An algebraically tunable NO-responsive magnetic resonance contrast agent system, preparation method and application thereof

Abstract

The application belongs to the technical field of biomedicine, and relates to an algebra-adjustable NO-responsive magnetic resonance contrast agent system, a preparation method and application thereof. The contrast agent system comprises one or more of a first-generation magnetic resonance contrast agent shown as formula I, a second-generation magnetic resonance contrast agent shown as formula II and a third-generation magnetic resonance contrast agent shown as formula III. By changing the algebra of the dendritic polymer, the particle size, the surface charge, the NO response efficiency, the T1 relaxation performance and the like of the contrast agent can be systematically adjusted, and the contrast agent with different particle sizes and different imaging signals can be flexibly selected according to the lesion requirements of different diseases. The contrast agent system of the application is suitable for multiple clinical scenarios.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an algebraically tunable NO-responsive magnetic resonance contrast agent system, its preparation method, and its application. Background Technology

[0002] Nitric oxide (NO) is an important gaseous signaling molecule in the body, widely involved in various physiological and pathological processes such as vasodilation, neurotransmission, immune regulation, and inflammatory responses. Under normal physiological conditions, low levels of NO produced by endothelial nitric oxide synthase and neuronal nitric oxide synthase help maintain vascular homeostasis and tissue microenvironment balance. However, under pathological conditions such as inflammation, ischemia-reperfusion injury, infection, and acute organ injury, the expression of inducible nitric oxide synthase is significantly increased, leading to excessive NO production. Excessive NO can react with superoxide anions to generate reactive nitrogen species such as peroxynitrite, further inducing oxidative stress, mitochondrial dysfunction, protein nitration, and cell damage, thereby exacerbating local tissue inflammation and organ dysfunction.

[0003] In diseases such as acute kidney injury, inflammatory bowel disease, the tumor inflammatory microenvironment, and neuroinflammation, abnormally elevated NO levels often precede tissue damage and changes in traditional functional indicators. Therefore, NO is not only an important participant in the inflammatory process but can also serve as a molecular marker reflecting early pathological states. Transforming the dynamic changes of NO within tissues into visual signals can facilitate non-invasive, real-time, and spatially localized diagnosis in the early stages of disease.

[0004] However, NO has characteristics such as a short half-life, limited diffusion distance, low tissue concentration, and highly dynamic spatiotemporal distribution, making it difficult for traditional detection methods to achieve real-time monitoring of deep tissues in vivo. While fluorescent probes offer high sensitivity, they are limited by tissue penetration depth and autofluorescence background, making them more suitable for cellular or superficial tissue imaging. Photoacoustic imaging and near-infrared imaging have improved some penetration issues, but still have limitations in quantitative capabilities, clinical applicability, and resolution of deep organs. Magnetic resonance imaging (MRI) offers advantages such as being non-invasive, having high soft tissue resolution, large imaging depth, and a mature clinical translational foundation. Therefore, developing NO-responsive MRI contrast agents is of great significance for the early diagnosis of inflammation-related diseases.

[0005] Currently, gadolinium-based contrast agents are the mainstay of clinical MRI contrast agents. These agents effectively shorten the longitudinal relaxation time of water protons and improve T1-weighted imaging signals, making them widely used in the diagnosis of tumors, vascular lesions, and inflammatory diseases. However, gadolinium ions are exogenous heavy metal ions, and their long-term safety remains a concern. Some gadolinium-based contrast agents may dissociate in vivo, leading to the deposition of free gadolinium ions in areas such as the kidneys, bones, and brain tissue. For patients with renal insufficiency, gadolinium-based contrast agents also pose a risk of inducing renal systemic fibrosis. Therefore, developing novel metal-free, low-toxicity, and metabolically clearable MRI contrast agents is an important direction in the field of biomedical imaging.

[0006] Organic nitroxide radicals are a class of stable free radical molecules with unpaired electrons. They can modulate the relaxation behavior of water protons through paramagnetism, thus enabling their use as MRI contrast agents. Compared to gadolinium-based contrast agents, organic radicals do not contain heavy metals and have better biocompatibility and degradation potential. Among them, the 2,2,6,6-tetramethylpiperidine-1-oxo radical and its derivatives have good chemical stability and paramagnetic properties, making them important candidate structures for constructing metal-free MRI contrast agents. However, small-molecule organic radical contrast agents still suffer from insufficient in vivo stability, susceptibility to quenching by biological reducing agents, low relaxation rates, and short cycle times.

[0007] Furthermore, different diseases place different demands on the physicochemical properties and in vivo behavior of contrast agents. For example, kidney diseases such as acute kidney injury require greater emphasis on the contrast agent's renal reach, rapid response, and renal safety; tumors and chronic inflammatory diseases may require longer lesion retention time and stronger local signal amplification; and imaging of vascular or neuroinflammatory diseases places different demands on material size, blood circulation stability, and tissue barrier penetration. Therefore, relying on a single contrast agent is insufficient to meet the differentiated needs for contrast agent size, response sensitivity, delivery efficiency, and safety in various disease scenarios.

[0008] Therefore, it is of great significance to develop a structurally stable, algebraically tunable NO-responsive organic nitrogen oxide radical MRI contrast agent. Summary of the Invention

[0009] To address the aforementioned problems in the prior art, the present invention aims to provide an algebraically tunable NO-responsive magnetic resonance imaging contrast agent system, its preparation method, and its application, thereby overcoming the shortcomings of the prior art. The present invention employs the following technical solutions to achieve its objective: One aspect of the present invention provides an algebraically tunable NO-responsive magnetic resonance imaging (MRI) contrast agent system, comprising one, two, or three of the following: a first-generation NO-responsive MRI contrast agent (G1-OLT) as shown in Formula I, a second-generation NO-responsive MRI contrast agent (G2-OLT) as shown in Formula II, and a third-generation NO-responsive MRI contrast agent (G3-OLT) as shown in Formula III. The first-generation, second-generation, and third-generation MRI contrast agents are respectively in the form of a first-generation polyamide-amine dendritic structure containing carboxyl-terminated groups. The core is a polymer (G1-PAMAM-COOH), with a second-generation polyamide-amine dendritic polymer (G2-PAMAM-COOH) containing carboxyl-terminated groups as the core, and a third-generation polyamide-amine dendritic polymer (G3-PAMAM-COOH) containing carboxyl-terminated groups as the core; the core is connected to L-lysine-derived structural units through amide bonds, and the L-lysine-derived structural units are further connected to o-phenylenediamine structural units and 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy free radical structural units through amide bonds; Formula I; Formula II; Formula III; In Equations I, II, and III, .

[0010] The first-generation polyamide-amine dendritic polymer with carboxyl-terminated ends, the second-generation polyamide-amine dendritic polymer with carboxyl-terminated ends, and the third-generation polyamide-amine dendritic polymer with carboxyl-terminated ends contain 8, 16, and 32 carboxyl functional groups on their surfaces, respectively. Therefore, the corresponding number of OLTs in Formula I, Formula II, and Formula III are 8, 16, and 32, respectively.

