A novel CEST polymer contrast agent and its preparation method

Abstract

The preparation method of the novel CEST polymer contrast agent provided by the present invention solves the technical problems in the prior art that the exogenous small molecule contrast agents are easily metabolized, have low accumulation efficiency and short accumulation time at the lesion site, and the multifunctional modification and purification steps of the small molecule contrast agents are cumbersome. The preparation method is: aminosalicylic acid is coupled with an acryloyl chloride compound or an acrylic isocyanate compound to prepare a salicylic acid derivative monomer, and under the action of a free radical initiator, the salicylic acid derivative monomer undergoes a polymerization reaction to prepare a novel CEST polymer contrast agent. The preparation process of the method is simple, and polymer contrast agents of different molecular weights and functions can be obtained by adjusting the free radical polymerization conditions and copolymerizing with other functional molecules. The polymer contrast agent of the present invention has high accumulation efficiency and long accumulation time at the lesion site, and is easier to modify and functionalize than small molecule contrast agents. It can be widely used in the field of biomedical technology.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, in particular to a novel CEST polymer contrast agent and a preparation method thereof. Background Art

[0002] Chemical exchange saturation transfer (CEST) imaging is a new type of MRI imaging technology derived from magnetization transfer. Its imaging is based on the exchange rate between water and protons. It detects exchangeable protons in endogenous or exogenous macromolecules at the molecular level. It is very sensitive to changes in the content of macromolecules such as free proteins in tissues. In addition to detecting endogenous signals, CEST technology can also perform CEST imaging through intravenous injection of exogenous contrast agents.

[0003] However, the current clinical application of exogenous contrast agents is very limited. Exogenous contrast agents mainly include small molecules with exchangeable protons such as porphyrins and salicylates. However, small molecule contrast agents are easily metabolized, have low accumulation efficiency and short accumulation time at the lesion site, and the multifunctional modification and purification steps of small molecule contrast agents are relatively cumbersome. A large number of literature reports have shown that polymer macromolecular contrast agents have longer circulation time and accumulation time at the lesion site in the body than small molecule contrast agents. Therefore, the construction of polymer CEST contrast agents is of great significance to improving the diagnostic effect of the disease, but there are currently few types of polymer contrast agents used for CEST imaging. Salicylic acid derivatives have good CEST imaging effects. For example, Michael T.McMahon et al. modified salicylic acid derivatives to the surface of hyperbranched polymers (PAMAM) by chemical coupling, and realized in vivo tracing of nanocarriers by CEST imaging (Nano Lett. 2016, 16, 2248-2253). However, the chemical modification process is complicated, the modification cost is high, and the yield is low, which limits its practical application and cannot meet the actual use demand of mass production. Therefore, a simple method is urgently needed to polymerize salicylic acid molecules and realize CEST imaging. Summary of the invention

[0004] In view of the shortcomings and deficiencies in the prior art, the present invention provides a novel CEST polymer contrast agent and its preparation method which has good CEST imaging effect, high accumulation efficiency and long accumulation time at the lesion site, is easier to modify and functionalize than small molecule contrast agents, has a simple preparation method, strong operability and a wide range of applications.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: The preparation method of the novel CEST polymer contrast agent provided by the present invention comprises the following steps:

[0006] (1) Modifying aminosalicylic acid to prepare salicylic acid derivative monomers:

[0007] Dissolving aminosalicylic acid in an organic solvent I, adding a reactant acryloyl chloride compound or acrylic isocyanate compound dropwise in an ice bath, and under the action of a catalyst, the amino group in the aminosalicylic acid and the reactant undergo a coupling reaction to obtain a salicylic acid derivative monomer;

[0008] (2) Preparation of polymer:

[0009] The salicylic acid derivative monomer obtained in step (1) is dissolved in an organic solvent II, and a novel CEST polymer contrast agent is prepared by an active free radical polymerization reaction under the action of a free radical initiator in an inert gas environment.

