CN108578698A - Purposes of the Prussian blue-Manganese Ferrite composite nano materials as magnetic heat/photo-thermal combination therapy agent - Google Patents

Abstract

The invention discloses the use of a Prussian blue-manganese ferrite composite nanomaterial as a magnetothermal/photothermal combined therapeutic agent. Wherein, the Prussian blue-manganese ferrite composite nanomaterial is PB-MnFe ₂ O ₄ nanocomposite material.

Description

Prussian blue-manganese ferrite composite nanomaterials as combined magnetothermal/photothermal therapeutic agents use

technical field

The invention relates to the field of new materials, in particular to a magnetothermal/photothermal combined therapeutic agent.

Background technique

With the aggravation of social problems such as environmental pollution and food safety, the incidence of cancer continues to rise, and cancer is becoming one of the major lethal diseases that threaten human health. 12.5%, the number is about 9 million. In 2010 alone, cancer caused nearly 570,000 deaths, and what is more serious is that the cancer population is getting younger and younger. The reason why the status quo of cancer is so serious is determined by its own nature. Cancer is also called malignant tumor, and its opposite is benign tumor, which is a disease caused by the loss of normal regulation and excessive proliferation of body cells. Excessively proliferating cells are commonly called cancer cells. Cancer cells are easy to invade surrounding tissues, transfer, multiply, and spread.

Contents of the invention

One of the purposes of the present invention is to provide a Prussian blue (PB)-manganese ferrite composite nanomaterial as a magnetothermal therapeutic agent,

Another object of the present invention is to provide the use of the Prussian blue-manganese ferrite composite nanomaterial as a photothermal therapy agent.

Another object of the present invention is to provide the use of the Prussian blue-manganese ferrite composite nanomaterial as a combination therapy of magneto-thermal and photothermal.

In the Prussian blue-manganese ferrite composite nanomaterial of the present invention, the mass ratio of PB and MnFe ₂ O ₄ can be 1:1-5.

Preferably, in the Prussian blue-manganese ferrite composite nanomaterial of the present invention, the mass ratio of PB and MnFe ₂ O ₄ is 1:2-3.

The preparation method of the Prussian blue-manganese ferrite composite nanomaterial of the present invention comprises: taking PB and MnFe ₂ O ₄ samples, mixing according to the ratio of mass concentration 1:1-5, then adjusting the pH value of the solution to 1-4, stirring After overnight, magnetic separation and washing were repeated until the solution was clear.

As a preference, the preparation method of the Prussian blue-manganese ferrite composite nanomaterial in the present invention comprises: taking PB and MnFe ₂ O ₄ samples, mixing according to the ratio of mass concentration 1:2-3, and then adjusting the pH value of the solution to 1.5- 2.5. After stirring overnight, repeat magnetic separation and washing until the solution is clear.

In the present invention, PB nanoparticles are selected as the substrate, and a layer of manganese ferrite nanoparticles is adhered to the outside, but the PB is not completely wrapped, and the combination ratio of the two is regulated to form a Prussian blue-manganese ferrite composite nanomaterial. And through experiments, it is found that after compounding, it can achieve the effect of "1+1>2", that is, this composite material not only has the photothermal effect of Prussian blue, but also can show the magnetic hyperthermia function of manganese ferrite. Composite nanomaterials that can realize the combination therapy of magneto-thermal and photothermal, and when the magnetic-thermal and photothermal are both used together, the effect of thermal therapy can be further improved, the temperature rises faster, the time required is less, and the effect is more obvious . Therefore, this Prussian blue-manganese ferrite composite nanomaterial can be used in the therapeutic field.

Description of drawings

The present invention will be further described below in conjunction with drawings and embodiments.

