Reagent for reducing liver radiation dose through in-vivo radiolabeling and application thereof

Abstract

The invention discloses a reagent for reducing the radiation dose of liver through in-vivo radiolabeling and application thereof, nanoparticles with surface modifiers and nuclide-labeled molecules are adopted, and the surface modifiers and the molecules can react to realize covalent binding, so that binding of the surface modifiers and the molecules in the liver is greatly reduced; and the tissue distribution difference between the liver and the focus part is obvious. Compared with a radioactive nano-material obtained through in-vitro labeling, the system is used for realizing in-vivo labeling of the nano-material, so that the liver can be better protected. Theoretical and experimental basis is provided for further development of application of nano materials in nuclear medicine, and the method has important significance in protecting non-target organs such as liver and other radiation-sensitive organs in nuclide diagnosis and treatment.

Description

Technical Field

[0001] The invention relates to a method for radioactively labeling nanomaterials and reducing liver radiation dose, specifically a reagent for reducing liver radiation dose by radioactively labeling in vivo, which can reduce the radiation dose of radionuclides to the liver compared with radioactively labeled nanomaterials in vitro. Background Art

[0002] Nanomaterials have attracted more and more attention as new carriers of radionuclides. Existing labeling methods mainly include direct labeling and indirect labeling. Direct labeling mainly involves directly incorporating the nuclide into the structure of the nanomaterial itself, while indirect labeling usually uses bifunctional groups chemically modified on the surface of the nanomaterial to coordinate and bind the nuclide to achieve labeling. However, no matter what method is used for labeling, these radioactive nanomaterials are often taken up in large quantities by reticuloendothelial organs such as the liver after intravenous injection and retained for a long time, thereby causing over-irradiation to these radiation-sensitive organs, hindering the application of nanomaterials in nuclear medicine.

[0003] Regarding the problem of excessive liver uptake of radioactive nanomaterials in tumor applications, existing research is devoted to studying the interaction between nanomaterials and the liver and tumors. At present, the design and synthesis of nanoparticles are guided by the results revealed by the interaction studies, such as surface modification of polyethylene glycol to escape macrophage uptake or modification of ion ligands that can be filtered by the kidney to promote renal metabolism of particles; or guiding the development of more sensible nanomaterial application methods, such as inhibiting macrophage activity to reduce uptake or pre-saturating macrophages. However, these studies have not ideally solved the problem of liver uptake of nanomaterials. For example, when using zwitterionic ligands, firstly, the design of the ligand needs to avoid causing the colloidal stability of the modified nanomaterial to decrease. Secondly, the precisely designed ligands often require special synthetic routes. For example, zwitterionic dopamine sulfonate requires two-step heating reaction for preparation. Macrophages are immune cells that clear foreign bodies from the body. Inhibiting their activity may disrupt the body's immune cell homeostasis. In addition, by pre-saturating macrophages, due to their huge phagocytic ability, it is often necessary to greatly increase the body's nanomaterial burden, which poses potential biosafety issues. Therefore, it is crucial to develop a safe method that can use nanomaterials as radionuclide carriers without causing a large amount of radionuclide retention in the liver. Summary of the invention

[0004] The technical problem to be solved by the present invention is that existing radioactive labeled nanomaterials are easily enriched in the liver in vivo, and it is necessary to reduce the radioactive exposure to the liver. Existing methods focus on changing the structural properties of materials or changing related functions of the body, and have not achieved a simple, safe and universal way to effectively solve the problem of liver enrichment of radioactive materials. The present invention improves and perfects the existing problems, and discloses an agent for reducing liver radiation dose by radioactive labeling in vivo, including nanoparticles with surface modifiers and radionuclide-labeled molecules, and the surface modifiers and molecules can undergo covalent binding reactions. The radiation agent is not easy to penetrate into the cell through the cell membrane, is quickly cleared from the blood and is rapidly metabolized by the kidneys, so that the radiation in the liver is greatly reduced, and single photon emission computed tomography imaging can be used to monitor its behavioral characteristics and biological distribution in the body.

[0005] The technical solution adopted by the present invention is: An in vivo radioactive labeling agent for reducing liver radiation dose comprises nanoparticles with surface modifiers and radionuclide-labeled molecules, wherein the modifiers and molecules can react. Further, the reaction comprises an in vitro reaction or an in vivo reaction.

[0006] In the present invention, the modifier and the molecule are a bio-orthogonal reaction system, so that the nanoparticles with surface modifiers and the molecules labeled with nuclides can react with the molecules through the modifier; preferably, the bio-orthogonal reaction system includes electron-rich diene structures and electron-deficient diene structures (such as trans-cyclooctene / tetrazine), metal ion-catalyzed alkyne structures and azide structures (such as copper ion-catalyzed phenylacetylene and azidobenzene), cycloalkyne structures and azide structures (such as dibenzocyclooctyne and azide), azide structures and phosphine (such as triphenylphosphine) or phosphite, biotin and avidin systems, and new bio-orthogonal reactions (under development, such as visible light-driven vicinal diketones and electron-rich olefin ethers) One or more. In the present invention, the nanoparticles include one or more of organic nanoparticles, inorganic nanoparticles, and hybrid nanoparticles; the nuclides include one or more of radioactive nuclides emitting helium nuclei, photons, electrons, and neutrons.

[0007] In the present invention, the surface modifier includes an electron-rich diene structure, such as trans-cyclooctene, and the molecule includes an electron-deficient diene structure, such as a tetrazine compound; or the molecule includes an electron-rich diene structure, such as trans-cyclooctene, and includes a surface modifier electron-deficient diene structure, such as a tetrazine compound.

