阅读说明：本技术 一种用于肿瘤t1-t2磁共振成像的介孔聚多巴胺纳米粒 (Mesoporous polydopamine nanoparticle for tumor T1-T2 magnetic resonance imaging ) 是由 曹众 吴英健 杨茜 于 2021-02-05 设计创作，主要内容包括：本发明属于生物医学材料技术领域,具体涉及一种用于肿瘤T1-T2磁共振成像的介孔聚多巴胺(MPDA)纳米粒,为制备出一种金属内掺杂且具有T1-T2双模态成像效果的介孔聚多巴胺纳米粒子,本发明采用聚合前掺杂方法,以盐酸多巴胺和Mn前体为基本原料,F127和TMB为模板制备得到一种锰-介孔聚多巴胺纳米粒,该纳米粒是一种金属锰内掺杂且具有介孔结构的聚多巴胺纳米粒,具有肿瘤微环境响应性,具有良好的光热转换能力,光热稳定性好；具有较好的生物相容性,可同时作为T1和T2造影剂用于肿瘤的磁共振成像。(The invention belongs to the technical field of biomedical materials, and particularly relates to Mesoporous Polydopamine (MPDA) nanoparticles for tumor T1-T2 magnetic resonance imaging, in order to prepare mesoporous polydopamine nanoparticles doped in metal and having a T1-T2 bimodal imaging effect, the invention adopts a doping method before polymerization, dopamine hydrochloride and Mn precursor are used as basic raw materials, F127 and TMB are used as templates to prepare manganese-mesoporous polydopamine nanoparticles, and the nanoparticles are polydopamine nanoparticles doped in metal manganese and having a mesoporous structure, have tumor microenvironment responsiveness, good photo-thermal conversion capability and good photo-thermal stability; has better biocompatibility and can be used as T1 and T2 contrast agents simultaneously for magnetic resonance imaging of tumors.)

1. A preparation method of manganese-mesoporous polydopamine nanoparticles is characterized in that a doping method before polymerization is adopted, dopamine hydrochloride and Mn are firstly mixed²⁺Chelating to generate a complex, and then preparing the manganese-mesoporous polydopamine nanoparticle by using manganese chelate-dopamine as a basic raw material and F127 and TMB as templates.

2. The preparation method of the manganese-mesoporous polydopamine nanoparticle as claimed in claim 1, wherein the preparation method comprises the following steps:

s1, dissolving dopamine hydrochloride and hydrated manganese sulfate in water, and stirring in a dark place to obtain a manganese chelate-dopamine solution;

s2, adding the F127 and the manganese chelate-dopamine solution obtained in the step S1 into a mixed solution of ethanol and water, and stirring in a dark place until the mixture is uniformly mixed;

s3, adding a TMB solution while shaking in water bath ultrasound, and continuously performing ultrasonic dispersion until the mixture is milky;

s4, dropwise adding a Tris aqueous solution under stirring, and stirring in a dark place;

s5, centrifuging, resuspending and washing the solution stirred in the step S4 to obtain the manganese-mesoporous polydopamine nanoparticles.

3. The preparation method of the manganese-mesoporous polydopamine nanoparticle as claimed in claim 2, wherein the mass ratio of dopamine hydrochloride to manganese sulfate hydrate in step S1 is (30-35): 1.

4. The preparation method of manganese-mesoporous polydopamine nanoparticles as claimed in claim 2, wherein the mass-to-volume ratio of hydrated manganese sulfate to water in step S1 is 1mg (1-1.5) mL.

5. The method for preparing manganese-mesoporous polydopamine nanoparticles as claimed in claim 2, wherein the mass-to-volume ratio of F127 to the manganese chelate-dopamine solution in step S2 is (100-150) mg:1 mL.

6. The preparation method of the manganese-mesoporous polydopamine nanoparticle as claimed in claim 2, wherein the volume ratio of the ethanol, water and manganese chelate-dopamine solution in step S2 is 20:20 (1-1.5).

7. The preparation method of the manganese-mesoporous polydopamine nanoparticle as claimed in claim 2, wherein the volume ratio of the TMB solution to the manganese chelate-dopamine solution in step S3 is (2-4): 3.

8. The method for preparing manganese-mesoporous polydopamine nanoparticles according to claim 2, wherein the concentration of the Tris aqueous solution in step S4 is 20mg/mL, and the volume ratio of the Tris aqueous solution to the TMB solution is 5 (1-2).

9. The manganese-mesoporous polydopamine nanoparticles prepared by the preparation method of any one of claims 1-8.

10. The use of the manganese-mesoporous polydopamine nanoparticles of claim 9 in magnetic resonance imaging of tumors T1-T2.