Figure 1: Photothermal curves of materials A) Photothermal curves of composite materials with different concentrations; B) Curves of materials with different power densities at the same concentration (100 μg/ml); C) Photothermal curves of different materials with the same concentration (100 μg/ml) Thermal curve; D) cyclic photothermal curve of the material: E) infrared thermal imaging pictures of materials with different concentrations;

Figure 2, the magnetocaloric heating curve of materials A) Materials with different concentrations; B) Different currents; C) Materials with different concentrations; D) Infrared thermal imaging pictures of materials with different concentrations; E) SAR values of materials and corresponding 3 temperature in minutes;

Figure 3, photothermal experiment of cells A) MTT diagram of composite material; B) Fluorescent staining diagram of cells, b1-b4) in turn without adding materials and no light, adding materials and no light, adding materials and adding light, and adding materials and adding illumination;

Figure 4, A) Magnetic-caloric MTT map of cells; B) Magnetic-caloric fluorescent staining of cells, b1-b4) are sequentially without material without magnetic heat, with material without magnetic heat, without material with magnetic heat, with material and magnetic hot;

Figure 5 is the photothermal flow data diagram of cells A) Negative control group; B) Positive control group; C-E) Material group, the concentration of the material is 50, 100, 200mg/ml in turn;

Figure 6 is the photothermal-magnetic-thermal heating curve of materials under different conditions; A is magnetothermal, 500KHz, 30A; B) is photothermal 1W/cm ² ; C) is magnetothermal (500KHz, 30A) and photothermal (1W/cm2) cm ² ) combined temperature rise curve; the concentration range of AC is 10-100μg/mlD) is when the sample concentration is 200μg/ml, magneto-thermal (500KHz, 20A), photothermal (1W/cm ² ) and magneto-thermal-photothermal combined temperature rise the curve;

Figure 7: Photothermal/Magnetothermal Combined Therapy Experiment of Mice: A) Infrared Thermography of Mice; B) Heating Curve of Tumor; C) Mass Change Curve of Mice; D) Volume Change Curve of Tumor; E) Before and After Treatment Photographs of mice; F) H&E-stained sections of tumors.

Fig. 8 is a picture of the pathological results, H&E stained sections of various organs of the mouse after 12 days.

Detailed ways

1. Preparation of PB

It is a prior art, and its synthesis method refers to relevant existing documents, which are briefly as follows: get 1.5g of PVP (K30) and dissolve it in 20mL of water, then ultrasonically stir until it is completely dissolved, then add dropwise HCl (6M/L) to The pH value was 3, and then 45mg of K3[Fe(CN) ₆ ] was added, and then stirred until completely dissolved, and then 0.192g of citric acid was added, and ultrasonicated until completely dissolved. Then pour it into the lining of the reaction kettle, then move it into the oven, 80°C, 2h, and then clean it after cooling at room temperature. Add acetone, mix the two according to the ratio of 1:1, centrifuge at a high speed at a speed of 12000rpm/min for 12min, then centrifuge with absolute ethanol, and finally centrifuge with ultrapure water, and then vacuum dry the sample at 60°C for 12h save.

2 Preparation of MnFe ₂ O ₄

As the prior art, the synthesis method of _MnFe2O4 refers to the existing literature, and the brief is as follows: take 0.01mM manganese _chloride tetrahydrate and pour it into a 50mL beaker, then add 25ml of ultrapure water, and ultrasonically shake it to dissolve it completely , determined as solution A, then poured 0.02mM ferric chloride hexahydrate into a 50mL beaker, then added 25mL ultrapure water, and ultrasonically oscillated to dissolve it completely, determined as solution B. Then introduce the two into a 100mL three-necked flask, stir mechanically at 80°C for 30min, and rotate at 600rpm/min, then add 0.08mM sodium hydroxide, continue stirring for 30min, then add 0.05g of polyethyleneimine, and continue stirring for 30min Cool at room temperature. Then the magnet separates and washes with ultrapure water until the solution is clear. Finally, it was dried under vacuum at 60°C for 12 hours and then stored.

3 Preparation of PB/MnFe ₂ O ₄

Take the PB and MnFe ₂ O ₄ samples synthesized above, mix them according to the ratio of mass concentration 1:2.3, then add 6M/L hydrochloric acid dropwise to make the pH value of the solution 2, then mechanically stir overnight at a speed of 300rpm/min, the solution is Dark green, then magnetic separation, cleaning with ultrapure water, repeated magnetic separation and cleaning until the solution is clear. Finally, it was dried under vacuum at 60°C for 12 hours and then stored.

