阅读说明：本技术 一种集磁共振成像和光热治疗于一体的高分子自愈凝胶及其制备与应用 (High-molecular self-healing gel integrating magnetic resonance imaging and photothermal therapy and preparation and application thereof ) 是由 蒋刚彪 朱水容 刘斌 胡甜 刘步宁 庞卯 于 2020-04-17 设计创作，主要内容包括：本发明公开了一种集磁共振成像和光热治疗于一体的高分子自愈凝胶的制备及应用。本发明以具有生物活性的Mn<Sup>2+</Sup>作为鳌合位点,与天然高分子形成三维网络结构,同时掺杂具有光热效果的纳米金棒,开发出一种安全、可降解、能促细胞生长和受损组织修复、且兼具磁共振成像诊断和光热治疗的医用水凝胶,实现“一次注射”便可满足诊疗的“双效果”。同时,结合自愈合的功能,该复合水凝胶在组织工程、病变诊断、定点监测与成像、光热治疗等领域具有可观的发展前景和潜在的医用价值。(The invention discloses a preparation method and application of a high-molecular self-healing gel integrating magnetic resonance imaging and photothermal therapy. The invention relates to Mn with biological activity 2+ As a chelating site, high score with the naturalThe nano-gold rods form a three-dimensional network structure and are doped with nano-gold rods with photo-thermal effects at the same time, so that the medical hydrogel which is safe, degradable, capable of promoting cell growth and repairing damaged tissues and has magnetic resonance imaging diagnosis and photo-thermal treatment is developed, and double effects of diagnosis and treatment can be met by one-time injection. Meanwhile, the composite hydrogel has considerable development prospect and potential medical value in the fields of tissue engineering, lesion diagnosis, fixed-point monitoring and imaging, photothermal therapy and the like by combining the self-healing function.)

1. A preparation method of a high-molecular self-healing gel integrating magnetic resonance imaging and photothermal therapy is characterized by comprising the following steps:

(1) adding p-aldehyde benzoic acid, 4-dimethylaminopyridine and dicyclohexylcarbodiimide into the chitosan solution, stirring for 5-30 min, then reacting for 1-4 days, precipitating, washing, and performing suction filtration to obtain aldehyde chitosan;

(2) at 25-40 ℃ to HAuCl₄Adding a surfactant into the solution, stirring for 5-60 s, and adding NaBH₄Stirring the solution for 5-60 s to obtain Au seed solution;

(3) at 25-40 ℃ to HAuCl₄Adding a surfactant into the solution, stirring for 5-60 s, and adding AgNO₃Stirring the solution for 5-60 s, adding a reducing agent solution, andstirring for 5-60 s to obtain a nano Au growth solution;

(4) adding the nano Au growth solution into the Au seed solution, stirring for 5-60 s at 25-40 ℃, standing for 6-18 h at normal temperature, centrifuging after the gold rod grows stably, washing, and performing suction filtration to obtain nano Au rod powder;

(5) dissolving the aldehyde chitosan in water, uniformly dispersing, adding the nano Au bar powder, and stirring for 1-3 h to obtain a nano Au bar mixed solution;

(6) adding a nano Au bar mixed solution into a carboxymethyl chitosan solution, stirring for 2-5 h, and adding MnCl₂·4H₂And stirring the O aqueous solution for 4-20 hours to obtain the high-molecular self-healing composite hydrogel.

2. The method for preparing the self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy according to claim 1, wherein the chitosan solution in the step (1) comprises chitosan, p-aldehyde benzoic acid, 4-dimethylaminopyridine and dicyclohexylcarbodiimide in a mass ratio of (5-25): (20-60): (0.6-1.2): (10-35);

HAuCl in the step (2)₄HAuCl in solution₄Surfactants and NaBH₄NaBH in solution₄The molar ratio of (5-9) mmol: (1.25-50) mol: (5-12) mmol.

3. The method for preparing self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy according to claim 1, wherein the HAuCl in step (3) is added₄HAuCl in solution₄Surfactant, AgNO₃AgNO in solution₃And the molar ratio of the reducing agent in the reducing agent solution is (10-15) mmol: (1.25-50) mol: (4.2-10.8) mmol: (80-150) mmol;

and (4) the volume ratio of the nano Au growth solution to the Au seed solution is (1-5): 1.