[0008] The present invention discloses a method for preparing the above-mentioned in vivo radioactive labeling agent for reducing liver radiation dose, comprising the following steps: combining a modifier on the surface of a nanoparticle to obtain a nanoparticle with a surface modifier; combining a nuclide on the molecular structure to obtain a nuclide-labeled molecule; the in vivo radioactive labeling agent for reducing liver radiation dose comprises a nanoparticle with a surface modifier and a nuclide-labeled molecule. Specifically, a nanoparticle with a surface modifier is obtained by covalently binding on the surface of the nanoparticle; a nuclide is introduced into the molecular structure by coordination binding to obtain a nuclide-labeled molecule; the in vivo radioactive labeling agent for reducing liver radiation dose comprises a nanoparticle with a surface modifier and a nuclide-labeled molecule.

[0009] In the present invention, the nanoparticles with surface modifiers and the radionuclide-labeled molecules will not be internalized by cells in large quantities at the lesion site, and the two can react and bind when in contact, thereby exerting the diagnostic and therapeutic function of the reagent. The lesion site includes the tumor site.

[0010] The present invention discloses the use of the above-mentioned in vivo radioactive labeling reagent for reducing liver radiation dose in the preparation of diagnostic and therapeutic drugs. Further, the present invention discloses the use of the above-mentioned in vivo radioactive labeling reagent for reducing liver radiation dose in the preparation of multimodal diagnostic and therapeutic drugs. Among them, diagnostic and therapeutic drugs refer to drugs for diagnosis and / or treatment. In addition to the radiation provided by the nuclide, imaging or therapeutic radiation, the diagnostic and therapeutic methods also include the functions provided by the nanoparticles themselves, such as iron oxide nanoparticles, which can have magnetic resonance imaging or magnetic thermal therapy. Therefore, the above-mentioned in vivo radioactive labeling reagent for reducing liver radiation dose has multimodal diagnostic and therapeutic potential.

[0011] The present invention discloses the use of the above-mentioned in vivo radioactive labeling agent for reducing liver radiation dose in the preparation of imaging agents, wherein the imaging includes in vivo imaging and in vitro imaging, preferably in vivo imaging; the imaging mode includes single photon emission computed tomography imaging.

[0012] The present invention discloses the use of the above-mentioned in vivo radioactive labeling agent for reducing liver radiation dose in the preparation of a drug for reducing liver accumulation or retention. Reducing liver accumulation or retention means that the agent of the present invention can reduce the accumulation or retention of the radiation agent in the liver compared with the existing imaging agent.

[0013] The present invention discloses the use of the above-mentioned reagent for reducing liver radiation dose by radioactive labeling in vivo in the preparation of a drug for increasing the ratio of radionuclide retention in the lesion site to the liver site. The reagent of the present invention can reduce the accumulation or retention of the reagent in the liver and exert the diagnostic and therapeutic function of the reagent in the lesion site.

[0014] The invention discloses a diagnostic and therapeutic drug, comprising the above-mentioned reagent for reducing liver radiation dose by in vivo radioactive labeling; and may also comprise a conventional pharmaceutically acceptable carrier or dispersion medium.

[0015] The invention discloses a method for reducing liver radiation dose by in vivo radioactive labeling, and uses the reagent for reducing liver radiation dose by in vivo radioactive labeling as a diagnostic and therapeutic drug.

[0016] The invention discloses a reagent for reducing liver radiation dose, comprising trans-cyclooctene-modified iron oxide nanoparticles Fe3O4@TCO and radioactive nuclides capable of undergoing bioorthogonal reactions in vivo and in vitro. 177 Lu-labeled tetrazine small molecule probe 177 Lu-Tz-DOTA can block the in vivo radioactive labeling of iron oxide nanoparticles in the liver and reduce the accumulation of radionuclides in the liver.

[0017] As an example of the present invention: a method for in vivo radiolabeling to reduce liver radiation dose, comprising the following steps: synthesizing nanoparticles, and modifying their surfaces to carry trans-cyclooctene with a bioorthogonal active group, to obtain Fe3O4@TCO; synthesizing a tetrazine small molecule (Tz-DOTA) capable of radionuclide labeling with complementary bioorthogonal activity, and carrying out radionuclide 177 Lu labeling 177 Lu-Tz-DOTA. The above Fe3O4@TCO, 177 Lu-Tz-DOTA undergoes an orthogonal reaction in vitro to obtain Fe3O4@ 177 Lu, which was injected intravenously and its radioactivity distribution was monitored by imaging as a control; the above Fe3O4@TCO was injected intravenously, and then, 177 Lu-Tz-DOTA, imaging monitoring of its radioactivity distribution, and quantitative evaluation and comparison of the signals of the two radioactivity monitoring schemes.

[0018] In the present invention, the nanomaterial model is iron oxide nanoparticles; nanomaterials can be divided into inorganic nanomaterials, organic nanomaterials and hybrid types based on their composition. Iron oxide nanoparticles are selected as model materials because they have the common characteristics of most nanomaterials and have relatively mature synthesis and surface chemical modification methods. They are also being developed as carriers of nuclides and have a broad application basis and prospects in disease diagnosis and treatment, which is conducive to simply verifying the method of the present invention and emphasizing that the method itself and its application are universal.

[0019] In the present invention, the reaction for achieving the formation of nanoparticles with surface modifications and nuclide-labeled molecules is the inverse electron demand Diels-Alder reaction (IEDDA), which is based on electron-deficient dienes (such as 1, 2, 4, 5-tetrazine) and electron-rich dienophiles (such as trans-cyclooctene). The reaction can be carried out at room temperature, physiological pH, and without a catalyst. The present invention uses the most active tetrazine (Tz) and trans-cyclooctene (TCO) system as an example, and its cycloaddition reaction rate exceeds 1000000 M −1 s −1 , surpassing other current reaction systems, such as azide and cycloalkyne systems.