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to mesoporous polydopamine nanoparticles for tumor T1-T2 magnetic resonance imaging.

Background

Magnetic Resonance Imaging (MRI) is receiving much attention due to its high temporal, spatial resolution, freedom from depth of penetration and freedom from radiation damage. Magnetic resonance contrast agents can enhance MRI signals by reducing the relaxation time of T1 or T2 water protons. T1 is called longitudinal relaxation time and is commonly used to improve visualization saturation, suppress magnetic resonance signal intensity or imaging intensity, and since tissue generally has a longer longitudinal relaxation time, T1 weighted images are suitable for viewing adipose tissue or anatomical structures. T2 is called transverse relaxation time, and the proton spins at the diseased tissue site generally have longer relaxation times, so the diseased tissue is generally identified with a T2 weighted image.

Magnetic Resonance Imaging (MRI) plays a crucial role in the early diagnosis and accurate treatment of tumors. Based on the principles of magnetic resonance, Magnetic Resonance Imaging (MRI) can provide high resolution images of soft tissue by monitoring the difference in relaxation rates of water protons in the tissue. The MRI contrast agent currently and generally used in clinic is T1 contrast agent, and although it has an advantage in recognizing tumor micro-infiltration compared with T2 contrast agent, its sensitivity is much lower than that of T2 contrast agent, and it is difficult to detect early stage tumor with diameter less than 1 cm. The T1-T2 bimodal MRI is carried out on the same instrument, and only the acquisition sequence in the MRI needs to be simply adjusted in the operation process, so that the accuracy and consistency of space-time imaging parameters in two times of imaging are ensured, and the condition that the imaging results are not matched in different modes can be effectively avoided. That is, the T1-T2 bimodal MRI can be used for accurate imaging of early tumors, vascular systems, central nervous systems and other parts, and the influence of MRI artifacts under single modality on a diagnosis result is avoided. Therefore, the construction of the multifunctional nano diagnosis and treatment agent with multiple anti-cancer effects and T1-T2 bimodal contrast capability has great significance for early diagnosis and accurate treatment of cancer.

The design and application of T1-T2 bimodal MRI contrast agents can be subdivided into the following categories: (1) combining a T1 contrast material with a T2 contrast material; (2) doping a T1 contrast material into a T2 contrast material;(3) preparing magnetic nanoparticles with appropriate size and magnetization; (4) compounding T1 contrast material with non-magnetic porous medium. For example, a scholars prepares a double-mesoporous silica sphere by a silica template method, and then performs an oxidation reduction reaction in the presence of potassium permanganate to obtain the manganese-core-shell structure double-mesoporous silica sphere Mn-DMSS, wherein manganese is mainly located in a shell region. Due to the fact that the microminiature manganese oxide nanoclusters are formed in DMSS pores, T2 relaxation time is shortened, and the unique double mesoporous structure can increase diffusion rate of water molecules in mesopores and enhance r₁Relaxivity, Mn-DMSS has T1-T2 contrast effect, developing a new class of bimodal contrast agents (DMCA) with a single component (Mn group) and no conflicting effects between the two functional units.

Studies have shown that geometric constraint is an effective strategy to improve relaxivity by adjusting the inner and outer sphere mechanisms. In particular, the interstitial framework structure allows the contrast agent to rotate freely and allows diffusion of surrounding water molecules to be limited, thereby significantly increasing the rotational correlation time (τ) of the contrast agent_R) Time (tau) associated with diffusion of water molecules_D) Thereby enhancing the T1 and T2 contrast effects simultaneously. But due to lack of clinical validation, the study of T1-T2 bimodal MRI is still in its infancy. Therefore, the biodegradable T1-T2 bimodal nano diagnosis and treatment agent capable of responding to the tumor microenvironment can provide a new idea for magnetic resonance imaging.

In recent years, Polydopamine (PDA) has good biocompatibility and biodegradability as a melanin-like substance, and can be used as a photothermal conversion agent for photothermal therapy (PTT) of cancer, and is widely used in a cancer diagnosis and treatment system. Studies have shown that the key cyclization product of Polydopamine (PDA), i.e. the 5, 6-dihydroxyindole unit, can be reduced by GSH, and that PDA is pH sensitive to the acidic microenvironment of the tumor. Thus, PDA can be degraded in acidic and GSH-rich tumor microenvironments, triggering drug release. In addition, the catechol linkage in PDA allows chelation with metal ions. Currently, the preparation of metal-doped polydopamine hybrid materials generally comprises two methods of doping after polymerization and doping before polymerization. The doping method before polymerization enables the PDA to have higher metal loading capacity and better MRI contrast effect under the same polydopamine concentration.

