A dual-pH threshold response two-order enhanced gadolinium-based polymer magnetic resonance contrast agent and application thereof

Abstract

This invention discloses a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response and its application. The contrast agent has an A- b -B-L-C(Gd), where A and B are anionic and cationic pH-sensitive polymer blocks, respectively, with the response threshold of block B being higher than that of block A; C(Gd) is the Gd-containing compound at the end of block B. 3+ Chelating groups. Contrast agents form first micelle states at physiological pH where water molecules have low contact ability with C(Gd). T 1. Low background signal; when pH is below the B-block response threshold, micelles depolymerize, significantly increasing the contact ability between water molecules and C(Gd), achieving first-order [response]. T 1. Enhancement: When the pH is below the A-block response threshold, the A-block response reassembles to form a second micelle state, maintaining the contact ability of water molecules with C(Gd) and prolonging the rotation time, thus achieving second-order [interaction / reaction]. T 1. Enhancement. This invention is applicable to pH gradient-responsive magnetic resonance imaging in acidic environments such as tumors, inflammation, and lysosomes. T 1. Imaging.

Description

Technical Field

[0001] This invention belongs to the technical field of magnetic resonance imaging contrast agents and stimulus-responsive polymer materials, specifically relating to a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) is an important medical imaging technique with advantages such as high spatial resolution, good soft tissue contrast, and no ionizing radiation. To improve the imaging contrast between lesions and normal tissues, MRI contrast agents are frequently used in clinical practice and research. Among them, gadolinium-based contrast agents are commonly used. T Contrast agents are widely studied and used because they can shorten the longitudinal relaxation time of water protons.

[0003] Traditional small-molecule gadolinium-based contrast agents, such as Gd-DTPA, Gd-DOTA, and their derivatives, typically possess good water solubility and rapid in vivo clearance. However, these small-molecule contrast agents generally suffer from insufficient lesion selectivity, high background signal in blood and normal tissues, and limited signal activation ability in the pathological microenvironment. Therefore, developing intelligent gadolinium-based contrast agents that can specifically enhance signals in the disease microenvironment while maintaining low background under normal physiological conditions is of great significance.

[0004] Pathological or subcellular environments such as tumor tissue, inflamed tissue, tumor-associated macrophages, intracellular endosomes, and lysosomes typically exhibit varying degrees of acidity. Based on this characteristic, pH-responsive magnetic resonance imaging (MRI) contrast agents have been used to improve the selectivity of lesion imaging. In previous studies, pH-responsive gadolinium-based polymer micelles have been shown to induce micelle disassembly and assembly through an acidic environment, exposing gadolinium chelates originally located in a hydrophobic core to an aqueous environment. This enhances the interaction between water molecules and the gadolinium centers, thereby strengthening MRI. T Signal 1.

[0005] However, most existing pH-responsive gadolinium-based polymer contrast agents rely on structural changes triggered by a single pH threshold, typically exhibiting a single-stage response process of "micelle-disassembly." These systems primarily enhance the contact ability between water molecules and gadolinium chelating groups. T The first signal makes it difficult to further utilize the pH gradient within the lesion, which progresses from a weakly acidic microenvironment to deeply acidic compartments such as endosomes and lysosomes, to achieve a more programmed and multi-stage process. T 1. Signal modulation.

[0006] Furthermore, pH-responsive contrast agents that rely solely on micellar disassembly typically remain in a dispersed or disassembled state in deeply acidic environments, making it difficult to simultaneously achieve both the contact capability between water molecules and gadolinium chelating groups and a relatively long molecular rotational correlation time. Based on gadolinium groups... T 1. The relaxation mechanism of contrast agents, water molecules and Gd3+ Both contact capability and rotational correlation time affect longitudinal relaxation rate. Therefore, if a contrast agent can be designed to first disassemble at a higher acid threshold to improve the contact capability between water molecules and gadolinium chelate groups, and then further reassemble at a lower acid threshold, achieving a longer rotational correlation time while maintaining the contact capability between water molecules and gadolinium chelate groups, it is hoped that multi-stage enhancement of gadolinium chelate relaxation rate can be achieved. T 1 signal, to achieve further improvement in MRI contrast.

[0007] Therefore, there is an urgent need to develop a novel gadolinium-based polymer magnetic resonance imaging agent that differs from the traditional single-pH-responsive micelle disassembly and assembly mechanism, enabling it to respond to multiple pH thresholds and achieve low background, staged enhancement, and lesion acid gradient response imaging through continuous structural reconstruction. Summary of the Invention

[0008] The present invention aims to provide a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response; this contrast agent is constructed by forming an anionic pH-sensitive polymer block A, a cationic pH-sensitive polymer block B, and a Gd-containing polymer block B. 3+ The general formula of the chelating group C(Gd) A- b -BLC(Gd) allows it to sequentially undergo a first micelle state, a disassembly state, and a second micelle state as the pH gradually decreases, thereby achieving magnetic resonance. T Two-stage enhancement of contrast.

[0009] Another object of the present invention is to provide the above-mentioned contrast agent in pH gradient responsive magnetic resonance imaging. T Applications in imaging, especially in acidic environments such as the weakly acidic microenvironment of tumors, intracellular endosomes, lysosomes, tumor-associated macrophages, inflammatory lesions, and atherosclerotic plaques.

[0010] To achieve the above objectives, the present invention provides the following technical solution.

[0011] This invention provides a gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response, having the following general formula structure: A- b -BLC(Gd).

[0012] Wherein, A is an anionic pH-sensitive polymer block, B is a cationic pH-sensitive polymer block, and "- b "-" indicates that block A and block B are linked by block copolymerization, L is the linking group or chemical bond, and C(Gd) indicates that it contains Gd. 3+ The chelating group. The C(Gd) is attached to the end of the B block via L.

[0013] The A segment is hydrophilic under physiological pH conditions and transitions to a hydrophobic state when the pH response threshold of the A segment is lower than that of the A segment. The B segment is hydrophobic under physiological pH conditions and transitions to a hydrophilic state when the pH response threshold of the B segment is lower than that of the B segment. The pH response threshold of the B segment is lower than that of the physiological pH value but higher than that of the A segment.

[0014] As the pH gradually decreases, the contrast agent sequentially forms a first micelle state, a disassembled state, and a second micelle state.

[0015] In the first micelle state, the B block, L linker group, or chemical bond and C(Gd) are located in the hydrophobic core, while the A block is located in the hydrophilic shell. The ability of water molecules to contact C(Gd) is limited, resulting in a lower magnetic resonance of the contrast agent under physiological pH conditions. T 1. Background signal.

[0016] In the disassembled state, block A remains hydrophilic, block B transforms into a hydrophilic state, and the L linker group or chemical bond is distributed in the aqueous phase, exposing C(Gd) to the aqueous environment. This enhances the interaction between C(Gd) and water molecules, thus achieving magnetic resonance. T The first stage of contrast enhancement.

[0017] In the second micelle state, the A block transforms into a hydrophobic state and drives the contrast agent to reassemble into a hydrophobic micelle core. The B block, L linker group, or chemical bonds and C(Gd) are located in the hydrophilic shell, micelle surface, or micelle-water interface of the micelle. This increases the rotational correlation time while maintaining the ability of water molecules to contact C(Gd), thereby achieving magnetic resonance. T The second stage of contrast enhancement.

[0018] Preferably, L is a chemical bond or a divalent linking group. The divalent linking group includes amide bonds, ester bonds, ether bonds, carbamate bonds, thioether bonds, disulfide bonds, triazole bonds, alkylene bonds, or combinations thereof.

[0019] Preferably, C(Gd) contains Gd. 3+ The macrocyclic or open-chain chelating group is selected from one or more of DOTA(Gd), DTPA(Gd), DO3A(Gd), Nota(Gd), TETA(Gd), DTPA-BMA(Gd), HP-DO3A(Gd), and their derivatives. More preferably, the C(Gd) is DOTA(Gd) or DTPA(Gd).

[0020] Preferably, the pH response threshold of segment A is 5.4-6.6, more preferably 5.8-6.3. The pH response threshold of segment B is 6.4-7.1, more preferably 6.6-7.0. The pH response threshold of segment B is higher than that of segment A, and the difference between the two is 0.4-1.6 pH units, more preferably 0.5-0.8 pH units.