4. the method for preparing the self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy according to claim 1, wherein the mass ratio of the aldehyde chitosan to the nano Au rod powder in the step (5) is (2-7): (1-10);

carboxymethyl chitosan in the carboxymethyl chitosan solution, and nano Au bars and MnCl in the nano Au bar mixed solution in the step (6)₂·4H₂MnCl in aqueous O solution₂·4H₂The mass ratio of O is (20-80): (1-10): (2-16).

5. The method for preparing the self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy according to claim 1, wherein the concentration of the chitosan solution in the step (1) is 1-5 wt%, the molecular weight of chitosan in the chitosan solution is 800-1000, and the degree of deacetylation is 75-85%;

adding p-aldehyde benzoic acid in the step (1) in the form of a p-aldehyde benzoic acid solution, wherein the concentration of the p-aldehyde benzoic acid solution is 10-30 wt%; the 4-dimethylaminopyridine is added in the form of a 4-dimethylaminopyridine solution, and the concentration of the 4-dimethylaminopyridine solution is 0.6-1.2 wt%; the dicyclohexylcarbodiimide is added in the form of a dicyclohexylcarbodiimide solution, and the concentration of the dicyclohexylcarbodiimide solution is 10-35 wt%.

6. The method for preparing self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy according to claim 1, wherein the HAuCl is obtained in the steps (2) and (3)₄The concentration of the solution is 0.5-5 mmol/L; NaBH in step (2)₄The concentration of the solution is 5-12 mmol/L; the surfactant in the steps (2) and (3) is cetyl trimethyl ammonium bromide, and is added in the form of surfactant solution, and the concentration of the surfactant solution is 0.05-2 mol/L;

the reducing agent in the reducing agent solution in the step (3) is vitamin C and NaBH₄At least one of; the concentration is 80-150 mmol/L; AgNO described in step (3)₃The concentration of the solution is 7-18 mmol/L.

7. The method for preparing a self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy according to claim 1, wherein the concentration of the solution formed by dissolving the aldehyde chitosan in water in step (5) is 2-7 wt%; the concentration of the nano Au bar powder in the system in the step (5) is 1-10 wt%;

the concentration of the carboxymethyl chitosan solution in the step (6) is 2-8 wt%, wherein the molecular weight of the carboxymethyl chitosan is 2000, and the substitution degree is 80-85%; MnCl in the step (6)₂·4H₂The concentration of the O aqueous solution is 1-8 wt%.

8. The method for preparing a self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy according to claim 1, wherein the stirring and the reaction in step (1) are both performed at room temperature; the solvent used in the reaction is dimethyl sulfoxide; the precipitation time is 12-48 h, the precipitating agent is deionized water, and the volume ratio of the reaction product mixed solution to the added precipitating agent is 9: (3-7);

the solvents of the solutions involved in the steps (2) and (3) are all water; the rotating speed of the centrifugation in the step (4) is 5000-8000 r/min, and the time is 10-20 min;

washing in the steps (1) and (4) refers to repeatedly washing with absolute ethyl alcohol and deionized water for 5-10 times until the solution is colorless; the uniform suction filtration is normal temperature and normal pressure suction filtration;

and (5) performing ultrasonic oscillation in the uniform dispersion mode, wherein the ultrasonic time is 5-10 min.

9. The self-healing polymer gel integrating magnetic resonance imaging and photothermal therapy, prepared by the method of any one of claims 1 to 8.

10. The use of the self-healing polymer gel according to claim 9 for integrating mri and photothermal therapy.

Technical Field

The invention belongs to the field of medical polymer materials, and particularly relates to a polymer self-healing gel integrating magnetic resonance imaging and photothermal therapy, and preparation and application thereof.

Background

Magnetic resonance imaging is an emission tomography, which obtains electromagnetic signals from a human body by using a magnetic resonance phenomenon so as to reconstruct a digital image of a human body system, and is widely used in the medical imaging aspect of injuries and lesions of spinal cords, craniocerebra, joints and the like at present, and particularly has remarkable effect in diagnosing spinal cavities, cranial neuropathy, brain tumors, angiography, soft tissue injuries and the like.

