dialysis bag with a molecular weight cutoff of 3500 to obtain a multimodal contrast agent (Liposome).

[0089] Example 2

[0090] This embodiment provides a multimodal contrast agent and its preparation method. The multimodal contrast agent comprises, by weight, 1 part precursor, 20 parts DSPE-PEG-DOTA contrast agent (Gd), 5 parts dipalmitoylphosphatidylcholine and 1 part cholesterol.

[0091] The specific preparation method of the multimodal contrast agent is as follows:

[0092] Accurately weigh DSPE-PEG-DOTA contrast agent (Gd), dipalmitoylphosphatidylcholine, cholesterol, and the fluorescent precursor, dissolve them in chloroform, disperse by sonication, remove the organic solvent by rotary evaporation under reduced pressure at 30-40℃, then add 10mL of pure water for homogeneous hydration, and cycle through ice bath and hot water bath five times, 10min each time, to obtain a blue liposome solution. Then, extrude the liposome solution 50-60 times using an extruder, and dialyze the extruded liquid in 1200mL of pure water for 48h using a dialysis bag with a molecular weight cutoff of 3500 to obtain a multimodal contrast agent.

[0093] Example 3

[0094] This embodiment provides a multimodal contrast agent and its preparation method. The multimodal contrast agent comprises, by weight, 1 part precursor, 10 parts DSPE-PEG-DOTA contrast agent (Gd), and 1 part cholesterol.

[0095] The specific preparation method of the multimodal contrast agent is as follows:

[0096] Accurately weigh DSPE-PEG-DOTA contrast agent (Gd), dipalmitoylphosphatidylcholine, cholesterol, and the fluorescent precursor, dissolve them in chloroform, disperse by sonication, remove the organic solvent by rotary evaporation under reduced pressure at 30-40℃, then add 10mL of pure water for homogeneous hydration, and cycle through ice bath and hot water bath five times, 10min each time, to obtain a blue liposome solution. Then, extrude the liposome solution 50-60 times using an extruder, and dialyze the extruded liquid in 1200mL of pure water for 48h using a dialysis bag with a molecular weight cutoff of 3500 to obtain a multimodal contrast agent.

[0097] Example 4

[0098] The multimodal contrast agent obtained in Example 1 was tested: the multimodal contrast agent prepared in Example 1 was imaged using transmission electron microscopy, and the results are as follows. Figure 6 As shown, the multimodal contrast agent prepared in Example 1 has a particle size of approximately 100 nm and exhibits a spherical morphology. The multimodal imaging contrast agent prepared in Example 1 was tested using a Malvern particle size analyzer, and the results are as follows... Figure 7 As shown, the hydrated particle size of the multimodal contrast agent prepared in Example 1 is approximately 140 nm, and it is uniformly distributed and dispersed. The multimodal contrast agent prepared in Example 1 was measured using a UV absorption spectrophotometer, and the results are as follows. Figure 8 As shown, the maximum absorption peak of the multimodal contrast agent prepared in Example 1 is around 750 nm. The multimodal contrast agent prepared in Example 1 was measured using fluorescence spectroscopy, and the results are as follows. Figure 9 As shown, the maximum emission peak of the multimodal contrast agent prepared in Example 1 is around 1050 nm.

[0099] Example 5

[0100] The concentration of the contrast agent obtained in Example 1 was tested: Based on the prepared multimodal contrast agent, the concentration of the multimodal imaging contrast agent was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Gd standard solutions of 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL were prepared by diluting the Gd standard solution with 2% nitric acid aqueous solution, and a standard curve was determined to establish the Gd concentration of the prepared multimodal contrast agent. The measured concentration of the prepared multimodal contrast agent was 90 μg / mL, and subsequent experiments were conducted at this concentration.

[0101] Example 6

[0102] Biocompatibility testing was performed on the multimodal contrast agent obtained in Example 1: the results are as follows Figures 10-12 As shown, the major organs of mice treated with the multimodal imaging contrast agent (200 μL of the prepared 0.1 mg / mL multimodal imaging contrast agent solution was injected into the tail vein of the mice, blood was collected from the orbital cavity 24 hours later, the mice were sacrificed, and organs such as the heart, liver, spleen, lungs, and kidneys were dissected and removed. After removal, the organs were immediately immersed in paraformaldehyde solution, and then paraffin sections were prepared for H&E staining) and the PBS (phosphate buffer saline) treatment group were not damaged. Blood routine and liver and kidney function tests were also within the normal range, indicating that the multimodal contrast agent constructed in this invention has good biocompatibility.