[0011] L-lysine-derived structural units are structural segments formed by linking the α-amino, carboxyl, and side-chain amino groups of L-lysine with carboxyl-terminated polyamide-amine dendritic polymers (GX-PAMAM-COOH), o-phenylenediamine, and 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radicals (4-carboxyl-TEMPO) via amide bonds.

[0012] Specifically, the L-lysine-derived structural unit is obtained from N-Boc-L-lysine methyl ester hydrochloride (Boc-Lys-OMe) through a reaction. N-Boc-L-lysine methyl ester hydrochloride (Boc-Lys-OMe) undergoes three types of reactions, resulting in three types of linkages: the α-amino group is linked to GX-PAMAM-COOH via an amide bond; the methyl ester in Boc-Lys-OMe is hydrolyzed to form a carboxyl group, which is linked to o-phenylenediamine via an amide bond; and after the Boc group is removed, the side chain amino group is linked to 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical (4-carboxyl-TEMPO) via an amide bond.

[0013] The o-phenylenediamine structural unit is a structural segment formed by amidation reaction of o-phenylenediamine (OPD) as a raw material, and the 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical structural unit is a structural segment formed by amidation reaction of 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical (4-carboxyl-TEMPO) as a raw material.

[0014] The structural formula of N-Boc-L-lysine methyl ester hydrochloride is shown in Formula IV below: Formula IV.

[0015] The structural formula of o-phenylenediamine is shown in formula V below: Formula V.

[0016] The structural formula of 4-carboxy-TEMPO is shown in Formula VI below: Formula VI.

[0017] The structures of the L-lysine-derived structural unit, the o-phenylenediamine structural unit, and the 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical structural unit are shown in Formula VII below.

[0018] Equation VII.

[0019] This invention uses a carboxyl-terminated polyamide-amine dendritic polymer as a carrier to couple the NO-responsive o-phenylenediamine (OPD) and the MRI imaging group 2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPO) to the same molecule via the intermediate N-Boc-L-lysine methyl ester, forming a NO-responsive magnetic resonance contrast agent. TEMPO endows the contrast agent with excellent magnetic resonance properties. O-phenylenediamine can respond to nitric oxide by undergoing charge reversal, causing the dendritic polymer to aggregate and thus altering the rotational correlation time, affecting the magnetic resonance signal. This allows the nitric oxide concentration to be correlated with the magnetic resonance signal, enabling the visual detection of nitric oxide concentration through changes in the magnetic resonance signal.

[0020] G1-OLT has a smaller size and better diffusion ability, which is beneficial for tissue penetration and in vivo clearance, but the NMR signal is weak; G3-OLT has more surface reaction sites, which can introduce higher density of NO response groups and MRI imaging groups, thereby enhancing NO response and magnetic resonance signal, resulting in a strong NMR signal, but the particle size is larger; G2-OLT is between the two, forming a better balance between imaging sensitivity, tissue delivery and metabolic clearance.

[0021] Preferably, the contrast agent system includes first-generation magnetic resonance imaging (MRI) contrast agents, second-generation MRI contrast agents, and third-generation MRI contrast agents. By configuring contrast agents with three different generations of substrates, the system can selectively choose the appropriate contrast agent for MRI detection of lesions based on differences in the lesion microenvironment, tissue barrier penetration ability, and in vivo metabolic requirements of the target disease leading to excessive NO production. This satisfies the differentiated needs of different disease scenarios for contrast agent size, response sensitivity, delivery efficiency, and safety. For example, when the target disease is a tumor inflammatory microenvironment or chronic vascular inflammatory disease that requires long lesion retention time and high local signal amplification and leads to excessive NO production, G3-OLT with large particle size and high functional group loading is preferentially selected as the contrast agent; when the target disease is a disease that requires rapid tissue penetration and rapid metabolic clearance and leads to excessive NO production, G1-OLT or G2-OLT with smaller molecular size is preferentially selected as the contrast agent.

[0022] Preferably, the average hydrated particle size of the first-generation magnetic resonance contrast agent is 1-15 nm, more preferably 3-10 nm. The average hydrated particle size of the second-generation magnetic resonance contrast agent is 20-90 nm, more preferably 30-80 nm, and even more preferably 40-70 nm. The average hydrated particle size of the third-generation magnetic resonance contrast agent is 95-200 nm, more preferably 100-150 nm.

[0023] Preferably, the first-generation, second-generation, and third-generation magnetic resonance contrast agents will aggregate after reacting with NO, thereby altering the T1-weighted magnetic resonance imaging signal; within the NO concentration range of 0~1000 mM, the intensity of the T1-weighted magnetic resonance imaging signal increases with increasing NO concentration.

[0024] Preferably, the preparation methods of the first-generation magnetic resonance imaging (MRI) contrast agent, the second-generation MRI contrast agent, and the third-generation MRI contrast agent each include the following steps: S1. N-Boc-L-lysine methyl ester was modified onto a carboxyl-terminated polyamide-amine dendritic polymer after carboxyl activation via an amidation reaction to obtain the G2-L intermediate. S2. Remove the methyl ester group from the G2-L intermediate to form a carboxyl group, and then activate the carboxyl group. Then, modify the o-phenylenediamine onto the demethylated and carboxyl-activated G2-L intermediate through an amidation reaction to obtain the G2-OL intermediate. S3. Remove the Boc group from the G2-OL intermediate, and then react it with the carboxyl-activated 4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxy free radical to obtain a NO-responsive magnetic resonance imaging agent. The carboxyl-terminated polyamide-amine dendritic polymers are first-generation polyamide-amine dendritic polymers, second-generation polyamide-amine dendritic polymers, or third-generation polyamide-amine dendritic polymers.

[0025] When the carboxyl-terminated polyamide-amine dendritic polymer is a first-generation polyamide-amine dendritic polymer with a carboxyl terminus, a first-generation magnetic resonance imaging (MRI) contrast agent is prepared; when the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation polyamide-amine dendritic polymer with a carboxyl terminus, a second-generation MRI contrast agent is prepared; and when the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer with a carboxyl terminus, a third-generation MRI contrast agent is prepared.

[0026] Preferably, the carboxyl activation step in steps S1-S3 includes: adding a carboxyl activator to the substance to be activated and reacting at 10-40 °C for 10-100 min. The carboxyl activator is preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS). Preferably, the molar ratio of the substance to be activated, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:7-50:7-50. Further, when the carboxyl-terminated polyamide-amine dendritic polymer is a first-generation carboxyl-terminated polyamide-amine dendritic polymer, the molar ratio of the carboxyl-terminated substance, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:7-15:7-15, more preferably 1:9:9. When the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of the substance to be activated by the carboxyl group, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:16~28:16~28, more preferably 1:18:18. When the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of the substance to be activated by the carboxyl group, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:30~50:30~50, more preferably 1:36:36.