[0010] Preferably, in the preparation method of the novel CEST polymer contrast agent of the present invention, in step (1), when the reactant is an acryloyl chloride compound, the catalyst is triethylamine, and the acryloyl chloride compound is methacryloyl chloride or acryloyl chloride; when the reactant is an acrylic isocyanate compound, the catalyst is dibutyltin dilaurate;

[0011] In the coupling reaction of step (1), the molar ratio of the added amount of the salicylic acid derivative and the reactant is 1:1.

[0012] Preferably, in the method for preparing the novel CEST polymer contrast agent of the present invention, the aminosalicylic acid in step (1) is 4-aminosalicylic acid, 3-aminosalicylic acid, or 5-aminosalicylic acid; and the organic solvent I is tetrahydrofuran.

[0013] Preferably, in the method for preparing the novel CEST polymer contrast agent of the present invention, in step (2), the free radical initiator is azobisisobutyl or azobisisoheptanenitrile; and the organic solvent II is N,N-dimethylformamide.

[0014] Preferably, in the method for preparing the novel CEST polymer contrast agent of the present invention, step (2) preparation of the polymer comprises (2.1) preparation of the homopolymer:

[0015] The salicylic acid derivative monomer, free radical initiator and RAFT chain transfer agent prepared in step (1) are dissolved in an organic solvent II, an inert gas is introduced into the system to remove oxygen for polymerization reaction, and the homopolymer obtained by the reaction is dialyzed and freeze-dried to obtain a polymer contrast agent;

[0016] The reaction molar ratio of the free radical initiator to the RAFT chain transfer agent is 1:0.2.

[0017] Preferably, the method for preparing the novel CEST polymer contrast agent of the present invention, step (2) preparation of the polymer, further comprises (2.2) preparation of the copolymer:

[0018] The salicylic acid derivative monomer, functional monomer, free radical initiator and RAFT chain transfer agent prepared in step (1) are dissolved in organic solvent II, an inert gas is introduced into the system to remove oxygen for polymerization reaction, and the copolymer obtained by the reaction is dialyzed and freeze-dried to obtain a copolymer contrast agent;

[0019] The reaction molar ratio of the free radical initiator to the RAFT chain transfer agent is 1:0.2.

[0020] Preferably, the method for preparing the novel CEST polymer contrast agent of the present invention, step (2) preparation of the polymer, further comprises (2.3) preparation of the block copolymer:

[0021] The salicylic acid derivative monomer, free radical initiator and macromolecular chain transfer agent prepared in step (1) are dissolved in organic solvent II, an inert gas is introduced into the system to remove oxygen for polymerization reaction, and the block copolymer obtained by the reaction is dialyzed and freeze-dried to obtain a block copolymer contrast agent;

[0022] The macromolecular chain transfer agent is a hydrophilic polymer or a hydrophobic polymer chain transfer agent;

[0023] Wherein, the reaction molar ratio of the free radical initiator to the macromolecular chain transfer agent is 1:0.2.

[0024] Preferably, the preparation method of the novel CEST polymer contrast agent of the present invention further comprises the preparation of a CEST nanoparticle contrast agent:

[0025] The block copolymer prepared in step (2.3) is dissolved in organic solvent II, stirred evenly, and then dropped into water, stirred sufficiently, placed in a dialysis bag, dialyzed to remove the reaction solvent, and the CEST nanoparticle contrast agent is collected.

[0026] Preferably, the preparation method of the novel CEST polymer contrast agent of the present invention further includes the preparation of a drug-loaded CEST nanoparticle contrast agent:

[0027] The block copolymer and hydrophobic drug prepared in step (2.3) are dissolved in organic solvent II, stirred evenly, and then dropped into water, stirred thoroughly, placed in a dialysis bag, dialyzed to remove the reaction solvent, and the drug-loaded CEST nanoparticle contrast agent is collected.

[0028] A novel CEST polymer contrast agent is prepared according to any one of the above methods for preparing a novel CEST polymer contrast agent.