As a novel PDA material, Mesoporous Polydopamine (MPDA) becomes an ideal drug carrier due to a regular pore structure and a higher specific surface area, and nanoparticles can effectively target tumor sites through Enhanced Permeation and Retention (EPR) effects, so that drug leakage in the drug release process is reduced. In the early work of the subject group, manganese carbonyl (MnCO) is loaded by utilizing the hydrophilic and hydrophobic action of high-porosity MPDA (MPDA), and the manganese carbonyl is in acidity and H₂O₂The Mn ions generated in situ can be used as an effective contrast agent of MRI, and can realize a bimodal-guided imaging effect by combining with photoacoustic imaging (PAI), and finally a multi-modal imaging anticancer diagnostic agent of CO gas treatment and PTT under the guidance of MRI/PAI is constructed. The anticancer diagnosis and treatment agent is prepared by wrapping a metal precursor, is not doped with metal, but does not have a T1-T2 bimodal imaging effect, so that the application of the anticancer diagnosis and treatment agent in tumor diagnosis and treatment integration is limited. Therefore, the preparation of the metal-doped nano-particle with the T1-T2 bimodal imaging effect is urgently needed.

Disclosure of Invention

In order to overcome the defects of the prior art, the manganese-mesoporous polydopamine nanoparticle with tumor microenvironment response, T1-T2 dual-mode magnetic resonance imaging effect and photothermal conversion property is prepared by adopting a doping method before polymerization, taking dopamine hydrochloride and Mn precursor as basic raw materials and F127 and TMB as templates.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a preparation method of manganese-mesoporous polydopamine nanoparticles, namely, a doping method before polymerization is adopted, and dopamine hydrochloride and Mn are firstly mixed²⁺Chelating to generate a complex, and then preparing the manganese-mesoporous polydopamine nanoparticle by using manganese chelate-dopamine as a basic raw material and F127 and TMB as templates.

As a preferred embodiment of the present invention, the preparation method of the manganese-mesoporous polydopamine nanoparticle specifically comprises the following steps:

s1, dissolving dopamine hydrochloride and hydrated manganese sulfate in water, and stirring in a dark place to obtain a manganese chelate-dopamine solution;

s2, adding the F127 and the manganese chelate-dopamine solution obtained in the step S1 into a mixed solution of ethanol and water, and stirring in a dark place until the mixture is uniformly mixed;

s3, adding a TMB solution (1,3, 5-trimethylbenzene) while shaking in water bath ultrasound, and continuously performing ultrasonic dispersion until the mixture is milky;

s4, dropwise adding a Tris (Tris) aqueous solution under stirring, and stirring in a dark place;

s5, centrifuging, resuspending and washing the solution stirred in the step S4 to obtain the manganese-mesoporous polydopamine nanoparticles (Mn-MPDA for short).

The invention adopts a doping method before polymerization, and dopamine hydrochloride is mixed with Mn before polymerization²⁺Chelating to generate a complex, and then preparing the Mn-MPDA by using manganese chelate-dopamine as a basic raw material and F127 and TMB as templates. The Mn-MPDA is polydopamine nanoparticles doped in metal manganese and having a mesoporous structure, and the Mn-MPDA retains a good photo-thermal effect of the MPDA. In addition, due to Mn²⁺The internal doping and the geometric constraint conformational action of the metal oxide can limit the free rotation of water molecules near Mn-MPDA and the diffusion of proximal water molecules, thereby shortening the T2 relaxation time and increasing r₁Relaxivity, thereby giving it a T1-T2 bimodal imaging function; the Mn-MPDA has tumor microenvironment responsiveness, can realize targeted release of the drug, has good biocompatibility, and can realize T1-T2 bimodal magnetic resonance imaging. The Mn-MPDA is expected to realize T1-T2 bimodal magnetic resonance imaging guide photothermal therapy, and provides a new idea for clinical cancer diagnosis and treatment.

In principle, Mn in the present invention²⁺Can be replaced by other metal elements such asFe²⁺That is, the metal elements which can achieve the same or similar effect as the present invention by using the method of the present invention are within the protection scope of the present invention.

Preferably, the mass ratio of the dopamine hydrochloride to the hydrated manganese sulfate in the step S1 is (30-35): 1. With the proportion, the Mn-MPDA can obtain the best doping effect.

Preferably, the mass-to-volume ratio of the hydrated manganese sulfate to water (generally ultrapure water) in step S1 is 1mg (1-1.5) mL.