[0027] Preferably, the amidation reaction step S1 includes: mixing the carboxyl-activated polyamide-amine dendritic polymer with carboxyl-terminated ends with N-Boc-L-lysine methyl ester hydrochloride to form a solution system, adjusting the pH of the solution system to 7-8, and reacting at 10-40 °C for 10-60 h.

[0028] Preferably, the molar ratio of the carboxyl-terminated polyamide-amine dendritic polymer with carboxyl groups activated to N-Boc-L-lysine methyl ester hydrochloride is 1:7 to 50. More preferably, the molar ratio of the carboxyl-terminated first-generation polyamide-amine dendritic polymer with carboxyl groups activated to N-Boc-L-lysine methyl ester hydrochloride is 1:7 to 15, more preferably 1:9; or, the molar ratio of the carboxyl-terminated second-generation polyamide-amine dendritic polymer with carboxyl groups activated to N-Boc-L-lysine methyl ester hydrochloride is 1:16 to 28, more preferably 1:18; or, the molar ratio of the carboxyl-terminated third-generation polyamide-amine dendritic polymer with carboxyl groups activated to N-Boc-L-lysine methyl ester hydrochloride is 1:30 to 50, more preferably 1:36.

[0029] Preferably, step S2, the step of removing the methyl ester group of the GX-L intermediate to form a carboxyl group, includes: under anaerobic conditions, reacting the GX-L intermediate with an alkaline solution in a deprotection solvent at 10-40 °C for 1-10 h, and then adding an acid neutralizer for neutralization.

[0030] Preferably, the alkaline solution is one or more of sodium hydroxide solution and potassium hydroxide solution, and the concentration of the alkaline solution is 1~10 mol / L. The molar ratio of GX-L intermediate to alkali in the alkaline solution is 1:1~15. Preferably, when the carboxyl-terminated polyamide-amine dendritic polymer is a first-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of GX-L intermediate to alkali in the alkaline solution is 1:1~3, more preferably 1:2.5; when the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of GX-L intermediate to alkali in the alkaline solution is 1:4~7, more preferably 1:5; when the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of GX-L intermediate to alkali in the alkaline solution is 1:8~15, more preferably 1:10.

[0031] The deprotecting solvent is one or more of the following: water, N,N-dimethylformamide (DMF), dimethyl sulfoxide, 1,4-dioxane, methanol, ethanol, tetrahydrofuran, acetonitrile, and acetone.

[0032] The acid neutralizing agent is one or more of the following: hydrochloric acid solution, sulfuric acid solution, formic acid solution, acetic acid solution, phosphoric acid solution, citric acid solution, lactic acid solution, oxalic acid solution, tartaric acid solution, and malic acid solution. The concentration of the acid neutralizing agent is 0.5~5 mol / L. The molar amount of acid in the acid neutralizing agent is equivalent to the molar amount of alkali in the alkaline solution.

[0033] Preferably, the amidation reaction step S2 includes: mixing the demethylated and carboxyl-activated GX-L intermediate with o-phenylenediamine to form a solution system, adjusting the pH of the solution system to 7-8, and reacting at 10-40 °C for 10-60 h.

[0034] Preferably, the molar ratio of the carboxyl-activated and demethylated GX-L intermediate to o-phenylenediamine is 1:7~50. Furthermore, when the carboxyl-terminated polyamide-amine dendritic polymer is a first-generation polyamide-amine dendritic polymer with a carboxyl-terminated group, the molar ratio of the carboxyl-activated and demethylated GX-L intermediate to o-phenylenediamine is 1:7 to 15, more preferably 1:9; when the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation polyamide-amine dendritic polymer with a carboxyl-terminated group, the molar ratio of the carboxyl-activated and demethylated GX-L intermediate to o-phenylenediamine is 1:16 to 28, more preferably 1:18; when the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer with a carboxyl-terminated group, the molar ratio of the carboxyl-activated and demethylated GX-L intermediate to o-phenylenediamine is 1:30 to 50, more preferably 1:36.

[0035] Preferably, step S3, the step of removing the Boc group from the GX-OL intermediate, includes: under anaerobic conditions, reacting the GX-OL intermediate with an acid solution in a deprotection solvent at 10-40 °C for 1-10 h, and then adding an alkaline neutralizing agent for neutralization.

[0036] Preferably, the acid solution is one or more of hydrochloric acid solution, trifluoroacetic acid (TFA) solution, and sulfuric acid solution, and the acid concentration is 1~10 mol / L. The molar ratio of GX-OL intermediate to acid in the acid solution is 1:1~15. Preferably, when the carboxyl-terminated polyamide-amine dendritic polymer is a first-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of GX-OL intermediate to acid in the acid solution is 1:1~3, more preferably 1:2.5; when the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of GX-OL intermediate to acid in the acid solution is 1:4~7, more preferably 1:5; when the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer with carboxyl-terminus, the molar ratio of GX-OL intermediate to acid in the acid solution is 1:8~15, more preferably 1:10.

[0037] The alkali neutralizing agent is one or more of the following: saturated sodium bicarbonate aqueous solution, saturated potassium bicarbonate aqueous solution, saturated sodium carbonate aqueous solution, saturated potassium carbonate aqueous solution, and triethylamine solution. The molar amount of alkali in the alkali neutralizing agent is equivalent to the molar amount of acid in the acid solution.

[0038] Preferably, the reaction in step S3 includes: mixing the GX-OL intermediate with the Boc group removed with the 2,2,6,6-tetramethylpiperidine-1-oxy free radical after carboxyl activation to form a solution system, adjusting the pH of the solution system to 7-8, and reacting at 10-40 °C for 10-60 h.

[0039] Preferably, the molar ratio of the Boc-removed GX-OL intermediate to the carboxyl-activated 2,2,6,6-tetramethylpiperidine-1-ox free radical is 1:7 to 50. Further, when the carboxyl-terminated polyamide-amine dendritic polymer is a first-generation carboxyl-terminated polyamide-amine dendritic polymer, the molar ratio of the Boc-removed GX-OL intermediate to the carboxyl-activated 2,2,6,6-tetramethylpiperidine-1-ox free radical is 1:7 to 15, more preferably 1:9. When the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation carboxyl-terminated polyamide-amine dendritic polymer, the molar ratio of the Boc-removed GX-OL intermediate to the carboxyl-activated 2,2,6,6-tetramethylpiperidine-1-ox free radical is 1:16 to 28, more preferably 1:18. When the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer containing carboxyl-terminated groups, the molar ratio of the GX-OL intermediate with the Boc group removed to the 2,2,6,6-tetramethylpiperidine-1-oxy free radical activated by the carboxyl group is 1:30~50, more preferably 1:36.