[0029] Beneficial effects of the present invention:

[0030] (1) The preparation method of the novel CEST polymer contrast agent of the present invention has simple steps, strong operability and wide application range. The novel CEST polymer contrast agent of the present invention is a macromolecular polymer, which is not easily metabolized, has high accumulation efficiency and long accumulation time at the lesion site, and is of great significance for improving the diagnosis effect of the disease.

[0031] The present invention discloses a method for preparing a novel CEST polymer contrast agent containing a salicylic acid derivative by using active free radical polymerization. Active free radical polymerization has good advantages in preparing polymers with complex structures and diverse functions. The method couples aminosalicylic acid with acryloyl chloride compounds or acrylic isocyanate compounds, modifies aminosalicylic acid to generate a freely polymerizable salicylic acid derivative monomer, and then obtains a CEST polymer contrast agent by free radical polymerization of the salicylic acid derivative monomer. At the same time, the method of the present invention does not damage the hydroxyl and carboxyl groups at the ortho position of the salicylic acid derivative monomer during the preparation and polymerization of the salicylic acid derivative monomer, so the prepared CEST polymer contrast agent retains the CEST imaging characteristics of the salicylic acid derivative.

[0032] (2) The preparation method of the novel CEST polymer contrast agent of the present invention can obtain CEST polymer contrast agents of different molecular weights by adjusting the free radical polymerization conditions. The control method is simple and has strong controllability, and can meet the requirements of different molecular weights.

[0033] CEST polymer contrast agents with different functions can also be prepared by copolymerization with other functional molecules; or other contrast agents can be introduced through post-modification to achieve multimodal imaging.

[0034] At the same time, block polymer contrast agents can be prepared through multi-step polymerization, and assembled nanoparticles can be obtained, thereby greatly enriching the types and uses of CEST polymer contrast agents. In addition, the CEST polymer contrast agent is easier to modify and functionalize than small molecule contrast agents, meeting a variety of usage requirements and modifications, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The H-NMR structural characterization diagram of the salicylic acid derivative monomer prepared by the present invention;

[0036] Figure 2 This is the H-NMR structural characterization diagram of the hydrophilic CEST block polymer contrast agent prepared in Example 3 of the present invention;

[0037] Figure 3 This is a CEST imaging signal diagram of the hydrophilic CEST block polymer contrast agent prepared in Example 3 of the present invention under different pH conditions;

[0038] Figure 4This is a CEST imaging signal diagram of the drug-loaded CEST nanoparticle contrast agent prepared in Example 6 of the present invention at the tumor site of tumor-bearing mice. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0040] Example 1

[0041] The novel CEST polymer contrast agent is prepared, and the specific steps include the following:

[0042] (1) Preparation of salicylic acid derivative monomers:

[0043] 3 g of 4-aminosalicylic acid was dissolved in 20 mL of tetrahydrofuran, 50 μL of catalyst dibutyltin dilaurate was added, 2.75 g of methyl methacrylate isocyanate was added dropwise with stirring at 0°C in an ice bath, and the reaction was continued with stirring at room temperature for 5 h. The reactant was purified by silica gel column to remove impurities, and a salicylic acid derivative monomer was obtained with a yield of 85%. The specific reaction equation is as follows:

[0044]

[0045] (2) Preparation of polysalicylic acid homopolymer:

[0046] 0.5 g of salicylic acid derivative monomer prepared in step (1), 0.53 mg of free radical initiator azobisisobutyl and 6.5 mg of RAFT chain transfer agent 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid were dissolved in 2 mL of N,N-dimethylformamide, argon was introduced into the system for 30 min to remove oxygen, and the system was placed in an oil bath for polymerization at 70° C. for 24 h. The homopolymer obtained by the reaction was dialyzed and freeze-dried to obtain a CEST polymer contrast agent with a yield of 89%.

