A tumor contrast agent for enhancing magnetic resonance transverse relaxation signal in a weak acid environment, and a preparation method and application thereof

Abstract

The application provides a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment, a preparation method and application thereof, and belongs to the technical field of biomedical engineering; the tumor contrast agent is formed by hydrophobic interaction between a comb-shaped anionic pH-sensitive macromolecule with a phase transition pH value of 6.5-6.8 and a surface oleic acid-stabilized hydrophobic superparamagnetic nanoparticle; the tumor contrast agent has a small hydration particle size (20nm-50nm), a low transverse relaxation rate (low R2) and good hydrophilicity in a physiological environment (pH value is 7.35-7.45), can be retained in tumor tissues through the EPR effect, and can leak into the inside of tumor tissues; the tumor contrast agent can improve the contrast ratio of tumor tissues / normal tissues in magnetic resonance imaging, and further improve the accuracy of early diagnosis of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering, and particularly relates to a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment and a preparation method and application thereof. BACKGROUND

[0002] Magnetic resonance imaging is an important method for current tumor imaging diagnosis, wherein a contrast enhancement agent plays a key role in improving the contrast between tumor tissue and normal tissue, shortening the imaging time, etc. Current magnetic resonance contrast agents can be divided into two imaging modes, namely longitudinal relaxation (T1) and transverse relaxation (T2), wherein a T2 contrast enhancement agent represented by a superparamagnetic nanoparticle (SPION) has been widely applied in clinical tumor diagnosis due to its high biocompatibility and good spin-spin relaxation enhancement effect.

[0003] The imaging effect of the SPION-based T2 tumor imaging depends on three aspects: the distribution of SPION in the tumor, the transverse relaxation rate R2 (1 / T2) of SPION, and the contrast ratio of SPION in the tumor tissue / normal tissue. For example, the distribution of SPION in the tumor is subject to many influencing factors, including but not limited to particle size, composition of surface hydrophilic ligands, etc. According to the EPR effect, the high permeability of tumor blood vessels can make 20-200 nm particle size nanoparticles penetrate the tumor blood vessels and concentrate in the tumor tissue. However, clinical studies have found that the pure EPR effect can only improve the concentration of nanoparticles in the tumor blood vessels. Due to the high intratumoral interstitial pressure of the tumor tissue, the tumor tissue forms a positive pressure in the direction of the blood vessels, and only nanoparticles with a particle size of less than 50 nm can effectively penetrate the tumor blood vessels and enter the interior of the tumor tissue, and the smaller the particle size, the deeper the penetration depth of the tumor tissue.

[0004] In addition to the large blood vessel permeability, the tumor tissue also has a common feature, which is the Warburg effect, so the tumor tissue has a lower pH value (pH=6.5-6.8) than the normal tissue and blood (pH=7.35-7.45). There have been some reports on pH-sensitive polymers, but most of them are cationic pH-sensitive polymers, and the common anionic pH-sensitive polymers generally have a response pH value lower than 6.0, which is not suitable for responding to the microenvironment of the tumor.

[0005] The transverse relaxation rate R2 (1 / T2) of the SPION is also affected by multiple factors, such as the particle size of the SPION, the shape of the SPION, the crystallinity, the element composition, and the size of the SPION cluster. Although the SPION cluster has a higher transverse relaxation rate R2, the higher R2 of the contrast agent is always on from the injection into the body to the metabolism out of the body, so the T2 imaging of the tumor tissue by the SPION is also affected by the contrast ratio of the SPION in the normal tissue / tumor tissue. The current T2 contrast enhancer still depends on the accumulation in the tumor tissue, and does not have the ability to improve the contrast in the tumor tissue.

[0006] Therefore, it is necessary to develop a new tumor contrast agent capable of enhancing the magnetic resonance transverse relaxation signal in a weak acid environment. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides a tumor contrast agent capable of enhancing the magnetic resonance transverse relaxation signal in a weak acid environment, and a preparation method and application thereof. The tumor contrast agent is formed by hydrophobic interaction between a comb-shaped anionic pH-sensitive macromolecule with a phase transition pH value of 6.5-6.8 and hydrophobic superparamagnetic nanoparticles stabilized by oleic acid on the surface. The tumor contrast agent has a small hydration particle size (20-50 nm), a low transverse relaxation rate (low R2), and good hydrophilicity in a physiological environment (pH value of 7.35-7.45), can be retained in the tumor tissue through the EPR effect, and can leak into the interior of the tumor tissue. The tumor contrast agent can improve the contrast ratio of the tumor tissue / normal tissue in magnetic resonance imaging, and thus improve the accuracy of early diagnosis of tumors.

[0008] In order to achieve the above technical purposes, the present application provides the following technical means:

[0009] The present application first provides a tumor contrast agent capable of enhancing the magnetic resonance transverse relaxation signal in a weak acid environment. The tumor contrast agent uses a comb-shaped anionic pH-sensitive macromolecule as a carrier, and encapsulates hydrophobic superparamagnetic nanoparticles stabilized by oleic acid on the surface in the interior. The comb structure on the comb-shaped anionic pH-sensitive macromolecule is 1-octadecene, and the 1-octadecene and the oleic acid on the surface of the superparamagnetic nanoparticles are interpenetrated.

[0010] Preferably, the phase transition pH value of the comb-shaped anionic pH-sensitive macromolecule is 6.5-6.8.

[0011] Preferably, the hydration particle size of the tumor contrast agent is 20-50 nm when the pH value is 7.35-7.45, and the particle size increases to 200-5000 nm in a weak acid environment with a pH value of 6.5-6.8.

[0012] The application also provides a preparation method of the tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment, comprising the following steps:

[0013] (1) Preparation of the linear structure pH-sensitive macromolecule:

[0014] The reaction reagents are polymerized by reversible addition fragmentation chain transfer (RAFT) in a solvent to obtain a linear structure pH-sensitive macromolecule with a dissociation constant (pK a ) of 6.0-6.2, using 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol as a chain transfer agent and 4,4'-azobis(cyanovaleric acid) as an initiator.

[0015] The reaction reagents include:

[0016] (a) any one of pH-sensitive monomers of sulfacetamide acrylamide (SCAA), sulfabenzamide acrylamide (SBAA) and sulfamethizole acrylamide (SMAA) and a regulating monomer N,N-dimethyl acrylamide (DMAM); or

[0017] (b) a pH-sensitive monomer of sulfadoxine acrylamide (SDAA);

[0018] (2) Preparation of the comb-shaped anionic pH-sensitive macromolecule:

[0019] The linear structure pH-sensitive macromolecule and poly(maleic anhydride-alt-1-octadecene) are reacted to obtain the comb-shaped anionic pH-sensitive macromolecule after the reaction.