[0040] Preferably, the preparation methods of the first-generation magnetic resonance imaging (MRI) contrast agent, the second-generation MRI contrast agent, and the third-generation MRI contrast agent more specifically include the following steps: S1. Add a carboxyl activator to a solution of a polyamide-amine dendritic polymer containing a carboxyl terminus, and react at 10-40℃ for 10-100 min. Then add an N-Boc-L-lysine methyl ester hydrochloride solution to form a solution system. Adjust the pH of the solution system to 7-8, and react at 10-40℃ for 10-60 h. After the reaction is completed, dialyze and dry to obtain the GX-L intermediate. S2. Under anaerobic conditions, the GX-L intermediate reacts with an alkaline solution in a deprotected solvent at 10–40 °C for 1–10 h. Then, an acid neutralizer is added to neutralize the reaction and remove the organic solvent, yielding a demethylated GX-L intermediate. A solvent and a carboxyl activator are then added to the demethylated GX-L intermediate, and the reaction is carried out at 10–40 °C for 10–100 min. Subsequently, an o-phenylenediamine solution is added to form a solution system, and the pH of the solution system is adjusted to 7–8. The reaction is carried out at 10–40 °C for 10–60 h. After the reaction is complete, the intermediate is dialyzed and dried to obtain the GX-OL intermediate. S3. Under anaerobic conditions, the GX-OL intermediate reacts with acid in a deprotected solvent at 10–40 °C for 1–10 h. Then, an alkaline neutralizing agent is added to neutralize the reaction and remove the organic solvent, yielding the GX-OL intermediate without the Boc group. A solvent and a carboxyl activator are added to a 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxo radical, and the reaction is carried out at 10–40 °C for 10–100 min. Subsequently, a solution of the GX-OL intermediate without the Boc group is added to form a solution system. The pH of the solution system is adjusted to 7–8, and the reaction is carried out at 10–40 °C for 10–60 h. After the reaction, the solution is dialyzed and dried to obtain a NO-responsive magnetic resonance imaging agent.

[0041] During the carboxyl activation process, the solvent added is a mixture of DMF and water, with a volume ratio of DMF to water of 1:0.3~3.

[0042] The second aspect of this invention provides a method for preparing an algebraically tunable NO-responsive magnetic resonance imaging (MRI) contrast agent system, comprising the following steps to prepare a first-generation MRI contrast agent, a second-generation MRI contrast agent, and a third-generation MRI contrast agent: S1. N-Boc-L-lysine methyl ester was modified onto a carboxyl-terminated polyamide-amine dendritic polymer after carboxyl activation via an amidation reaction to obtain the G2-L intermediate. S2. Remove the methyl ester group from the G2-L intermediate to form a carboxyl group, and then activate the carboxyl group. Then, modify the o-phenylenediamine onto the demethylated and carboxyl-activated G2-L intermediate through an amidation reaction to obtain the G2-OL intermediate. S3. Remove the Boc group from the G2-OL intermediate, and then react it with the carboxyl-activated 4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxy free radical to obtain a NO-responsive magnetic resonance imaging agent. When preparing the first-generation magnetic resonance contrast agent, the carboxyl-terminated polyamide-amine dendritic polymer is the first-generation polyamide-amine dendritic polymer containing carboxyl-terminated groups; When preparing second-generation magnetic resonance contrast agents, the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation polyamide-amine dendritic polymer containing carboxyl-terminated groups; When preparing third-generation magnetic resonance contrast agents, the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer containing carboxyl-terminated groups.

[0043] A third aspect of the present invention provides a NO-responsive magnetic resonance imaging (MRI) contrast agent having a structural formula as shown in Formula I or Formula III, wherein the core is a first-generation polyamide-amine dendritic polymer or a third-generation polyamide-amine dendritic polymer containing a carboxyl terminus, the core being connected to an L-lysine-derived structural unit via an amide bond; the L-lysine-derived structural unit is further connected to an o-phenylenediamine structural unit and a 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical structural unit via an amide bond.

[0044] A fourth aspect of this invention provides a method for screening NO-responsive magnetic resonance imaging contrast agents, comprising the following steps: NO-responsive first-generation magnetic resonance contrast agent as shown in Formula I, NO-responsive second-generation magnetic resonance contrast agent as shown in Formula II, and NO-responsive third-generation magnetic resonance contrast agent as shown in Formula III were prepared respectively. The parameters of three magnetic resonance imaging agents were tested, including particle size, longitudinal relaxation rate, and nitric oxide response sensitivity. Based on the differentiated needs of the target disease, select the appropriate contrast agent according to the parameters.

[0045] The fifth aspect of this invention provides the application of the algebraically tunable NO-responsive magnetic resonance contrast agent system in the preparation of reagents for quantitative detection of NO concentration in vivo or in vitro.

[0046] The sixth aspect of the present invention provides the application of the algebraically tunable NO-responsive magnetic resonance contrast agent system in the preparation of magnetic resonance imaging diagnostic reagents.

[0047] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses G1, G2, and G3 third-generation carboxylated polyamide-amine dendritic polymers as the core, and couples the NO-responsive group OPD and the MRI imaging group TEMPO to the same molecule via the intermediate Nε-Boc-L-lysine methyl ester to form a NO-responsive magnetic resonance contrast agent with a well-defined structure and tunable generation. By changing the generation of the dendritic polymer, the particle size, surface charge, functional group loading, NO response efficiency, T1 relaxation properties, tissue delivery behavior, and in vivo metabolic characteristics of the contrast agent can be systematically adjusted, thereby providing a screenable, optimizable, and scalable metal-free MRI contrast agent platform for different inflammation-related diseases.

[0048] (2) In this invention, o-phenylenediamine is used as a NO-specific response unit. After the contrast agent comes into contact with NO, charge reversal occurs and particles aggregate, which effectively changes the T1-weighted magnetic resonance imaging signal. In the NO concentration range of 0~1000 mM, the imaging signal intensity increases regularly with the increase of NO concentration, and the imaging signal shows a good linear relationship with the concentration. Moreover, the detection limits of the three contrast agents are all at a low level, with a detection limit ≤30.23 μM, which can realize accurate quantitative detection of NO concentration in vivo and in vitro.

[0049] (3) The present invention uses organic nitrogen and oxygen free radicals such as 4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxy free radical as magnetic resonance imaging unit, which belongs to metal-free contrast system and completely avoids the safety hazards of heavy metal dissociation, tissue deposition and induction of renal systemic fibrosis of traditional gadolinium-based contrast agents; at the same time, the material has good cell uptake capacity and biodegradability, good biocompatibility and wider applicability.