[0020] In the present invention, the surface of iron oxide nanoparticles is modified with trans-cyclooctene TCO. First, hydrophobic iron oxide nanoparticles are prepared by thermal decomposition reaction. The particles are characterized by electron microscopy to be round particles with a diameter of about 9 nanometers and uniform particle size distribution. The particles are modified with polyethylene glycol. Polyethylene glycol (MW~2K) having a bisphosphonic acid group at one end and a maleimide at the other end is used. After modification, a click reaction occurs with mercaptodiethylamine to modify the free amino groups on the surface of the nanoparticles. Trans-cyclooctene carboxyl active ester and the free amino groups on the surface of iron oxide are used to undergo an amide reaction under alkaline conditions. After further purification, iron oxide nanoparticles Fe3O4@TCO carrying the bio-orthogonal active group trans-cyclooctene are obtained. The entire synthesis and modification process is relatively mature and simple. Characterization of the particle size and morphology of Fe3O4@TCO also shows that this process does not affect the nanoparticles themselves. The present invention introduces TCO on the surface of nanomaterials for subsequent orthogonal labeling, and does not need to introduce nuclides at the beginning, thus avoiding the problem of specially designing the synthesis of nanomaterials for achieving special properties and functions. For example, the prior art requires the nuclides to be incorporated into the particle lattice under very harsh and delicate operations, or the nanomaterials are placed in a specific solution system (high temperature or acidic conditions) for nuclide labeling, which may damage the nanomaterials themselves. As for the problem to be solved by the present invention, the method of reducing radioactive accumulation in the present invention is not to prevent the nanomaterials from going to the liver, so there is no need to finely design its surface features to escape immune uptake or change its metabolic pathway. For example, the prior art designs surface ligands with special properties, screens the molecular weight of polyethylene glycol, and considers the influence of different structures formed on the surface by polyethylene glycols of different molecular weights on their distribution and uptake.

[0021] In the present invention, a carboxyl group is introduced into 1,4-bipyridine-tetrazineamine through an anhydride ring-opening reaction, a polyethylene glycol chain (MW=644) with a protected amino group at the end is further introduced through an amide reaction, the amino group is deprotected in a trifluoroacetic acid mixed solution to obtain a free amino group, and finally a nuclide coordination structure 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is introduced under alkaline conditions to obtain a non-nuclide labeled tetrazine small molecule Tz-DOTA, and further heated in vitro 177 Lu labeling 177 Lu-Tz-DOTA is rapidly cleared from the blood and metabolized by the kidneys. It has excellent hydrophilicity and uses the macrocyclic structure of DOTA to carry nuclides. It has a labeling rate of nearly 100% and excellent in vivo stability.

[0022] In the present invention, by virtue of the characteristics of bioorthogonal reactions, the method can be carried out both in vitro and in vivo, and the difference in the radioactive distribution of the same nanomaterial labeled in vitro and in vivo can be directly compared. By utilizing the detectability of nuclides, the radiation dose of the liver can be non-invasively monitored. According to the traditional method, bioorthogonal labeling is carried out in vitro, and the above Fe3O4@TCO and 177 Lu-Tz-DOTA oscillation reaction to obtain Fe3O4@ 177 Lu, injected it intravenously, and monitored the mice with single photon emission computed tomography (SPECT), and found that the heart and liver signals were high, which indicated that the radioactive nanoparticles stayed in the blood for a long time, and most of them were enriched in the liver with blood transport, and the liver uptake increased first and then slowly decreased over time. This result is consistent with the general trend of radioactive nanomaterials, that is, they are captured by the reticuloendothelial organ and retained for a long time. In the present invention, the reagent for reducing the radiation dose of the liver by radioactive labeling in vivo, including nanoparticles with surface modifiers and radionuclide-labeled molecules, adopts a two-step injection method, first intravenously injecting Fe3O4@TCO, you can wait for a period of time, such as 1 to 10 hours, preferably 2 to 8 hours, and more preferably 3 to 6 hours, and then intravenously injecting 177 Lu-Tz-DOTA, and then the imaging monitoring was also performed. At this time, it was observed that the signal in the blood was quickly cleared and the liver did not produce significant signals, which suggested that the Fe3O4@TCO in the liver did not interact with the blood. 177Lu-Tz-DOTA is combined, so that no nuclides are trapped. Comparison of the two imaging monitoring results shows that the reagent for reducing liver radiation dose by in vivo radiolabeling with nanoparticles with surface modifiers and nuclide-labeled molecules effectively reduces liver radioactive uptake. This method can theoretically be applied to any nanomaterial, which means that when using nanomaterials as nuclide carriers, there is no need to specially design the nanomaterials or interfere with the natural state of the body. This method is simple, safe and universal, and is an effective way to develop nanomaterials as nuclide delivery carriers and avoid excessive liver irradiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Reaction route for TCO modification of iron oxide nanoparticle surfaces.

[0024] Figure 2 TEM and particle size statistics of hydrophobic iron oxide nanoparticles Fe3O4 (a, b) and surface-modified TCO iron oxide nanoparticles Fe3O4@TCO (c, d).

[0025] Figure 3 Characterization of the hydrated particle size of Fe3O4 nanoparticles before and after TCO modification.

[0026] Figure 4 This is the organic synthesis route of Tz-DOTA.

[0027] Figure 5 Mass spectrometric characterization of Tz-COOH, the synthetic intermediate of Tz-DOTA.

[0028] Figure 6 Tz-PEG is the synthetic intermediate of Tz-DOTA 10 Mass spectrometric characterization of -NH2-Boc.