Preferably, the mass-to-volume ratio of the F127 to the manganese chelate-dopamine solution in the step S2 is (100-150) mg:1 mL.

Preferably, the volume ratio of the ethanol, the water (generally ultrapure water) and the manganese chelate-dopamine solution in the step S2 is 20:20 (1-1.5).

Preferably, the volume ratio of the TMB solution to the manganese chelate-dopamine solution in step S3 is (2-4): 3. Further, the volume ratio of the TMB solution to the manganese chelate-dopamine solution is 1: 1.5.

Preferably, the concentration of the Tris aqueous solution in the step S4 is 20mg/mL, and the volume ratio of the Tris aqueous solution to the TMB solution is 5 (1-2).

Preferably, the stirring of step S1 is 150-200rpm for 22-26 h.

Preferably, the stirring of step S2 is 150-200rpm for 15-25 min.

Preferably, the frequency of the ultrasound in step S3 is (3-5) kHz, and the time is 2-5 min. Further, the frequency of the ultrasound is 4kHz, and the time is 3.5 min.

Preferably, the stirring of step S4 is 150-200rpm for 5-8 h.

Preferably, the centrifugation in step S5 is 13000rpm for 7 min.

Preferably, the solvent used in the resuspension in step S5 is absolute ethanol, and the resuspension process is repeated at least 2 times by using water bath ultrasound, wherein the frequency of the water bath ultrasound is (3-5) kHz, and the time is 25-30 min; further, the frequency of the water bath ultrasonic wave is 4kHz, and the time is 28 min.

Preferably, the solvent used for the washing in step S5 is water (typically ultrapure water), and the number of washing is not less than 2.

The invention also provides the manganese-mesoporous polydopamine nanoparticle prepared by the preparation method.

The invention also provides application of the manganese-mesoporous polydopamine nanoparticle in magnetic resonance imaging of tumors T1-T2.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a preparation method of manganese-mesoporous polydopamine nanoparticles, namely, a doping method before polymerization is adopted, and dopamine hydrochloride and Mn are firstly mixed²⁺Chelating to generate a complex, and then preparing the manganese-mesoporous polydopamine nanoparticle (Mn-MPDA) by using manganese chelate-dopamine as a basic raw material and F127 and TMB as templates. The Mn-MPDA has uniform particle size, a uniformly distributed mesoporous structure, good dispersibility in water and high Mn element doping efficiency and chelation; the drug has the responsiveness of tumor microenvironment, and can realize the targeted release of the drug; the photo-thermal conversion capability is good, and the photo-thermal stability is good; has better biocompatibility and can be used as T1 and T2 contrast agents for magnetic resonance imaging.

The Mn-MPDA has high doping efficiency and chelation, good dispersibility in water and good contrast effect, so that the problems of low metal ion loading efficiency and incompatibility with low-water-solubility metal ions in the preparation of the metal-loaded polydopamine hybrid material by doping metal salt after polymerization are solved. Meanwhile, the Mn-MPDA can realize T1-T2 bimodal magnetic resonance imaging, so that an MRI result is more accurate, and the problem of influence of MRI artifacts existing in a single mode in the magnetic resonance imaging on a diagnosis result is solved; in addition, the Mn-MPDA disclosed by the invention is an integrated nano diagnosis and treatment agent (contrast agent) which has tumor microenvironment response, good biocompatibility and photo-thermal capability and can perform T1-T2 bimodal imaging, can provide a new idea for cancer treatment, and solves the problems of poor targeting, large side effect and the need of auxiliary instruments for diagnosis in conventional chemotherapy, radiotherapy, surgery and other therapies.

Drawings

FIG. 1 is a diagram showing a change of a mixed solution during the preparation of Mn-MPDA nanoparticles;

FIG. 2 is a transmission electron micrograph of Mn-MPDA nanoparticles prepared in example 1;

FIG. 3 is a transmission electron micrograph of Mn-MPDA nanoparticles prepared in example 2;

FIG. 4 is a transmission electron micrograph of Mn-MPDA nanoparticles prepared in example 3;

FIG. 5 is a graph showing a distribution of the particle size of Mn-MPDA nanoparticles;

FIG. 6 is a scanning electron microscope image of Mn-MPDA nanoparticles (A is an electron microscope image of nanoparticles; B is an electron microscope analysis image [ O, N, Mn, C ] of each element in nanoparticles);

FIG. 7 is an X-ray energy spectrum (EDS) analysis of Mn-MPDA nanoparticles;