[0020] (3) Preparation of the tumor contrast agent:

[0021] The comb-shaped anionic pH-sensitive macromolecule is dispersed in an organic solvent A to obtain a dispersion A; and hydrophobic superparamagnetic nanoparticles stabilized by oleic acid on the surface are dispersed in an organic solvent B to obtain a dispersion B.

[0022] The dispersion A and the dispersion B are uniformly mixed to obtain a mixed solution C, then the mixed solution C is heated to the boiling point of the organic solvent B to volatilize the organic solvent B, and then heated to the boiling point of the organic solvent A to perform a reflux reaction, and the reaction is cooled to room temperature after the reaction, and the organic solvent A is removed by dialysis with a phosphate buffer with a pH value of 7.35-7.45, to obtain a pH-sensitive superparamagnetic nanomicelle, i.e., the tumor contrast agent.

[0023] The organic solution A is miscible with water and organic solvent B, and has a higher boiling point than the organic solvent B; the organic solvent B is immiscible with water.

[0024] Preferably, in the step (1), the solvent is dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF);

[0025] When the reactant is (a), the molar ratio of the pH-sensitive monomer and the regulating monomer is 1:5-5:1;

[0026] The molar ratio of the 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol, 4,4'-azobis(cyanovaleric acid) and the reactant is 1:0.02:20-1:0.1:100;

[0027] The reversible addition-fragmentation chain transfer polymerization is carried out at 50-80℃ for 2-10h;

[0028] The linear pH-sensitive macromolecule has a molecular weight of 3000-5000.

[0029] Preferably, in the step (2), the molar ratio of the linear pH-sensitive macromolecule and poly(maleic anhydride-alt-1-octadecene) is 15-50:1;

[0030] The phase transition pH value of the comb-like anionic pH-sensitive macromolecule is 6.5-6.8.

[0031] Preferably, in the step (3), the surface oleic acid-stabilized hydrophobic superparamagnetic nanoparticle is prepared by the high-temperature precursor decomposition method disclosed in the prior art, and the preparation method comprises:

[0032] Dibenzyl ether is used as the solvent, and iron tris(acetylacetonate) Fe(acac)3, 1,2-hexadecanediol, oleic acid and oleylamine are added in a molar ratio of 1:5:3:3, and then stirred uniformly under the protection of argon, heated to 200℃ and kept for 2h, continuously heated to 300℃, and refluxed at the temperature for 1h, and then cooled to room temperature, and the product is blended with anhydrous ethanol, precipitated and centrifuged three times, and then vacuum dried at room temperature, dispersed in n-hexane and stored at -20℃.

[0033] The organic solvent A comprises dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF); and the organic solvent B comprises methylcyclohexane or toluene;

[0034] The organic solvent A and the organic solvent B are mixed in a volume ratio of 2-100:1 to obtain the mixed solution C.

[0035] The mass ratio of the comb-shaped anionic pH-sensitive macromolecule to the superparamagnetic nanoparticles in the mixed solution C is 20-100:1.

[0036] The reflux reaction time is 0.5-3h.

[0037] Preferably, the preparation steps of the tumor contrast agent further comprise purification of the tumor contrast agent, and the steps are as follows:

[0038] The supernatant is removed by centrifugation of the dialysate in step (3) for the first time, and then the supernatant is removed by centrifugation for the second time at a higher speed, and the precipitate is dispersed in a phosphate buffer solution to obtain the purified tumor contrast agent.

[0039] Preferably, the first centrifugation is performed at a speed of 10,000 rpm for 10-30 minutes at 4°C.

[0040] The second centrifugation is performed at a speed of 20,000-50,000 rpm for 30-60 minutes at 4°C.

[0041] The pH value of the phosphate buffer solution is 7.35-7.45.

[0042] The application also provides application of the tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment to tumor imaging.

[0043] Compared with the prior art, the application has the following beneficial effects:

[0044] The existing tumor magnetic resonance T2 contrast enhancer is a nano magnetic fluid with SPION as the core, can be accumulated in tumor tissues through EPR effect, but generally has a large particle size (>50 nm) and cannot effectively penetrate tumor tissues, and does not have tumor weak acid environment response capability, and cannot specifically enhance the T2 imaging contrast of tumor tissues. Therefore, the existing tumor magnetic resonance T2 contrast enhancer can only rely on increasing the content of SPION in tumor tissues to enhance the MRI effect of tumors.

[0045] Compared with the prior art, the tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid (pH=6.5-6.8) environment can improve the MRI effect of tumors from the following three aspects.

[0046] ①Due to the low agglomeration or no agglomeration of SPION, a lower transverse relaxation rate (low R2) can be presented. Compared with the preparation of large particle size SPION agglomerates by ultrasonic assisted dispersion in the prior art, the comb-shaped anionic pH-sensitive macromolecules are first prepared in the present application. Due to the comb-shaped structure, the oil acid on the surface of the superparamagnetic nanoparticles can be interpenetrated. At the same time, the double solvent evaporation reflux method is used in the present application, and the high temperature (~ 180℃) reflux is used to help the oil acid on the surface of the SPION to be fully intercalated with the 1-octadecene on the comb-shaped anionic pH-sensitive macromolecule, so as to form small particle size micelles containing only a small amount of SPION, even a single SPION. The particle size of the prepared tumor contrast agent for enhancing magnetic resonance transverse relaxation signal in weak acid (pH = 6.5-6.8) environment under physiological conditions is 20-50 nm. The particle size is not only suitable for the particle size of EPR effect, but also helps the penetration of the contrast agent in the tumor tissue.

[0047] ②The tumor contrast agent for enhancing magnetic resonance transverse relaxation signal in weak acid (pH = 6.5-6.8) environment prepared by the present application has high colloidal stability in physiological environment (pH = 7.35-7.45), and can be kept in the range of 20-50 nm for a long time. According to the report in the literature, the R2 value of 4 nm SPION is 25.1 mM -1 -1 The R2 value of the agglomerate with larger particle size of 16 nm SPION is 471 mM -1 -1 Compared with the single particle state of the developer, the R2 is greatly improved, and the former is 18.7 times the latter, greatly improving the development sensitivity of T2.

[0048] The tumor contrast agent for enhancing magnetic resonance transverse relaxation signal in weak acid (pH = 6.5-6.8) environment described in the present patent keeps the pH-sensitive superparamagnetic nanomicelles with particle size of 20-50 nm after injection in vivo, and the pH-sensitive superparamagnetic nanomicelles keep a low transverse relaxation rate (low R2) in blood (physiological environment).