[0021] Preferably, the A block is an anionic pH-sensitive polymer block containing a weak acidic group, wherein the weak acidic group includes a sulfonamide group, a carboxyl group, or a combination thereof.

[0022] Preferably, the B block is a cationic pH-sensitive polymer block containing a protonable basic group, wherein the protonable basic group includes a tertiary amine group, an imidazole group, a pyridinyl group, or a combination thereof.

[0023] The gadolinium-based polymer magnetic resonance contrast agent exhibits the following longitudinal relaxation rate change relationship during pH decrease: r 1 (Physiological pH)< r 1-B (B-block response pH)< r 1-A (A-block response to pH); where... r 1 This refers to the longitudinal relaxation rate at physiological pH. r 1-B This refers to the longitudinal relaxation rate of the B-block response at pH. r 1-A This refers to the longitudinal relaxation rate of the A-block at pH; physiological pH corresponds to the first micelle state, the B-block at pH corresponds to the disassembly state, and the A-block at pH corresponds to the second micelle state. Magnetic Resonance T The phased enhancement of contrast includes: a first-stage enhancement caused by the increased contact ability between water molecules and C(Gd), and a second-stage enhancement caused by maintaining the contact ability between water molecules and C(Gd) and increasing the rotation time.

[0024] The gadolinium-based polymer magnetic resonance contrast agent of this invention exhibits pH gradient-responsive magnetic resonance imaging. T 1. Application in imaging contrast agents; specifically, the gadolinium-based polymer magnetic resonance contrast agent is used in the weakly acidic microenvironment of tumors, tumor cell endosomes, tumor cell lysosomes, tumor-associated macrophage phagolysosomes, inflammatory macrophage phagolysosomes, acidic microenvironment of inflammatory tissues, or acidic microenvironment of atherosclerotic plaques. T 1. Magnetic resonance imaging.

[0025] Beneficial effects: (1) The contrast agent provided by this invention is not a gadolinium-based polymer micelle with a single pH response, but rather a general formula structure A- with dual pH threshold response characteristics. b -BLC(Gd). Among them, the response pH of the B block is higher than that of the A block, which allows the contrast agent to sequentially undergo B block response and A block response as the pH gradually decreases, thereby achieving ordered structural evolution.

[0026] (2) This invention achieves continuous reconstruction between the first micelle state, the disassembled state, and the second micelle state through the opposite hydrophilic-hydrophobic transitions of the A-block and the B-block. This structural evolution process differs from the single-stage "micelle-disassembled" change of traditional single pH-responsive micelles.

[0027] (3) This invention can programmatically regulate the microenvironment in which C(Gd) resides. Under physiological pH conditions, C(Gd) is located in a hydrophobic core, limiting the contact ability between water molecules and C(Gd), which helps reduce background signals; under B-block response pH conditions, C(Gd) is exposed to the aqueous environment, increasing the contact ability between water molecules and C(Gd), thus achieving the first stage. T 1. Enhancement: Under pH responsiveness of the A-block, the contrast agent reassembles, allowing C(Gd) to be located in the hydrophilic shell, micelle surface, or micelle-water interface, while maintaining the relationship between water molecules and Gd. 3+ While increasing contact capability, the rotational correlation time is also increased to achieve the second stage. T 1. Enhancement.

[0028] (4) This invention is applicable to the continuous pH gradient response from the tumor microenvironment to the intracellular acidic compartment. Its response process can correspond to different acidic environments such as the weakly acidic tumor microenvironment, endosomes, lysosomes, and phagolysosomes, which is beneficial for improving magnetic resonance imaging. T 1. Lesion selectivity and signal contrast in imaging.

[0029] (5) This invention protects various anionic pH-sensitive blocks, cationic pH-sensitive blocks, and Gd-containing blocks through a general formula structure. 3+ The combination of chelating groups has good structural scalability and application adaptability. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the general formula structure of a contrast agent, wherein the contrast agent has A- b -BLC(Gd) structure.

[0031] Figure 2 This is a schematic diagram illustrating the structural evolution of the contrast agent of the present invention as it forms a first micelle state, a disassembly state, and a second micelle state during the gradual decrease of pH.

[0032] Figure 3This invention relates to the changes in the contact ability of water molecules with C(Gd) and rotation-dependent time of the contrast agent as pH decreases, as well as the corresponding longitudinal relaxation rate. r 1 Diagram showing the changes.

[0033] Figure 4 The contrast agent of this invention is used in physiological environments, the weakly acidic microenvironment of tumors, lysosomes and endosomes, or inflammatory acidic lesions on magnetic resonance imaging. T 1. Schematic diagram of imaging applications.

[0034] Figure 5 The corresponding test graphs in Example 1 are shown, where (A) is the pH-sensitive transmittance change curve of pSMTMA; (B) is the pH titration curve of pC7AMA; and (C) is the pH titration curve of pSMTMA- b -pC7AMA-DOTA(Gd) particle size as a function of pH; (D) represents pSMTMA- b -pC7AMA-DOTA(Gd) as a function of pH r 1.

[0035] Figure 6 The corresponding test graphs in Example 2 are shown, where (A) is the pH-sensitive transmittance change curve of pSDXAA; (B) is the pH-sensitive transmittance change curve of pSDXAA; and (C) is the pH-sensitive transmittance change curve of pSDXAA. b -pC7AMA-DTPA(Gd) particle size as a function of pH; (D) represents the particle size of pSDXAA-b-pC7AMA-DTPA(Gd) as a function of pH. r 1.

[0036] Figure 7 The corresponding test graphs in Example 3 are shown, where (A) is the pH-sensitive transmittance change curve of pSDXMA; (B) is the pH titration curve of pEPAMA; and (C) is the pH titration curve of pSDXMA- b -pEPAMA-DO3A(Gd) particle size as a function of pH; (D) represents the particle size of pSDXMA-b-pEPAMA-DO3A(Gd) as a function of pH. r 1.

[0037] Figure 8 The corresponding test graphs in Example 4 show (A) the pH-sensitive transmittance change curve of pSMTAA; and (B) the pH-sensitive transmittance change curve of p(DPAMA- co pH titration curve of (C7AMA); (C) pSMTAA- b -p(DPAMA- co -C7AMA)-DOTA(Gd) particle size as a function of pH; pSMTAA- b -p(DPAMA- co-C7AMA)-DOTA(Gd) as a function of pH r 1.

[0038] Figure 9 The corresponding test graphs in Example 5 show (A) the pH-sensitive transmittance change curve of pSBZMA; and (B) the pH-sensitive transmittance change curve of p(DPAMA- co pH titration curve of (-EPAMA); (C) pSBZMA- b -p(DPAMA- co -EPAMA)-DTPA(Gd) particle size as a function of pH; (D)pSBZMA- b -p(DPAMA- co -EPAMA)-DTPA(Gd) variation with pH r 1.

[0039] Figure 10 This is the test diagram corresponding to Example 6, where (A)p(SDXAA- co (A) pH-sensitive transmittance curve of p(SDXAA); (B) pH titration curve of pEPAMA; (C) pH-sensitive transmittance curve of p(SDXAA- co -SMTAA)-b-pEPAMA-DTPA(Gd) particle size as a function of pH; (D)p(SDXAA- co -SMTAA)-b-pEPAMA-DTPA(Gd) as a function of pH r 1.

[0040] Figure 11 This is the test diagram corresponding to Example 7, where (A)p(MAA- co (A) pH-sensitive transmittance curve of pEPAMA; (B) pH titration curve of pEPAMA; (C) pH-sensitive transmittance curve of pEPAMA. co -BMA)- b -pEPAMA-NOTA(Gd) particle size as a function of pH; (D)p(MAA- co -BMA)- b -pEPAMA-NOTA(Gd) as a function of pH r 1. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Various modifications and equivalent substitutions can be made to the present invention by those skilled in the art without departing from the concept of the present invention, and all such modifications and substitutions should be included within the scope of protection of the present invention.