FIG. 8 is (A) X-ray photoelectron spectroscopy and (B) nitrogen adsorption and desorption isotherms of Mn-MPDA nanoparticles;

fig. 9 is a transmission electron microscope image of Mn-MPDA nanoparticles incubated in PBS solutions of different pH values and GSH contents for 24h (a: pH 7.4, GSH 0 mM; B: pH 5.0, GSH 0 mM; C: pH 7.4, GSH 10 mM; D: pH 5.0, GSH 10 mM);

FIG. 10 is a temperature rise curve of Mn-MPDA solutions of (A) different concentrations and (B) different laser powers under irradiation of near-infrared laser; (C) the change curve of the temperature of the Mn-MPDA solution along with time under the irradiation of near-infrared laser; (D) a photo-thermal effect graph of the Mn-MPDA solution under near-infrared laser irradiation (an inset graph is a negative logarithmic graph of cooling time and temperature);

FIG. 11 is a graph of (A) longitudinal relaxation rate (1/T1) and (B) transverse relaxation rate (1/T2) of Mn-MPDA as a function of carrier concentration; (ii) the (C) longitudinal relaxation rate (1/T1) and (D) transverse relaxation rate (1/T2) of MPDA-Mn as a function of carrier concentration;

FIG. 12 cell viability after incubation of different concentrations of Mn-MPDA with HUVEC cells for 24h and 48 h.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 preparation of manganese-mesoporous polydopamine nanoparticles by Pre-polymerization doping method

(1) Dissolving 90mg of dopamine hydrochloride and 3mg of hydrated manganese sulfate in 3mL of ultrapure water (the mass ratio of dopamine hydrochloride to hydrated manganese sulfate is 30: 1; the mass-volume ratio of hydrated manganese sulfate to ultrapure water is 1mg:1mL), stirring at 180rpm in a dark place for 24 hours to obtain a manganese chelate-dopamine solution;

(2) adding 36mg of surfactant F127 and 0.3mL of manganese chelate-dopamine solution obtained in the step (1) into a mixed solution of 6mL of ethanol and 6mL of ultrapure water (the mass-volume ratio of the F127 to the manganese chelate-dopamine solution is 120mg:1mL, and the volume ratio of the ethanol, the ultrapure water to the manganese chelate-dopamine solution is 20:20:1), and stirring at 180rpm for 20min in a dark place; as shown in fig. 1(1), the manganese chelate-dopamine solution and F127 are dispersed in a mixed solution of ethanol and water to form a white transparent solution;

(3) adding 0.2mL of 1,3, 5-Trimethylbenzene (TMB) solution into a water bath ultrasonic wave (4kHz) at 25 ℃ while shaking, and continuously performing ultrasonic dispersion for 3.5min to obtain milk white; as shown in fig. 1(2), after the organic template agent TMB is gradually added under the water bath ultrasound, the solution gradually turns to milky white;

(4) dropwise adding 1mL of Tris aqueous solution with the concentration of 20mg/mL under the magnetic stirring of 180rpm, and stirring for 6 hours at 180rpm in a dark place; as shown in FIG. 1(3), after adding alkaline Tris buffer, due to Mn²⁺The solution rapidly turns pink and gradually deepens;

(5) and finally, the obtained solution is subpackaged in a 2mL centrifuge tube, centrifugation is carried out at 13000rpm for 7min, the obtained precipitate is resuspended in absolute ethyl alcohol, water bath ultrasound (4kHz) at 25 ℃ is carried out for 28min, the repetition is carried out for 2 times, and then the washing is carried out for 2 times by using ultrapure water under the same condition, so that the obtained product is the manganese-mesoporous polydopamine nanoparticle (Mn-MPDA for short). As shown in fig. 1(4), after 6 hours of reaction, a black nanoparticle dispersion is finally formed, and the product Mn-MPDA nanoparticles can be obtained after centrifugal cleaning.

Example 2 preparation of manganese-mesoporous polydopamine nanoparticles by Pre-polymerization doping method

(1) Dissolving 99mg of dopamine hydrochloride and 3mg of hydrated manganese sulfate in 3.75mL of ultrapure water (the mass ratio of dopamine hydrochloride to hydrated manganese sulfate is 33: 1; the mass-volume ratio of hydrated manganese sulfate to ultrapure water is 1mg:1.25mL), stirring at 150rpm in a dark place for 26h to obtain a manganese chelate-dopamine solution;

(2) adding 30mg of surfactant F127 and 0.3mL of the manganese chelate-dopamine solution obtained in the step (1) into a mixed solution of 4.8mL of ethanol and 4.8mL of ultrapure water (the mass-volume ratio of the F127 to the manganese chelate-dopamine solution is 100mg:1mL, and the volume ratio of the ethanol to the ultrapure water to the manganese chelate-dopamine solution is 20:20:1.25), and stirring at 150rpm in a dark place for 25 min;