[0029] Figure 7 Mass spectrometry characterization of the final product Tz-DOTA.

[0030] Figure 8 a is the radiolabeling process of Tz-DOTA; b is 177 Lu-Tz-DOTA radiochemical purity characterization; c is 177 Lu-Tz-DOTA radiostability characterization; d is the specific activity optimization process; e is 177 Lu-Tz-DOTA oil-water partition coefficient; f is 177 Lu-Tz-DOTA blood clearance rate; g is 177 Distribution of Lu-Tz-DOTA in vivo.

[0031] Fig. 9a is Fe3O4@TCO in vitro 177 Lu orthogonal labeling efficiency; b is Fe3O4@ 177 Lu ligand stability in vitro.

[0032] Fig.10 The results of SPECT-CT in vivo monitoring of normal mice, a is Fe3O4@ 177 a) The distribution of Lu, b) and c) are the signal circle values ​​of the heart and liver, respectively; d) is the in vivo biological orthogonal validation imaging result, e) and f) are the signal circle values ​​of the liver and injection site, respectively.

[0033] Fig.11 SPECT-CT in vivo monitoring imaging of tumor mice, a is Fe3O4@ 177 The distribution of Lu, c, d are the corresponding liver and tumor signal circles, g is the in vitro anatomical biodistribution result; b is Fe3O4@TCO+ 177 The distribution of Lu-Tz-DOTA, e and f are the corresponding liver and tumor signal circle values; h is the in vitro anatomical biodistribution result. DETAILED DESCRIPTION

[0034] At present, there are few reports on the use of bioorthogonal reactions to achieve in vivo radioactive labeling of nanoparticles. Existing reports mainly focus on the development of nano-platforms that can achieve tumor imaging or treatment, and do not pay attention to the effects of the developed systems, especially the radionuclide systems, on the liver. There is no work on the reasonable design and verification of the bioorthogonal systems used by the liver. As for the problem of liver enrichment of radioactive materials, there is no relevant report on the use of bioorthogonal systems to improve and solve it. Currently, the main methods used are surface modification of nanomaterials or regulation of immune phagocytosis, and there is no safe and effective way to overcome liver problems at the same time. The present invention proposes to develop and utilize bioorthogonal system nanosystems to reduce the radioactive burden of the liver, especially creatively proposes to use the cell membrane in the liver to separate the nanomaterial and the nuclide part to block the orthogonal reaction. It is unexpectedly found that the system can greatly reduce the liver dose. This system is not only suitable for the iron oxide nanoparticles used in the present invention, but also can be used for other materials that are easily enriched in the liver and retained for a long time.

[0035] The invention discloses an in vivo radioactive labeling reagent for reducing liver radiation dose, comprising nanoparticles with bio-orthogonal active groups modified on the surface and molecules with complementary reaction activity labeled with radionuclides, wherein the surface-modified nanoparticles and the molecules labeled with radionuclides can undergo covalent binding reaction.

[0036] Furthermore, the bioorthogonal active groups and their complementary reactive molecules include electron-rich diene structures and electron-deficient diene structure molecules, (cyclo)alkyne structures and azide structures, azide structures and phosphines (such as triphenylphosphine) or phosphites, biotin and avidin systems, and other novel bioorthogonal reactions; nanoparticles include organic nanoparticles, inorganic nanoparticles, hybrid nanoparticles; nuclides include radioactive nuclides that emit helium nuclei, photons, and electrons.

[0037] As an example, the present invention discloses an in vivo radioactively labeled reagent for reducing liver radiation dose, including nanoparticles with surface modified electron-rich diene structures and electron-deficient diene structure molecules labeled with radionuclides, wherein the surface-modified nanoparticles and the radionuclide-labeled molecules can undergo a covalent binding reaction.

[0038] Furthermore, the electron-rich diene structure includes trans-cyclooctene; and the electron-deficient diene structure molecule includes tetrazine.

[0039] The preparation method of the above-mentioned in vivo radioactive labeling agent for reducing liver radiation dose of the present invention comprises the following steps: chemically modifying the electron-rich dienophile structure on the surface of nanoparticles to obtain nanoparticles with the electron-rich dienophile structure modified on the surface; introducing a nuclide chelating group on the electron-deficient diene molecule structure to obtain a radionuclide-labeled electron-deficient diene structure molecule. The nanoparticles with the electron-rich dienophile structure modified on the surface and the radionuclide-labeled electron-deficient diene structure molecule constitute the in vivo radioactive labeling agent for reducing liver radiation dose.

[0040] As a specific embodiment, the present invention takes trans-cyclooctene-modified iron oxide nanoparticles Fe3O4@TCO as an example, the surface of which is modified with trans-cyclooctene having a bio-orthogonal active group, which can be bio-orthogonally bound to substances having a tetrazine structure in vivo and in vitro; a radionuclide-labeled tetrazine small molecule is also disclosed. 177 Lu-Tz-DOTA, with radionuclides 177 Lu emits single photons and electrons, can produce internal irradiation and in vitro radioactivity detection, can rapidly and safely covalently bind to substances with a trans-cyclooctene structure both in vivo and in vitro, has high hydrophilicity, is rapidly cleared from the blood and rapidly metabolized by the kidneys.

[0041] In the present invention, the preparation method of trans-cyclooctene-modified iron oxide nanoparticles Fe3O4@TCO comprises the following steps: preparing hydrophobic iron oxide nanoparticles through thermal decomposition reaction, modifying them with polyethylene glycol (MW~2K) having bisphosphonic acid at one end and maleimide at the other end, performing click reaction with mercaptodiethylamine to modify free amino groups on the surface of the nanoparticles, performing amide reaction with trans-cyclooctene carboxyl active ester and free amino groups on the surface of iron oxide under alkaline conditions, and obtaining Fe3O4@TCO through further purification.