[0042] Figure 1This is a schematic diagram of the general formula structure of a contrast agent, wherein the contrast agent has A- b -BLC(Gd) structure. Figure 2 This is a schematic diagram illustrating the structural evolution of the contrast agent of the present invention as it forms a first micelle state, a disassembly state, and a second micelle state during the gradual decrease of pH. Figure 3 This invention relates to the changes in the contact ability of water molecules with C(Gd) and rotation-dependent time of the contrast agent as pH decreases, as well as the corresponding longitudinal relaxation rate. r 1. Schematic diagram of the changes. Figure 4 The contrast agent of this invention is used in physiological environments, the weakly acidic microenvironment of tumors, lysosomes and endosomes, or inflammatory acidic lesions on magnetic resonance imaging. T 1. Schematic diagram of imaging application. See the embodiment for detailed operation.

[0043] Example 1: A dual pH threshold-responsive polymer contrast agent consisting of an anionic pH-sensitive block (phase transition point 6.1) with poly(hexamethyleneimine)-ethyl methacrylate as segment A, a cationic pH-sensitive block (phase transition point 6.9) with segment B, and DOTA(Gd) linked to the end of the cationic block.

[0044] (1) Preparation of poly(methacrylamide) sulfamethoxazole The monomer of sulfamethoxymethyl methacrylamide (SMT-MA) was subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain thioester-terminated poly(sulfamethoxymethyl methacrylamide) (pSMTMA). The preparation of pSMTMA is RAFT polymerization. For specific steps, please refer to reference 1 [Abel Brooks A., Sims Michael B., McCormick Charles L. Tunable pH- and CO2-Responsive Sulfonamide-Containing Polymers by RAFTPolymerization [J]. Macromolecules, 2015, 48(16): 5487-5495].

[0045] (2) Preparation of poly(hexamethyleneimino)-ethyl methacrylate 2-(hexamethyleneimino)-ethyl methacrylate monomer (C7A-MA) and a carboxyl-terminated RAFT chain transfer agent were subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain poly(2-(hexamethyleneimino)-ethyl methacrylate) (pC7AMA) with carboxyl and thioester groups at their ends. The preparation method of pC7AMA is RAFT polymerization, and the specific steps can be found in reference 2 ([Wang Sheng, Yu Guocan, Wang Zhantong, et al. Hierarchical Tumor Microenvironment-Responsive Nanomedicine for Programmed Delivery of Chemotherapeutics [J]. Advanced Materials, 2018, 30(40):1803926).

[0046] (3) Coupling of DOTA (Gd) DOTA modified with monomaleimide, i.e., DOTA-Mal, and pC7AMA with carboxyl and thioester groups at their ends, respectively, were modified using typical click chemistry to obtain pC7AMA-DOTA. Specific steps can be found in reference 3 [MoadGraeme, Rizzardo Ezio, Thang San H. End-functional polymers, thiocarbonylthiogroup removal / transformation and reversible addition-fragmentation-chaintransfer (RAFT) polymerization [J]. Polymer International, 2011, 60(1): 9-25]. After obtaining pC7AMA-DOTA, it was further combined with Gd... 3+The chelation yields pC7AMA-DOTA(Gd). For specific steps, please refer to reference 4 [Fu SX, Cai ZY, Liu L., et al. Gadolinium(III) Complex-Backboned Branched Polymers as Imaging Probes for Contrast-Enhanced MagneticResonance Angiography [J]. Acs Applied Materials & Interfaces, 2023, 15(14):18311-18322].

[0047] (4) Preparation of a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response First, pC7AMA-DOTA(Gd) was coupled with maleimide (Mal) via esterification to obtain Mal-pC7AMA-DOTA(Gd); the specific steps of this esterification reaction can be found in reference 5 [Munawar Saba, Zahoor AmeerFawad, Hussain Syed Makhdoom, et al. Steglich esterification: A versatile synthetic approach toward the synthesis of natural products, their analogues / derivatives [J]. Heliyon, 2024, 10(1): e23416.]. Then, pSMTMA and Mal-pC7AMA-DOTA(Gd), which retain the terminal thioester, were modified by typical click chemistry to obtain pSMTMA- b -pC7AMA-DOTA(Gd) is a gadolinium-based polymeric magnetic resonance imaging agent with dual pH threshold responses and two-stage enhancement. The reaction procedure for this step is the same as the click chemistry for the coupling of DOTA(Gd) in this embodiment, which can be found in reference 3.

[0048] (5) Characterization of contrast agents ① Characterization of the phase transition point of poly(methacrylamide)thiadiazole sulfamethoxymethylacrylamide 10 mg of pSMTMA was dispersed in 10 ml of water at pH 7.4. While stirring, a 1 mM HCl solution was added dropwise. During the titration, the transmittance and pH value of the test solution were measured using a UV spectrophotometer and a pH meter after each addition of HCl solution. A curve was plotted with transmittance on the ordinate and pH value on the abscissa. Figure 5 (A) shows that the transmittance of pSMTMA drops sharply between pH 6.2 and 6.0. Taking a 50% decrease in transmittance as its phase transition point, the phase transition point of pSMTAA prepared in this embodiment is pH 6.1.

[0049] ②Characteristics of the phase transition point of poly(hexamethyleneimino)-ethyl methacrylate 10 mg of pC7AMA was dispersed in 10 ml of water with pH 7.4. A 1 mM HCl solution was added dropwise while stirring. During the titration, the pH value of the test solution was measured with a pH meter after each addition of HCl solution. The pH value was plotted on the ordinate, and V... HCl Plot a curve for the x-axis. Based on... Figure 5 (B) The results showed that the pH change of pC7AMA was very slow between pH=6.94 and pH=6.86. The midpoint between the start and end of the process was taken as its phase transition point. Therefore, the phase transition point of pC7AMA prepared in this example was pH=6.9.

[0050] ③pSMTMA- b Determination of Gd content in pC7AMA-DOTA(Gd) 2 mg of DOTA (Gd) was placed in 8 ml of 1 M HNO3 solution and shaken overnight to fully release Gd. 3+ The binding efficiency of Gd in DOTA(Gd) was then calculated using an ICP-OES system; 10 mg of pC7AMA-DOTA(Gd) was placed in 8 ml of 1 M HNO3 solution and shaken overnight to fully release Gd. 3+ The coupling efficiency of DOTA(Gd) in pC7AMA-DOTA(Gd) was then determined and calculated using an ICP-OES system. A standard ICP-OES curve was established using standard Gd reagents. The binding efficiency of Gd in DOTA(Gd) was 100%, and the coupling efficiency of DOTA(Gd) in pC7AMA-DOTA(Gd) exceeded 95%.

[0051] ④ Characterization of the two-phase transition point of pSMTMA-b-pC7AMA-DOTA(Gd) 10 mg pSMTMA-b -pC7AMA-DOTA(Gd) was dispersed in 10 ml of water at pH 7.4 to obtain the test solution. Then, 1 mM HCl solution was added dropwise to adjust the pH of the test solution. The particle size was measured by a particle size analyzer at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.1, 5.9 and 5.6. Figure 5 (C) shows that as the pH value decreases, pSMTMA- b The particle size of -pC7AMA-DOTA(Gd) exhibits a change process that conforms to "from the first micelle state to the disassembled state and then to the second micelle state", and has a two-phase transition point that conforms to pSMTMA and pC7AMA.

[0052] ⑤pSMTMA- b -pC7AMA-DOTA(Gd) T 1. Characterization of pH variation 10 mg pSMTMA- b -pC7AMA-DOTA(Gd) was dispersed in 10 ml of water at pH 7.4 to obtain the test solution. Then, 1 mM HCl solution was added dropwise to adjust the pH of the test solution. Based on the MRI scan results at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.1, 5.9, and 5.6, the corresponding pSMT-DOTA(Gd) concentrations were determined. b -pC7A-DOTA(Gd) r 1. Figure 5 (D) shows that as the pH value decreases, pSMTMA- b -pC7AMA-DOTA(Gd) r A second-level enhancement has appeared, which is consistent with pSMTMA- b The structural changes of -pC7AMA-DOTA(Gd) are observed. At pH > 6.9, DOTA(Gd) is encapsulated within micelles, resulting in weaker contact with water molecules. T 1 is relatively weak; when pH > 6.9 > 6.1, DOTA(Gd) can fully contact water molecules to achieve enhanced performance. T 1. At pH < 6.1, DOTA(Gd) maintains its ability to contact water molecules while increasing its rotation time, thus achieving further enhancement. T 1.