(3) adding 0.3mL of 1,3, 5-Trimethylbenzene (TMB) solution into a water bath ultrasonic wave (4kHz) at 25 ℃ while shaking, and continuously performing ultrasonic dispersion for 2min to obtain milk white;

(4) dropwise adding 1mL of Tris aqueous solution with the concentration of 20mg/mL under magnetic stirring at 180rpm, and stirring at 150rpm in a dark place for 8 hours;

(5) and finally, the obtained solution is subpackaged in a 2mL centrifuge tube, centrifugation is carried out at 13000rpm for 7min, the obtained precipitate is resuspended in absolute ethyl alcohol, water bath ultrasound (4kHz) at 25 ℃ is carried out for 25min, the process is repeated for 3 times, and then ultrapure water is used for washing for 3 times under the same condition, so that the obtained product is the manganese-mesoporous polydopamine nanoparticle (Mn-MPDA for short).

Example 3 preparation of manganese-mesoporous polydopamine nanoparticles by Pre-polymerization doping method

(1) Dissolving 105mg of dopamine hydrochloride and 3mg of hydrated manganese sulfate in 4.5mg of ultrapure water (the mass ratio of dopamine hydrochloride to hydrated manganese sulfate is 35: 1; the mass-volume ratio of hydrated manganese sulfate to ultrapure water is 1mg:1.5mL), stirring at 200rpm in a dark place for 22 hours to obtain a manganese chelate-dopamine solution;

(2) adding 45mL of surfactant F127 and 0.3mL of manganese chelate-dopamine solution obtained in the step (1) into a mixed solution of 4mL of ethanol and 4mL of ultrapure water (the mass-volume ratio of the F127 to the manganese chelate-dopamine solution is 150mg:1mL, and the volume ratio of the ethanol, the ultrapure water to the manganese chelate-dopamine solution is 20:20:1.5), and stirring at 150-200rpm for 25min in a dark place;

(3) adding 0.4mL of 1,3, 5-Trimethylbenzene (TMB) solution into a water bath ultrasonic wave (4kHz) at 25 ℃ while shaking, and continuously performing ultrasonic dispersion for 5min to obtain milk white;

(4) dropwise adding 1mL of Tris aqueous solution with the concentration of 20mg/mL under magnetic stirring at 180rpm, and stirring at 200rpm in a dark place for 5 hours;

(5) and finally, the obtained solution is subpackaged in a 2mL centrifuge tube, centrifugation is carried out at 13000rpm for 7min, the obtained precipitate is resuspended in absolute ethyl alcohol, water bath ultrasound (4kHz) at 25 ℃ is carried out for 30min, the repetition is carried out for 2 times, and then the washing is carried out for 2 times by using ultrapure water under the same condition, so that the obtained product is the manganese-mesoporous polydopamine nanoparticle (Mn-MPDA for short).

Comparative example 1 preparation of mesoporous polydopamine nanoparticles of manganese (MPDA-Mn) by doping method after polymerization

(1) Dissolving 90mg of dopamine hydrochloride and 36mg of surfactant F127 in a mixed solution of 6mL of ethanol and 6mL of ultrapure water (the mass ratio of the dopamine hydrochloride to the surfactant F127 is 1: 4; and the mass-volume ratio of the dopamine hydrochloride to the ultrapure water is 3mg:2mL), stirring at 180rpm in a dark place for 20min until the dopamine hydrochloride and the surfactant F127 are uniformly mixed;

(2) adding 0.2mL of 1,3, 5-Trimethylbenzene (TMB) solution into a water bath ultrasonic wave (4kHz) at 25 ℃ while shaking, and continuously performing ultrasonic dispersion for 4min to obtain milk white;

(3) slowly adding 1mL of Tris aqueous solution (20mg/mL) dropwise into the solution obtained in the step (2) under magnetic stirring at 180rpm (the volume ratio of the solution obtained in the step (2) to Tris aqueous solution is 12.3:1), and stirring at 180rpm for 6 hours in a dark place;

(4) the obtained solution is subpackaged in a 2mL centrifuge tube, centrifuged at 13000rpm for 7min, the obtained precipitate is resuspended in absolute ethyl alcohol, water bath ultrasound (4kHz) is carried out at 25 ℃ for 28min, washing is repeated for 2 times, and then washing is carried out for 2 times by using ultrapure water under the same condition, and the obtained product is MPDA nano particles;

(5) the MPDA nanoparticles are resuspended in 5mL of ultrapure water, 0.3mL of hydrated manganese sulfate aqueous solution (1mg/mL) is added, magnetic stirring is carried out for 24 hours at 180rpm under the condition of keeping out of the sun, finally the mixture is subpackaged in 2mL centrifuge tubes, centrifugation is carried out for 7 minutes at 13000rpm, the obtained precipitate is resuspended in the ultrapure water, water bath ultrasound (4kHz) at 25 ℃ is carried out for 28 minutes, and the MPDA-Mn nanoparticles can be obtained after washing for 2 times.