[0042] In the present invention, the radionuclide-labeled tetrazine small molecule 177 The preparation method of Lu-Tz-DOTA comprises the following steps: introducing a carboxyl group into 1,4-dipyridine-tetrazine amine through an anhydride ring-opening reaction, further introducing a polyethylene glycol chain (MW=644) with a protected amino group at the end through an amide reaction, deprotecting the amino group in a trifluoroacetic acid mixed solution to obtain a free amino group, and finally introducing a nuclide coordination structure 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid under alkaline conditions to obtain a non-nuclide labeled tetrazine small molecule Tz-DOTA, and further heating the reaction mixture in vitro. 177 Lu labeling 177 Lu-Tz-DOTA.

[0043] The invention discloses a method for reducing liver radiation dose by in vivo radioactive labeling, which comprises intravenous injection of iron oxide nanoparticles Fe3O4@TCO and radionuclide labeled tetrazine small molecules in two steps. 177 Lu-Tz-DOTA, the two can combine in vivo and realize in vivo radiolabeling to reduce liver radiation dose. The first injection of Fe3O4@TCO is internalized by cells in the liver and can be combined with subsequent blood in the lesion site. 177 Lu-Tz-DOTA binds to each other in contact, and its behavioral characteristics and biodistribution in the body can be monitored using single-photon emission computed tomography imaging; it significantly reduces the radiation dose to the liver, while increasing the ratio of radionuclide retention in the lesion site to that in the liver.

[0044] As a control, trans-cyclooctene-modified iron oxide nanoparticles Fe3O4@TCO can be reacted with radionuclide-labeled tetrazine small molecules using bioorthogonal reactions. 177 Lu-Tz-DOTA covalently binds in vitro to directly obtain radionuclide-labeled iron oxide nanoparticles Fe3O4@ 177 Lu;Fe3O4@ 177 Lu has radionuclides 177Lu emits single photons and electrons, which can cause internal irradiation and in vitro radioactivity detection. After intravenous injection, single photon emission computed tomography imaging can be used to monitor its behavioral characteristics and biological distribution in the body. It will also be taken up and internalized by macrophages. After intravenous injection, it will be taken up in large quantities by the liver, and its retention time in the liver is very long (>44h), thus causing continuous internal irradiation to the liver.

[0045] The specific implementation modes of the present invention are further described below in conjunction with the accompanying drawings. The raw materials used are all existing products, the specific preparation operations and performance tests are all conventional techniques, and the data analysis is a conventional technique.

[0046] All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Soochow University and were performed in accordance with the National Laboratory Animal Care and Use Guide. The mice used in the animal experiments were 6-week-old male nude mice weighing approximately 20 g and purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Tumor-bearing mouse modeling: HepG2 (human hepatocellular carcinoma cell line) was cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin at 37°C and 5% CO2 / 95% air. HepG2 cells (6×10 6 ) was injected subcutaneously into 6-week-old specific pathogen-free (SPF) BALB / c nude mice to construct a HepG2 tumor model. When the tumor volume reached 200-300 mm 3 When the tumor is detected, tumor imaging studies are performed.

[0047] Example 1 Synthesis and characterization of nanoparticles. The specific process is as follows.

[0048] Synthesis of 9.0 nm hydrophobic iron oxide nanoparticles: 3.6 g (4 mmol) of iron oleate and 3.39 g (4 mmol) of oleic acid were dissolved in 25 mL of 1-octadecene to obtain a solution; the solution was heated from room temperature to 310°C at a rate of 3.3°C / min, and then maintained at 310°C for 30 min under nitrogen protection; it was then naturally cooled to room temperature, and the reaction solution was precipitated with ethanol to obtain nanoparticles, which were collected by centrifugation and washed three times with ethanol to obtain hydrophobic iron oxide nanoparticles, which were redispersed in tetrahydrofuran or cyclohexane for further experiments.

[0049] See also Figure 1, prepare iron oxide nanoparticles coated with polyethylene glycol (DP-PEG2000-MAL) with bisphosphonic acid at one end and maleimide at the other end: 120 mg of DP-PEG-MAL was dissolved in 2 mL of tetrahydrofuran containing 12 mg of hydrophobic iron oxide nanoparticles to obtain a reaction mixture; the reaction mixture was then stirred at 700 rpm at 60 °C for 20 h, then precipitated with cyclohexane to obtain particles, which were continued to be washed with cyclohexane for 3 times, and then vacuum dried at room temperature for 2 hours, and then the PEGylated iron oxide nanoparticles were dissolved in Milli-Q water, and ultrafiltered using a 30 kDa MWCO ultrafiltration tube at 2200 g for 10 minutes each time, and purified by ultrafiltration 3 times to obtain coated iron oxide nanoparticles (Fe3O4@PEG).

[0050] Cysteamine-functionalized Fe3O4@PEG nanoparticles were synthesized by thiol-maleimide click reaction: 800 μL of HEPES-buffered saline (1 M) was added to a mixed solution of cysteamine (0.6 mg) and TCEP (50 mM tris(2-carboxyethyl)phosphine) to adjust the pH to 7.0, and finally Fe3O4@PEG nanoparticles (5 mg) were added. After shaking at 37°C for 4 h, the reaction solution was purified by ultrafiltration and centrifugation at 2500 g three times to remove free cysteamine and TCEP, and then the pH was further adjusted to 8 with 0.1 M sodium carbonate. Next, 280 μL of 2 mg / mL TCO NHS ester ((4E)-trans-cyclooctene-succinimide ester, dissolved in dimethyl sulfoxide) was added to the product, and shaking was continued for 2 h. Finally, the reaction solution was purified by ultrafiltration and centrifugation at 2500 g three times to remove free TCO and other small molecule derivatives to obtain the final product Fe3O4@TCO nanoparticles.