[0049] Moreover, the tumor contrast agent for enhancing magnetic resonance transverse relaxation signal in weak acid (pH = 6.5-6.8) environment described in the present application can respond to the weak acid environment of the tumor tissue after reaching the tumor tissue, and quickly agglomerate to improve the transverse relaxation rate R2, so that the MRI contrast of the tumor is specifically enhanced.

[0050] ​​The particle size of the tumor contrast agent for enhancing magnetic resonance transverse relaxation signal in the weak acid (pH = 6.5-6.8) environment is 20-50 nm under physiological conditions, and is 200-5000 nm in tumor tissues. Therefore, the tumor contrast agent has only a low transverse relaxation rate R2 under physiological conditions, and has a transverse relaxation rate R2 in tumor tissues, which can further improve the contrast ratio of tumor tissues / normal tissues in magnetic resonance imaging, and improve the accuracy of early diagnosis of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 Schematic diagram of the tumor contrast agent for enhancing magnetic resonance transverse relaxation signal in response to the weak acid environment.

[0052] Figure 2 UV-vis transmittance curve of p(SCAA-co-DMAM) under different pH conditions.

[0053] Figure 3 UV-vis transmittance curve of the comb-shaped anionic pH-sensitive macromolecule with a phase transition pH value of 6.52 in different pH environments.

[0054] Figure 4 Particle size change diagram of the pH-sensitive superparamagnetic nanomicelles of Example 1 in different pH environments.

[0055] Figure 5 UV-vis transmittance curve of p(SBAA-co-DMAM) under different pH conditions.

[0056] Figure 6 UV-vis transmittance curve of the comb-shaped anionic pH-sensitive macromolecule with a phase transition pH value of 6.62 in different pH environments.

[0057] Figure 7 Particle size change diagram of the pH-sensitive superparamagnetic nanomicelles of Example 2 in different pH environments.

[0058] Figure 8 UV-vis transmittance curve of p(SMAA-co-DMAM) under different pH conditions.

[0059] Figure 9 UV-vis transmittance curve of the comb-shaped anionic pH-sensitive macromolecule with a phase transition pH value of 6.71 in different pH environments.

[0060] Figure 10 Particle size change diagram of the pH-sensitive superparamagnetic nanomicelles of Example 3 in different pH environments.

[0061] Figure 11Figure 6. UV-vis transmittance curves of pSDAA at different pH values.

[0062] Figure 12 Figure 7. UV-vis transmittance curves of comb-like anionic pH-sensitive macromolecule with phase transition pH value of 6.65 in different pH environments.

[0063] Figure 13 Figure 8. Particle size change of pH-sensitive superparamagnetic nanomicelles of Example 4 in different pH environments.

[0064] Figure 14 Figure 9. T2 map (A) and fitting curve (B) of pH-sensitive superparamagnetic nanomicelles of Example 4 in different pH environments.

[0065] Figure 15 Figure 10. Contrast-enhanced effect of pH-sensitive superparamagnetic nanomicelles of Example 4 in 4T1 tumor model. DETAILED DESCRIPTION

[0066] The present application is further described in conjunction with the accompanying drawings and specific examples, but the scope of the present application is not limited thereto. In the following examples, the specific conditions are not specified, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified, and are conventional products that can be purchased on the market. Unless otherwise specified, the present application uses the existing technology in the field.

[0067] Example 1: Preparation of a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in weak acid environment

[0068] (1) Preparation of pH-sensitive macromolecules with linear structure:

[0069] 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol: The molar ratio of sulfacetamide acrylamide is 1:100. In this reaction, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol is used as a chain transfer agent, sulfacetamide acrylamide (SCAA) is used as a pH-sensitive monomer, and N,N-dimethylacrylamide (DMAM) is used as a regulating monomer to prepare linear pH-sensitive macromolecule P(SCAA-co-DMAM). The specific steps are as follows:

[0070] According to the molar ratio of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol: sulfacetamide acrylamide (SCAA) 1:100, sulfacetamide acrylamide (SCAA): N, N-dimethyl acrylamide (DMAM) 1:5, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol (1 mmol), sulfacetamide acrylamide (SCAA, 100 mmol), N, N-dimethyl acrylamide (DMAM, 500 mmol) were added in dimethyl sulfoxide as the reaction medium, vacuum / argon replacement was carried out for 3 times, and then the reaction was carried out at 60°C for 2 hours under argon protection to obtain a linear structure pH-sensitive macromolecule with a molecular weight of 5000 and a dissociation constant (pK a ) of 6.16, which is denoted as P(SCAA-co-DMAM).

[0071] The UV-vis transmittance curve of the linear structure pH-sensitive macromolecule in different pH environments is shown in Figure 2 From the figure, it can be seen that the pH-sensitive polymer maintains a transmittance close to 100% at pH 7.4; when the pH is adjusted to the phase transition point of the linear structure pH-sensitive macromolecule, the transmittance is close to 50%, that is, when the pH is about 6.16, the polymer undergoes a change in hydrophilic-hydrophobic form, that is, the transmittance decreases obviously and presents a rapid mutation in a cliff-like manner. The above results can show that the linear structure pH-sensitive macromolecule has the ability of environmental response, and the linear structure pH-sensitive macromolecule with a molecular weight of 5000.

[0072] (2) Preparation of a comb-shaped anionic pH-sensitive macromolecule:

[0073] Dimethyl sulfoxide (DMSO) was used as the solvent, p(SCAA-co-DMAM) with a pK a of 6.16 and poly(maleic anhydride-alt-1-octadecene) were added according to a molar ratio of 15:1, stirring was carried out at room temperature for 48 hours, and then dialysis was carried out to obtain a comb-shaped anionic pH-sensitive macromolecule.

[0074] The UV-vis transmittance of the comb-shaped anionic pH-sensitive macromolecule in different pH environments was investigated to evaluate the phase transition pH, and the investigation results are shown in Figure 3 At pH 7.4, the comb-shaped anionic pH-sensitive macromolecule maintains a transmittance close to 100%; when the pH is adjusted to the phase transition point of the comb-shaped anionic pH-sensitive macromolecule, the transmittance is close to 50%, that is, when the pH is about 6.52, the polymer undergoes a change in hydrophilic-hydrophobic form, that is, the transmittance decreases obviously and presents a rapid mutation in a cliff-like manner. The above results can show that the comb-shaped anionic pH-sensitive macromolecule has the ability of environmental response. (3) Preparation of a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment:

[0075] The stepwise high temperature precursor decomposition method was adopted, and Fe(acac)3, 1,2-hexadecyl glycol, oleic acid, and oleylamine were added in a molar ratio of 1:5:3:3, and then dibenzyl ether was added as a solvent, and the mixture was stirred uniformly under the protection of argon. The reaction environment was replaced with an oxygen-free environment, the mixture was heated to 200°C and kept for 2 hours, and then the temperature was continuously increased to 300°C, and the reaction was refluxed at this temperature for 1 hour. After the reaction was completed, the reaction system was cooled to room temperature. The product was blended with anhydrous ethanol, precipitated and centrifuged three times to obtain the product, which was vacuum dried at room temperature to obtain superparamagnetic Fe3O4 nanoparticles with a particle size of 18 nm, which were dispersed in n-hexane and stored at -20°C. Transmission electron microscopy and dynamic light scattering determination showed that the superparamagnetic Fe3O4 nanoparticles had a particle size of 18 nm, were spherical, had monodispersity and superparamagnetism, and had a saturation magnetization of 98 emu / g.