Experimental example 1 characterization of Mn-MPDA morphology and particle size

The Mn-MPDA prepared in the examples 1-3 is prepared into a sample solution of 100. mu.g/mL, 10. mu.L of the sample solution is dripped on the front surface of a carbon-supported membrane copper mesh, the sample solution is naturally dried in a drier at room temperature, and then the microscopic morphology (morphology, particle size and dispersion condition) of the Mn-MPDA is observed by using a transmission electron microscope under the voltage condition of 120 KV.

As can be seen from FIGS. 2 to 4, the prepared Mn-MPDA has a narrow particle size distribution range, is spherical, has a uniform particle size of about 130nm, and has a uniformly distributed mesoporous structure.

Meanwhile, the Mn-MPDA nanoparticles prepared in example 1 were resuspended in ultrapure water, diluted to 50 μ g/mL and ultrasonically homogenized, and the hydrated particle size, polydispersity index and Zeta potential of the nanoparticle sample were measured by a dynamic light scattering method.

As shown in Table 1 and FIG. 5, the Mn-MPDA nanoparticles have good dispersibility, a hydrated particle diameter of about 201.4nm, and are electrically neutral. The transmission electron microscopy and Dynamic Light Scattering (DLS) measurements differ because the latter result in a hydrated nanoparticle size, and the solvent effect causes the nanoparticles to exhibit a larger hydrated particle size.

TABLE 1 dispersibility, particle size and Zeta potential of Mn-MPDA nanoparticles

In addition, the experimental results of examples 2 to 3 were similar to those of example 1 and will not be described herein.

Experimental example 2 SEM scanning Electron microscopy characterization and EDS energy Spectroscopy analysis of Mn-MPDA

The Mn-MPDA prepared in the example 1 is prepared into a 50 mu G/mL Mn-MPDA ethanol solution, a small amount of Mn-MPDA nano-particle ethanol solution with a certain concentration is dripped on an aluminum foil, the dried Mn-MPDA nano-particle ethanol solution is placed under a G500 high-resolution field emission scanning electron microscope (Gemini500, Zeiss/Bruker) to observe the particle morphology, and the relative content of each element in the Mn-MPDA is determined through X-ray energy spectrum analysis (EDS).

As shown in fig. 6, it can be visually observed that the Mn-MPDA nanoparticles are in a three-dimensional spherical shape with a uniform particle size and have a surface mesoporous structure consistent with a transmission electron microscope image. As shown in fig. 7, an element map of Mn-MPDA nanoparticles was obtained by EDS analysis, and a uniform distribution of C, O, N, Mn elements was observed, indicating that Mn elements were uniformly doped in the interior of Mn-MPDA nanoparticles.

In addition, the experimental results of examples 2 to 3 were similar to those of example 1 and will not be described herein.

Experimental example 3X-ray photoelectron spectroscopy analysis and nitrogen adsorption curve characterization of Mn-MPDA

An appropriate amount of the Mn-MPDA sample prepared in example 1 was lyophilized and then uniformly spread on an aluminum foil with double-sided adhesive tape (about 2mm × 2mm), and the Mn element content and valence state in the sample were measured by an X-ray photoelectron spectrometer (Nexsa, Thermo Fisher) after being pressed flat by a tablet press.

As shown in FIG. 8A, the Mn valence in the Mn-MPDA sample is Mn as compared with the standard spectrum of Mn element²⁺. The nitrogen adsorption-desorption isotherm is shown in FIG. 8B, and the BET specific surface area of the Mn-MPDA is calculated to be 42.2571m²(ii)/g, about twice as large as MPDA having a particle size of about 200nm, indicating that Mn-MPDA having a smaller particle size has a higher specific surface area.

In addition, the experimental results of examples 2 to 3 were similar to those of example 1 and will not be described herein.

Experimental example 4 study on pH and Glutathione (GSH) responsiveness of Mn-MPDA

An appropriate amount of the Mn-MPDA sample prepared in example 1 was dispersed in PBS buffers with different pH values and GSH contents, and diluted to 200. mu.g/mL. The specific grouping is as follows: a pH 7.4, GSH 0mM (control); b, pH is 5.0, GSH is 0 mM; c, pH 7.4, GSH 10 mM; d, pH is 5.0, GSH is 10 mM. The above solutions were incubated in a constant temperature shaker for 24h in the dark (37 ℃ C., 100rpm), 10. mu.L of each was dropped onto a copper mesh, dried in the air and observed for morphological changes with a transmission electron microscope.