[0053] Example 2: A dual pH threshold-responsive polymer contrast agent consisting of an anionic pH-sensitive block (phase transition point 6.1) with polysulfonamide octanoic acid as segment A, a cationic pH-sensitive block (phase transition point 6.9) with poly(hexamethyleneimino)-ethyl methacrylate as segment B, and DTPA(Gd) linked to the end of the cationic block.

[0054] (1) Preparation of polysulfonamide polyacrylamide The sulfadiazine acrylamide monomer (SDX-AA) was subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain thioester-terminated polysulfadiazine acrylamide pSDXAA. The polymerization method used in the preparation of pSDXAA was a conventional and well-known operation, and the preparation process and reaction conditions were the same as those in Reference 1 in Example 1.

[0055] (2) The preparation of poly(hexamethyleneimino)-ethyl methacrylate pC7AMA is the same as in Example 1.

[0056] (3) The coupling of DTPA is the same as in Example 1, except that DOTA-Mal in Example 1 is replaced with DTPA-Mal, and finally pC7AMA-DTPA(Gd) is obtained.

[0057] (4) Preparation of a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response Maleimide (Mal) modified Mal-pC7AMA-DTPA(Gd) was obtained using the same method as in Example 1. Further, following the method corresponding to Example 1, pSDXAA- was obtained. b -pC7AMA-DTPA(Gd) is a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response. The click chemistry method used is a conventional and well-known operation, as described in Reference 3 of Example 1.

[0058] (5) Characterization of contrast agents ①Characteristics of polysulfamine polyacrylamide The pSDXAA phase transition point was characterized using the transmittance method described in Example 1. Figure 6 (A) shows that the transmittance of pSDXAA decreases sharply between pH 6.2 and 6.0. Taking a 50% decrease in transmittance as its phase transition point, the phase transition point of pSDXAA prepared in this embodiment is pH 6.1.

[0059] ②Characteristics of the phase transition point of poly(hexamethyleneimino)-ethyl methacrylate The phase transition point of pC7AMA was characterized using the pH titration method described in Example 1. Figure 6 (B) The results showed that the pH change of pC7AMA was very slow between pH=6.92 and pH=6.88. The midpoint between the start and end of the process was taken as its phase transition point. Therefore, the phase transition point of pC7AMA prepared in this example was pH=6.9.

[0060] ③pSDXAA- bDetermination of Gd content in -pC7AMA-DTPA(Gd) The binding efficiency of Gd in DTPA(Gd) and the coupling rate of DTPA(Gd) were determined and calculated using the ICP-OES detection method described in Example 1. The binding efficiency of Gd in DTPA(Gd) was 100%, and the coupling efficiency of DOTA(Gd) in pC7AMA-DTPA(Gd) exceeded 95%.

[0061] ④pSDXAA- b Characterization of the two-phase transition point of -pC7AMA-DTPA(Gd) The pSDXAA- particle size was determined according to the particle size analyzer detection method described in Example 1. b -pC7AMA-DTPA(Gd) particle size at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9 and 5.6. Figure 6 (C) shows that as the pH value decreases, pSDXAA- b The particle size of -pC7AMA-DTPA(Gd) exhibits a change process that conforms to "from the first micelle state to the disassembled state and then to the second micelle state", and has a two-phase transition point that conforms to pSDXAA and pC7AMA.

[0062] ⑤pSDXAA- b -pC7AMA-DTPA(Gd) T 1. Characterization of ability changes with pH pSDXAA- was measured using the MRI detection method described in Example 1. b -pC7AMA-DTPA(Gd) at pH 7.4, 7.1, 6.8, 6.5, 6.2, 5.9 and 5.6 r 1. Figure 6 (D) shows that as the pH value decreases, pSDXAA- b -pC7AMA-DTPA(Gd) r 1. Secondary enhancement has occurred, which is consistent with pSDXAA- b The structural changes of -pC7AMA-DTPA(Gd) are observed. At pH > 6.9, DTPA(Gd) is encapsulated within micelles, resulting in weaker contact with water molecules. T 1. The ability of DTPA(Gd) to contact water molecules is relatively weak when pH > 6.9 > 6.1, thus achieving an enhanced form. T 1. At pH < 6.1, DTPA(Gd) achieves further enhancement by increasing its rotation time while maintaining its ability to contact water molecules. T 1.

[0063] Example 3: A dual pH threshold responsive polymer contrast agent consisting of an anionic pH-sensitive block (phase transition point 6.5) with polysulfonamide octyl methacrylamide as segment A, a cationic pH-sensitive block (phase transition point 7.0) with poly(2-isopropylaminomethyl methacrylate) as segment B, and HP-DO3A(Gd) linked to the end of the cationic block.

[0064] (1) Preparation of polysulfonamide polyoctyl methacrylamide Sulfamethoxymethylacrylamide (SDX-MA) was polymerized by a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain thioester-terminated polysulfamethoxymethylacrylamide pSDXMA. The polymerization method used in the preparation of pSDXMA was a conventional and well-known operation, and the preparation process and reaction conditions were the same as those in Reference 1 in Example 1.

[0065] (2) Preparation of poly(2-isopropylaminomethacrylate) 2-Isopropylaminomethyl methacrylate (EPA-MA) and a hydroxyl-terminated RAFT chain transfer agent were subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain poly(2-isopropylaminomethyl methacrylate) pEPAMA with carboxyl groups and thioesters at the two ends. The polymerization method used in the preparation of pEPAMA was a conventional and well-known operation, and the preparation process and reaction conditions were the same as those in Reference 2 in Example 1.

[0066] (3) Coupling of HP-DO3A(Gd) The coupling of DO3A is the same as in Example 1, except that DOTA-Mal in Example 1 is replaced with Mal-(CH2). n -HP-DO3A or Mal-(CH2CH2O) n -HP-DO3A (n=2~5) replaces pC7AMA in Example 1 with pEPAMA, and finally obtains pEPAMA-DO3A(Gd).

[0067] (4) Preparation of a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response Maleimide (Mal) modified Mal-pEPAMA-DO3A(Gd) was obtained using the same method as in Example 1. Further, using the same method as in Example 1, pSDXMA- b -pEPAMA-DO3A(Gd), a two-stage enhanced gadolinium-based polymeric magnetic resonance contrast agent with dual pH threshold response, is produced using a click chemistry method that is a conventional and well-known procedure, as described in Reference 3 of Example 1.

[0068] (5) Characterization of contrast agents ①Characteristics of polysulfonamide polyoctyl methacrylamide The pSDXMA phase transition point was characterized using the transmittance method described in Example 1. Figure 7 (A) shows that the transmittance of pSDXMA decreases sharply between pH 6.6 and 6.4. Taking a 50% decrease in transmittance as its phase transition point, the phase transition point of pSDXMA prepared in this embodiment is pH 6.5.

[0069] ②Characteristics of the phase transition point of poly(2-isopropylaminomethylmethacrylate) The phase transition point of pEPAMA was characterized using the pH titration method described in Example 1. Figure 7 (B) The results showed that the pH of pEPAMA changed very slowly between pH 6.95 and 7.05. The midpoint between the start and end of the process was taken as its phase transition point. Therefore, the phase transition point of the pEPAMA prepared in this example was pH 7.0.

[0070] ③pSDXMA- b Determination of Gd content in pEPAMA-DO3A(Gd) The binding efficiency of Gd in HB-DO3A(Gd) and the coupling rate of DTPA(Gd) in pEPAMA-DO3A(Gd) were determined and calculated using the ICP-OES detection method described in Example 1. The binding efficiency of Gd in HB-DO3A(Gd) was 100%, and the coupling efficiency of DO3A(Gd) in pEPAMA-DO3A(Gd) exceeded 95%.

[0071] ④pSDXMA- b Characterization of the two-phase transition point of -pEPAMA-DO3A(Gd) The pSDXMA- particle size was determined according to the particle size analyzer detection method described in Example 1. b -pEPAMA-DO3A(Gd) particle size at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2 and 5.9. Figure 7 (C) shows that as the pH value decreases, pSDXMA- b The particle size of -pEPAMA-DO3A(Gd) exhibits a change process that conforms to "from the first micelle state to the disassembled state and then to the second micelle state", and has a two-phase transition point that conforms to pSDXMA and pEPAMA.