[0076] Then, the comb-shaped anionic pH-sensitive macromolecule with a phase transition pH of 6.52 was lyophilized, and 1 g was dispersed in 10 mL of DMSO to obtain dispersion A; 50 mg of the superparamagnetic Fe3O4 nanoparticles with a particle size of 18 nm was dried under argon and dispersed in 5 mL of toluene to obtain dispersion B.

[0077] The dispersion A and the dispersion B were mixed, and a mixed solution C was obtained by mechanical stirring at room temperature for 1 hour. Then, the mixed solution C was heated to the boiling point of toluene (110°C) under normal pressure for 10 minutes to volatilize the toluene. Subsequently, the mixed solution C was continuously heated to the boiling point of DMSO (189°C) under normal pressure to sufficiently reflux for 30 minutes. After the reflux was completed, the mixed solution C was dialyzed under a reflux state using a dialysis bag with a molecular weight cut-off of 50,000, and the dialysis liquid was a phosphate buffer with a pH of 7.35-7.45. The dialysis was performed for 24 hours to remove the DMSO, and a pH-sensitive superparamagnetic nanomicelle was obtained, which was a tumor contrast agent that specifically enhanced the magnetic resonance transverse relaxation signal in an acidic (pH = 6.5-6.8) environment, and a schematic diagram thereof is shown in Figure 1

[0078] (4) Purification of the tumor contrast agent:

[0079] The dialyzed solution obtained in step (3) was centrifuged at a speed of 10,000 rpm at 4°C for 10 minutes to obtain the supernatant, and then centrifuged at a speed of 21,000 rpm at 4°C for 30 minutes to remove the supernatant. The precipitate was dispersed in a phosphate buffer with a pH of 7.35-7.45 to obtain a purified tumor contrast agent that specifically enhanced the magnetic resonance transverse relaxation signal in an acidic (pH = 6.5-6.8) environment.

[0080] ​The raw pH value of the tumor contrast agent obtained above was measured by a pH meter, and then the pH was adjusted by 0.1M dilute hydrochloric acid or sodium hydroxide to obtain a nanomicelle solution with a physiological pH of about 7.4 and a phase transition point of about 6.5. The particle size of the micelle solution at two pH values was determined by a high-sensitivity particle size analyzer, as shown in Figure 4 .

[0081] Figure 4 The particle size change diagram of the pH-sensitive superparamagnetic nanomicelle of Example 1 in different pH environments can be seen from the figure. When the contrast agent is in a physiological environment (pH 7.4), it presents a uniform state and has a small particle size of 31 nm and a PDI of 0.105. When the environmental conditions present a phase transition point of about 6.5, the tumor weak acid-responsive nanomagnetic fluid exhibits its high sensitivity to the environment, and deformation and aggregation occur, and the particle size also increases significantly to 256.6 nm with a PDI of 0.169. The sudden change in particle size under different pH conditions can prove that the contrast agent synthesized in the experimental process has the ability to respond to the tumor weak acid microenvironment.

[0082] Example 2: Preparation of a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment

[0083] (1) Preparation of a linear structure pH-sensitive macromolecule:

[0084] 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol: The molar ratio of benzoyl sulfanilamide acrylamide is 1:50. In this reaction, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol is used as a chain transfer agent, benzoyl sulfanilamide acrylamide (SBAA) is used as a pH-sensitive monomer, and N,N-dimethyl acrylamide (DMAM) is used as a regulating monomer to prepare a linear pH-sensitive macromolecule P(SBAA-co-DMAM). The specific steps are as follows:

[0085] According to the molar ratio of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol: benzoyl sulfanilamide (SBAA) 1:50, sulfanilamide acrylamide (SBAA): N,N-dimethyl acrylamide (DMAM) 1:2, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol (1 mmol), benzoyl sulfanilamide (SBAA, 50 mmol), and N,N-dimethyl acrylamide (DMAM, 100 mmol) were added in dimethyl sulfoxide as the reaction medium. After vacuum / argon replacement for 3 times, the reaction was carried out at 55°C for 3 hours under argon protection to obtain a linear structure pH-sensitive macromolecule with a molecular weight of 4500 and a dissociation constant (pK a ) of 6.09, denoted as P(SBAA-co-DMAM).

[0086] The UV-vis transmittance curves of the linear structured pH-sensitive macromolecule under different pH environments are shown below. Figure 5 As shown in the figure, under pH 7.4 conditions, the linear pH-sensitive macromolecule maintains a permeability close to 100%; when the pH is adjusted to the phase transition point of the linear pH-sensitive macromolecule, the permeability is close to 50%, that is, when the pH is around 6.09, the polymer undergoes a change in hydrophilic and hydrophobic morphology, that is, the permeability decreases significantly and exhibits a rapid and abrupt change. The above results can be used to show that the comb-shaped anionic pH-sensitive macromolecule has the ability to respond to the environment, and a linear pH-sensitive macromolecule with a molecular weight of 4500 is obtained. (2) Preparation of comb-shaped anionic pH-sensitive macromolecule:

[0087] Using DMSO as a solvent, pK a (SBAA-co-DMAM) with a p-value of 6.09 and poly(maleic anhydride-alt-1-octadecene) were fed at a molar ratio of 20:1, stirred at room temperature for 48 hours, and then dialyzed to obtain a comb-shaped anionic pH-sensitive macromolecule.

[0088] The phase transition pH of comb-shaped anionic pH-sensitive macromolecules was evaluated by examining their UV-vis transmittance under different pH environments. The results are as follows: Figure 6 As shown. From Figure 6 As can be seen, under pH 7.4 conditions, the comb-shaped anionic pH-sensitive macromolecule maintains a permeability close to 100%. When the pH is adjusted to the phase transition point of the comb-shaped anionic pH-sensitive macromolecule, the permeability approaches 50%, that is, at pH around 6.62, the polymer undergoes a change in hydrophilic / hydrophobic morphology, i.e., the permeability decreases significantly and exhibits a rapid, abrupt drop. These results demonstrate that the comb-shaped anionic pH-sensitive macromolecule possesses environmental responsiveness.

