CN111821281A

CN111821281A - Gd/Tm-PB nano-particles, preparation method thereof, multifunctional composite material, and preparation method and application thereof

Info

Publication number: CN111821281A
Application number: CN202010759054.1A
Authority: CN
Inventors: 李玲; 许明悦; 王应席
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-10-27

Abstract

The invention discloses Gd/Tm-PB nano particles and a preparation method thereof, and a multifunctional composite material and a preparation method and application thereof. The Gd/Tm-PB nano-particle is formed by gadolinium and thulium co-doped Prussian blue nano-particles, the nano-particle is spherical or cubic, and the particle size of the nano-particle is 200-500 nm. The Gd/Tm-PBA composite material wrapped by the ZIF-8/PDA takes the Gd/Tm-PB nano particles as cores and the ZIF-8/PDA as shells, and the particle size of the nano particles is 200-550 nm. The Gd/Tm-PB nano-particles have a photo-thermal adjustable effect and can be used for photo-thermal treatment in a tumor treatment process. The Gd/Tm-PBA composite material wrapped by the ZIF-8/PDA can be used as a contrast agent in MRI multimode imaging, can also be used as a release carrier of an antitumor drug adriamycin, can be used for early diagnosis and later treatment of tumors, and has important significance for tumor treatment.

Description

Gd/Tm-PB nano-particles, preparation method thereof, multifunctional composite material, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to Gd/Tm-PB nano particles and a preparation method thereof, and a multifunctional composite material and a preparation method and application thereof.

Background

Cancer is one of three major diseases threatening human health, about 5-6 million people die of cancer every year, and how to improve diagnostic and therapeutic methods and effectively treat tumors has been a long-standing focus of research in the fields of materials, chemistry and medicine.

Chemotherapy, a common cancer treatment, inhibits the growth and proliferation of tumor cells. However, poor selectivity of chemotherapeutic drugs not only kills tumor cells but also normal cells, and chemotherapy inevitably results in drug resistance, impairing the therapeutic effect. To overcome the side effects of chemotherapy, targeted drug carriers are aimed at controlling the release of the drug. Because the pH value and the Glutathione (GSH) concentration of the cancer cells are different from those of normal cells, the pH/GSH double-target drug carrier can accurately target the drug to the tumor part, and avoids the toxic and side effects of the drug on the normal cells.

In order to compensate for the problem of multidrug resistance caused by chemotherapy, chemotherapy is often combined with other therapies for synergistic treatment. As a new generation of cancer treatment, photothermal therapy (PTT) has received much attention, which kills cancer cells at a tumor site using light-induced heat. Therefore, in clinical treatment, combination of photothermal therapy and chemotherapy is expected to improve the treatment efficiency.

With the development of sophisticated medical conceptsVisual observation is needed in the treatment process, so that the treatment effect is emphasized, and the treatment scheme is adjusted to achieve the optimal treatment effect. Magnetic Resonance Imaging (MRI) is widely used in the medical and biological fields, providing a good auxiliary function for cancer detection. Magnetic Resonance Imaging (MRI) has become one of the important tools for clinical disease diagnosis due to its advantages of higher tissue resolution, multi-parameter and multi-directional imaging. However, there is usually little difference in signal intensity between benign and malignant tumor tissue and between tumor tissue and normal tissue. Therefore, MRI contrast agents are important in order to change the signal intensity at the tumor site and improve the accuracy of MRI diagnosis. T is₁/T₂The dual-mode MRI utilizes the signal intensity difference generated by hydrogen protons under different scanning sequence conditions, can realize different imaging results, and is helpful to improve the accuracy of tumor diagnosis. Thus, at T₁-T₂The best treatment effect is expected to be obtained by performing the cooperative treatment under the guidance of MRI bimodal imaging.

Traditionally, prussian blue particles were used for T₂Photothermal properties of MRI imaging and simple fluorescence display, the uncontrollable photothermal properties greatly limit its application in the biomedical field.

As a new type of hybrid organic-inorganic material, MOFs have received a great deal of attention in drug loading applications due to their high porosity, large specific surface area, surface modification and other properties. From Zn²⁺ZIF-8, which is composed of 2-methylimidazole, is sensitive to a slightly acidic environment, which is consistent with the slightly acidic environment in the tumor environment, and helps to release the drug to cancer cells. However, some tissues also show weak acidity. The release of the pH-responsive drug is not precise enough, which will cause some damage to normal tissues during the release process. Due to the high Glutathione (GSH) content in the tumor cells, the drug can be accurately released by the pH/GSH double reaction. PDA has good biocompatibility and can be applied to the surface of almost any material. Oxidation occurs during polymerization of dopamine, which makes it possible for reduced glutathione to disrupt its oxidation and trigger degradation of polydopamine, and thus it is well suited for use in tumor environment designResponse to high GSH.

Therefore, the development of the prussian blue and PDA diagnosis and treatment integrated nano-carrier has important significance for tumor treatment.