[0051] In order to determine the morphology and core size of hydrophobic iron oxide nanoparticles and Fe3O4@TCO nanoparticles, TEM images were taken using a TalosF200S G2 transmission electron microscope at an accelerating voltage of 200 kV, and the particle size distribution of the nanoparticles in the electron microscope images was statistically analyzed using Image J software; the hydrated particle size and Zeta potential of the two were measured at 25°C using a Malvern ZS90 Zeta meter and a solid-state He-Ne laser (λ = 633 nm).

[0052] like Figure 2 As shown in Figure 2, electron microscopy results show that the particle morphology and size remain unchanged before and after TCO modification, and the particles are round particles of about 9 nm. Figure 3 The hydrated particle size shown is slightly reduced after modification, which is due to the fact that the TCO groups carried on the surface are hydrophobic groups.

[0053] Embodiment 2 For the organic synthesis of Tz-DOTA, see Figure 4 , the specific process is as follows.

[0054] The commercially purchased starting materials 1,4-dipyridine-tetrazineamine (16.55 mg, 0.066 mmol) and glutaric anhydride (37.58 mg, 0.33 mmol) were dissolved in ultra-dry tetrahydrofuran (2 mL) and heated in a sealed bottle (25 mL) in an oil bath at 70°C for 18 hours. After naturally cooling to room temperature, a precipitate was obtained. The precipitate was washed twice with dichloromethane (10 mL) and ethyl acetate (10 mL) to obtain the carboxylation product as a purple solid (9.5 mg, 39.51%).

[0055] The purple solid (12 mg, 0.033 mmol) obtained above and the amide condensation agent BOP reagent (benzotriazole-1-epoxy) tris(dimethylamino)phosphine hexafluorophosphate (16.4 mg, 0.037 mmol) were dissolved in 746 μL of ultra-dry N,N-dimethylformamide, N,N-diisopropylethylamine (71 μL, 0.30 mmol) was added, and then the polyethylene glycol derivative NH2-PEG with one end amino protected was added dropwise. 10 -NH-Boc (24 mg, 0.037 mmol), after stirring at room temperature for 24 hours, the mixture was evaporated and the product was purified by column chromatography on silica with a gradient elution of methanol: dichloromethane (0-10%) and concentrated by rotary evaporation to give a purple solid (17.2 mg, 52.15%).

[0056] The purple solid (19.6 mg, 0.019 mmol) obtained by column chromatography purification was stirred at room temperature for 30 min in a mixed solution of TFA (0.98 mL of trifluoroacetic acid) and DCM (3.27 mL of dichloromethane), then the solution was removed, dissolved in acetonitrile, precipitated with ether, and the precipitate was dissolved in DMF (3 mL), triethylamine (56 μL, 0.37 mmol) was added, and then DOTA-mono-NHS ester (12 mg of cyclopentane tetraacetic acid, 0.024 mmol), stirred at room temperature for 2 h; then, after rotary evaporation to remove the solution, the mixture was washed with tetrahydrofuran three times and dried in vacuo. The obtained purple solid was further dissolved in water and freeze-dried using preparative HPLC (mobile phase A: 0.1% trifluoroacetic acid + pure water, phase B: 0.1% trifluoroacetic acid + acetonitrile, flow rate: 9 mL / min, 0-3 min: 80% phase A + 20% phase B, 3-37 min: 70% phase + 30% phase B, 37-40 min: 80% phase A + 20% phase B, UV monitoring at 254 nm, collecting the product peak at 31 min) to obtain the final product Tz-DOTA as a purple solid (11.1 mg, 45.71%).

[0057] The above three purple products were characterized by mass spectrometry and hydrogen spectrum to confirm that their molecular weight and structure were the target products. Figure 5 , Figure 6 as well as Figure 7 The mass spectrum showed that the product (2) in positive ion mode had a quasi-molecular ion peak of 366.29 [M+H] + , product (3) in positive ion mode is 1014.606, which is the quasi-molecular ion peak of the target product [M+Na] + , product (4) in positive ion mode 1278.735 is the target product quasi-molecular ion peak [M+H] + . 1 H NMR (400 MHz, [D6]DMSO) showed: δ=10.60 (s, 1H), 9.22 (d, J=2.4 Hz, 1H), 9.10 (dd, J1=4.7 Hz, J2=1.1 Hz, 1H), 8.78 (d, J=8.7 Hz, 1H), 8.75 (d, J=7.8 Hz, 1H), 8.59 (dd, J1=2.4 Hz, J2=8.7Hz, 1H), 8.32 (td,J1=7.8 Hz, J2=1.7 Hz, 1H), 8.08 (ddd, J1=1.1 Hz, J2= 4.7Hz, J3=7.8 Hz), 3.52 (d, J = 3.0 Hz, 19H), 3.51 (d, J = 1.6 Hz, 44H),3.28 (d, J = 5.6 Hz, 2H),3.22 (q, J = 5.8 Hz, 2H), 3.13 (m, 8H), 2.60 (t, S6 J=7.3 Hz, 2H), 2.34 (t, J=7.3 Hz, 2H), 2.02 (q, J=7.3 Hz, 2H).

[0058] Example 3 Radiochemistry of Tz-DOTA, the specific process is as follows.