[0089] (3) Preparation of tumor contrast agents that enhance transverse relaxation signals in a weakly acidic environment:

[0090] The stepwise high temperature precursor decomposition method is adopted, and Fe(acac)3, 1,2-hexadecyl glycol, oleic acid, and oleylamine are added in a molar ratio of 1:5:3:3, and then dibenzyl ether is added as a solvent, and stirred uniformly under the protection of argon. The reaction environment is replaced into an oxygen-free environment, the mixture is heated to 200°C for 2h, and then continuously heated to 300°C, and refluxed at the temperature for 1h. After the reaction is completed, the reaction system is cooled to room temperature. The product is blended with anhydrous ethanol, precipitated and centrifuged three times to obtain the product, and vacuum dried at room temperature to obtain superparamagnetic Fe3O4 nanoparticles with a particle size of 12nm, which are dispersed in n-hexane and stored at -20°C. The superparamagnetic Fe3O4 nanoparticles have a particle size of about 12nm, are spherical, have monodispersity and superparamagnetism, and have a saturation magnetization of 59emu / g, as determined by transmission electron microscopy and dynamic light scattering.

[0091] Then, the comb-shaped anionic pH-sensitive macromolecule with a phase transition pH value of 6.62 is lyophilized, and 10g is dispersed in 500mL of DMSO to obtain dispersion A; 100mg of the superparamagnetic Fe3O4 nanoparticles with a particle size of 12nm is dispersed in 5mL of toluene to obtain dispersion B.

[0092] The dispersion A and the dispersion B are mixed, and a mixed solution C is obtained by mechanical stirring at room temperature for 2h. Then, the mixed solution C is heated to the boiling point of toluene (110°C) under normal pressure for 15min to volatilize the toluene. Subsequently, the mixed solution C is continuously heated to the boiling point of DMF (153°C) under normal pressure to sufficiently reflux for 3h. After the reflux is completed, the mixed solution C is dialyzed under a reflux state by using a dialysis bag with a molecular weight cut-off of 50000, and the dialysis liquid is a phosphate buffer with a pH value of 7.35-7.45. The dialysis is performed for 24h to remove the DMF, and a pH-sensitive superparamagnetic nanomicelle, i.e., a tumor contrast agent that specifically enhances the magnetic resonance transverse relaxation signal in an acidic (pH=6.5-6.8) environment, is obtained.

[0093] (4) Purification of the tumor contrast agent:

[0094] The dialyzed solution obtained in step (3) is centrifuged at a speed of 10000r / min at 4°C for 30min to obtain the supernatant, and then centrifuged at a speed of 25000r / min at 4°C for 60min to remove the supernatant. The precipitate is dispersed in a phosphate buffer with a pH value of 7.35-7.45 to obtain a purified tumor contrast agent that specifically enhances the magnetic resonance transverse relaxation signal in an acidic (pH=6.5-6.8) environment.

[0095] The raw pH value of the tumor contrast agent obtained above was measured by a pH meter, and then the pH was adjusted by 0.1M dilute hydrochloric acid or sodium hydroxide to obtain a nanomicelle solution with a physiological pH of about 7.4 and a phase transition point of about 6.6. The particle size of the micelle solution at two pH values was determined by a high-sensitivity particle size analyzer, as shown in Figure 7 .

[0096] Figure 7 The particle size change diagram of the pH-sensitive superparamagnetic nanomicelle of Example 2 in different pH environments shows that when the contrast agent is in a physiological environment (pH 7.4), it presents a uniform state and has a small particle size of 43 nm and a PDI of 0.158; when the environmental condition presents a phase transition point of about 6.6, the tumor weak acid-responsive nanomagnetic fluid exhibits its high sensitivity to the environment, and the particle size also significantly increases to 226.8 nm with a PDI of 0.172. The sudden change in particle size under different pH conditions can prove that the contrast agent synthesized in the experimental process has the ability to respond to the tumor weak acid microenvironment.

[0097] Example 3: Preparation of a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment

[0098] (1) Preparation of a linear structure pH-sensitive macromolecule:

[0099] 4-cyano-4-[(dodecyl sulfonamido thio carbonyl) sulfonamido] pentanol: the molar ratio of sulfadiazine acrylamide is 1:20. In this reaction, 4-cyano-4-[(dodecyl sulfonamido thio carbonyl) sulfonamido] pentanol is used as a chain transfer agent, sulfadiazine acrylamide (SMAA) is used as a pH-sensitive monomer, and N,N-dimethyl acrylamide (DMAM) is used as a regulating monomer to prepare a linear pH-sensitive macromolecule P(SMAA-co-DMAM). The specific steps are as follows:

[0100] According to the molar ratio of 4-cyano-4-[(dodecyl sulfonamido thio carbonyl) sulfonamido] pentanol to sulfadiazine acrylamide (SMAA) of 1:20 and the molar ratio of sulfadiazine acrylamide (SMAA) to N,N-dimethyl acrylamide (DMAM) of 5:1, 4-cyano-4-[(dodecyl sulfonamido thio carbonyl) sulfonamido] pentanol (1 mmol), sulfadiazine acrylamide (SMAA, 20 mmol), and N,N-dimethyl acrylamide (DMAM, 4 mmol) were added in a reaction medium of DMF. After vacuum / argon replacement for 3 times, the reaction was carried out at 80°C for 2 hours under argon protection to obtain a linear structure pH-sensitive macromolecule with a molecular weight of 3700 and a dissociation constant (pK a ) of 6.01, which is denoted as P(SMAA-co-DMAM).

[0101] The UV-vis transmittance curves of the linear structured pH-sensitive macromolecule under different pH environments are shown below. Figure 8 As shown in the figure, at pH 7.4, the linear pH-sensitive macromolecule maintains a permeability close to 100%. When the pH is adjusted to the phase transition point of the linear pH-sensitive macromolecule, the permeability approaches 50%, i.e., at around pH 6.01. At this point, the polymer undergoes a change in hydrophilic / hydrophobic morphology, resulting in a significant and rapid drop in permeability. These results demonstrate that the comb-like anionic pH-sensitive macromolecule possesses environmental responsiveness, yielding a linear pH-sensitive macromolecule with a molecular weight of 3700.

[0102] (2) Preparation of comb-shaped anionic pH-sensitive macromolecules:

[0103] Using DMSO as a solvent, pK a SDAA-co-DMAM (p=6.01) and poly(maleic anhydride-alt-1-octadecene) were fed at a molar ratio of 40:1, stirred at room temperature for 48 hours, and then dialyzed to obtain comb-shaped anionic pH-sensitive macromolecules.