Disclosure of Invention

Based on the prior art, the invention provides Gd/Tm-PB nano particles and a preparation method thereof, a multifunctional composite material and a preparation method and application thereof, and the Gd/Tm-PB nano particles have a photo-thermal adjustable effect and can be used for photo-thermal treatment in a tumor treatment process.

The Gd/Tm-PB nano-particle is simple in preparation method and low in preparation cost.

The Gd/Tm-PBA composite material wrapped by the ZIF-8/PDA can be used as a contrast agent in MRI multimode imaging, can also be used as a release carrier of an antitumor drug adriamycin, can be used for early diagnosis and later treatment of tumors, and has important significance for tumor treatment.

The ZIF-8/PDA-wrapped Gd/Tm-PBA composite material is simple in preparation method and low in preparation cost.

The technical scheme adopted for realizing the above purpose of the invention is as follows:

a Gd/Tm-PB nanoparticle characterized by: the nano-particles are formed by gadolinium and thulium co-doped Prussian blue nano-particles, the nano-particles are spherical or cubic, and the particle size of the nano-particles is 200-500 nm.

A preparation method of Gd/Tm-PB nanoparticles comprises the following steps:

1. uniformly mixing ethanol and deionized water to obtain a mixed solvent, dissolving a thulium source and a gadolinium source in the mixed solution to obtain the mixed solution, wherein the molar ratio of the thulium element to the gadolinium element is 1: 1 to 95;

2. dropwise adding the mixed solution into a potassium ferricyanate solution, wherein the molar ratio of potassium ferricyanate to thulium gadolinium is 0.8-1.5:1, after dropwise adding is completed, adding polyvinylpyrrolidone, wherein the mass ratio of polyvinylpyrrolidone to potassium ferricyanate is 3.7-24:1, then heating to 140-160 ℃, carrying out hydrothermal reaction for 24-36h at 140-160 ℃, after the reaction is completed, centrifuging, washing the obtained solid, and finally drying to obtain the Gd/Tm-PB nano-particles, which are recorded as Gd/Tm-PB.

Further, the thulium source is thulium chloride, and the gadolinium source is gadolinium nitrate.

Further, the concentration of thulium in the mixed solution is 0.128-0.854 mmol/L, and the concentration of gadolinium is 1.28-2.00 mmol/L.

Furthermore, the concentration of the potassium ferricyanate is 0.0017-0.025 mol/L.

The multifunctional composite material is a core-shell structure nanoparticle with Gd/Tm-PB nanoparticle as a core and ZIF-8/PDA as a shell, and the particle size of the nanoparticle is 200-550 nm.

A preparation method of a multifunctional composite material comprises the following steps:

1. dispersing Gd/Tm-PB as defined in claim 1 in methanol or deionized water to obtain turbid liquid A, dissolving a zinc source in the turbid liquid A, wherein the ratio of zinc element to Gd/Tm-PB is 0.3-8:1 to obtain turbid liquid B;

2. dissolving 2-methylimidazole in methanol or deionized water to obtain a 2-methylimidazole solution, dropwise adding the 2-methylimidazole solution into the turbid solution B, wherein the molar ratio of 2-methylimidazole to zinc is 1-15: 1, after dropwise adding, stirring at room temperature for reaction for 20-60min, after the reaction is finished, centrifuging, washing and drying the obtained precipitate to obtain a Gd/Tm-PB nanoparticle material wrapped by ZIF-8, and marking the Gd/Tm-PB nanoparticle material as Gd/Tm-PB @ ZIF-8;

3. dispersing Gd/Tm-PB @ ZIF-8 in a dopamine hydrochloride solution, stirring the dopamine hydrochloride solution and the Gd/Tm-PB @ ZIF-8 in a mass ratio of 2.5-5:1 and at a concentration of 0.05-0.2mg/mL for 2-12h to obtain a turbid solution D, centrifuging the turbid solution D at a rotation speed of 8000-00 rpm for 15-20min, washing and drying the obtained precipitate to obtain the Gd/Tm-PBA composite material wrapped by ZIF-8/PDA, which is recorded as Gd/Tm- @ ZIF-8/PDA.

The application of a multifunctional composite material in MRI multimode imaging.

An application of multifunctional composite material in serving as an anticancer drug release carrier.

Further, the anticancer drug is adriamycin.

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

1. the Gd/Tm-PB can adjust the morphology of the gadolinium-Tm-PB by adjusting the doping ratio of the gadolinium to the thulium so as to obtain proper particle size and uniform morphology and facilitate entering the interior of a tumor.

2. The Gd/Tm-PB can adjust the fluorescence property of the Gd/Tm-PB by adjusting the doping ratio of the gadolinium to the thulium, and improve the non-adjustable property of Prussian blue fluorescence.

3. The Gd/Tm-PB can adjust the photo-thermal performance of the Gd/Tm-PB by adjusting the doping ratio of the gadolinium to the thulium, can design the photo-thermal performance of the Gd/Tm-PB aiming at the treatment of different tumor cells, and provides scientific basis for the photo-thermal treatment of tumors.