[0072] ⑤pSDXMA- b -pEPAMA-DO3A(Gd) T 1. Characterization of ability changes with pH pSDXMA- was measured using the MRI detection method described in Example 1. b-pEPAMA-DO3A(Gd) at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, and 5.9 r 1. Figure 7 (D) shows that as the pH value decreases, pSDXMA- b -pEPAMA-DO3A(Gd) r A second-level enhancement has appeared, which is consistent with pSDXMA- b The structural changes of -pEPAMA-DO3A(Gd) are observed. At pH > 7.0, DO3A(Gd) is encapsulated within micelles, resulting in weaker contact with water molecules. T 1. The ability of DO3A(Gd) to contact water molecules is relatively weak when pH > 7.0 > 6.5, thus achieving an enhanced form. T 1. At pH < 6.5, DO3A(Gd) achieves further enhancement by increasing its rotation time while maintaining its ability to contact water molecules. T 1.

[0073] Example 4: A dual pH threshold responsive polymer contrast agent consisting of an anionic pH-sensitive block (phase transition point 5.7) of poly(methoxydiazole sulfamethacrylamide) as segment A, a cationic pH-sensitive block (phase transition point 6.6) of the non-regression copolymer of ethyl 2-(diisopropylamino)methacrylate and ethyl 2-(hexamethyleneimino)-ethyl methacrylate as segment B, and DOTA(Gd) linked to the end of the cationic block.

[0074] (1) Preparation of poly(methathiazole sulfonamide) acrylamide The preparation of poly(methoxythiazolidinyl sulfonamide) acrylamide was as described in Example 1, yielding poly(methoxythiazolidinyl sulfonamide) acrylamide, pSMTAA. The preparation process and reaction conditions were the same as those in Reference 1 in Example 1.

[0075] (2) Preparation of the free copolymer of 2-(diisopropylamino)methacrylate and 2-(hexamethyleneimino)-ethylmethacrylate 2-(diisopropylamino)ethyl methacrylate (DPA-MA) and 2-(hexamethyleneimino)-ethyl methacrylate (C7A-MA) were fed in a 1 / 1 molar ratio with a carboxyl-terminated RAFT chain transfer agent via a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain a non-regressed copolymer of 2-(diisopropylamino)ethyl methacrylate and 2-(hexamethyleneimino)-ethyl methacrylate with carboxyl and thioester groups respectively at their ends. co -C7AMA), p(DPAMA- coThe polymerization method used in the preparation of -C7AMA) is a conventional and well-known operation, and the preparation process and reaction conditions are the same as those in Reference 2 in Example 1.

[0076] (3) DOTA's association The coupling of DOTA is the same as in Example 1, except that DOTA-Mal is replaced with DOTA-(CH2)2-Mal in Example 1, and pC7AMA is replaced with p(DPAMA- co -C7AMA), ultimately yielding p(DPAMA- co -C7AMA)-DOTA(Gd).

[0077] (4) Preparation of a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response Maleimide (Mal) modified Mal-p(DPAMA-) was obtained by the same method as in Example 1. co -C7AMA)-DOTA(Gd). Further, using the method corresponding to Example 1, pSMTAA- b -p(DPAMA- co -C7AMA)-DOTA(Gd), a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response, is produced using a click chemistry method that is a conventional and well-known procedure, as described in Reference 3 of Example 1.

[0078] (5) Characterization of contrast agents ① Characterization of poly(methathiazole sulfonamide) acrylamide The pSMTAA phase transition point was characterized using the transmittance method described in Example 1. According to... Figure 8 (A) shows that the transmittance of pSMTAA decreases sharply between pH 5.8 and 5.6. Taking a 50% decrease in transmittance as its phase transition point, the phase transition point of pSMTAA prepared in this embodiment is pH 5.7.

[0079] ② Characterization of the phase transition point of the non-regression copolymer of poly(diisopropylamino)methacrylate and 2-(hexamethyleneimino)-ethyl methacrylate The pH titration method described in Example 1 was used to perform p(DPAMA-) titration. co Characterization of the phase transition point (-C7AMA). Based on Figure 8 (B) results p(DPAMA- co The pH change of p(DPAMA-C7AMA) is very slow between pH 6.64 and 6.57. Taking the midpoint between the start and end of this process as its phase transition point, the p(DPAMA-C7AMA) prepared in this embodiment... co -C7AMA), its phase transition point is pH=6.6.

[0080] ③pSMAAT- b -p(DPAMA- co Determination of Gd content in -C7AMA)-DOTA(Gd) DOTA(Gd) and p(DPAMA-) were determined and calculated using the ICP-OES detection method described in Example 1. co The binding efficiency of Gd in DOTA(Gd) and the coupling rate of DOTA(Gd). The binding efficiency of Gd in DOTA(Gd) is 100%, p(DPAMA- co In -C7AMA)-DOTA(Gd), the coupling efficiency of DOTA(Gd) exceeds 95%.

[0081] ④pSMAAT- b -p(DPAMA- co Characterization of the dual phase transition point of -C7AMA)-DOTA(Gd) pSMTAA- was determined according to the particle size analyzer detection method described in Example 1. b -p(DPAMA- co The particle size of -C7AMA)-DOTA(Gd) at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9, 5.6 and 5.3. Figure 8 (C) shows that as the pH value decreases, pSMTAA- b -p(DPAMA- co The particle size of -C7AMA)-DOTA(Gd) exhibits a change process conforming to "from the first micelle state to the disassembled state and then to the second micelle state", and has properties consistent with pSMTAA and p(DPAMA- co The biphase transition point of -C7AMA).

[0082] ⑤pSMAAT- b -p(DPAMA- co -C7AMA)-DOTA(Gd) T 1. Characterization of ability changes with pH pSMAAT- was measured using the MRI detection method described in Example 1. b -p(DPAMA- co -C7AMA)-DOTA(Gd) at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9, 5.6 and 5.3 r 1. Figure 8 (D) shows that as the pH value decreases, pSMTAA- b -p(DPAMA- co-C7AMA)-DOTA(Gd) r 1. Secondary enhancement has occurred, which is consistent with pSMTAA- b -p(DPAMA- co The structural changes of -C7AMA)-DOTA(Gd) lead to weaker contact between DOTA(Gd) and water molecules at pH > 6.7, as DOTA(Gd) is encapsulated within micelles. T 1. The ability of DOTA(Gd) to contact water molecules is relatively weak when pH > 6.7 > 5.7, thus achieving an enhanced form. T 1. At pH < 5.7, DOTA(Gd) achieves further enhancement by increasing its rotation time while maintaining its ability to contact water molecules. T 1.

[0083] Example 5: A dual pH threshold responsive polymer contrast agent consisting of an anionic pH-sensitive block (phase transition point 5.5) with polysulfonamide benzoylmethacrylamide as segment A, a cationic pH-sensitive block (phase transition point 6.7) with the non-regression copolymer of 2-(diisopropylamino)methacrylate and 2-isopropylaminomethacrylate as segment B, and DTPA(Gd) linked to the end of the cationic block.

[0084] (1) Preparation of polysulfonamide benzoyl methylacrylamide The sulfabenzoyl methyl acrylamide monomer (SBZ-MA) was subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain thioester-terminated polysulfabenzoyl methyl acrylamide pSBZMA. The polymerization method used in the preparation of pSBZMA was a conventional and well-known operation, and the preparation process and reaction conditions were the same as those in Reference 1 in Example 1.

[0085] (2) Preparation of the free copolymer of 2,2-(diisopropylamino)methacrylate and 2-isopropylaminomethacrylate 2-(diisopropylamino)ethyl methacrylate (DPA-MA) and 2-isopropylaminoethyl methacrylate (EPA-MA) were fed in a 1 / 1 molar ratio with a carboxyl-terminated RAFT chain transfer agent via a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain an unregressed copolymer p(DPAMA-MA) of 2-(diisopropylamino)ethyl methacrylate and 2-isopropylaminoethyl methacrylate with carboxyl groups and thioesters at their respective ends. co -EPAMA), p(DPAMA- co The polymerization method used in the preparation of EPAMA is a conventional and well-known operation, and the preparation process and reaction conditions are the same as those in Reference 2 in Example 1.

[0086] (3) Coupling of DTPA(Gd) DTPA Coupling: Same as Example 2, except that DTPA-Mal in Example 2 is replaced with DTPA-(CH2)2-Mal, and pC7AMA is replaced with p(DPAMA- co -EPAMA), ultimately yielding p(DPAMA- co -EPAMA)-DTPA.