[0104] The phase transition pH of comb-shaped anionic pH-sensitive macromolecules was evaluated by examining their UV-vis transmittance under different pH environments. The results are as follows: Figure 9 As shown. From Figure 9 As can be seen, under pH 7.4 conditions, the comb-shaped anionic pH-sensitive macromolecule maintains a permeability close to 100%. When the pH is adjusted to the phase transition point of the comb-shaped anionic pH-sensitive macromolecule, the permeability approaches 50%, i.e., around pH 6.71. At this point, the polymer undergoes a change in hydrophilic / hydrophobic morphology, resulting in a significant and rapid drop in permeability. These results demonstrate that the comb-shaped anionic pH-sensitive macromolecule possesses environmental responsiveness. Therefore, the phase transition pH value of the comb-shaped anionic pH-sensitive macromolecule is 6.71.

[0105] (3) Preparation of tumor contrast agents that enhance transverse relaxation signals in a weakly acidic environment:

[0106] Fe(acac)3, 1,2-hexadecanediol, oleic acid, oleylamine with a molar ratio of 1:5:3:3, and then dibenzyl ether was added as a solvent, and stirred uniformly under the protection of argon. The reaction environment was replaced into an oxygen-free environment, the mixture was heated to 200°C for 2h, and then continuously heated to 300°C, and refluxed at the temperature for 1h. After the reaction was completed, the reaction system was cooled to room temperature. The product was blended with anhydrous ethanol, precipitated and centrifuged three times to obtain the product, and vacuum dried at room temperature to obtain superparamagnetic Fe3O4 nanoparticles with a particle size of about 15nm, which were dispersed in n-hexane and stored at -20°C. The superparamagnetic Fe3O4 nanoparticles had a particle size of about 15nm, were spherical, had monodispersity and superparamagnetism, and had a saturation magnetization of 69emu / g, as determined by transmission electron microscopy and dynamic light scattering.

[0107] Then, the comb-like anionic pH-sensitive macromolecule with a phase transition pH of 6.71 was lyophilized, and 5g was dispersed in 250mL of DMSO to obtain dispersion A; 100mg of the superparamagnetic Fe3O4 nanoparticles with a particle size of about 15nm was dried by blowing argon, and then dispersed in 10mL of toluene to obtain dispersion B.

[0108] The dispersion A and the dispersion B were mixed, and a mixed solution C was obtained by mechanical stirring at room temperature for 2h. Then, the mixed solution C was heated to the boiling point of methylcyclohexane (101°C) under normal pressure for 20min to volatilize the methylcyclohexane. Subsequently, the mixed solution C was continuously heated to the boiling point of DMF (153°C) under normal pressure to sufficiently reflux for 1h. After the reflux was completed, the mixed solution C was dialyzed under a reflux state using a dialysis bag with a molecular weight cut-off of 50000, and the dialysis liquid was a phosphate buffer with a pH of 7.35-7.45. The dialysis was performed for 24h to remove the DMF, and a pH-sensitive superparamagnetic nanomicelle was obtained, which was a tumor contrast agent that specifically enhanced the magnetic resonance transverse relaxation signal in an acidic (pH=6.5-6.8) environment.

[0109] (4) Purification of the tumor contrast agent:

[0110] The dialyzed solution obtained in step (3) was centrifuged at a speed of 10000r / min at 4°C for 20min to obtain the supernatant, and then centrifuged at a speed of 30000r / min at 4°C for 50min to remove the supernatant. The precipitate was dispersed in a phosphate buffer with a pH of 7.35-7.45 to obtain a purified tumor contrast agent that specifically enhanced the magnetic resonance transverse relaxation signal in an acidic (pH=6.5-6.8) environment.

[0111] The raw pH value of the tumor contrast agent obtained above was measured by a pH meter, and then the pH was adjusted by 0.1M dilute hydrochloric acid or sodium hydroxide to obtain a nanomicelle solution with a physiological pH of about 7.4 and a phase transition point of about 6.7. The particle size of the micelle solution at two pH values was determined by a high-sensitivity particle size analyzer, as shown in Figure 10 .

[0112] Figure 10 The particle size change diagram of the pH-sensitive superparamagnetic nanomicelle of Example 3 in different pH environments can be seen from the figure. When the contrast agent is in a physiological environment (pH 7.4), it presents a uniform state and a small particle size of 33 nm, and the PDI is 0.124. When the environmental conditions present a phase transition point of about 6.7, the tumor weak acid responsive nanomagnetic fluid exhibits its high sensitivity to the environment, and the particle size also increases significantly to 250 nm, and the PDI is 0.147. The sudden change in particle size under different pH conditions can prove that the contrast agent synthesized in the experimental process has the ability to respond to the tumor weak acid microenvironment.

[0113] Example 4: Preparation of a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment

[0114] (1) Preparation of a linear structure pH-sensitive macromolecule:

[0115] Since the pH-sensitive monomer is sulfadoxine acrylamide (SDAA), its dissociation constant is close to 6.0-6.2, which is 6.03, so it is not necessary to add a regulating monomer. In this reaction, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol is used as a chain transfer agent, and sulfadoxine acrylamide (SDAA) is used as a pH-sensitive monomer to prepare a linear pH-sensitive macromolecule pSMAA. The specific steps are as follows:

[0116] According to the molar ratio of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol to sulfadoxine acrylamide (SDAA) of 1:40, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol (1 mmol) and sulfadoxine acrylamide (SDAA, 40 mmol) were added in a reaction medium DMF. After vacuum / argon replacement for 3 times, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol (1 mmol) and sulfadoxine acrylamide (SDAA, 40 mmol) were added in a reaction medium DMF. After vacuum / argon replacement for 3 times, under argon protection, 70°C reaction for 6 hours, a linear structure pH-sensitive macromolecule with a molecular weight of 4000 and a dissociation constant (pKa) of 6.03 was obtained, which was denoted as pSDAA.

[0117] The UV-vis transmittance curve of the linear structure pH-sensitive macromolecule in different pH environments is as followsFigure 11 As can be seen from the figure, under the condition of pH 7.4, the linear pH-sensitive macromolecule keeps the transmittance close to 100%; when the pH is adjusted to the phase transition point of the linear pH-sensitive macromolecule, the transmittance is close to 50%, i.e. when the pH is about 6.03, the polymer undergoes a change in hydrophilic-hydrophobic morphology, i.e. the transmittance obviously decreases and presents a rapid mutation in a cliff-like manner. The above results can show that the linear pH-sensitive macromolecule has the ability of environmental response.

[0118] The linear anionic pH-sensitive macromolecule was prepared by stirring pSDAA with pKa of 6.03 and poly(maleic anhydride-alt-1-octadecene) at a molar ratio of 50:1 for 48 hours at room temperature, followed by dialysis.