4. The Gd/Tm-PB @ ZIF-8/PDA can be used as a contrast agent, can change the signal intensity of a tumor part, and can be used for T_1/T₂MRI bimodal imaging, improving the accuracy of MRI diagnosis.

5. The Gd/Tm-PB @ ZIF-8/PDA disclosed by the invention can be used as a carrier of an antitumor drug adriamycin, has a PH/GSH dual response, and enables the drug adriamycin to be released in a weak acid environment and a high glutathione concentration environment, the release is lower in a neutral environment and a low glutathione concentration environment, and the toxic and side effects of the drug adriamycin on normal cells are reduced.

6. The Gd/Tm-PB @ ZIF-8/PDA disclosed by the invention has good photo-thermal stability, and can be used for photo-thermal treatment in a tumor treatment process.

Drawings

FIG. 1 is a TEM image of a ZIF-8/PDA wrapped Gd/Tm-PBA composite prepared in example 1.

FIG. 2 is an XRD pattern of a ZIF-8/PDA encapsulated Gd/Tm-PBA composite prepared in example 1.

FIG. 3 is an infrared spectrum of a ZIF-8/PDA encapsulated Gd/Tm-PBA composite prepared in example 1.

FIG. 4 is an SEM image of Gd/Tm-PB nanoparticles prepared in examples 1-6.

Wherein FIG. 4(a) is an SEM picture of Gd/Tm-PB-II prepared in example 2, FIG. 4(b) is an SEM picture of Gd/Tm-PB-III prepared in example 3, FIG. 4(c) is an SEM picture of Gd/Tm-PB-IV prepared in example 4, FIG. 4(d) is an SEM picture of Gd/Tm-PB-I prepared in example 1, FIG. 4(e) is an SEM picture of Gd/Tm-PB-V prepared in example 5, FIG. 4(f) is an SEM picture of Gd/Tm-PB-VI prepared in example 6, and FIG. 4(g) is an SEM picture of Gd-PB prepared in comparative example 1.

FIG. 5 is a fluorescence plot of Gd/Tm-PB nanoparticles prepared in examples 1-6 at an excitation wavelength of 365 nm.

FIG. 6 is a graph of photothermal effects of the Gd/Tm-PB nanoparticles prepared in examples 1-6 under near infrared irradiation.

FIG. 7 is MRI images of mice at different time points before and after injection of the ZIF-8/PDA encapsulated Gd/Tm-PBA composite material prepared in example 1.

Wherein, FIG. 7(a) shows T before and after injecting the ZIF-8/PDA-encapsulated Gd/Tm-PBA composite material prepared in example 1 into a mouse₁MRI image, FIG. 7(b) shows T before and after mouse injection of the ZIF-8/PDA-wrapped Gd/Tm-PBA composite prepared in example 1₂-MRI images.

FIG. 8 is T at different time points before and after mice were injected with the ZIF-8/PDA-encapsulated Gd/Tm-PBA composite prepared in example 1₁、T₂The imaged signal profiles are weighted.

Wherein FIG. 8(a) shows T before and after mouse injection of the ZIF-8/PDA-encapsulated Gd/Tm-PBA composite material prepared in example 1₁FIG. 8(b) is a graph showing the T-values before and after injecting the ZIF-8/PDA-encapsulated Gd/Tm-PBA composite material prepared in example 1 into a mouse₂The imaged signal profiles are weighted.

FIG. 9 is a graph showing the effect of the ZIF-8/PDA encapsulated Gd/Tm-PB composite material prepared in example 1 on doxorubicin loading.

FIG. 10 is a graph showing the effect of the ZIF-8/PDA encapsulated Gd/Tm-PB composite material prepared in example 1 on the controlled release of doxorubicin at different pH and GSH concentrations.

FIG. 11 is a graph showing the effect of controlled release of doxorubicin from the ZIF-8/PDA encapsulated Gd/Tm-PB composites prepared in example 1 under near infrared irradiation and at different pH and GSH concentrations.

FIG. 12 is a graph of photothermal stability of the ZIF-8/PDA encapsulated Gd/Tm-PB composite prepared in example 1.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

1. Uniformly mixing 5ml of ethanol and 2.5ml of deionized water to obtain a mixed solvent, dissolving 0.01624g of gadolinium nitrate hexahydrate and 0.0046g of thulium chloride hexahydrate in the mixed solvent to obtain a mixed solution, dissolving 0.01317g of potassium ferricyanide in 2.5ml of deionized water to obtain a potassium ferricyanate solution, slowly dropwise adding the potassium ferricyanate solution into the mixed solution while stirring at room temperature, adding 0.2g of PVP (polyvinylpyrrolidone) after dropwise adding is finished, stirring at room temperature for 30min, transferring the obtained mixed solution into a polytetrafluoroethylene autoclave, heating to 140 ℃, reacting at 140 ℃ for 24h, cooling to room temperature after the reaction is finished, centrifuging the obtained mixed product, collecting precipitate, washing the precipitate with distilled water (3 times) and ethanol (2 times) in sequence, drying the obtained solid in an oven at 60 ℃ for 24h to obtain Gd/Tm-PB nanoparticles, is marked as Gd/Tm-PB-I, wherein the molar ratio of the thulium element to the thulium gadolinium is 25 percent;