[0087] (4) Preparation of a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response Maleimide (Mal) modified Mal-p(DPAMA-) was obtained by the same method as in Example 1. co -EPAMA)-DTPA(Gd). Further, following the method corresponding to Example 1, pSBZMA- b -p(DPAMA- co -EPAMA)-DTPA(Gd), a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response, was prepared using a click chemistry method that is a conventional and well-known procedure, as described in Reference 3 of Example 1.

[0088] (5) Characterization of contrast agents ①Characteristics of polysulfonamide benzoylmethylacrylamide The pSBZMA phase transition point was characterized using the transmittance method described in Example 1. Figure 9 (A) shows that the transmittance of pSBZMA decreases sharply between pH 5.8 and 5.6. Taking a 50% decrease in transmittance as its phase transition point, the phase transition point of pSBZMA prepared in this embodiment is pH 5.5.

[0089] ②Characteristics of the phase transition point of the non-regression copolymer of 2-(diisopropylamino)methacrylate and 2-(hexamethyleneimino)-ethylmethacrylate The p(DPAMA-) titration was performed according to the pH titration method described in Example 1. co Characterization of the phase transition point (EPAMA). Based on Figure 9 (B) results p(DPAMA- co The pH change of p(DPAMA-) is very slow between pH 6.73 and 6.67. Taking the midpoint between the start and end of this process as its phase transition point, the p(DPAMA-) prepared in this embodiment... co -EPAMA), with a phase transition point of pH=6.7.

[0090] ③pSBZMA- b -p(DPAMA- co Determination of Gd content in EPAMA-DTPA(Gd) The DTPA(Gd) and p(DPAMA-) were determined and calculated using the ICP-OES detection method described in Example 1. co The binding efficiency of Gd in DTPA(Gd) and the coupling rate of DTPA(Gd). The binding efficiency of Gd in DTPA(Gd) is 100%, p(DPAMA- co The coupling efficiency of DTPA(Gd) in -EPAMA)-DTPA(Gd) exceeds 95%.

[0091] ④pSBZMA- b -p(DPAMA- co Characterization of the dual phase transition point of -EPAMA)-DTPA(Gd) The pSBZMA- was determined according to the particle size analyzer detection method described in Example 1. b -p(DPAMA- co Particle sizes of -EPAMA)-DTPA(Gd) at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9, 5.6, 5.3 and 5.0. Figure 9 (C) shows that as the pH value decreases, pSBZ- b -p(DPAMA- co The particle size of -EPAMA)-DTPA(Gd) exhibits a change process conforming to "from the first micelle state to the disassembled state and then to the second micelle state", and has properties consistent with pSBZMA and p(DPAMA- co -EPAMA) biphase transition point.

[0092] ⑤pSBZMA- b -p(DPAMA- co -EPAMA)-DTPA(Gd) T 1. Characterization of ability changes with pH pSBZMA- was measured using the MRI detection method described in Example 1. b -p(DPAMA- co -EPAMA)-DTPA(Gd) at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9, 5.6, 5.3 and 5.0 r 1. Figure 9 (D) shows that as the pH value decreases, pSBZMA- b -p(DPAMA- co -EPAMA)-DTPA(Gd) r 1. A second-order enhancement has appeared, which is consistent with pSBZMA- b -p(DPAMA- coThe structural changes of -EPAMA)-DTPA(Gd) result in DTPA(Gd) being encapsulated within micelles at pH > 6.7, leading to weaker contact with water molecules. T 1. The ability of DTPA(Gd) to contact water molecules is relatively weak when pH > 6.7 > 5.5, thus achieving an enhanced form. T 1. When pH < 5.5, DTPA(Gd) maintains its ability to contact water molecules while increasing the rotation time to achieve further enhancement. T 1.

[0093] Example 6: The anionic pH-sensitive block (phase transition point 5.9) is composed of an anionic copolymer of sulfadiazine acrylamide and methadiazol sulfadiazine acrylamide, and the cationic pH-sensitive block (phase transition point 7.0) is composed of poly(2-isopropylaminomethylmethacrylate) as the B-segment. A linker is attached at the end of the cationic block. DOTA(Gd) dual pH threshold responsive polymeric contrast agent.

[0094] (1) Preparation of a non-regression copolymer of sulfadiazine acrylamide and methylthiazolidinyl sulfadiazine. Sulfamethoxam-acrylamide (SDX-AA) and methylthiazolium sulfamethacrylamide (SMT-AA) were subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction at a molar ratio of 1 / 1 to obtain an unregressed copolymer p(SDXAA-) of thioester-terminated sulfamethoxam-acrylamide and methylthiazolium sulfamethacrylamide. co -SMTAA), p(SDXAA- co The polymerization method used in the preparation of SMTAA is a conventional and well-known operation, and the preparation process and reaction conditions are the same as those in Reference 1 in Example 1.

[0095] (2) Preparation of poly(2-isopropylaminomethacrylate) The preparation of poly(2-isopropylaminomethyl methacrylate) (pEPAMA), a polymer of 2-isopropylaminomethyl methacrylate (EPA-MA) with carboxyl and thioester ends respectively, was the same as in Example 3, and the preparation process and reaction conditions were the same as in Reference 2 in Example 1.

[0096] (3) Coupling of DTPA(Gd) The coupling of DTPA is the same as in Example 3, except that HP-DO3A in Example 3 is replaced with DTPA-(CH2)2-Mal, and finally pEPAMA-DTPA(Gd) is obtained.

[0097] (4) Preparation of a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response Maleimide (Mal) modified Mal-pEPAMA-DTPA(Gd) was obtained using the same method as in Example 1. Furthermore, following the corresponding method in Example 1, p(SDXAA- co -SMTAA)- b -pEPAMA-DTPA(Gd), a two-stage enhanced gadolinium-based polymeric magnetic resonance contrast agent with dual pH threshold response, was prepared using a click chemistry method that is a conventional and well-known procedure, as described in Reference 3 of Example 1.

[0098] (5) Characterization of contrast agents ①Characteristics of the phase transition point of the non-regression copolymer of sulfadiazine acrylamide and methadiazol sulfadiazine acrylamide The transmittance method described in Example 1 was used to measure p(SDXAA-) co Characterization of the phase transition point (SMTAA). Based on Figure 10 (A) result p(SDXAA- co The transmittance of p(SDXAA-) decreases drastically between pH 6.0 and 5.8. Taking a 50% decrease in transmittance as its phase transition point, the p(SDXAA-) prepared in this embodiment... co -SMTAA), with a phase transition point of pH=5.9.

[0099] ②Characteristics of the phase transition point of poly(2-isopropylaminomethylmethacrylate) The phase transition point of pEPAMA was characterized using the pH titration method described in Example 1. Figure 10 (B) The results showed that the pH change of pEPAMA was very slow between pH=7.02 and pH=6.97. The midpoint between the start and end of the process was taken as its phase transition point. Therefore, the phase transition point of the pEPAMA prepared in this example was pH=7.0.

[0100] ③p(SDXAA- co -SMTAA)- b Determination of Gd content in pEPAMA-DTPA(Gd) The binding efficiency of Gd in DOTA(Gd) and the coupling rate of DTPA(Gd) were determined and calculated using the ICP-OES detection method described in Example 1. The binding efficiency of Gd in DOTA(Gd) was 100%, and the coupling efficiency of DOTA(Gd) in pEPAMA-DTPA(Gd) exceeded 95%.

[0101] ④p(SDXAA- co -SMTAA)- b Characterization of the -pEPAMA-DTPA(Gd) dual phase transition point The particle size analyzer detection method described in Example 1 was used to determine p(SDXAA-) co -SMTAA)- b -pEPAMA-DTPA(Gd) particle size at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9 and 5.6. Figure 10 (C) shows that as the pH value decreases, p(SDXAA-) co -SMTAA)- b The particle size of -pEPAMA-DTPA(Gd) exhibits a change process conforming to "from the first micelle state to the disassembled state and then to the second micelle state", and has a consistency with p(SDXAA- co The biphase transition point of SMTAA and pEPAMA.