[0059] See also Figure 8 a. Dissolve the purple solid Tz-DOTA in 1M ammonium acetate aqueous solution (pH 7.0) (1 mg / mL). Take 5 µL of the dissolved Tz-DOTA solution (1 µg / µL) and place it in a 1.5 mL EP tube. Add 20 µL of NaOAc buffer (0.2 M, pH 4.1) and then add [ 177Lu]LuCl30.01M hydrochloric acid solution (18-37 MBq, see below for details), and the mixture was shaken at 60°C for 30 minutes.

[0060] By radio-HPLC and radioactive thin layer chromatography iTLC ( Figure 8 b. Figure 8 c and Figure 8 d) Confirm that the method achieves the desired labeling efficiency and radiochemical purity. 177 The specific activity (Specific Activity SA) of Lu-Tz-DOTA solution is 3600 kBq / μg (5 μg Tz-DOTA+18 MBq [ 177 Lu]LuCl3); SA used for imaging experiments was 7.4MBq / μg (5μg Tz-DOTA+37 MBq [ 177 Lu]LuCl3).

[0061] 177 The oil-water partition coefficient of Lu-Tz-DOTA is determined as follows: Determined 177 n-Octanol / PBS distribution coefficient of Lu-Tz-DOTA (logP value, Figure 8 e), 177 The specific activity of Lu-Tz-DOTA is 9.5 MBq / nmol. 177 Lu-Tz-DOTA (2 μL; ∼480 kBq) was mixed with PBS pH 7.4 (1498 μL) and n-octanol (1500 μL). Phase separation was performed by vigorous vortexing for 1 min and then centrifugation at 2500 rpm for 6 min. The radioactivity concentrations of the two phases were measured in a gamma counter. The distribution coefficient was the logarithm of the ratio of counts per minute (cpm) measured in an equal volume of n-octanol phase to the cpm measured in the PBS phase. Each experiment was repeated three times and the result was calculated to be LogP = -3.4, which suggests that 177 Lu-Tz-DOTA has excellent hydrophilicity and is not easy to penetrate the cell membrane.

[0062] 177 The blood clearance rate of Lu-Tz-DOTA is determined as follows: Will 177 Lu-Tz-DOTA was injected into the tail vein, and blood was collected from the mice retro-orbitally at the predetermined time points. The blood quality and the corresponding radioactivity counts were measured to obtain the time activity curve ( Figure 8 f), and the first-order decay fitting is performed on the curve to calculate 177 The blood elimination half-life of Lu-Tz-DOTA is approximately 10 minutes.

[0063] 177 The in vivo distribution of Lu-Tz-DOTA was monitored as follows: In tumor mice, the tail vein was injected 177 Lu-Tz-DOTA, obtain its SPECT-CT images for 5 minutes, 10 minutes, and 1 hour, reconstruct the SPECT-CT images, and analyze them with PMOD software. The images ( Figure 8 g) This indicates that the probe was rapidly excreted through the kidney within 5 minutes, which is consistent with the results of the blood clearance experiment.

[0064] Example 4 In vitro labeling of iron oxide and characterization of its ligand stability pass 177 The bioorthogonal reaction of Lu-Tz-DOTA and Fe3O4@TCO converts radioactivity 177 Lu was labeled onto the iron oxide nanoparticles. 200ɥCi 177 Lu-Tz-DOTA was added to Fe3O4@TCO solution (containing 0.2 mg Fe) and reacted at 37°C for 30 min. After the reaction was completed, the solution was filtered using a 30 kDa MWCO ultrafiltration tube. 177 Lu-labeled iron oxide nanoparticles were purified by ultrafiltration to obtain Fe3O4@ 177 The radiolabeling yield of Lu nanoparticles was calculated according to the following formula: Radiolabeling yield = R NPs / R IN *100%, where R NPs and R IN respectively refer to the synthesized radiolabeled nanoparticles and the introduced 177 The radioactivity of Lu-Tz-DOTA showed that the labeling activity of iron oxide was linearly related to the specific activity (SA) of the probe ( Fig. 9 a), but the labeling reaction efficiency is about 50%. The obtained labeled iron oxide Fe3O4@ 177 Lu was incubated in water at 37°C, and samples were taken for ultrafiltration purification at different time points. The filtrate and supernatant were measured to determine the stability of the particle surface ligand. The results showed that it had good 24-hour stability ( Fig. 9 b).

[0065] Example 5 In vivo bioorthogonal reaction, the specific process is as follows.

[0066] In normal mice, first in the control group, Fe3O4@ 177Lu nanoparticles were injected into the tail vein (containing 0.2 mg Fe, ~3.7 MBq), and SPECT-CT images were obtained at different time points (30 min, 2 hours, and 4 hours) after injection; in the experimental group, Fe3O4@TCO nanoparticles were injected into one leg of mice, and then the SPECT-CT images were taken immediately. 177 Lu-Tz-DOTA was injected into the tail vein (18 MBq, 3600 kBq / ug), and the mice were anesthetized by gas. SPECT-CT images were obtained at different time points after injection (after injection, 1 hour, and 2 hours). During the imaging experiment, the mice were kept anesthetized with 2% isoflurane (400 mL / min). After imaging was completed, the SPECT-CT images were reconstructed using the software package provided by MILabs and analyzed using PMOD software. Using the quantitative tool of PMOD software, the desired organ was selected as the volume of interest (VOI) for quantification. Fig.10 As shown, where a is Fe3O4@ 177 The distribution of Lu, b and c are the signal circle values ​​of the heart and liver respectively, d is the in vivo biological orthogonal validation imaging result, e and f are the signal circle values ​​of the liver and the injection site respectively. In the control group, i.e., mice directly labeled with iron oxide, it was found that the radioactive signal was basically enriched in the liver and kidneys, while in the experimental group, i.e., the in vivo labeling method, a significant signal was obtained at the injection site, and the liver was negatively imaged, indicating that the two parts of the material can be effectively combined in the biological environment of the body.