2. 50mg of Gd/Tm-PB-I is weighed firstly and added into 20ml of methanol, ultrasonic dispersing for 30min at room temperature to obtain turbid solution A, weighing 0.12g zinc nitrate, dissolving in the turbid solution A, obtaining turbid solution B, then weighing 0.6212g of 2-methylimidazole and dissolving in 5ml of methanol to obtain 2-methylimidazole methanol solution, then slowly dripping the 2-methylimidazole methanol solution into the human turbid solution C while stirring at room temperature, after finishing dripping, magnetically stirring at room temperature for 30min, centrifuging the obtained mixed product, collecting precipitate, sequentially washing the precipitate with distilled water (3 times) and ethanol (2 times), drying the obtained solid in an oven at 60 ℃ for 24h, and marking the material of Gd/Tm-PB nano particles wrapped by ZIF-8 as Gd/Tm-PB @ ZIF-8;

3. weighing 0.023g of Gd/Tm-PB @ ZIF-8, adding the weighed Gd/Tm-PB @ ZIF-8 into 20mL of 0.1mg/mL hydrochloric acid dopamine solution, performing ultrasonic dispersion for 2 hours at room temperature to obtain turbid liquid C, centrifuging the turbid liquid C for 20 minutes under the condition of 10000r/min, collecting precipitates after the centrifugation is finished, sequentially washing the precipitates with distilled water (3 times) and ethanol (2 times), and performing vacuum drying on the obtained solids to obtain the multifunctional composite material, wherein the mark is Gd/Tm-PB @ ZIF-8/PDA.

The Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 was scanned by a scanning electron microscope, and the SEM image obtained is shown in FIG. 1, wherein in FIG. 1, black on the inside is core Gd/Tm-PB nanoparticles, the Gd/Tm-PB nanoparticles are similar to spherical or cubic, the average particle size is 200nm, and light gray on the outside is coated with ZIF-8/PDA.

The Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 was subjected to X-ray diffraction analysis, and the obtained XRD pattern is shown in fig. 2, and as can be seen from fig. 2, the synthesized Gd/Tm-PB @ ZIF-8 and Gd/Tm-PB @ ZIF-8/PDA had characteristic diffraction peaks similar to those of Gd/Tm-PB, which indicates that they have substantially the same crystal structure, and it is noted that, after wrapping of Gd/Tm-PB @ ZIF-8, a new diffraction peak appears at 2 θ ═ 7.35 ° which confirms the presence of a shell of ZIF-8, whereas, after wrapping of Gd/Tm-PB @ ZIF-8/PDA, two peaks respectively appear at 2 θ ═ 7.65 °, 9.94 ° which confirms the successful wrapping.

The Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 was subjected to infrared spectroscopic analysis, and the obtained infrared spectrum is shown in FIG. 3, from which it can be seen that Gd/Tm-PB is 2074cm^-1The characteristic peak in the vicinity is attributed to-CN at 601cm^-1The characteristic peak in the vicinity is attributed to the tensile vibration peak of v (Fe-CN). As can be seen from the Gd/Tm-PB @ ZIF-8 spectrum, the absorption spectrum of Gd/Tm-PB @ ZIF-8 is similar to that of Gd/Tm-PB, and is 1639cm^-1A vibration peak of C ═ N bond in 2-methylimidazole appears nearby, which indicates that the coupling of ZIF-8 and Gd/Tm-PB is successful at 1580cm^-1The characteristic peak of PDA benzene ring skeleton is detected, which confirms the successful encapsulation of PDA.

Comparative example 1

Uniformly mixing 5ml of ethanol and 2.5ml of deionized water to obtain a mixed solvent, dissolving 0.02166g of gadolinium nitrate hexahydrate in the mixed solvent, to obtain a mixed solution, 0.01317g of potassium ferricyanide was dissolved in 2.5ml of deionized water, obtaining potassium ferricyanate solution, slowly dripping the potassium ferricyanate solution into the mixed solution at room temperature while stirring, adding 0.2g of PVP (polyvinylpyrrolidone) after finishing dripping, then stirring for 30min at room temperature, transferring the obtained mixed solution into a polytetrafluoroethylene autoclave, heating to 140 ℃, reacting at 140 ℃ for 24h, cooling to room temperature after the reaction is finished, centrifuging the obtained mixed product, collecting precipitate, sequentially washing the precipitate with distilled water (3 times) and ethanol (2 times), and drying the obtained solid in an oven at 60 ℃ for 24h to obtain Gd-doped Prussian blue analogue nanoparticles which are marked as Gd-PB.

Example 2

Much the same as the procedure and operation of example 1, except that the mole ratio of thulium element to thulium gadolinium is different when preparing Gd/Tm-PB nanoparticles in step 1, in this example, the amount of gadolinium nitrate hexahydrate is 0.02036g, the amount of thulium chloride hexahydrate is 0.0011g, Gd/Tm-PB nanoparticles prepared at this mole ratio of thulium element to thulium gadolinium element is denoted as Gd/Tm-PB-ii, wherein the mole ratio of thulium element to thulium gadolinium element is 6%.