[0102] ⑤p(SDXAA- co -SMTAA)- b -pEPAMA-DTPA(Gd) T 1. Characterization of ability changes with pH p(SDXAA-) was measured using the MRI detection method described in Example 1. co -SMTAA)- b -pEPAMA-DTPA(Gd) at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9 and 5.6 r 1. Figure 10 (D) shows that as the pH value decreases, p(SDXAA-) co -SMTAA)- b -pEPAMA-DTPA(Gd) r 1. A second-order enhancement occurred, which conforms to p(SDXAA- co -SMTAA)- b The structural changes of -pEPAMA-DTPA(Gd) are observed. At pH > 7.0, DTPA(Gd) is encapsulated within micelles, resulting in weaker contact with water molecules. T 1. The ability of DTPA(Gd) to contact water molecules is relatively weak when pH > 7.0 > 5.9, thus achieving an enhanced form. T 1. When pH < 5.9, DTPA(Gd) maintains its ability to contact water molecules while increasing the rotation time to achieve further enhancement. T 1.

[0103] Example 7: A dual pH threshold-responsive polymer contrast agent consisting of an anionic pH-sensitive block (phase transition point 5.4) with an anionic pH-sensitive block (phase transition point 7.0) with a poly(2-isopropylaminomethylmethacrylate) ethyl ester (P2-) as the A-segment and NOA(Gd) as the B-segment, with NOA(Gd) attached to the end of the cationic block.

[0104] (1) Preparation of non-regressed copolymer of methacrylic acid and butyl methacrylate Methacrylic acid (MAA) and butyl methacrylate (BMA) were subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction at a molar ratio of 9 / 1 to obtain a thioester-terminated methacrylic acid and butyl methacrylate regressive copolymer p(MAA- co -BMA), p(MAA- co The preparation process of MAA-BMA uses RAFT polymerization. For specific experimental steps, please refer to reference 6 [Zhan K., Zhang H., Li M., et al. Charges of soluble amphiphiles and particles: random and diblock copolymerizations of MAA / AAm, MAA / St, and MAA / 4VP in ethanol [J]. COLLOID AND POLYMER SCIENCE, 2014, 292(7): 1553-1565].

[0105] (2) Preparation of poly(2-isopropylaminomethacrylate) 2-Isopropylaminomethyl methacrylate (EPA-MA) and a hydroxyl-terminated RAFT chain transfer agent were subjected to a typical reversible addition-fragmentation chain transfer polymerization reaction to obtain poly(2-isopropylaminomethyl methacrylate) pEPAMA, which has hydroxyl and thioester ends on both sides. The polymerization method used to prepare pEPAMA is a conventional and well-known operation, and the preparation process and reaction conditions are the same as those in Reference 2 in Example 1.

[0106] (3) Coupling of Nota (Gd) Coupled with NOTA: Same as in Example 3, except that HP-DO3A in Example 1 is replaced with NOTA-Mal, resulting in pEPAMA-NOTA(Gd).

[0107] (4) Preparation of a two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response Maleimide (Mal) modified Mal-pEPAMA-NOTA(Gd) was obtained using the same method as in Example 1. Further, using the same method as in Example 1, p(MAA- co -BMA)- b -pEPAMA-NOTA(Gd), a two-stage enhanced gadolinium-based polymeric magnetic resonance contrast agent with dual pH threshold response, was prepared using a click chemistry method that is a conventional and well-known procedure, as described in Reference 3 of Example 1.

[0108] (5) Characterization of contrast agents ①Characteristics of the phase transition point of the non-regression copolymer of methacrylic acid and butyl methacrylate The p(MAA-) method was performed according to the transmittance method described in Example 1. co Characterization of the phase transition point (BMA). Based on Figure 11 (A) results p(MAA- co The transmittance of p(MAA-BMA) decreases sharply between pH 5.6 and 5.2. Taking a 50% decrease in transmittance as its phase transition point, the p(MAA-BMA) prepared in this embodiment... co -BMA), with a phase transition point of pH=5.4.

[0109] ②Characteristics of the phase transition point of poly(2-isopropylaminomethylmethacrylate) The phase transition point of pEPAMA was characterized using the pH titration method described in Example 1. Figure 11 (B) The results showed that the pH change of pEPAMA was very slow between pH=7.04 and pH=6.97. The midpoint between the start and end of the process was taken as its phase transition point. Therefore, the phase transition point of the pEPAMA prepared in this example was pH=7.0.

[0110] ③p(MAA- co -BMA)- b Determination of Gd content in pEPAMA-NOTA(Gd) The binding efficiency of Gd in NOTA(Gd) and the coupling rate of NOTA(Gd) in pEPAMA-NOTA(Gd) were determined and calculated using the ICP-OES detection method described in Example 1. The binding efficiency of Gd in NOTA(Gd) was 100%, and the coupling efficiency of NOTA(Gd) in pEPAMA-NOTA(Gd) exceeded 95%.

[0111] ④p(MAA- co -BMA)- b Characterization of the -pEPAMA-NOTA(Gd) dual phase transition point The p(MAA-) particle size analyzer detection method described in Example 1 was used to determine the particle size distribution.co -BMA)- b -pEPAMA-NOTA(Gd) particle size at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9, 5.6, 5.3 and 5.0. Figure 11 (C) shows that as the pH value decreases, p(MAA-) co -BMA)- b The particle size of pEPAMA-NOTA(Gd) exhibits a change process consistent with "agglomeration to dispersion and then agglomeration", and has the characteristics of p(MAA- co The biphase transition point of BMA and pEPAMA.

[0112] ⑤p(MAA- co -BMA)- b -pEPAMA-NOTA(Gd) T 1. Characterization of ability changes with pH p(MAA-) was measured using the MRI detection method described in Example 1. co -BMA)- b -pEPAMA-NOTA(Gd) at pH values ​​of 7.4, 7.1, 6.8, 6.5, 6.2, 5.9, 5.6, 5.3 and 5.0 r 1. Figure 11 (D) shows that as the pH value decreases, p(MAA-) co -BMA)- b -pEPAMA-NOTA(Gd) r 1. Second-order enhancement occurred, which is consistent with p(MAA- co -BMA)- b Structural changes in -pEPAMA-NOTA(Gd). At pH > 7.0, NOA(Gd) is encapsulated within micelles, resulting in weaker contact with water molecules. T 1. The ability of NOA(Gd) to contact water molecules is relatively weak when pH > 7.0 > 5.4, thus achieving an enhanced form. T 1. How can the rotation time of Nota(Gd) be increased to achieve further enhancement while maintaining its ability to contact water molecules at pH < 5.4? T 1.

[0113] Table 1 Phase transition points of Examples 1-7

[0114] Test Example 1: This test case examines the pSMTAA- at different DPA-MA / C7A-MA molar ratios. b -p(DPAMA- co-C7AMA)-DOTA(Gd) biblock phase transition point.

[0115] According to the same pSMTAA- as in Example 4 b -p(DPAMA- co The preparation method of -C7AMA)-DOTA(Gd) yielded pH dual-sensitive contrast agents with different B-block phase transition points (Table 2).

[0116] Table 2. pSMTAA with different DPA-MA / C7A-MA molar ratios b -p(DPAMA- co -C7AMA)-DOTA(Gd) block phase transition point

[0117] Data shows that with pSMTAA- b -p(DPAMA- co As the proportion of DPA-MA in the B-block of pSMTAA-DOTA(Gd) gradually increases, the phase transition point of the B-block gradually decreases, while the phase transition point of the A-block remains unaffected. This indicates that adjusting the molar ratio of DPA-MA / C7A-MA in the B-block can effectively change the phase transition point of pSMTAA-DOTA(Gd). b -p(DPAMA- co -C7AMA)-DOTA(Gd) B-block phase transition point.

[0118] The above explains that the B-block obtained by combining DPA-MA and C7A-MA can have its phase transition point artificially controlled by adjusting the molar ratio of DPA-MA / C7A-MA.

[0119] Test Example 2: This test case examines pSBZMA with different DPA-MA / EPA-MA molar ratios. b -p(DPAMA- co The biblock phase transition point of -EPAMA)-DTPA(Gd).

[0120] According to the same example 5, pSBZMA- b -p(DPAMA- co The preparation method of -EPAMA)-DTPA(Gd) yielded pH dual-sensitive contrast agents with different B-block phase transition points (Table 3).