[0047] Example 2

[0048] The novel CEST polymer contrast agent is prepared, and the specific steps include the following:

[0049] (1) Preparation of salicylic acid derivative monomers:

[0050] 3 g of 4-aminosalicylic acid was dissolved in 20 mL of tetrahydrofuran, 4 mL of catalyst triethylamine was added, and 2.04 g of methacryloyl chloride was added dropwise with stirring at 0°C in an ice bath. After the addition, the reaction was continued under stirring at room temperature for 5 h. The reactant was purified by a silica gel column to remove impurities, and a salicylic acid derivative monomer was obtained with a yield of 80%. The reaction equation is as follows:

[0051]

[0052] (2) Preparation of polysalicylic acid homopolymer:

[0053] 0.5 g of salicylic acid derivative monomer prepared in step (1), 0.53 mg of free radical initiator azobisisobutyl and 3.25 mg of RAFT chain transfer agent 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid were dissolved in 2 mL of N,N-dimethylformamide, argon was introduced into the system for 30 min to remove oxygen, and the system was placed in an oil bath for polymerization at 70° C. for 24 h. The homopolymer obtained by the reaction was dialyzed and freeze-dried to obtain a CEST polymer contrast agent with a yield of 82%.

[0054] Example 3

[0055] The preparation of a hydrophilic CEST block polymer contrast agent comprises the following specific steps:

[0056] (1) Preparation of salicylic acid derivative monomers:

[0057] 3 g of 4-aminosalicylic acid was dissolved in 20 mL of tetrahydrofuran, 50 μL of catalyst dibutyltin dilaurate was added, 2.75 g of methyl methacrylate isocyanate was added dropwise with stirring at 0°C in an ice bath, and the reaction was continued with stirring at room temperature for 5 h. The reactant was purified using a silica gel column to remove impurities, and a salicylic acid derivative monomer was obtained with a yield of 85%.

[0058] (2) Preparation of hydrophilic block copolymers:

[0059] 0.5 g of salicylic acid derivative monomer prepared in step (1), 0.8 mg of free radical initiator azobisisobutyl and 0.038 g of hydrophilic polymer macromolecular chain transfer agent mPEG-CDTPA were dissolved in 4 mL of N,N-dimethylformamide, argon was introduced into the system for 30 min to remove oxygen, and the system was placed in an oil bath for polymerization at 70° C. for 24 h. The block copolymer obtained by the reaction was dialyzed and freeze-dried to obtain a hydrophilic block polymer contrast agent (mPEG-b-PSA) with a yield of 78%.

[0060] Example 4

[0061] The preparation of the amphiphilic CEST block polymer contrast agent comprises the following specific steps:

[0062] (1) Preparation of salicylic acid derivative monomers:

[0063] 3 g of 4-aminosalicylic acid was dissolved in 20 mL of tetrahydrofuran, 50 μL of catalyst dibutyltin dilaurate was added, 2.75 g of methyl methacrylate isocyanate was added dropwise with stirring at 0°C in an ice bath, and the reaction was continued with stirring at room temperature for 5 h. The reactant was purified using a silica gel column to remove impurities, and a salicylic acid derivative monomer was obtained with a yield of 85%.

[0064] (2) Preparation of amphiphilic block copolymers:

[0065] 0.5 g of salicylic acid derivative monomer prepared in step (1), 0.8 mg of free radical initiator azobisisobutyl and 0.038 g of polycaprolactone-CDTPA chain transfer agent were dissolved in 4 mL of N,N-dimethylformamide, argon was introduced into the system for 30 min to remove oxygen, and the system was placed in an oil bath for polymerization at 70° C. for 24 h. The block copolymer obtained by the reaction was dialyzed and freeze-dried to obtain an amphiphilic block polymer contrast agent (PCL-b-PSA) with a yield of 90%.

[0066] Example 5

[0067] The novel CEST nanoparticle polymer contrast agent is prepared, and the specific steps include the following:

[0068] 5 mg PCL-b-PSA prepared in Example 4 was dissolved in 1 mL N,N-dimethylformamide under stirring, and then dropped into 3 mL water under stirring. After dropping, the mixture was stirred for 30 min, placed in a dialysis bag, and dialyzed overnight to remove dimethylformamide. The CEST nanoparticle polymer contrast agent was collected with a yield of 98%. The particle size of the prepared nanoparticles was 50 nm-150 nm.