Example 3

Much the same as the procedure and operation of example 1, except that the mole ratio of thulium element to thulium gadolinium is different when preparing Gd/Tm-PB nanoparticles in step 1, in this example, the amount of gadolinium nitrate hexahydrate is 0.01999g, the amount of thulium chloride hexahydrate is 0.0015g, and Gd/Tm-PB nanoparticles prepared at this mole ratio of thulium element to thulium gadolinium element is denoted as Gd/Tm-PB-iii, wherein the mole ratio of thulium element to thulium gadolinium element is 8%.

Example 4

Much the same as the steps and operations of example 1, except that the molar ratio of thulium element to thulium gadolinium is different when preparing Gd/Tm-PB nanoparticles in step 1, in this example, the amount of gadolinium nitrate hexahydrate is 0.0194g, the amount of thulium chloride hexahydrate is 0.00184g, and Gd/Tm-PB nanoparticles prepared at this molar ratio of thulium element to thulium gadolinium element are denoted as Gd/Tm-PB-iv, wherein the molar ratio of thulium element to thulium gadolinium element is 10%.

Example 5

Much the same as the procedure and operation of example 1, except that the mole ratio of thulium element to thulium gadolinium is different when preparing Gd/Tm-PB nanoparticles in step 1, in this example, the amount of gadolinium nitrate added is 0.01516g, the amount of thulium chloride added is 0.0055g, and Gd/Tm-PB nanoparticles prepared at this mole ratio of thulium element to thulium gadolinium are denoted as Gd/Tm-PB-v, wherein the mole ratio of thulium element to thulium gadolinium element is 30%.

Example 6

Much the same as the procedure and operation of example 1, except that the mole ratio of the thulium element to the thulium gadolinium element is not the same when preparing Gd/Tm-PB nanoparticles in step 1, in this example, the amount of gadolinium nitrate hexahydrate is 0.013g, the amount of thulium chloride hexahydrate is 0.0074g, and Gd/Tm-PB nanoparticles prepared at this mole ratio of the thulium element to the thulium gadolinium element are denoted as Gd/Tm-PB-vi, wherein the mole ratio of the thulium element to the thulium gadolinium element is 40%.

First, the Gd/Tm-PB morphology regulation performance test

The test method comprises the following steps:

0.05mg of Gd-PB, Gd/Tm-PB-I, Gd/Tm-PB-II, Gd/Tm-PB-III, Gd/Tm-PB-IV, Gd/Tm-PB-V and Gd/Tm-PB-VI are respectively weighed, then the mixture is respectively dispersed in 5mL of ethanol solution, one to two drops of the seven turbid liquids are respectively dripped on seven silicon chips to prepare silicon chip samples for SEM test, and the microscopic morphology of the silicon chip samples is observed by an electronic scanner, as shown in figure 4.

And (3) test results:

as can be seen from FIG. 4, the Gd-PB prepared in comparative example 1 is not suitable for in vivo use because of only doping Gd, and the particle size of Gd/Tm-PB-II, Gd/Tm-PB-III, Gd/Tm-PB-IV, Gd/Tm-PB-I, Gd/Tm-PB-V and Gd/Tm-PB-VI is 200-500nm, especially when the Tm doping ratio reaches 25%, the obtained Gd/Tm-PB-I has uniform particle size, and the particle size is 200-300nm, which is suitable for the nano-scale particle size of tumor, thereby showing that the Gd/Tm-PB synthesized by doping of Tm element has a role in regulating and controlling the appearance.

Experiment II, fluorescence regulation performance experiment of Gd/Tm-PB

The test method comprises the following steps:

0.1mg of Gd/Tm-PB-I, Gd/Tm-PB-II, Gd/Tm-PB-III, Gd/Tm-PB-IV, Gd/Tm-PB-V and Gd/Tm-PB-VI are respectively weighed, then the mixture is respectively dispersed in 5mL of ethanol solution, the fluorescence performance test is carried out under the excitation wavelength of 365nm, and the result is shown in figure 5.

And (3) test results:

as shown in FIG. 5, it can be seen from FIG. 5 that the Gd/Tm-PB prepared in examples 1 to 6 has enhanced fluorescence with increasing Tm doping ratio, and when the Tm doping ratio reaches 25%, the Tm doping ratio is further increased, and the fluorescence intensity is not substantially changed, showing the controllability of Gd/Tm-PB for fluorescence. Different Tm³⁺The reason for the enhanced fluorescence intensity of Gd/Tm-PB in the ratio can be explained by changing the cation, and the electronic structure of PB is also changed, so that the band gap is also found to be changed along with the cation, the width is in positive correlation with the cation radius, and Tm is caused by the contraction of lanthanide³⁺Radius less than Gd³⁺This will help the electrons jump and increase the fluorescence intensity.