As shown in fig. 9, the Mn-MPDA sample morphology in control a was unchanged; partial degradation of the nanoparticles was observed for the samples of group B/C; meanwhile, the acidic condition of pH value of 5.0 and the D group sample of 10mM reduced glutathione are completely degraded, which shows that Mn-MPDA has tumor microenvironment responsiveness and lays a foundation for realizing the targeted release of the drug later.

In addition, the experimental results of examples 2 to 3 were similar to those of example 1 and will not be described herein.

Experimental example 5 in vitro photothermal Properties of Mn-MPDA

Mn-MPDA prepared in example 1 was formulated into Mn-MPDA aqueous solutions of various concentrations (0ug/mL, 100ug/mL, 200ug/mL, 400ug/mL), which were then placed in 2mL cuvettes and irradiated with 808nm near-infrared laser (0.5W/cm)²、1.0W/cm²、1.5W/cm²、2.0W/cm²) And characterizing the temperature rise curve, the photo-thermal stability and the photo-thermal conversion efficiency of the Mn-MPDA nano particles.

As shown in fig. 10A/B, the Mn-MPDA material exhibited concentration, laser power dependent temperature rise changes, and the results indicate that Mn-MPDA still has good photothermal conversion capability, and can efficiently convert near-infrared light energy into thermal energy. As shown in FIG. 10C, the temperature change (Δ T) of the Mn-MPDA solution in the four temperature increase and decrease cycles is not much different, indicating that the Mn-MPDA has good photo-thermal stability. The photothermal conversion efficiency (η) of Mn-MPDA was then calculated, and as shown in fig. 10D, it was calculated by equation (1) to have a high photothermal conversion rate, with the η value of 67.97%, and τ s in the equation was linearly fitted to 256.44.

In addition, the experimental results of examples 2 to 3 were similar to those of example 1 and will not be described herein.

Experimental example 6 magnetic resonance imaging study of Mn-MPDA

The Mn-MPDA prepared in example 1 and the MPDA-Mn prepared in comparative example 1 were prepared as aqueous solutions, and then subjected to gradient dilution (0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35mM) respectively, 150. mu.L of each solution was added to a 96-well plate, and scanned under a 1.5T MRI machine to characterize the T1-weighted and T2-weighted MRI imaging effects.

As shown in FIGS. 11A/B, Mn-MPDA has a higher longitudinal relaxation rate (147.27 mM) than MPDA-Mn (FIG. 11C/D)^-1S^-1) And transverse relaxation Rate (63.67 mM)^-1S^-1). As shown in the map image of fig. 11, as the Mn concentration increases, the T1 weighting signal of Mn-MPDA gradually decreases and the T2 weighting signal gradually increases, indicating that Mn-MPDA can be used for magnetic resonance imaging as both T1 and T2 contrast agents.

In addition, the experimental results of examples 2 to 3 were similar to those of example 1 and will not be described herein.

EXAMPLE 7 in vitro biocompatibility study of Mn-MPDA

The effect of different concentrations (9.375, 18.75, 37.5, 75, 150, 300. mu.g/mL) of Mn-MPDA (exemplified in example 1) on the viability of HUVEC cells (human umbilical vein endothelial cells) was investigated by thiazole blue (MTT) colorimetry. The absorbance A at 570nm was measured with a microplate reader. Cell viability was calculated according to equation (2):

as shown in FIG. 12, the viability of HUVEC cells remained essentially unchanged, all approaching 100%, as the sample concentration increased. When the concentration of Mn-MPDA is as high as 300 mu g/mL, the survival rate of HUVEC cells after 48h of co-incubation is still over 80 percent, which indicates that Mn-MPDA has better biocompatibility.

In addition, the experimental results of examples 2 to 3 were similar to those of example 1 and will not be described herein.

It can be seen from the comprehensive experimental examples 1-7 that the Mn-MPDA prepared by the invention has the advantages of uniform particle size, uniform mesoporous structure, good dispersibility in water, high Mn element doping efficiency and chelation effect, and narrow spherical particle size distribution; the drug has the responsiveness of tumor microenvironment, and can realize the targeted release of the drug; the photo-thermal conversion capability is good, and the photo-thermal stability is good; has better biocompatibility; can be used as T1 and T2 contrast agents for magnetic resonance imaging.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.