[0069] Example 6

[0070] The preparation of drug-loaded CEST nanoparticle polymer contrast agent includes the following specific steps:

[0071] 5 mg PCL-b-PSA prepared in Example 4 and 1 mg hydrophobic drug paclitaxel were dissolved in 1 mL N,N-dimethylformamide, and dropped into 3 mL water under stirring. After dropping, the mixture was stirred for 30 min, placed in a dialysis bag, and dialyzed overnight to remove N,N-dimethylformamide. The drug-loaded CEST nanoparticle polymer contrast agent with integrated diagnosis and treatment was collected, with a yield of 98%. The particle size of the drug-loaded nanomicelles was 50 nm-150 nm, and the drug loading was 5%-9%.

[0072] Example 7

[0073] The preparation of the functional CEST polymer contrast agent comprises the following specific steps:

[0074] (1) Preparation of salicylic acid derivative monomers:

[0075] 3 g of 4-aminosalicylic acid was dissolved in 20 mL of tetrahydrofuran, 50 μL of catalyst dibutyltin dilaurate was added, 2.75 g of methyl methacrylate isocyanate was added dropwise with stirring at 0°C in an ice bath, and the reaction was continued with stirring at room temperature for 5 h. The reactant was purified using a silica gel column to remove impurities, and a salicylic acid derivative monomer was obtained with a yield of 85%.

[0076] (2) Preparation of post-modified polysalicylic acid:

[0077] 0.5 g of salicylic acid derivative monomer prepared in step (1), 0.5 g of glycidyl methacrylate, 0.8 mg of free radical initiator azobisisobutyl and 0.038 g of RAFT chain transfer agent 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid were dissolved in 4 mL of N,N-dimethylformamide, argon was introduced into the system for 30 min to remove oxygen, and the system was placed in an oil bath for polymerization at 70° C. for 24 h. The homopolymer obtained by the reaction was dialyzed and freeze-dried to obtain a CEST polymer contrast agent (P(SA-co-GMA)), with a yield of 80%.

[0078] (3) Post-modification of polymers:

[0079] 1 g of P(SA-co-GMA) prepared in step (2) was dissolved in 5 mL of N,N-dimethylformamide, 10 mg of 6-aminofluorescein with an amino molecule was added, the mixture was reacted at room temperature overnight, the mixture was dialyzed in a dialysis bag, and freeze-dried to obtain a functional CEST polymer contrast agent, which can be used for CEST and fluorescence imaging.

[0080] Example 8

[0081] The preparation of CEST / CT dual-mode imaging nanoparticle polymer contrast agent includes the following specific steps:

[0082] (1) Preparation of salicylic acid derivative monomers:

[0083] 3 g of 4-aminosalicylic acid was dissolved in 20 mL of tetrahydrofuran, 50 μL of catalyst dibutyltin dilaurate was added, 2.75 g of methyl methacrylate isocyanate was added dropwise with stirring at 0°C in an ice bath, and the reaction was continued with stirring at room temperature for 5 h. The reactant was purified using a silica gel column to remove impurities, and a salicylic acid derivative monomer was obtained with a yield of 85%.

[0084] (2) Preparation of amphiphilic block polymers:

[0085] 0.5 g of salicylic acid derivative monomer prepared in step (1), 0.8 mg of free radical initiator azobisisobutyl and 0.038 g of PTIMA-CDTPA chain transfer agent (poly methacrylate 2-(2', 3', 5'-triiodobenzoyl)ethyl ester chain transfer agent) were dissolved in 4 mL of N,N-dimethylformamide, argon was introduced into the system for 30 min to remove oxygen, and the system was placed in an oil bath for polymerization at 70° C. for 24 h. The block copolymer obtained by the reaction was dialyzed and freeze-dried to obtain a CT and CEST dual-mode imaging polymer contrast agent (PTIMA-b-PSA), with a yield of 90%.