[0067] Example 6 SPECT-CT monitoring imaging, the specific process is as follows.

[0068] In tumor mice, Fe3O4@ 177 Lu group: Fe3O4@ 177 Lu nanoparticles, tail vein injection (containing 0.2 mg Fe, ~3.7MBq), SPECT-CT images were obtained at different time points after injection (30 min, 2 hours, 4 hours, 8 hours, 20 hours, 44 hours); Fe3O4@TCO+ 177 Lu-Tz-DOTA group: Fe3O4@TCO nanoparticles (containing 0.2 mgFe) were injected into the tail vein. Four hours later, 177 Lu-Tz-DOTA (~11.1MBq), SPECT-CT images were obtained at different time points (30 min, 2 h, 4 h, 8 h, 20 h, 44 h) after injection.

[0069] The mice in the above two groups were sacrificed, and the terminal biodistribution was performed. The tumors and major organs were collected, and their weights and radioactivity counts were measured. SPECT-CT images were reconstructed using the software package provided by MILabs, and the quantitative tool of PMOD software was used to select the desired organs as the volume of interest (VOI) for quantification.

[0070] See also Fig.11 , where a is Fe3O4@ 177 The distribution of Lu, c, d are the corresponding liver and tumor signal circles, g is the in vitro anatomical biodistribution result; b is Fe3O4@TCO+ 177 The distribution of Lu-Tz-DOTA, e and f are the corresponding liver and tumor signal circle values; h is the in vitro anatomical biodistribution result. 177 Lu obtained Fe3O4@ 177 Lu, after intravenous injection, single photon emission computed tomography imaging found that the liver took up a large amount of radioactive signals and the signal lasted for a long time (>44h). Further comparison with the orthogonal system, first intravenous injection of Fe3O4@TCO, then intravenous injection 177 Lu-Tz-DOTA, single photon emission computed tomography (SPECT) imaging found that compared with Fe3O4@ 177 Lu results showed that the liver signal was significantly reduced, and the signal decreased over time, which indicated that the two parts of the material had a very low degree of binding in the liver, inhibiting the accumulation and retention of radionuclides. The radioactivity counting results of the biodistribution also indicated that the tumor to liver signal ratio was effectively improved by bioorthogonal in vivo labeling. Therefore, the present invention provides a theoretical and experimental basis for further developing the application of nanomaterials in nuclear medicine, which is of great significance for protecting non-target organs such as the liver and other radiation-sensitive organs in nuclear diagnosis and treatment.

[0071] The present invention discloses a method for reducing liver radiation dose by in vivo radioactive labeling. The present invention mainly relates to a method for reducing liver enrichment of radionuclides by in vivo radioactive labeling of nanomaterials mediated by bioorthogonal reactions. Compared with general radioactive labeling, the labeling process mediated by bioorthogonal reactions reduces the labeling condition requirements and can even occur in vivo, thereby reducing the retention of radionuclides. The implementation of this process only requires two-step injection, and does not require the nanomaterials to be pre-finely designed and synthesized in order to reduce tissue retention, nor does it require intervention in the natural biological properties of the body. Therefore, the method of the present invention is simple, safe and universal.

Claims

1. An in vivo radiolabeled agent for reducing liver radiation dose, characterized in that: The invention comprises nanoparticles with surface modifications and molecules labeled with nuclides, wherein the modifications can react with the molecules.

2. The agent for reducing liver radiation dose by in vivo radiolabeling according to claim 1, characterized in that: The reactions include in vitro reactions or in vivo reactions.

3. The agent for reducing liver radiation dose by in vivo radiolabeling according to claim 1, characterized in that: The nanoparticles include one or more of organic nanoparticles, inorganic nanoparticles, and organic-inorganic nanoparticles; the nuclides include one or more of radioactive nuclides that emit helium nuclei, photons, electrons, and neutrons.

4. The agent for reducing liver radiation dose by in vivo radiolabeling according to claim 1, characterized in that: The modifier and the molecule constitute a bioorthogonal reaction system.

5. The agent for reducing liver radiation dose by in vivo radioactive labeling according to claim 4, characterized in that: The bioorthogonal reaction system includes one or more of the electron-rich diene structure and electron-deficient diene structure system, the alkyne structure and azide structure system, the cycloalkyne structure and azide structure system, the azide structure and phosphine compound, the azide structure and phosphite, the biotin and avidin system, and other novel bioorthogonal reactions.

6. The method for preparing the reagent for reducing liver radiation dose by in vivo radioactive labeling according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: combining a modifier on the surface of a nanoparticle to obtain a nanoparticle with a surface modifier; combining a nuclide on a molecular structure to obtain a nuclide-labeled molecule; and the reagent for reducing liver radiation dose by in vivo radioactive labeling comprises nanoparticles with a surface modifier and a nuclide-labeled molecule.

7. Use of the in vivo radiolabeled agent for reducing liver radiation dose as claimed in any one of claims 1 to 5 in the preparation of diagnostic and therapeutic drugs or imaging agents.

8. Use of the reagent for reducing liver radiation dose by in vivo radioactive labeling as claimed in any one of claims 1 to 5 in the preparation of a drug for increasing the ratio of radionuclide retention in the lesion site to that in the liver site.

9. A diagnostic or therapeutic drug or imaging agent, comprising the agent for reducing liver radiation dose by in vivo radioactive labeling as claimed in any one of claims 1 to 5.

10. A method for reducing liver radiation dose by in vivo radioactive labeling, characterized in that: The agent for reducing liver radiation dose by in vivo radioactive labeling as claimed in any one of claims 1 to 5 is used as a diagnostic drug or imaging agent.

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