At present, most of traditional magnetic resonance contrast agents are heavy metal complexes or nano oxides, and although the contrast agents have good contrast effect and are applied to clinical treatment, various defects still exist, such as common heavy metal complex Gd³⁺The toxicity is high, and even the brain allergy and the nerve cell death can be caused; the commonly used nano Fe₃O₄The particles are small, flow with blood in the body, and are difficult to be discharged from the human metabolic system. In addition, the contrast agent is mostly injected in the form of solution, and has strong fluidity, short imaging time and unobvious effect. Therefore, in order to overcome the defects of high toxicity, difficult degradation and short diagnosis time of the traditional magnetic resonance contrast agent, a safe and degradable magnetic resonance imaging medical material with long imaging time needs to be developed.

The hydrogel prepared from the natural chitosan and the derivatives thereof becomes a good medical carrier material due to the advantages of high safety, strong plasticity, good biocompatibility and flexibility, easy degradation and the like. Recent studies have shown that Mn²⁺Can be used as a manganese-enhanced magnetic resonance diagnostic agent, can enhance longitudinal relaxation imaging signals, and replace the traditional high-toxicity contrastAnd (3) preparing. In addition, manganese also has certain biological activity and can promote cartilage formation. Gold nano-materials have been generally proved to be a more common photo-thermal reagent due to the plasma resonance characteristic.

Therefore, the present invention uses Mn²⁺The hydrogel is combined with nano Au to develop a medical hydrogel integrating magnetic resonance imaging and photothermal therapy, and simultaneously, the hydrogel self-healing function is combined, so that the hydrogel has great development prospect and potential medical value in the fields of tissue engineering, lesion diagnosis, fixed-point monitoring and imaging, photothermal therapy and the like.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a preparation method of a high-molecular self-healing gel integrating magnetic resonance imaging and photothermal therapy. Taking chitosan derivative from natural sources as a carrier substrate, adding gold nano material, and introducing Mn²⁺And (4) crosslinking the system to prepare the self-healing hydrogel composite material. The photothermal therapy of Au and the magnetic resonance imaging function of Mn are integrated, and the medical composite hydrogel dual diagnosis and treatment means is provided.

The invention also aims to provide the high-molecular self-healing gel which integrates the magnetic resonance imaging and the photothermal therapy.

The invention further aims to provide application of the high-molecular self-healing gel integrating magnetic resonance imaging and photothermal therapy.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a high-molecular self-healing gel integrating magnetic resonance imaging and photothermal therapy comprises the following steps:

(1) adding p-aldehyde benzoic acid, 4-Dimethylaminopyridine (DMAP) and Dicyclohexylcarbodiimide (DCC) into the chitosan solution, stirring for 5-30 min, reacting for 1-4 days, precipitating, washing, and performing suction filtration to obtain aldehyde chitosan (OCS);

(2) at 25-40 ℃ to HAuCl₄Adding a surfactant into the solution, stirring for 5-60 s, and adding NaBH₄Solution and stirring 5E60s, obtaining Au seed liquid;

(3) at 25-40 ℃ to HAuCl₄Adding a surfactant into the solution, stirring for 5-60 s, and adding AgNO₃Stirring the solution for 5-60 s, adding a reducing agent solution, and stirring for 5-60 s to obtain a nano Au growth solution;

(4) adding the nano Au growth solution into the Au seed solution, stirring for 5-60 s at 25-40 ℃, standing for 6-18 h at normal temperature, centrifuging after the gold rod grows stably, washing, and performing suction filtration to obtain nano Au rod powder;

(5) dissolving aldehyde chitosan (OCS) in water, uniformly dispersing, adding nano Au bar powder, and stirring for 1-3 h to obtain a nano Au bar mixed solution;

(6) adding a nano Au bar mixed solution into a carboxymethyl chitosan solution, stirring for 2-5 h, and adding MnCl₂·4H₂And stirring the O aqueous solution for 4-20 hours to obtain the high-molecular self-healing composite hydrogel.

Preferably, the concentration of the chitosan solution in the step (1) is 1-5 wt% (w/w), the molecular weight of chitosan in the chitosan solution is 800-1000, the degree of deacetylation is 75-85%, and the solvent is at least one of dimethyl sulfoxide, acetone, methanol, ethanol and water.

Preferably, the mass ratio of the chitosan to the p-aldehyde benzoic acid, the 4-dimethylaminopyridine and the dicyclohexylcarbodiimide in the chitosan solution in the step (1) is (5-25): (20-60): (0.6-1.2): (10-35).