[0121] Table 3. pSMTAA with different DPA-MA / EPA-MA molar ratios b -p(DPAMA- co -EPAMA)-DTPA(Gd) block phase transition point

[0122] Data shows that with pSMTAA- b -p(DPAMA- co As the proportion of DPA-MA in the β-block of pSMTAA-(Gd) gradually increases, the phase transition point of the β-block gradually decreases, while the phase transition point of the A-block remains unaffected. This indicates that adjusting the molar ratio of DPA-MA / EPA-MA in the β-block can effectively alter the pSMTAA- b -p(DPAMA- co -C7AMA)-DOTA(Gd) B-block phase transition point.

[0123] The above explains that the B-block obtained by combining DPA-MA and EPA-MA can have its phase transition point artificially controlled by adjusting the DPA-MA / EPA-MA (molar ratio).

[0124] Test Example 3: This test case examines p(SDXAA-) with different SDX-AA / SMT-AA molar ratios. co -SMTAA)- b The biblock phase transition point of -pEPAMA-DTPA(Gd).

[0125] According to the same example 6, p(SDXAA- co -SMTAA)- b The preparation method of -pEPAMA-DTPA(Gd) yielded pH dual-sensitive contrast agents with different A-block phase transition points (Table 4).

[0126] Table 4. p(SDXAA-) with different SDX-AA / SMT-AA molar ratios co -SMTAA)- b Block phase transition point of -pEPAMA-DTPA(Gd)

[0127] Data shows that as p(SDXAA- co -SMTAA)- b As the proportion of SDX-MA in the A-block of -pEPAMA-DTPA(Gd) gradually increases, the phase transition point of the A-block also gradually increases, while the phase transition point of the B-block remains unaffected. This indicates that adjusting the molar ratio of DPA-MA / EPA-MA in the A-block can effectively change the p(SDXAA- co -SMTAA)- b The A-block phase transition point of -pEPAMA-DTPA(Gd).

[0128] The above explains that the phase transition point of the A-block obtained by combining SDX-AA and SMT-AA can be artificially controlled by adjusting the molar ratio of SDX-AA / SMT-AA.

[0129] In summary, this invention first combines anionic pH-sensitive polymer block A, cationic pH-sensitive polymer block B, and Gd-containing polymer block A. 3+ The chelating group C(Gd) was applied to the preparation of a gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response. In this method, the anionic pH-sensitive polymer block A and the cationic pH-sensitive polymer block B altered the phase transition point by adjusting the monomer ratio within the blocks. The resulting gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response exhibited excellent responsiveness to ambient pH values. By sequentially passing through a first micelle state, a disassembly state, and a second micelle state during a gradual decrease in pH, the magnetic resonance response was influenced by ambient pH. T 1 Two-stage enhancement of contrast.

[0130] It should be noted that the above description is only a preferred embodiment of the present invention. Any changes made according to the concept of the present invention that do not exceed the spirit covered by the specification should be within the scope of the present invention.

[0131] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0132] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

Claims

1. A two-stage enhanced gadolinium-based polymer magnetic resonance contrast agent with dual pH threshold response, characterized in that, The gadolinium-based polymer magnetic resonance contrast agent has the following general formula: A- b -BLC(Gd); Wherein, A is an anionic pH-sensitive polymer block, B is a cationic pH-sensitive polymer block, and "- b "-" indicates that block A and block B are linked by block copolymerization, L is the linking group or chemical bond, and C(Gd) indicates that it contains Gd. 3+ chelating groups; The C(Gd) is connected to the end of the B segment via L; The A-block is hydrophilic under physiological pH conditions and transforms into a hydrophobic state when the pH response threshold of the A-block is lower than that of the A-block. The B-block is hydrophobic under physiological pH conditions and transforms into a hydrophilic state when the pH response threshold of the B-block is lower than that of the B-block. The pH response threshold of the B block is lower than the physiological pH value and higher than the pH response threshold of the A block; The gadolinium-based polymer magnetic resonance contrast agent sequentially forms a first micelle state, a disassembled state, and a second micelle state during the pH decrease process; In the first micelle state, the B block, L linking group or chemical bond and C(Gd) are located in the hydrophobic core, and the A block is located in the hydrophilic shell, thus limiting the ability of water molecules to contact C(Gd); In the disassembled state, block A remains hydrophilic, block B transforms into a hydrophilic state, and the L linker groups or chemical bonds are distributed in the aqueous phase, exposing C(Gd) to the aqueous environment, thereby achieving magnetic resonance imaging of the gadolinium-based polymer magnetic resonance contrast agent. T The first stage of contrast enhancement; In the second micelle state, block A transitions to a hydrophobic state and drives the gadolinium-based polymer magnetic resonance contrast agent to reassemble and form a hydrophobic micelle core. Block B, L linker groups or chemical bonds, and C(Gd) are located in the hydrophilic shell, micelle surface, or micelle-water interface of the micelle. By increasing the rotational correlation time of C(Gd), magnetic resonance imaging of the gadolinium-based polymer magnetic resonance contrast agent is achieved. T 1 The second stage of contrast enhancement.

2. The gadolinium-based polymer magnetic resonance imaging agent according to claim 1, characterized in that, The L is a chemical bond or a divalent linking group; the divalent linking group includes amide bonds, ester bonds, ether bonds, carbamate bonds, thioether bonds, disulfide bonds, triazole bonds, alkylene bonds, or combinations thereof.

3. The gadolinium-based polymer magnetic resonance imaging agent according to claim 1, characterized in that, The C(Gd) is a Gd-containing... 3+ The macrocyclic or open-chain chelating group is selected from one or more of DOTA(Gd), DTPA(Gd), HP-DO3A(Gd), Nota(Gd), TETA(Gd), DTPA-BMA(Gd) and their derivatives.

4. The gadolinium-based polymer magnetic resonance contrast agent according to claim 3, characterized in that, The C(Gd) is DOTA(Gd), DTPA(Gd), DO3A(Gd), or Nota(Gd).

5. The gadolinium-based polymer magnetic resonance contrast agent according to any one of claims 1-4, characterized in that, The pH response threshold of the A block is 5.4-6.6; the pH response threshold of the B block is 6.4-7.1; and the pH response threshold of the B block is higher than that of the A block, with a difference of 0.4-1.6 pH units.

6. The gadolinium-based polymer magnetic resonance contrast agent according to claim 5, characterized in that, The pH response threshold of the A block is 5.8-6.3; the pH response threshold of the B block is 6.6-7.0; and the pH response threshold of the B block is higher than that of the A block, with a difference of 0.5-0.8 pH units.

7. The gadolinium-based polymer magnetic resonance imaging agent according to claim 1, characterized in that, The A block is an anionic pH-sensitive polymer block containing a weakly acidic group, including sulfonamide, carboxyl, or a combination thereof; the B block is a cationic pH-sensitive polymer block containing a protonable basic group, including tertiary amine, imidazole, pyridinium, or a combination thereof.

8. The gadolinium-based polymer magnetic resonance contrast agent according to any one of claims 1-7, characterized in that, The gadolinium-based polymer magnetic resonance contrast agent exhibits the following longitudinal relaxation rate change relationship during pH decrease: r 1 < r 1-B < r 1-A ,in, r 1 This refers to the longitudinal relaxation rate at physiological pH. r 1-B This refers to the longitudinal relaxation rate of the B-block response at pH. r 1-A This refers to the longitudinal relaxation rate of the A-block at pH. Physiological pH corresponds to the first micelle state, B-block response pH corresponds to the disassembly state, and A-block response pH corresponds to the second micelle state. Magnetic Resonance T The phased enhancement of contrast includes: a first-stage enhancement caused by the increased contact ability between water molecules and C(Gd), and a second-stage enhancement caused by maintaining the contact ability between water molecules and C(Gd) and increasing the rotation time.

9. The gadolinium-based polymer magnetic resonance contrast agent according to any one of claims 1-7 in pH gradient-responsive magnetic resonance imaging. T 1. Application in imaging contrast agents, characterized in that, The gadolinium-based polymer magnetic resonance contrast agent is used for the weakly acidic microenvironment of tumors, tumor cell endosomes, tumor cell lysosomes, tumor-associated macrophage phagolysosomes, inflammatory macrophage phagolysosomes, acidic microenvironment of inflamed tissues, or acidic microenvironment of atherosclerotic plaques. T 1. Magnetic resonance imaging.

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