Third, photo-thermal regulation performance test of Gd/Tm-PB nano particles

The test method comprises the following steps:

1. respectively weighing 0.1mg of Gd/Tm-PB-I, Gd/Tm-PB-II, Gd/Tm-PB-III, Gd/Tm-PB-IV, Gd/Tm-PB-V and Gd/Tm-PB-VI, then respectively dispersing in 5mL of deionized water solution to prepare Gd/Tm-PB-I, Gd/Tm-PB-II, Gd/Tm-PB-III, Gd/Tm-PB-IV, Gd/Tm-PB-V and Gd/Tm-PB-VI solutions with the concentration of 0.02mg/mL, and using near infrared light (808nm,1.5 Wcm) to obtain near infrared light^-2) Respectively irradiating the 6 parts of Gd/Tm-PB solution with different gadolinium-thulium doping ratios for 10min, measuring the temperature change of the 6 parts of Gd/Tm-PB solution in real time in the near infrared light irradiation process, and drawing a temperature change curve.

And (3) test results:

the temperature change curve (i.e. photo-thermal effect graph) of Gd/Tm-PB solution with different gadolinium-thulium doping ratio under near-infrared laser irradiation is shown in FIG. 6, and it can be seen from FIG. 6 that Gd/Tm-PB solutionThe photo-thermal effect of the Gd/Tm-PB is changed along with different doping ratios of Tm elements, and the photo-thermal adjustable performance of the Gd/Tm-PB is shown. With Tm³⁺The reason why the photothermal properties become better with an increase in the ratio can be explained by the change in the electron density of the cyanide bond, because Tm is a change in the relative electron density of the cyanide bond³⁺With Fe³⁺Different.

Experiment four, the multifunctional composite material of the invention is used for MRI multimode imaging experiment

The test method comprises the following steps:

1. 0.1mL of cell suspension (containing 1X 10 cells)^-74T₁Mouse breast cancer cells) is injected into the thigh area of a female mouse (the weight is 15-20g) subcutaneously to establish a mouse model with subcutaneous tumor, and the mouse is used for scanning and imaging by a 3TMRI scanner (Siemens magnetic Trio 3.0T) about 15 days after inoculation to obtain a pre-injection T₁/T₂-MRI multimodal images;

2. the Gd/Tm-PB @ ZIF-8/PDA prepared in the example 1 is added into a 0.1mLPBS solution to prepare 5mg/mL Gd/Tm-PB @ ZIF-8/PDA turbid solution, the Gd/Tm-PB @ ZIF-8/PDA turbid solution (the weight of each gram of mouse is about 15-20mg) is injected through the tail vein, and 30min, 1h, 2h, 3h and 4h after intravenous injection are scanned and imaged by an MRI scanner (Siemens Magnetom Trio 3.0T) in sequence to obtain T of the corresponding time point₁/T₂-MRI dual mode images;

and (3) test results:

as shown in FIGS. 7 and 8, FIG. 7(a) is T of mice 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours before and after intravenous injection of Gd/Tm-PB @ ZIF-8/PDA₁MRI imaging graph, as can be seen from FIG. 7(a), the image of the tumor site becomes brighter with time as compared with the image of the tumor site before no Gd/Tm-PB @ ZIF-8/PDA injection, and FIG. 7(b) is T of the mice 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours before and after the Gd/Tm-PB @ ZIF-8/PDA injection₂MRI imaging, it can be seen from FIG. 7(b) that the image of the tumor site becomes darker over time as compared to the image of the tumor site before no Gd/Tm-PB @ ZIF-8/PDA injection.

From FIG. 8(a)) It can be seen that T at the tumor site is observed over time as compared to the signal at the tumor site before no Gd/Tm-PB @ ZIF-8/PDA injection₁MRI images show enhanced signals due to T₁Contrast-like agents in T₁Weighting the intensity of the signal in imaging to increase T₁-MRI image brightening; as can be seen from FIG. 8(b), the trend of the signal at the tumor site decreased with time, compared to the signal at the tumor site before no Gd/Tm-PB @ ZIF-8/PDA injection, due to T₂Contrast-like agents in T₂Reducing signal strength in weighted imaging, thereby enabling T₂Darkening of MRI images, it can be shown that the synthesized Gd/Tm-PB @ ZIF-8/PDA of the invention can be used as a contrast agent for T1/T2-MRI dual-mode imaging in vivo.

Fifth, the effect test of the multifunctional composite material of the invention as the drug adriamycin carrier

The experimental method comprises the following steps:

preparing 20ml of 0.1g/L adriamycin solution, adding 0.1g of Gd/Tm-PB @ ZIF-8/PDA prepared in the embodiment 1 into the adriamycin solution, standing for 3 days, taking supernatant liquor every 24 hours, measuring absorbance of the supernatant liquor, and obtaining the concentration of the adriamycin in the supernatant liquor according to the absorbance of the supernatant liquor so as to calculate the drug loading rate of the Gd/Tm-PB @ ZIF-8/PDA to the adriamycin at 24 hours, 48 hours and 72 hours;

the experimental results are as follows:

as shown in FIG. 9, it can be seen from FIG. 9 that the drug loading of the ZIF-8/PDA-wrapped Gd/Tm-PBA composite material prepared in example 1 on doxorubicin was about 63mg/g at 24 hours, the drug loading of the ZIF-8/PDA-wrapped Gd/Tm-PBA composite material prepared in example 1 on doxorubicin was about 75mg/g at 48 hours, and the drug loading of the ZIF-8/PDA-wrapped Gd/Tm-PBA composite material prepared in example 1 on doxorubicin was about 81mg/g at 72 hours, and it can be seen that the drug loading of the ZIF-8/PDA-wrapped Gd/Tm-PBA composite material of the present invention on doxorubicin increased with time.