Preferably, the p-aldehyde benzoic acid in the step (1) is added in the form of a p-aldehyde benzoic acid solution, and the concentration of the p-aldehyde benzoic acid solution is 10-30 wt% (w/w); the 4-dimethylaminopyridine is added in the form of a 4-dimethylaminopyridine solution, and the concentration of the 4-dimethylaminopyridine solution is 0.6-1.2 wt% (w/w); the dicyclohexylcarbodiimide is added in the form of a dicyclohexylcarbodiimide solution, and the concentration of the dicyclohexylcarbodiimide solution is 10-35 wt% (w/w); the solvents of the p-formaldehyde benzoic acid solution, the 4-dimethylaminopyridine solution and the dicyclohexylcarbodiimide solution are the same as those of the chitosan solution.

Preferably, the stirring and the reaction of step (1) are both carried out at room temperature.

Preferably, the precipitation time in the step (1) is 12-48 h, the precipitating agent is deionized water, and the volume ratio of the reaction product mixed solution to the added precipitating agent is 9: (3-7); the washing is to repeatedly wash the solution for 5-10 times by using absolute ethyl alcohol and deionized water until the solution is colorless; the suction filtration is normal temperature and normal pressure suction filtration.

Preferably, the HAuCl of steps (2) and (3)₄The concentration of the solution is 0.5-5 mmol/L, and the solvent is water.

Preferably, the HAuCl of step (2)₄HAuCl in solution₄Surfactants and NaBH₄NaBH in solution₄The molar ratio of (5-9) mmol: (1.25-50) mol: (5-12) mmol.

Preferably, the surfactant in steps (2) and (3) is Cetyl Trimethyl Ammonium Bromide (CTAB), the surfactant is added in the form of surfactant solution, the concentration of the surfactant solution is 0.05-2 mol/L, and the solvent is water.

Preferably, the NaBH of step (2)₄The concentration of the solution is 5-12 mmol/L, and the solvent is water.

Preferably, the HAuCl of step (3)₄HAuCl in solution₄Surfactant, AgNO₃AgNO in solution₃And the molar ratio of the reducing agent in the reducing agent solution is (10-15) mmol: (1.25-50) mol: (4.2-10.8) mmol: (80-150) mmol.

Preferably, the reducing agent in the reducing agent solution in the step (3) is vitamin C (Vc) and NaBH₄At least one of; the concentration is 80-150 mmol/L.

Preferably, the AgNO of step (3)₃The concentration of the solution is 7-18 mmol/L, and the solvent is water.

Preferably, the volume ratio of the nano Au growth solution to the Au seed solution in the step (4) is (1-5): 1.

preferably, the rotating speed of the centrifugation in the step (4) is 5000-8000 r/min, and the time is 10-20 min; the washing is to repeatedly wash the solution for 5-10 times by using absolute ethyl alcohol and deionized water until the solution is colorless; the suction filtration is normal temperature and normal pressure suction filtration.

Preferably, the concentration of the solution formed by dissolving the aldehyde chitosan in the step (5) in water is 2-7 wt%.

Preferably, the mass ratio of the aldehyde chitosan in the step (5) to the nano Au bar powder is (2-7): (1-10).

Preferably, the concentration of the nano Au bar powder in the step (5) in the system is 1-10 wt%.

Preferably, the uniform dispersion in the step (5) is ultrasonic oscillation, and the ultrasonic time is 5-10 min.

Preferably, the concentration of the carboxymethyl chitosan solution in the step (6) is 2-8 wt%, wherein the molecular weight of the carboxymethyl chitosan is 2000, the substitution degree is 80-85%, and the solvent is water.

Preferably, the carboxymethyl chitosan in the carboxymethyl chitosan solution of step (6), the nano Au bars and the MnCl in the mixed solution of the nano Au bars₂·4H₂MnCl in aqueous O solution₂·4H₂The mass ratio of O is (20-80): (1-10): (2-16).

Preferably, the MnCl of the step (6)₂·4H₂The concentration of the O aqueous solution is 1-8 wt%.

The high-molecular self-healing gel prepared by the method integrates magnetic resonance imaging and photothermal therapy.

The application of the high-molecular self-healing gel integrates magnetic resonance imaging and photothermal therapy.