[0050] (4) The magnetic resonance imaging relaxation performance of the contrast agent of the present invention is positively correlated with the generation of the dendritic polymer. The longitudinal relaxation rates r1 of G1-OLT, G2-OLT and G3-OLT are 0.4035, 0.7699 and 1.025 respectively. As the generation increases, the nitrogen oxide free radical loading increases and the T1-weighted imaging signal is enhanced synchronously. All three contrast agents have excellent MRI imaging effects, and G3-OLT has the strongest MRI signal. The average hydrated particle size of the three contrast agents is 1~15 nm, 20~90 nm and 95~200 nm respectively, with a clear size gradient. Contrast agents with different particle sizes and imaging signals can be flexibly selected according to the lesion requirements of different diseases such as acute kidney injury, tumor inflammation and neuroinflammation. The contrast agent system of the present invention is suitable for a variety of clinical scenarios.

[0051] (5) The present invention adopts a stepwise amidation reaction combined with selective deprotection synthesis process to sequentially complete the graft coupling of N-Boc-L-lysine methyl ester, o-phenylenediamine and 4-carboxyl-TEMPO. The reaction temperature is controlled at 10~40 ℃ throughout the process. The reaction conditions are mild, the operation is simple and the raw materials are readily available. The prepared product has a concentrated particle size distribution and stable structure. The synthesis route is highly reproducible, which is conducive to subsequent standardization and large-scale production. Attached Figure Description

[0052] Figure 1 Dynamic light scattering and Zeta potential diagrams for G1-OLT, G2-OLT, and G3-OLT; Figure 2 Electron paramagnetic resonance spectra of G1-OLT, G2-OLT, and G3-OLT; Figure 3 The hydrogen nuclear magnetic resonance spectra of G1-OLT, G2-OLT, and G3-OLT are shown. Figure 4 Infrared spectra of G1-OLT, G2-OLT, and G3-OLT; Figure 5 MRI imaging and relaxation rate fitting diagrams of G1-OLT, G2-OLT, and G3-OLT; Figure 6 MRI imaging and quantitative signals of G1-OLT, G2-OLT, and G3-OLT in response to nitric oxide; Figure 7 Calculate the NO response limit for G1-OLT, G2-OLT, and G3-OLT; Figure 8 This is a diagram showing the cellular uptake capacity of G1-OLT, G2-OLT, and G3-OLT. Detailed Implementation

[0053] In the description of this invention, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, and includes both a and b. "Multiple" includes two or more types, and can be two, three, four, five, or more.

[0054] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0055] In the following embodiments: The G1-PAMAM-COOH solution was prepared by dissolving G1-PAMAM-COOH in a DMF:water ratio of 1:1, with a concentration of 10 mg / mL; it was purchased from Hangzhou Xinqiao Biotechnology Co., Ltd.

[0056] The G2-PAMAM-COOH solution was prepared by dissolving G2-PAMAM-COOH in a DMF:water ratio of 1:1, with a concentration of 10 mg / mL; it was purchased from Hangzhou Xinqiao Biotechnology Co., Ltd.

[0057] The G3-PAMAM-COOH solution was prepared by dissolving G3-PAMAM-COOH in a DMF:water ratio of 1:1, with a concentration of 10 mg / mL; it was purchased from Hangzhou Xinqiao Biotechnology Co., Ltd.

[0058] The Boc-Lys-OMe solution was formed by dissolving N-Boc-L-lysine methyl ester hydrochloride (CAS No.: 2389-48-2) in DMF:water = 1:1 solvent, with a concentration of 32.1 mg / mL; purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0059] The OPD solution is formed by dissolving OPD in a DMF:water ratio of 1:1, with a concentration of 47.2 mg / mL.

[0060] The room temperature is approximately 20°C.

[0061] Example 1 In this embodiment, the G1-OLT contrast agent was prepared through the following steps: S1. G1-PAMAM-COOH solution (1 equivalent) was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (9 equivalents) and N-hydroxysuccinimide (9 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature to activate the carboxyl groups. Then, Boc-Lys-OMe solution (9 equivalents) was slowly added dropwise using a constant pressure dropping funnel, and the pH of the final solution was adjusted to 7-8 (pH paper test) with triethylamine (TEA). The reaction was carried out at room temperature for 24 h. After the reaction was completed, the product was purified by dialyzing in water using a 3000 Da dialysis bag for 72 h. After freeze-drying, the dendritic polymer intermediate (G1-L) was obtained.

[0062] S2. Under anaerobic conditions, using 1,4-dioxane as a solvent, G1-L (1 equivalent) and 4 mol / L sodium hydroxide solution (2.5 equivalent) were magnetically stirred at 500 rpm for 2 h at room temperature. Then, 4 mol / L hydrochloric acid (2.5 equivalent) was added to neutralize the mixture. The organic solvent was removed using a rotary evaporator (100 mbar, 100 rpm, 50℃, 30 min). The bottom of the flask was purged with nitrogen for 10 min to remove as much unevaporated organic solvent as possible. This step removes methyl esters. After deprotection, G1-L was re-added with a DMF:water volume ratio of 1:1. G1-L (1 equivalent) was then mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (9 equivalents) and N-hydroxysuccinimide (9 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature. Subsequently, OPD solution (9 equivalents) was slowly added dropwise using a constant-pressure dropping funnel, and the pH of the final solution was adjusted to 7-8 using TEA (pH paper test). The reaction was carried out at room temperature for 24 h. After the reaction was complete, the product was purified by dialyzing in water using a 3000 Da dialysis bag for 72 h, and then freeze-dried to obtain G1-OL.

[0063] S3. Under anaerobic conditions, 1,4-dioxane was selected as the solvent. G1-OL (1 equivalent) and 4 mol / L hydrochloric acid solution (2.5 equivalent) were magnetically stirred at 500 rpm for 2 h at room temperature. Saturated sodium bicarbonate aqueous solution (2.5 equivalent) was added for neutralization. The organic solvent was removed using a rotary evaporator (100 mbar, 100 rpm, 30℃, 30 min). The bottom of the flask was purged with nitrogen for 10 min to remove as much unevaporated organic solvent as possible. This step removes Boc. Using DMF:water at a volume ratio of 1:1 as the solvent, 4-carboxy-TEMPO (9 equivalents) was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (9 equivalents) and N-hydroxysuccinimide (9 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature to activate the carboxyl groups. Subsequently, a Boc-deprotected G1-OL solution (dissolved in DMF:water at a volume ratio of 1:1; 1 equivalent) was slowly added dropwise using a constant-pressure dropping funnel. The pH of the final solution was adjusted to 7-8 using TEA (pH paper test). The reaction was carried out at room temperature for 24 h. After the reaction, the product was purified by dialyzing in water for 72 h using a 3000 Da dialysis bag. The purified product was then freeze-dried to obtain the probe (G1-OLT).

[0064] Example 2 In this embodiment, the G2-OLT contrast agent was prepared through the following steps: S1. G2-PAMAM-COOH solution (1 equivalent) was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (18 equivalents) and N-hydroxysuccinimide (18 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature to activate the carboxyl groups. Then, Boc-Lys-OMe solution (18 equivalents) was slowly added dropwise using a constant-pressure dropping funnel, and the pH of the final solution was adjusted to 7-8 (pH paper test) with triethylamine. The reaction was carried out at room temperature for 24 h. After the reaction was complete, the product was purified by dialyzing in water for 72 h using a 3000 Da dialysis bag. The purified product was then freeze-dried to obtain the dendritic polymer intermediate (G2-L).