[0103] Example 7

[0104] Fluorescence imaging tests were performed on the multimodal contrast agent obtained in Example 1: Based on the prepared multimodal contrast agent, it was diluted several times to obtain contrast agent solutions with concentrations of 60 μg / mL, 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, and 0 μg / mL, respectively. Two-zone fluorescence imaging images were obtained using small animal NIRF imaging (1 mL of each of the prepared 60 μg / mL, 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, and 0 μg / mL contrast agent solutions were placed in 1.5 mL EP tubes, and then placed under a small animal NIRF imaging instrument to obtain two-zone fluorescence images). The results are as follows: Figure 13 As shown, the fluorescence imaging effects of different concentrations of multimodal contrast agents are presented by... Figure 13 It can be seen that the fluorescence intensity increases with the increase of concentration, and the bright near-infrared II fluorescence is beneficial for cell tracking in vivo.

[0105] Example 8

[0106] The T1 values ​​of the multimodal contrast agent obtained in Example 1 (T1 is one of the important parameters of MRI, representing the longitudinal relaxation time. T1 indicates the time required for the magnetization vector in the magnetization direction to recover to 63% of its original amplitude. T1 is related to contrast calculation, and contrast enhancement is measured by the relaxation rate Ri = 1 / Ti(s-1), where i = 1 or 2. In the T1-weighted image, the shorter the T1 relaxation time, the brighter the tissue contrast and the more obvious the imaging effect) and the T1 values ​​of the commercial gadolinium ethanol in Comparative Example 1 were determined: Based on gadolinium ethanol and the prepared multimodal contrast agent, gadolinium ethanol solutions and contrast agent solutions with concentrations of 60 μg / mL, 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, and 0 μg / mL were obtained by dilution several times, and the T1 values ​​and T1-weighted images were obtained by MRI testing. The results are as follows: Figures 14-16 As shown, MRI imaging results of different concentrations of multimodal contrast agents and commercially available gadolinium ethanol are presented. Figures 14-16 It is known that, at the same concentration, the multimodal contrast agent of the present invention has a higher relaxation rate r1 value, a lower T1 value, and a stronger T1-weighted image, which indicates that it can improve the contrast of the imaging results and enhance the cell tracking effect.

[0107] Comparative Example 1

[0108] Commercial gadoteridol, manufactured by Prohex, contains excipients such as catelide calcium, aminobutanetriol, and water for injection. Its chemical name is 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid.

[0109] The applicant declares that this invention illustrates a multimodal contrast agent, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials in the product, addition of auxiliary components, and selection of specific methods, all fall within the protection and disclosure scope of this invention.

Claims

1. A multimodal contrast agent, characterized in that, The multimodal contrast agent comprises, by weight, 1 part of a fluorescent precursor, 10-15 parts of the contrast agent, 5-8 parts of dipalmitoylphosphatidylcholine and 1-3 parts of cholesterol, wherein the fluorescent precursor has the structure shown in Formula 1; the contrast agent comprises distearate phosphatidylethanolamine-polyethylene glycol-gadolinium contrast agent. 。 2. The multimodal contrast agent according to claim 1, characterized in that, The fluorescent precursor is prepared by the following method, the method comprising: 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole was reacted with triphenylamine 4-boronic acid in the presence of a palladium catalyst to obtain the fluorescent precursor.

3. The multimodal contrast agent according to claim 2, characterized in that, The molar ratio of 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole to triphenylamine 4-boronic acid is 1:(2-3).

4. The multimodal contrast agent according to claim 2, characterized in that, The palladium catalyst comprises tetra(triphenylphosphine)palladium.

5. The multimodal contrast agent according to claim 4, characterized in that, The molar ratio of 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole to tetra(triphenylphosphine)palladium is 1:(0.1-0.3).