The aldehyde chitosan (OCS) in the raw materials used in the invention can stably wrap the nano gold rod, and the amino group on the carboxymethyl chitosan (CMC) molecular chain and the aldehyde group on the OCS molecular chain can generate Schiff base action, and simultaneously, the macromolecule physical winding and hydrogen bond action exist, so that a first layer of cross-linked network is formed. In addition, the amino and carboxyl groups on the molecular chain of CMC are mixed with Mn²⁺Coordination chelation exists between the two layers, and a second layer of cross-linked network is formed. The formation of a double-layer polymer cross-linked network greatly stabilizes the internal structure of the hydrogel; the nano gold rod has certain photo-thermal effect and chelated active substance Mn²⁺The magnetic resonance imaging material has the characteristics of manganese enhancement and magnetic resonance imaging; the composite medical hydrogel with good stability, high safety and two diagnosis and treatment means can be finally prepared.

The self-healing hydrogel with a three-dimensional network structure is formed by taking chitosan derivatives as main macromolecular chains, cooperating with the dynamic covalent bond formed by Schiff base bonds and the non-covalent bond formed by hydrogen bonds and metal coordination bonds and combining with the physical winding effect among the macromolecular chains, and meanwhile, nano gold rods are taken as nano fillers to be uniformly dispersed in the hydrogel network, so that not only are the rigid sites increased, but also the internal structure of the hydrogel is stabilized; but also has certain photo-thermal effect. Recombination of chelated active substance Mn²⁺The magnetic resonance imaging system can promote the repair of damaged nerves and has the characteristic of manganese-enhanced magnetic resonance imaging.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the polymer gel provided by the invention is simple to prepare and high in safety. The gel can be prepared by mixing and stirring the raw materials in one pot at normal temperature without an initiator. In addition, compared with artificially synthesized macromolecules, the chitosan derivative is prepared from chitosan derivatives of natural sources, is degradable, biocompatible and higher in safety, and is more suitable for human medical treatment.

2. The polymer gel provided by the invention provides a new entry point for searching a substitute of a magnetic resonance imaging contrast agent. The traditional contrast agent has high toxicity, unstable in vivo flow and difficult degradation, and the natural polymer hydrogel has a three-dimensional network structure and is a good medical carrier; while a low concentration of Mn²⁺But also is a trace element required by human bodies, has certain biological activity and can promote the repair of damaged cartilage; therefore, the chelated Mn prepared by the invention²⁺The hydrogel has biological functions of promoting cell growth, repairing tissue and the like, and is expected to replace the existing high-toxicity contrast medium to be applied to human bodies.

3. The polymer gel provided by the invention provides a more convenient dual diagnosis and treatment means. Mn has been demonstrated to have manganese-enhanced magnetic resonance imaging properties and can be used as a contrast diagnostic agent; meanwhile, the system also comprises a nano gold rod, and can be applied to photo-thermal treatment; therefore, the composite hydrogel prepared by the invention can integrate magnetic resonance imaging and photothermal therapy, can meet the diagnosis and treatment effects by one-time injection, and provides a more accurate and convenient method for medical diagnosis and treatment.

4. The invention develops a research idea of combining multiple functions of magnetic resonance imaging plus, and although only relates to the research of the aspects of magnetic resonance imaging plus photothermal therapy, aiming at different functional requirements, the oriented preparation of the multifunctional composite material can be realized by adding raw materials with different characteristics, such as conductive materials, photothermal materials, imaging materials and the like, so that the aim of integrating multiple effects is fulfilled.

Drawings

Fig. 1 is a Scanning Electron Microscope (SEM) image of the composite hydrogel prepared in example 1 after lyophilization.

Fig. 2 is a swelling curve of the composite hydrogel prepared in example 2 at 37 ℃ in PBS buffer at pH 7.4.

Fig. 3 is a degradation curve of the composite hydrogel prepared in example 2 at 37 ℃ in PBS buffer at pH 7.4.

FIG. 4 is a graph showing the in vitro cytotoxic effect of the composite hydrogel prepared in example 5.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

Those who do not specify specific conditions in the examples of the present invention follow conventional conditions or conditions recommended by the manufacturer. The raw materials, reagents and the like which are not indicated for manufacturers are all conventional products which can be obtained by commercial purchase.