[0065] S2. Under anaerobic conditions, using 1,4-dioxane as a solvent, G2-L (1 equivalent) and 4 mol / L sodium hydroxide solution (5 equivalents) were magnetically stirred at 500 rpm for 2 h at room temperature. Then, 4 mol / L hydrochloric acid (5 equivalents) was added to neutralize the mixture. The organic solvent was removed using a rotary evaporator (100 mbar, 100 rpm, 50℃, 30 min). The bottom of the flask was purged with nitrogen for 10 min to remove as much unevaporated organic solvent as possible. This step removes methyl esters. After deprotection, G2-L was re-added with a DMF:water volume ratio of 1:1. G2-L (1 equivalent) was then mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (18 equivalents) and N-hydroxysuccinimide (18 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature. Subsequently, OPD solution (18 equivalents) was slowly added dropwise using a constant-pressure dropping funnel, and the pH of the final solution was adjusted to 7-8 (pH paper test) with triethylamine (TEA). The reaction was allowed to proceed for 24 h at room temperature. After the reaction was complete, the product was purified by dialyzing in water for 72 h using a 3000 Da dialysis bag. The purified product was then freeze-dried to obtain G2-OL.

[0066] S3. Under anaerobic conditions, 1,4-dioxane was selected as the solvent. G2-OL (1 equivalent) and 4 mol / L hydrochloric acid solution (5 equivalents) were magnetically stirred at 500 rpm for 2 h at room temperature. Saturated sodium bicarbonate aqueous solution (5 equivalents) was added for neutralization. The organic solvent was removed by rotary evaporation (100 mbar, 100 rpm, 30℃, 30 min). The bottom of the flask was purged with nitrogen for 10 min to remove as much unevaporated organic solvent as possible. This step removes Boc. Using DMF:water at a volume ratio of 1:1 as the solvent, 4-carboxy-TEMPO (18 equivalents) was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (18 equivalents) and N-hydroxysuccinimide (18 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature to activate the carboxyl groups. Subsequently, a Boc-deprotected G2-OL solution (dissolved in DMF:water at a volume ratio of 1:1; 1 equivalent) was slowly added dropwise using a constant-pressure dropping funnel. The pH of the final solution was adjusted to 7-8 using TEA (pH paper test). The reaction was carried out at room temperature for 24 h. After the reaction, the product was purified by dialyzing in water for 72 h using a 3000 Da dialysis bag. The purified product was then freeze-dried to obtain the probe (G2-OLT).

[0067] Example 3 In this embodiment, the G3-OLT contrast agent was prepared through the following steps: S1. G3-PAMAM-COOH solution (1 equivalent) was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (36 equivalents) and N-hydroxysuccinimide (36 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature to activate the carboxyl groups. Then, Boc-Lys-OMe solution (36 equivalents) was slowly added dropwise using a constant-pressure dropping funnel, and the pH of the final solution was adjusted to 7-8 (pH paper test) with triethylamine. The reaction was carried out at room temperature for 24 h. After the reaction was complete, the product was purified by dialyzing in water for 72 h using a 3000 Da dialysis bag. The purified product was then freeze-dried to obtain the dendritic polymer intermediate (G3-L).

[0068] S2. Under anaerobic conditions, using 1,4-dioxane as a solvent, G3-L (1 equivalent) and 4 mol / L sodium hydroxide solution (10 equivalents) were magnetically stirred at 500 rpm for 2 h at room temperature. Then, 4 mol / L hydrochloric acid (10 equivalents) was added to neutralize the mixture. The organic solvent was removed using a rotary evaporator (100 mbar, 100 rpm, 50℃, 30 min). The bottom of the flask was purged with nitrogen for 10 min to remove as much unevaporated organic solvent as possible. This step removes methyl esters. After deprotection, G3-L was re-added with a DMF:water volume ratio of 1:1. G3-L (1 equivalent) was then mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (36 equivalents) and N-hydroxysuccinimide (36 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature. Subsequently, OPD solution (36 equivalents) was slowly added dropwise using a constant-pressure dropping funnel, and the pH of the final solution was adjusted to 7-8 using TEA (pH paper test). The reaction was allowed to proceed for 24 h at room temperature. After the reaction was complete, the product was purified by dialyzing in water for 72 h using a 3000 Da dialysis bag. The purified product was then freeze-dried to obtain G3-OL.

[0069] S3. Under anaerobic conditions, 1,4-dioxane was selected as the solvent. G3-OL (1 equivalent) and 4 mol / L hydrochloric acid solution (10 equivalents) were magnetically stirred at 500 rpm for 2 h at room temperature. Saturated sodium bicarbonate aqueous solution (10 equivalents) was added for neutralization. The organic solvent was removed by rotary evaporation (100 mbar, 100 rpm, 30℃, 30 min). The bottom of the flask was purged with nitrogen for 10 min to remove as much unevaporated organic solvent as possible. This step removed Boc. Using DMF:water at a volume ratio of 1:1 as the solvent, 4-carboxy-TEMPO (36 equivalents) was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (36 equivalents) and N-hydroxysuccinimide (36 equivalents) and magnetically stirred at 700 rpm for 30 min at room temperature to activate the carboxyl groups. Subsequently, a Boc-deprotected G3-OL solution (dissolved in DMF:water at a volume ratio of 1:1; 1 equivalent) was slowly added dropwise using a constant-pressure dropping funnel. The pH of the final solution was adjusted to 7-8 using TEA (pH paper test). The reaction was carried out at room temperature for 24 h. After the reaction, the product was purified by dialyzing in water for 72 h using a 3000 Da dialysis bag. The purified product was then freeze-dried to obtain the probe (G3-OLT).

[0070] The hydration size distribution and potential of the GX-OLT nanoparticles (prepared as 1 mg / mL aqueous solution) prepared in Examples 1-3 were characterized using a nanoparticle size analyzer. Figure 1 As shown, G1-OLT, G2-OLT, and G3-OLT all exhibit relatively concentrated particle size distributions, indicating that dendritic polymers of different generations can still maintain good dispersibility in aqueous systems after OPD and TEMPO modification. With increasing dendritic polymer generation, the overall particle size increases, with average particle sizes of 3 nm, 53 nm, and 135 nm for G1-OLT, G2-OLT, and G3-OLT, respectively, demonstrating that the generation of PAMAM can effectively control the hydrodynamic size of the contrast agent. Zeta potential results show that unmodified G1, G2, and G3 generation PAMAM exhibits a negative potential due to surface carboxyl ionization; after modification with lysine linkers, OPD response units, and TEMPO imaging units, the surface potentials of G1-OLT, G2-OLT, and G3-OLT change significantly, tending towards positive potentials or increasing in potential. This result indicates that functional groups were successfully introduced into the dendritic polymer surface and altered the surface charge characteristics of the material. The above results demonstrate that the present invention can achieve controllable adjustment of contrast agent particle size and surface charge by adjusting the PAMAM generation.