[0086] (3) CEST / CT dual-mode imaging nanoparticle polymer contrast agent:

[0087] 0.5 g of PTIMA-b-PSA prepared in step (2) was dissolved in 1 mL of N,N-dimethylformamide, and dropped into 3 mL of water under stirring. After dropping, the mixture was stirred for 30 min, placed in a dialysis bag, and dialyzed overnight to remove the dimethylformamide. The CEST / CT dual-mode imaging nanoparticle polymer contrast agent was collected, with a yield of 98% and a particle size of 50 nm-150 nm.

[0088] Example 9

[0089] The preparation of the functional CEST polymer contrast agent comprises the following specific steps:

[0090] (1) Preparation of salicylic acid derivative monomers:

[0091] 3 g of 4-aminosalicylic acid was dissolved in 20 mL of tetrahydrofuran, 50 μL of catalyst dibutyltin dilaurate was added, 2.75 g of methyl methacrylate isocyanate was added dropwise with stirring at 0°C in an ice bath, and the reaction was continued with stirring at room temperature for 5 h. The reactant was purified using a silica gel column to remove impurities, and a salicylic acid derivative monomer was obtained with a yield of 85%.

[0092] (2) Preparation of functional block copolymers:

[0093] 0.5 g of salicylic acid derivative monomer prepared in step (1), 0.1 g of camptothecin methacrylate monomer, 0.8 mg of free radical initiator diisobutyl azo and 0.038 g of mPEG-CDTPA chain transfer agent were dissolved in 4 mL of N,N-dimethylformamide, argon was introduced into the system for 30 min to remove oxygen, and the system was placed in an oil bath for polymerization at 70° C. for 24 h. The functional block copolymer obtained by the reaction was dialyzed to obtain a functional CEST copolymer contrast agent (mPEG-bP(SA-co-CPT), with a yield of 80%.

[0094] The performance of the CEST polymer contrast agent prepared in the examples of the present invention was tested below.

[0095] Test Example 1

[0096] Synthesis and determination of monomers of salicylic acid derivatives

[0097] Testing method: The salicylic acid derivative monomer prepared in the embodiment of the present invention is subjected to nuclear magnetic resonance mass spectrometry analysis to obtain its H-NMR structural characterization diagram.

[0098] Depend on Figure 1 The conclusions of the H-NMR analysis of the salicylic acid derivative monomers are as follows: Figure 1 It can be seen that the characteristic chemical shift peaks of methacrylate and salicylic acid are present, the peak at about 1.9 ppm is attributed to the methyl hydrogen of methacrylate, and the peaks at 6.5, 6.8, and 7.2 ppm are attributed to the hydrogen of the benzene ring of salicylic acid. It can be seen that the present invention successfully prepares a salicylic acid derivative monomer having an active free group by modifying a salicylic acid derivative.

[0099] Test Example 2

[0100] Performance determination of CEST block polymer contrast agent

[0101] Testing method: The hydrophilic CEST block polymer contrast agent prepared in Example 3 of the present invention was subjected to nuclear magnetic resonance mass spectrometry analysis to obtain its H-NMR structural characterization diagram, and its CEST imaging signal under different pH conditions was measured.

[0102] Depend on Figure 2 The H-NMR graph shows that the characteristic chemical shift peaks of mPEG and salicylic acid monomers are present. The peak at the chemical shift of 3.5 ppm is attributed to the methylene group of mPEG, while the peak at the chemical shift of 6.5-8.0 ppm is attributed to the benzene ring hydrogen of salicylic acid, indicating the successful preparation of the hydrophilic CEST block polymer contrast agent. Figure 3 It can be seen that under different environmental pH conditions, the hydrophilic block polymer contrast agent (mPEG-b-PSA) prepared by the present invention has CEST imaging performance and has a significant CEST signal at a chemical shift of about 9 ppm, which indicates that by adopting the method of the present invention, the hydroxyl group and the carboxyl group at the ortho position of the modified salicylic acid derivative monomer are not destroyed during the preparation process, so the prepared CEST polymer contrast agent retains the CEST imaging characteristics of the salicylic acid derivative. The generated polymer contrast agent has CEST imaging capability and can be used as a macromolecular exogenous contrast agent. Its macromolecular structure is not easily metabolized, and it has high accumulation efficiency and long accumulation time at the lesion site.