[0119] The UV-vis transmittance of the comb-like anionic pH-sensitive macromolecule in different pH environments was investigated to evaluate the phase transition pH, and the investigation results are shown in Figure 12 As can be seen from the figure, under the condition of pH 7.4, the comb-like anionic pH-sensitive macromolecule keeps the transmittance close to 100%; when the pH is adjusted to the phase transition point of the comb-like anionic pH-sensitive macromolecule, the transmittance is close to 50%, i.e. when the pH is about 6.65, the polymer undergoes a change in hydrophilic-hydrophobic morphology, i.e. the transmittance obviously decreases and presents a rapid mutation in a cliff-like manner. The above results can show that the comb-like anionic pH-sensitive macromolecule has the ability of environmental response. Therefore, the phase transition pH value of the comb-like anionic pH-sensitive macromolecule is 6.65. Figure 12 (3) Preparation of a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in weak acid environment:

[0120] The stepwise high-temperature precursor decomposition method was adopted, and Fe(acac)3, 1,2-hexadecanediol, oleic acid, and oleylamine were added at a molar ratio of 1 / 5 / 3 / 3, and then dibenzyl ether was added as a solvent, and stirring was uniformly conducted under the protection of argon. The reaction environment was replaced into an oxygen-free environment, the mixture was heated to 200°C for 2h, and then the temperature was continuously increased to 300°C, and the reaction was refluxed at the temperature for 1h. After the reaction was completed, the reaction system was cooled to room temperature. The product was blended with anhydrous ethanol, precipitated and centrifuged three times, and the product was obtained, and vacuum drying was conducted at room temperature, thereby obtaining superparamagnetic Fe3O4 nanoparticles with a particle size of 6nm, which were dispersed in n-hexane and stored at -20°C. Transmission electron microscopy and dynamic light scattering determination showed that the superparamagnetic Fe3O4 nanoparticles had a particle size of 6nm, were spherical, had monodispersity and superparamagnetism, and had a saturation magnetization of 72emu / g.

[0121]

[0122] ​Then, the comb-like anionic pH-sensitive macromolecule with phase transition pH value of 6.65 was lyophilized and 3 g of it was dispersed in 100 mL of DMSO to obtain dispersion A; 50 mg of the superparamagnetic Fe3O4 nanoparticles with particle size of 6 nm was dried by argon blowing and then dispersed in 5 mL of toluene to obtain dispersion B.

[0123] The dispersion A and the dispersion B were mixed and mechanically stirred at room temperature for 6 hours to obtain mixed solution C, and then the mixed solution C was heated at normal pressure to the boiling point of methylcyclohexane (101°C) for 15 minutes to volatilize the methylcyclohexane. Subsequently, the mixed solution C was continuously heated at normal pressure to the boiling point of DMSO (189°C) for sufficient reflux for 1.5 hours, and then was dialyzed in a dialysis bag with a molecular weight cut-off of 50000 under reflux after the reflux was ended, using a phosphate buffer with pH value of 7.35-7.45 as the dialysis liquid, and the dialysis was performed for 24 hours to remove the DMSO, thereby obtaining the pH-sensitive superparamagnetic nanomicelles, i.e. the tumor contrast agent which specifically enhances the magnetic resonance transverse relaxation signal in weak acid (pH=6.5-6.8) environment.

[0124] (4) Purification of the tumor contrast agent:

[0125] The dialyzed solution obtained in step (3) was centrifuged at a speed of 10000 rpm at 4°C for 25 minutes to remove the supernatant, and then was centrifuged at a speed of 50000 rpm at 4°C for 60 minutes to remove the supernatant, and the precipitate was dispersed in a phosphate buffer with pH value of 7.35-7.45, thereby obtaining the purified tumor contrast agent which specifically enhances the magnetic resonance transverse relaxation signal in weak acid (pH=6.5-6.8) environment.

[0126] The original pH value of the tumor contrast agent obtained above was measured by a pH meter, and then the pH value was adjusted by 0.1M dilute hydrochloric acid or sodium hydroxide to obtain a nanomicelle solution with physiological pH of about 7.4 and phase transition point of about 6.65, and the particle sizes of the micelles with two pH values were measured by a high-sensitivity particle size analyzer, as shown in Figure 10 .

[0127] Figure 13The particle size changes of the pH-sensitive superparamagnetic nanomicelles of Example 4 in different pH environments are shown in the figure. As can be seen from the figure, when the contrast agent is in a physiological environment (pH 7.4), it presents a uniform state of dispersion, the particle size is small, at 36 nm, and the PDI is 0.116; when the environmental conditions present a phase transition point of about 6.65, the tumor weak acid-responsive nanomagnetic fluid exhibits its high sensitivity to the environment, and the particle size also significantly increases, at 249.7 nm, and the PDI is 0.139. The sudden change in particle size under different pH conditions can prove that the contrast agent synthesized in the experimental process has the ability to respond to the tumor weak acid microenvironment.

[0128] Example 5: In vitro magnetic resonance imaging enhancement of the tumor contrast agent

[0129] In this example, the in vitro magnetic resonance imaging enhancement of the tumor contrast agent prepared by the method described in Example 4 was investigated by the following means, and the specific investigation method is shown as follows:

[0130] According to the actual metal content determined by ICP-MS, a certain amount of tumor contrast agent specific to enhance the magnetic resonance transverse relaxation signal in the weak acid (pH = 6.5-6.8) environment was dissolved in PBS with pH 7.41 and 6.65 to prepare pH-sensitive superparamagnetic nanomicelles with a concentration of 1070 μM, and gradient dilution was performed to finally obtain pH-sensitive superparamagnetic nanomicelles containing C Fe+Mn : 33, 67, 135, 268, 535, 1070 μM of pH-sensitive superparamagnetic nanomicelles.

[0131] The T2 map of the pH-sensitive superparamagnetic nanomicelles containing gradient metal content under two pH environments (pH = 7.41 / 6.65) was scanned by a 3.0T nuclear magnetic resonance instrument, and the scanning results are shown in Figure 14 A.

[0132] As can be seen from Figure 14 A, with the increase of the gradient of the metal element concentration, the T2 imaging presents a trend of becoming darker and darker, and the magnetic resonance imaging ability under the physiological environment is far less than that under the weak acid condition, which presents a trend of becoming brighter in a macroscopic way.

[0133] The 1 / T2 value was calculated, a straight line related to the concentration and the reciprocal of the relaxation time was fitted, and the r2 value was calculated. The T2 weighted imaging conditions are as follows: TR = 5000 ms, TE = 10-90 ms, layer thickness = 3 mm, flip angle = 150°, matrix size = 256 x 256, field of view = 100 mm, echo length = 8, and the fitting curve is shown in Figure 14 B.