Test VI test of the controlled drug Release Effect of the multifunctional composite Material of the present invention

The test method comprises the following steps:

1. preparing 20ml of 0.1g/L adriamycin solution, adding 0.1g of Gd/Tm-PB @ ZIF-8/PDA prepared in the embodiment 1, standing for 3 days, centrifuging, and drying the centrifuged solid at 60 ℃ for 12 hours to obtain a Gd/Tm-PB @ ZIF-8/PDA loaded with adriamycin, which is called a drug-loaded sample for short;

2. taking four dialysis bags (USA import dialysis bag, Cat No: MD10, MWCO: 14000D, and Nominal Flat Width:10mm) with length of 3-4cm, placing in deionized water, and boiling;

3. 4 parts of PBS buffer solutions with different pH values and GSH concentrations are prepared and put into four culture bottles, wherein the pH value of the 1 st part of the PBS buffer solution is 5.8, the GSH concentration is 5mmol/L, the pH value of the 2 nd part of the PBS buffer solution is 5.8, the GSH concentration is 20mmol/L, the pH value of the 3 rd part of the PBS buffer solution is 7.4, the GSH concentration is 5mmol/L, and the pH value of the 4 th part of the PBS buffer solution is 7.4, and the GSH concentration is 20 mmol/L.

4. Weighing four 0.01g drug-loaded samples, respectively placing the four 0.01g drug-loaded samples into four dialysis bags, and respectively adding 1mL of pH 5.8GSH 5mmol/L into the four dialysis bags; (ii) pH 5.8GSH 20 mmol/L; (ii) pH 7.4GSH 5 mmol/L; (pH 7.4) GSH 20mmol/L buffer.

5. Respectively immersing 4 dialysis bags into 4 culture bottles, wherein the pH value and the GSH concentration of each culture bottle and a buffer solution immersed in the dialysis bag are the same, respectively putting the 4 culture bottles into a THZ-series constant-temperature shaking table for shaking, setting the constant-temperature shaking table to be at a constant temperature of 37 ℃, and simulating the temperature of a human body;

6. in the process of constant-temperature shaking, deionized water is used as a reference, the absorbance of the solution in the culture flask is measured by a UV-Vis spectral method every half hour, when the data change is not large, the absorbance is measured every 1 hour, when the data change is not large, the absorbance is measured every half day until the absorbance value does not change any more. The content of doxorubicin released from the culture flask was calculated by a regression equation of a standard curve prepared by a standard curve method using an ultraviolet spectrophotometer, and plotted against the cumulative drug release rate at time intervals, to obtain doxorubicin release curves in PBS buffer solutions of different pH (pH 5.8, pH 7.4) and GSH concentrations (5mM, 20 mM).

7. The above test was repeated according to the above procedure with the only difference: at 2h, 6h and 10h respectivelyUsing near infrared light (808nm,1.5W cm)^-2) The four flasks were irradiated for 5min and the absorbance was measured.

And (3) test results:

1. the controlled release effect of Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 on doxorubicin under different pH values and GSH concentrations without near infrared light irradiation is shown in FIG. 10. from FIG. 10, it can be seen that the Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 has higher release efficiency on doxorubicin under acidic and high GSH concentration conditions, that is, the Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 can more easily release loaded doxorubicin under acidic and high GSH concentration conditions.

Based on the difference between the pH environment and the GSH concentration environment of cancer cells and normal cells, the Gd/Tm-PBA composite material wrapped by ZIF-8/PDA can perform double-control specific drug release under the conditions of acidity and high GSH concentration, and can provide a basis for reducing the damage to normal cells in actual treatment, thereby providing an effective actual reference basis for an anticancer tumor drug loading system.

2. Under the irradiation of near infrared light of 808nm, the controlled release effect of Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 on adriamycin under different PH values and GSH concentrations is shown in figure 11, and as can be seen from figure 11, the release rate of the Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 on adriamycin is obviously increased compared with that without the irradiation of the near infrared light every time the Gd/Tm-PB @ ZIF-8/PDA is irradiated by the near infrared light. Moreover, the Gd/Tm-PB @ ZIF-8/PDA prepared in example 1 released doxorubicin at the highest rate under the same conditions of near infrared irradiation and under acidic and high GSH concentration conditions. Therefore, the near infrared light can also induce the Gd/Tm-PBA composite material wrapped by the ZIF-8/PDA to release the adriamycin, and the pH/GSH dual-control performance is reflected again.