6. The multimodal contrast agent according to claim 2, characterized in that, The reaction is carried out in the presence of an alkaline substance.

7. The multimodal contrast agent according to claim 6, characterized in that, The alkaline substance includes potassium carbonate.

8. The multimodal contrast agent according to claim 7, characterized in that, The molar ratio of the 4,8-bis(5-bromo-(2-ethylhexyl)-2-thienyl)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole to potassium carbonate is 1:(2.5-3.5).

9. The multimodal contrast agent according to claim 2, characterized in that, The reaction is carried out in the presence of a solvent.

10. The multimodal contrast agent according to claim 9, characterized in that, The volume of the solvent is 30-50 mL, based on 1 mol of 4,8-bis(5-bromo-(2-ethylhexyl)-2-thiophene)-2λ4δ2-benzo[1,2-c:4,5-c']bis[1,2,5]thiazole.

11. The multimodal contrast agent according to claim 9, characterized in that, The solvent includes a combination of toluene, ethanol and water.

12. The multimodal contrast agent according to claim 11, characterized in that, The volume ratio of toluene to ethanol is (15-20):(0.5-1.5).

13. The multimodal contrast agent according to claim 11, characterized in that, The volume ratio of toluene to water is (15-20):(0.5-1.5).

14. The multimodal contrast agent according to claim 2, characterized in that, The reaction temperature is 90-110 ℃.

15. The multimodal contrast agent according to claim 2, characterized in that, The reaction time is 10-12 h.

16. The multimodal contrast agent according to claim 2, characterized in that, The reaction also includes post-processing.

17. The multimodal contrast agent according to claim 16, characterized in that, The post-processing includes concentration and column chromatography separation.

18. The multimodal contrast agent according to claim 17, characterized in that, The developing solvent for column chromatography separation is a combination of n-hexane and ethyl acetate.

19. The multimodal contrast agent according to claim 18, characterized in that, The volume ratio of n-hexane to ethyl acetate is (250-300):

1.

20. A method for preparing a multimodal contrast agent as described in any one of claims 1-19, characterized in that, The preparation method includes: A fluorescent precursor, contrast agent, dipalmitoylphosphatidylcholine, and cholesterol are mixed and dispersed with a first solvent to obtain a first solution. The solvent in the first solution is removed to form a liposome membrane. The liposome membrane is mixed with a second solvent and hydrated to obtain a liposome solution. The liposome solution is repeatedly extruded and purified to obtain the multimodal contrast agent.

21. The preparation method according to claim 20, characterized in that, The first solvent is an organic solvent.

22. The preparation method according to claim 20, characterized in that, The first solvent includes chloroform.

23. The preparation method according to claim 20, characterized in that, The dispersion is carried out under ultrasonic conditions.

24. The preparation method according to claim 23, characterized in that, The ultrasound session lasted 5-10 minutes.

25. The preparation method according to claim 23, characterized in that, The temperature of the ultrasound is 25-35 ℃.

26. The preparation method according to claim 20, characterized in that, Methods for removing solvent from the first solution include rotary evaporation under reduced pressure.

27. The preparation method according to claim 20, characterized in that, The second solvent is water.

28. The preparation method according to claim 20, characterized in that, The hydration method includes alternating ice baths and hot water baths.

29. The preparation method according to claim 28, characterized in that, The alternation is repeated 4-6 times.

30. The preparation method according to claim 28, characterized in that, The ice bath time is 5-15 minutes.

31. The preparation method according to claim 28, characterized in that, The hot water bath should last for 5-15 minutes.

32. The preparation method according to claim 28, characterized in that, The temperature of the hot water bath is 90-100 ℃.

33. The preparation method according to claim 20, characterized in that, The number of repeated extrusions is 50-60 times.

34. The preparation method according to claim 20, characterized in that, The purification method includes dialysis.

35. The preparation method according to claim 34, characterized in that, The dialysis time is 40-50 hours.

36. The preparation method according to claim 34, characterized in that, The molecular weight cutoff of the dialysis bag used for dialysis is 3000-4000.

37. The use of a multimodal contrast agent as described in any one of claims 1-19 in the preparation of cell imaging reagents.

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