[0071] The characteristic signal of TEMPO nitrogen-oxygen radicals in GX-OLT was characterized by electron paramagnetic resonance (EPR). The EPR image of GX-OLT is shown below. Figure 2As shown, typical TEMPO nitroxide radical triplet signals were observed in G1-OLT, G2-OLT, and G3-OLT, indicating that 4-carboxyl-TEMPO was successfully coupled to PAMAM dendrimers of different generations. Simultaneously, as the dendrimer generation increased from G1 to G3, the EPR signal intensity generally increased, suggesting that higher-generation PAMAMs have more surface reaction sites and can load more TEMPO imaging units. These results demonstrate that this invention can achieve adjustable organic nitroxide radical loading by controlling the dendrimer generation, providing a foundation for subsequent generation-dependent regulation of MRI imaging performance.

[0072] Hydrogen nuclear magnetic resonance (HMR) spectroscopy is used to verify whether OPD and TEMPO-related structural units have been successfully introduced into dendritic polymers of different generations. Figure 3 The figures show the 1H NMR spectra of G1-OLT, G2-OLT, and G3-OLT. As can be seen from the figures, G1-OLT, G2-OLT, and G3-OLT all exhibit characteristic signals attributed to the protons of the aromatic ring of OPD, indicating that the o-phenylenediamine NO-responsive unit has been successfully modified into the dendritic polymer structure.

[0073] Infrared spectroscopy was used to further verify the formation of amide bonds, OPD aromatic structures, and TEMPO nitroxide radical-related structures in GX-OLTs of different generations. Figure 4 The figures show the infrared spectra of G1-OLT, G2-OLT, and G3-OLT. As can be seen from the figures, the wavelength range is 1650–1700 cm⁻¹. -1 An absorption peak of amide I appears nearby, corresponding to the C=O stretching vibration; at approximately 1540 cm⁻¹ -1 The presence of an absorption peak in the vicinity of the amide II band corresponds to the bending vibration of NH and the stretching vibration of CN, indicating that a new amide bond was formed during the reaction.

[0074] A series of GX-OLT solutions with varying concentration gradients were prepared to characterize the material's T1 imaging performance (magnetic resonance imaging parameters: TE = 3.1 ms, TR = 132 ms, FOV = 55 mm × 35 mm, Slicer = 1 mm, Flipangle = 25°). Figure 5As shown, under the same mass concentration gradient, G1-OLT, G2-OLT, and G3-OLT all produced concentration-dependent T1-weighted signal enhancement, indicating that the TEMPO-modified dendritic polymer has effective T1 MRI imaging capabilities. At the same mass concentration, the MRI signal intensity was: G1-OLT < G2-OLT < G3-OLT; and the relaxation fitting results showed that the longitudinal relaxation efficiencies r1 for G1-OLT, G2-OLT, and G3-OLT were 0.4035, 0.7699, and 1.025, respectively. These results indicate that as the generation number of the PAMAM dendritic macromolecule increases, the r1 of the contrast agent increases, and the MRI imaging signal is enhanced.

[0075] A series of NO donors at varying concentrations (PAPA NONOate (CAS: 146672-58-4)) were prepared and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Figure 6 The concentration of NO in the solution (which is the NO donor concentration) was used to simulate the NO release process. GX-OLT material (with a GX-OLT material solution concentration of 0.08 mM) was added to the NO donor to characterize the nitric oxide response imaging performance of GX-OLT. From... Figure 6 As can be seen, under different NO concentration conditions, the T1-weighted MRI signals of G1-OLT, G2-OLT and G3-OLT all showed concentration-dependent changes, indicating that this type of contrast agent can respond to NO and convert changes in NO concentration into detectable MRI signals.

[0076] In the NO response experiment, different GX-OLT samples showed varying detection sensitivities to NO. By plotting a standard curve and calculating the limit of detection (LOD), such as... Figure 7 The results showed that G2-OLT had the lowest LOD (Level of Detection) at 11.54 μM, indicating that it was the most sensitive to NO. The LODs of G1-OLT and G3-OLT were 24.48 μM and 30.23 μM, respectively, with slightly lower sensitivities than G2-OLT. These data indicate that G2-OLT can generate a measurable signal even in low-concentration NO environments, demonstrating high detection potential.

[0077] Cellular uptake assays are used to evaluate the cellular uptake capacity of GX-OLTs at different passages and their imaging applications. Figure 8 It can be seen that at 0 min, the intracellular fluorescence signal of each group of cells is weak; as the incubation time is extended to 15 min and 30 min, the intracellular fluorescence signal of G1-OLT, G2-OLT and G3-OLT gradually increases, indicating that GX-OLT of different generations can be taken up by cells and have a certain intracellular delivery ability.

[0078] In summary, this invention uses carboxylated PAMAM dendrimers of different generations as its core, and by selecting G1, G2, or G3 generation dendrimers, it provides 8, 16, or 32 surface functional group sites, respectively, thereby achieving adjustable loading of NO-responsive units and MRI imaging units. Compared to single-structure contrast agents, this invention can adjust the generation of the dendrimer, the density of functional groups, and the free radical loading according to different detection requirements, thereby optimizing particle size, surface charge, relaxation properties, and in vivo metabolic behavior. Furthermore, a more suitable contrast agent can be selected according to the disease requirements.

[0079] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0080] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0081] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. An algebraically tunable NO-responsive magnetic resonance imaging contrast agent system, characterized in that, The contrast agent includes one or more of the following: a first-generation NO-responsive magnetic resonance imaging (MRI) contrast agent as shown in Formula I, a second-generation NO-responsive MRI contrast agent as shown in Formula II, and a third-generation NO-responsive MRI contrast agent as shown in Formula III. The first-generation, second-generation, and third-generation MRI contrast agents are respectively based on a first-generation polyamide-amine dendritic polymer with a carboxyl terminus as the core, a second-generation polyamide-amine dendritic polymer with a carboxyl terminus as the core, and a third-generation polyamide-amine dendritic polymer with a carboxyl terminus as the core. The core is connected to an L-lysine-derived structural unit through an amide bond. The L-lysine-derived structural unit is further connected to an o-phenylenediamine structural unit and a 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical structural unit through an amide bond. Equation I; Formula II; Formula III; In Equations I, II, and III, .

2. The algebraically tunable NO-responsive magnetic resonance contrast agent system according to claim 1, characterized in that, The average hydrated particle sizes of the first-generation, second-generation, and third-generation magnetic resonance imaging (MRI) contrast agents are 1–15 nm, 20–90 nm, and 95–200 nm, respectively.