[0103] Test Example 3

[0104] Performance test of drug-loaded CEST nanoparticle contrast agent

[0105] Test method: The drug-loaded CEST nanoparticle contrast agent prepared in Example 6 of the present invention was injected into osteosarcoma-bearing mice through the tail vein, and osteosarcoma-bearing mice injected with small molecule salicylic acid were used as the control group. CEST imaging detection was performed on the tumor site of the injected mice.

[0106] Combination Figure 4 It can be seen from the CEST imaging signal that: CEST signal appeared in the mouse tumor site 4 hours after the injection of the drug-loaded CEST nanoparticle contrast agent of the present invention, and the imaging signal gradually increased with the extension of time. The CEST signal in the mouse tumor site reached the maximum at about 24 hours, while the CEST signal in the tumor site of the control group mice injected with small molecule salicylic acid gradually weakened with the extension of time, and there was almost no CEST signal at 24 hours. This result shows that the drug-loaded CEST nanoparticle contrast agent prepared by the present invention, the salicylic acid derivative monomer in the polymer molecule has significant CEST tumor imaging ability, and its macromolecular chain structure has good tumor accumulation ability, and the CEST signal increases with time.

[0107] Moreover, through the test of organ tissue sections and blood routine tests of test mice, no obvious toxicity was found in the injected drug-loaded CEST nanoparticle contrast agent, which is safe for use. In addition, the invented drug-loaded CEST nanoparticle contrast agent can significantly inhibit tumor growth through continuous administration, realize the integrated function of tumor diagnosis and treatment, and has a wide range of applications and strong practicality.

[0108] In summary, the preparation method of the novel CEST polymer contrast agent of the present invention does not destroy the hydroxyl and carboxyl groups at the ortho position of the salicylic acid derivative monomer and the polymerization process, so the prepared novel CEST polymer contrast agent retains the CEST imaging characteristics of the salicylic acid derivative. The preparation process of the method is simple, and polymer contrast agents with different molecular weights and functions can be obtained by adjusting the free radical polymerization conditions and copolymerizing with other functional molecules, thereby greatly enriching the types and uses of CEST polymer contrast agents. The CEST polymer contrast agent of the present invention has high accumulation efficiency and long accumulation time at the lesion site, and is easier to modify and functionalize than small molecule contrast agents, has a wide range of applications, and is highly practical.

[0109] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. For example, in the embodiments of the present invention, the salicylic acid derivative monomer is purified by silica gel column to remove impurities; 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid is used as RAFT chain transfer agent; mPEG-CDTPA chain transfer agent and polycaprolactone-CDTPA are used as macromolecular chain transfer agent; paclitaxel is used as hydrophobic drug; argon is used as inert gas. In practical applications, the purpose of the present invention can be achieved by using other parameters defined in the present invention or conventional replacement.

[0110] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a novel CEST polymer contrast agent, characterized in that: The following steps are involved: (1) Modifying aminosalicylic acid to prepare salicylic acid derivative monomers: The aminosalicylic acid is dissolved in an organic solvent I, and a reactant acryloyl chloride compound or an acrylic isocyanate compound is added dropwise in an ice bath, and under the action of a catalyst, the amino group in the aminosalicylic acid and the reactant undergo a coupling reaction to obtain a salicylic acid derivative monomer; the organic solvent I is tetrahydrofuran; when the reactant is an acryloyl chloride compound, the catalyst is triethylamine, and the acryloyl chloride compound is methacryloyl chloride or acryloyl chloride; when the reactant is an acrylic isocyanate compound, the catalyst is dibutyltin dilaurate; (2) Preparation of polymer: The salicylic acid derivative monomer obtained in step (1) is dissolved in an organic solvent II, and in an inert gas environment, under the action of a free radical initiator, a living free radical polymerization reaction is carried out to prepare a novel CEST polymer contrast agent; the organic solvent II is N,N-dimethylformamide; the free radical initiator is azobisisobutyl or azobisisoheptonitrile; When the polymer is (2.1) a homopolymer, the preparation method comprises: The salicylic acid derivative monomer, free radical initiator and RAFT chain transfer agent prepared in step (1) are dissolved in an organic solvent II, an inert gas is introduced into the system to remove oxygen for polymerization reaction, and the homopolymer obtained by the reaction is dialyzed and freeze-dried to obtain a polymer contrast agent; the RAFT chain transfer agent is 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid.