[0134] As can be seen from Figure 14As can be seen in B, under physiological conditions (pH 7.41), the r2 value is 86.72 mM -1 s -1 ; weakly acidic microenvironment (pH 6.65), the r2 value is 202.76 mM -1 s -1 The change in pH significantly increases the r2 value, indicating that the tumor contrast agent can significantly reduce the relaxation time, increase the r2 value, and increase the contrast degree of the tumor region, i.e., the weakly acidic environment, and the normal tissue, i.e., the physiological environment, which is conducive to the early diagnosis of small tumors.

[0135] Example 6: In vivo magnetic resonance imaging enhancement of the tumor contrast agent

[0136] In this example, the in vivo magnetic resonance imaging enhancement of the tumor contrast agent prepared by the method described in Example 4 was investigated by the following means, and the specific investigation method is shown as follows:

[0137] The non-targeting carrier (PEG-PCL) loaded with the same superparamagnetic nanoparticles was used as the control group, and the tumor contrast agent prepared in Example 4 was used as the test group. The nanomicelles with an iron concentration of 5 mg / kg were prepared. The preparation method of the non-targeting carrier (PEG-PCL) loaded with the same superparamagnetic nanoparticles is as follows: the micelles were prepared according to the mass ratio of PEG-PCL to superparamagnetic nanoparticles Fe3O4 of 7:3, PEG-PCL and superparamagnetic nanoparticles were dispersed in DMF and tetrahydrofuran, then they were mixed, and an excess of pure water was added dropwise under ultrasonic conditions. After removing the organic solvent by dialysis, the supernatant was obtained by centrifugation at 2000 rpm for 10 min, and finally freeze-drying was performed to obtain the non-targeting nanomicelles.

[0138] 4T1 cells (purchased from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences) were used to inoculate the 4th pair of mammary glands of 6-8 week old female BALB / c mice (about 25 g) purchased from the Experimental Animal Center of Jiangsu University to obtain experimental mice. About a week after inoculation, when the tumor volume reached 150 mm 3 , 0.15 mL of nanomicelles in the test group and the control group were injected into the experimental mice through the tail vein, respectively. At 0, 2, and 12 hours after injection, the T2 imaging was performed by a 3.0T magnetic resonance instrument, and the difference in the contrast degree between the test group and the control group was compared before and after injection. Before the magnetic resonance imaging, anesthetic (0.1 mL chloral hydrate) was injected into the abdominal cavity of the mice. The T2 weighted imaging conditions are as follows: TR=5000 ms, TE=10-90 ms, slice thickness=3 mm, flip angle=150°, matrix size=256x256, field of view=100 mm, echo length=8.

[0139] The in vivo magnetic resonance imaging enhancement investigation results are as follows:Figure 15 As shown in the figure, at 0h, the contrast between tumor region (red circle) and surrounding tissue is very weak, and the position of tumor is basically indistinguishable. Figure 15 However, 2h after injection of the tumor contrast agent, the pH-sensitive group has obvious T2 imaging effect, and macroscopically, the tumor region presents a darker color, and has obvious contrast with the normal tissue beside it; at 12h, the enhanced contrast degree is more obvious, and it can be seen that the tumor region is darker, while in the non-pH-sensitive group, these phenomena do not appear in 12h, which proves the responsiveness and targeting of pH sensitivity in the tumor microenvironment, because the material deforms and aggregates in the tumor region, increasing the R2 value, so that the tumor contrast enhancer has good T2 imaging effect.

[0140] In summary, the tumor contrast agent described in the present application can improve the contrast ratio of tumor tissue / normal tissue in magnetic resonance imaging, and further improve the accuracy of early diagnosis of tumors.

[0141] The above embodiments are preferred embodiments of the present application, but the present application is not limited to the above embodiments, and any obvious improvements, replacements or modifications made by those skilled in the art without departing from the essential content of the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weakly acidic environment, characterized by, The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. With poly(maleic anhydride- The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. (-1-octadecene) is the main body of a comb-like anionic pH-sensitive macromolecule, which combines a linear pH-sensitive macromolecule with poly(maleic anhydride- The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The reaction of -1-octadecene yields a comb-like anionic pH-sensitive macromolecule after the reaction is complete. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent.

2. The method of claim 1, wherein the method is performed in the presence of a weak acid environment. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent.

3. The method for preparing a tumor contrast agent that enhances transverse relaxation signals in a weakly acidic environment according to claim 1, characterized in that, In step (2), the linear structure of the pH-sensitive macromolecule and poly(maleic anhydride- The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The molar ratio of 1-octadecene to 15-50:1; The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent.

4. The method of claim 1, wherein the method is characterized by: The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent.

5. The method for preparing a tumor contrast agent that enhances transverse relaxation signals in a weakly acidic environment according to claim 1, characterized in that, The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. 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The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. 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The application relates to a preparation method of a linear pH-sensitive macromolecule, a preparation method of a comb-shaped anionic pH-sensitive macromolecule and a preparation method of a tumor contrast agent. The application relates to a preparation method of 6. The method of preparing a tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weakly acidic environment according to claim 5, wherein The first centrifugation is carried out at 10000 rpm for 10-30 minutes at 4℃; The second centrifugation is carried out at 20000-50000 rpm for 30-60 minutes at 4℃; The pH value of the phosphate buffer solution is 7.35-7.

45.

7. The tumor imaging agent for enhancing magnetic resonance transverse relaxation signals in a weakly acidic environment prepared by the method according to any one of claims 1 to 6, characterized in that, The tumor contrast agent uses a comb-like anionic pH-sensitive macromolecule as a carrier, and internally encapsulates a hydrophobic superparamagnetic nanoparticle stabilized by oleic acid on the surface; the comb structure on the comb-like anionic pH-sensitive macromolecule is 1-octadecene; the 1-octadecene and the oleic acid on the surface of the superparamagnetic nanoparticle are interpenetrated.

8. The tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weakly acidic environment according to claim 7, wherein The phase transition pH value of the comb-like anionic pH-sensitive macromolecule is 6.5-6.

8.

9. The tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weakly acidic environment according to claim 7, wherein The hydrated particle size of the tumor contrast agent is 20-50 nm at a pH value of 7.35-7.

45. The particle size increases to 200-5000 nm in a weak acid environment with a pH value of 6.5-6.

8.

10. Use of the tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment according to any one of claims 7-9, or the tumor contrast agent for enhancing magnetic resonance transverse relaxation signals in a weak acid environment prepared by the method according to any one of claims 1-6 in the preparation of a tumor imaging reagent.

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