3. The algebraically tunable NO-responsive magnetic resonance contrast agent system according to claim 1, characterized in that, First-generation, second-generation, and third-generation magnetic resonance imaging (MRI) contrast agents react with NO and aggregate, altering the T1-weighted MRI signal. Within the NO concentration range of 0–1000 mM, the intensity of the T1-weighted MRI signal increases with increasing NO concentration.

4. The algebraically tunable NO-responsive magnetic resonance contrast agent system according to claim 1, characterized in that, The preparation methods for first-generation, second-generation, and third-generation magnetic resonance imaging (MRI) contrast agents respectively include the following steps: S1. N-Boc-L-lysine methyl ester was modified onto a carboxyl-terminated polyamide-amine dendritic polymer after carboxyl activation via an amidation reaction to obtain the GX-L intermediate. S2. Remove the methyl ester group from the GX-L intermediate to form a carboxyl group, and activate the carboxyl group. Then, modify the o-phenylenediamine onto the demethylated and carboxyl-activated GX-L intermediate through an amidation reaction to obtain the GX-OL intermediate. S3. Remove the Boc group from the GX-OL intermediate, and then react it with the carboxyl-activated 4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxy radical to obtain a NO-responsive magnetic resonance imaging agent. The carboxyl-terminated polyamide-amine dendritic polymers are first-generation polyamide-amine dendritic polymers, second-generation polyamide-amine dendritic polymers, or third-generation polyamide-amine dendritic polymers.

5. The algebraically tunable NO-responsive magnetic resonance contrast agent system according to claim 4, characterized in that, The amidation reaction step S1 includes: mixing the carboxyl-activated polyamide-amine dendritic polymer with carboxyl-terminated ends with N-Boc-L-lysine methyl ester hydrochloride to form a solution system, adjusting the pH of the solution system to 7-8, and reacting at 10-40 °C for 10-60 h; And / or, the amidation reaction step S2 includes: mixing the demethylated and carboxyl-activated GX-L intermediate with o-phenylenediamine to form a solution system, adjusting the pH of the solution system to 7-8, and reacting at 10-40 °C for 10-60 h; And / or, the reaction in step S3 includes: mixing the GX-OL intermediate with the Boc group removed with the carboxyl-activated 4-carboxy-2,2,6,6-tetramethylpiperidine-1-ox free radical to form a solution system, adjusting the pH of the solution system to 7-8, and reacting at 10-40 °C for 10-60 h.

6. The algebraically tunable NO-responsive magnetic resonance contrast agent system according to claim 4, characterized in that, Step S2, the removal of the methyl ester group from the GX-L intermediate to form a carboxyl group, includes: under anaerobic conditions, the GX-L intermediate reacts with an alkaline solution in a deprotection solvent at 10-40 °C for 1-10 h, and then neutralizes with an acid neutralizing agent; The alkaline solution is one or more of sodium hydroxide solution and potassium hydroxide solution, and the concentration of the alkaline solution is 1~10 mol / L; The deprotecting solvent is one or more of the following: water, N,N-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, methanol, ethanol, tetrahydrofuran, acetonitrile, and acetone. The acid neutralizing agent is one or more of the following: hydrochloric acid solution, sulfuric acid solution, formic acid solution, acetic acid solution, phosphoric acid solution, citric acid solution, lactic acid solution, oxalic acid solution, tartaric acid solution, and malic acid solution; Step S3, the removal of the Boc group from the GX-OL intermediate, includes: under anaerobic conditions, the GX-OL intermediate reacts with acid in a deprotection solvent at 10-40 °C for 1-10 h, and then neutralizes with an alkaline neutralizing agent; The acid solution is one or more of hydrochloric acid solution, trifluoroacetic acid solution, and sulfuric acid solution, and the acid concentration is 1~10 mol / L; The alkali neutralizing agent is one or more of the following: saturated sodium bicarbonate aqueous solution, saturated potassium bicarbonate aqueous solution, saturated sodium carbonate aqueous solution, saturated potassium carbonate aqueous solution, and triethylamine solution.

7. A method for preparing an algebraically tunable NO-responsive magnetic resonance contrast agent system as described in claim 1, characterized in that, First-generation, second-generation, and third-generation magnetic resonance imaging (MRI) contrast agents were prepared using the following steps: S1. N-Boc-L-lysine methyl ester was modified onto a carboxyl-terminated polyamide-amine dendritic polymer after carboxyl activation via an amidation reaction to obtain the GX-L intermediate. S2. Remove the methyl ester group from the GX-L intermediate to form a carboxyl group, and activate the carboxyl group. Then, modify the o-phenylenediamine onto the demethylated and carboxyl-activated GX-L intermediate through an amidation reaction to obtain the GX-OL intermediate. S3. Remove the Boc group from the GX-OL intermediate, and then react it with the carboxyl-activated 4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxy radical to obtain a NO-responsive magnetic resonance imaging agent. When preparing the first-generation magnetic resonance contrast agent, the carboxyl-terminated polyamide-amine dendritic polymer is the first-generation polyamide-amine dendritic polymer containing carboxyl-terminated groups; When preparing second-generation magnetic resonance contrast agents, the carboxyl-terminated polyamide-amine dendritic polymer is a second-generation polyamide-amine dendritic polymer containing carboxyl-terminated groups; When preparing third-generation magnetic resonance contrast agents, the carboxyl-terminated polyamide-amine dendritic polymer is a third-generation polyamide-amine dendritic polymer containing carboxyl-terminated groups.

8. A NO-responsive magnetic resonance imaging contrast agent, characterized in that, The magnetic resonance imaging agent has a structure as shown in Formula I or Formula III, with a core consisting of a first-generation polyamide-amine dendritic polymer or a third-generation polyamide-amine dendritic polymer containing a carboxyl terminus. The core is connected to an L-lysine-derived structural unit via an amide bond. The L-lysine-derived structural unit is further connected to an o-phenylenediamine structural unit and a 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical structural unit via an amide bond.

9. A method for screening NO-responsive magnetic resonance imaging contrast agents, characterized in that, Includes the following steps: The first-generation magnetic resonance contrast agent with NO response as shown in Formula I, the second-generation magnetic resonance contrast agent with NO response as shown in Formula II, and the third-generation magnetic resonance contrast agent with NO response as shown in Formula III were prepared respectively. The parameters of three magnetic resonance imaging agents were tested, including particle size, longitudinal relaxation rate, and nitric oxide response sensitivity. Based on the differentiated needs of the target disease, select the appropriate contrast agent according to the parameters.

10. The application of the algebraically tunable NO-responsive magnetic resonance contrast agent system as described in claim 1 or the NO-responsive magnetic resonance contrast agent as described in claim 8, characterized in that, The application is any of the following: Application in the preparation of reagents for quantitative detection of NO concentration in vivo or in vitro; Application in the preparation of magnetic resonance imaging diagnostic reagents.

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