2. The method for preparing a novel CEST polymer contrast agent according to claim 1, characterized in that: In the coupling reaction of step (1), the molar ratio of the added amount of the salicylic acid derivative and the reactant is 1:

1.

3. The method for preparing a novel CEST polymer contrast agent according to claim 2, characterized in that: The aminosalicylic acid in step (1) is 4-aminosalicylic acid, 3-aminosalicylic acid, or 5-aminosalicylic acid.

4. The method for preparing a novel CEST polymer contrast agent according to claim 1, characterized in that: Step (2.1) Preparation of homopolymer, wherein the reaction molar ratio of the free radical initiator to the RAFT chain transfer agent is 1:0.

2.

5. The method for preparing a novel CEST polymer contrast agent according to claim 4, characterized in that: It also includes step (2.2) preparation of copolymer, When (2.2) the method for preparing the functional copolymer comprises: The salicylic acid derivative monomer, functional monomer, free radical initiator and RAFT chain transfer agent prepared in step (1) are dissolved in organic solvent II, an inert gas is introduced into the system to remove oxygen for polymerization reaction, and the copolymer obtained by the reaction is dialyzed and freeze-dried to obtain a copolymer contrast agent; The RAFT chain transfer agent is a RAFT chain transfer agent 4-cyano-4-[(dodecylsulfanylsulfanylcarbonyl)sulfanyl]pentanoic acid; The functional monomer is camptothecin methacrylate monomer or glycidyl methacrylate; The reaction molar ratio of the free radical initiator to the RAFT chain transfer agent was 1:0.

2.

6. The method for preparing a novel CEST polymer contrast agent according to claim 5, characterized in that: It also includes step (2.3) preparation of block copolymers, When (2.3) the method for preparing the block copolymer comprises: The salicylic acid derivative monomer, free radical initiator and macromolecular chain transfer agent prepared in step (1) are dissolved in organic solvent II, an inert gas is introduced into the system to remove oxygen for polymerization reaction, and the block copolymer obtained by the reaction is dialyzed and freeze-dried to obtain a block copolymer contrast agent; The macromolecular chain transfer agent is PTIMA-CDTPA chain transfer agent (polymethacrylate 2-(2', 3', 5'-triiodobenzoyl)ethyl ester chain transfer agent, mPEG-CDTPA chain transfer agent, polycaprolactone-CDTPA chain transfer agent; The reaction molar ratio of the free radical initiator to the macromolecular chain transfer agent is 1:0.

2.

7. The method for preparing a novel CEST polymer contrast agent according to claim 6, characterized in that: The preparation of CEST nanoparticle contrast agents is also included: The block copolymer prepared in step (2.3) is dissolved in organic solvent II, stirred evenly, and then dropped into water, stirred thoroughly, placed in a dialysis bag, dialyzed to remove the reaction solvent, and the CEST nanoparticle contrast agent is collected.

8. The method for preparing a novel CEST polymer contrast agent according to claim 7, characterized in that: It also includes the preparation of drug-loaded CEST nanoparticle contrast agents: The block copolymer and hydrophobic drug prepared in step (2.3) are dissolved in organic solvent II, stirred evenly, and then dropped into water, stirred thoroughly, placed in a dialysis bag, dialyzed to remove the reaction solvent, and the drug-loaded CEST nanoparticle contrast agent is collected.

9. A novel CEST polymer contrast agent, characterized in that: The novel CEST polymer contrast agent is prepared according to the preparation method of any one of claims 1 to 8.

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