Seventh test for testing photothermal stability of the multifunctional composite Material of the present invention

The test method comprises the following steps:

dispersing Gd/Tm-PB @ ZIF-8/PDA into deionized water, preparing Gd/Tm-PB @ ZIF-8/PDA turbid liquid with the concentration of 0.20mg/mL, irradiating the Gd/Tm-PB @ ZIF-8/PDA turbid liquid for 5min by using near infrared, naturally cooling for 5min after the temperature of the Gd/Tm-PB @ ZIF-8/PDA turbid liquid rises to 45 ℃, and circulating the process for 4 times.

And (3) test results:

the result is shown in fig. 12, and it can be seen from fig. 12 that the composite material of Gd/Tm-PBA wrapped with ZIF-8/PDA prepared in example 1 is subjected to 4 photo-thermal cycles, and Gd/Tm-PB @ ZIF-8/PDA can still exceed the survival limit temperature of cancer cells during each cycle, so that it can be shown that the composite material of Gd/Tm-PBA wrapped with ZIF-8/PDA prepared in example 1 has better photo-thermal stability and has a good application prospect in photo-thermal treatment of tumor cells.

Claims

1. A Gd/Tm-PB nanoparticle characterized by: the nano-particles are formed by gadolinium and thulium co-doped Prussian blue nano-particles, the nano-particles are spherical or cubic, and the particle size of the nano-particles is 200-500 nm.

2. A method for preparing Gd/Tm-PB nanoparticles according to claim 1, characterized by comprising the steps of:

2.1, uniformly mixing ethanol and deionized water to obtain a mixed solvent, dissolving a thulium source and a gadolinium source in the mixed solution to obtain a mixed solution, wherein the molar ratio of the thulium element to the gadolinium element is 1: 1 to 95;

2.2, dropwise adding the mixed solution into a potassium ferricyanate solution, wherein the molar ratio of potassium ferricyanate to thulium gadolinium is 0.8-1.5:1, after the dropwise adding is completed, adding polyvinylpyrrolidone, the mass ratio of polyvinylpyrrolidone to potassium ferricyanate is 3.7-24:1, then heating to 140-160 ℃, carrying out hydrothermal reaction at 140-160 ℃ for 24-36h, after the reaction is completed, centrifuging, washing the obtained solid, and finally drying to obtain the Gd/Tm-PB nano particles, wherein the Gd/Tm-PB nano particles are marked as Gd/Tm-PB.

3. The Gd/Tm-PB nanoparticles of claim 2, wherein: the thulium source is thulium chloride, and the gadolinium source is gadolinium nitrate.

4. The Gd/Tm-PB nanoparticles of claim 2, wherein: the concentration of thulium in the mixed solution is 0.128-0.854 mmol/L, and the concentration of gadolinium is 1.28-2.00 mmol/L.

5. The Gd/Tm-PB nanoparticles of claim 2, wherein: the concentration of the potassium ferricyanate is 0.0017-0.025 mol/L.

6. A multifunctional composite material characterized by: the composite material is the nano-particle with a core-shell structure, wherein the Gd/Tm-PB nano-particle is taken as a core and ZIF-8/PDA is taken as a shell in the claim 1, and the particle size of the nano-particle is 200-550 nm.

7. A method of preparing the ZIF-8/PDA wrapped Gd/Tm-PBA composite of claim 6, characterized by the steps of:

7.1, dispersing the Gd/Tm-PB of claim 1 in methanol or deionized water to obtain turbid liquid A, dissolving a zinc source in the turbid liquid A, wherein the ratio of zinc element to Gd/Tm-PB is 0.3-8:1 to obtain turbid liquid B;

7.2, dissolving 2-methylimidazole in methanol or deionized water to obtain a 2-methylimidazole solution, dropwise adding the 2-methylimidazole solution into the turbid solution B, wherein the molar ratio of 2-methylimidazole to zinc elements is (1-15): 1, after dropwise adding, stirring at room temperature for reaction for 20-60min, after the reaction is finished, centrifuging, washing and drying the obtained precipitate to obtain a Gd/Tm-PB nanoparticle material wrapped by ZIF-8, and marking the Gd/Tm-PB nanoparticle material as Gd/Tm-PB @ ZIF-8;

7.3, dispersing Gd/Tm-PB @ ZIF-8 in a dopamine hydrochloride solution, wherein the mass ratio of the dopamine hydrochloride to the Gd/Tm-PB @ ZIF-8 is 2.5-5:1, the concentration of the dopamine hydrochloride solution is 0.05-0.2mg/mL, stirring for 2-12h to obtain a turbid liquid D, centrifuging the turbid liquid D, washing and drying the obtained precipitate to obtain the Gd/Tm-PBA composite material wrapped by the ZIF-8/PDA, and marking the Gd/Tm-PB @ ZIF-8/PDA.

8. Use of the multifunctional composite of claim 6 in MRI multimodal imaging.

9. Use of the multifunctional composite material of claim 6 as a carrier for the release of anticancer drugs.

10. Use of the multifunctional composite according to claim 9, characterized in that: the anticancer drug is adriamycin.