4. Cell photothermal and magnetothermal experiments

In order to verify the photothermal and magnetothermal properties of the material from the cellular level, the steps are as follows: 1) Select the cells in the logarithmic growth phase, digest with trypsin, wash twice with PBS buffer, and then use the concentration of 5×10 ⁵ mL-1 Inoculated in 12-well plates, divided into two groups, one group was tested by MTT method, the other group was stained with Calcein AM/PI, the volume of each hole was 1.5mL, and then moved into a constant temperature incubator for incubation for 24h; 2), and then transferred In addition to the original culture solution, PBS buffer was washed twice, and then the culture solution containing different material concentrations (400 μg/mL, 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, no material group) was added, and then Move them into a constant temperature incubator and incubate for 4 hours; then carry out photothermal irradiation experiment, magnetothermal experiment and photothermal magnetothermal combined treatment experiment respectively, and then move them into a constant temperature incubator for incubation again, MTT group for 24 hours, dyeing group for 4 hours; then MTT group added 20ml of MTT, continue to culture for 4h, then remove the supernatant, dissolve the precipitate with DMSO, and then measure the absorbance at 570nm with a spectrophotometer, and use the group without material as the control group to calculate the cell survival rate. After the other group was incubated for 4 hours, 5 mL of Calcein AM and PI in the supernatant were removed, and then placed on a horizontal shaker at 20 r/min for 10 minutes in the dark, and incubated for another 30 minutes. Then take it out again, remove the supernatant, wash gently twice with PBS buffer, add 100 μL of culture solution to each well, and finally observe and take pictures under a fluorescent microscope.

5. Cell flow experiments

Treat the cells, collect the treated cells, fix them overnight with 70% cold ethanol, clear them twice with PBS buffer the next day, and stain them with Calcein AM and PI staining solution in the dark for 30 minutes at room temperature, and finally detect cell apoptosis by flow cytometry Mortality rate, and the group without material was the blank control group.

6. Tumor implantation

The 4T1 cell line was subcultured and expanded to prepare a cell suspension of 1×10 ⁸ mL-1. Then the mice were divided into 8 groups, 3 in each group, which were pure PBS group, PBS+MFH group, PBS+PTT group, PBS+MFH+PTT group, material group, material+MFH group, material+PTT group And the material+MFH+PTT group. Then respectively in the chest wall 5-6 intercostal space of mice and the cell suspension of side abdominal wall subcutaneous inoculation corresponding concentration, then observe the growth situation of mouse living diet and inoculation site tumor piece every day, as tumor body appearance time, tumor body volume (V =ab2/2, a is the long diameter of the tumor, b is the short diameter), the weight of the mouse, etc., and draw the curve. After the experiment was completed, tumor tissues were taken for HE staining for pathological observation.

7. Animal treatment

In order to verify the therapeutic function of the material at the animal level, mainly referring to the magnetocaloric properties, photothermal properties and the superimposed properties of magnetothermal and photothermal properties of the material, Blab/C mice were selected as the experimental subjects. Firstly, the magnetothermal experiment and photothermal experiment of the mouse were carried out separately. After the mouse was anesthetized, 50μl, 200μg/ml material was injected in situ, and then the mouse was placed under the magnetic heating equipment and laser, and the tumor of the mouse was recorded with an infrared thermal imager Changes in the temperature of the area. Then, the magnetothermal photothermal superposition experiment was carried out, the laser and the magnetic heating equipment were combined and matched, the mouse was anesthetized, the material was injected in situ, and then the mouse was put into the built instrument, and the temperature of the tumor area of the mouse was recorded with an infrared thermal imager. According to the previous grouping of the mice (pure PBS group, material+MFH group, material+PTT group and material+MFH+PTT group), the detection was performed sequentially. Take pictures every day and detect the changes in the tumors and body weight of the mice, and then stop the treatment according to the disappearance of the tumors in the mice.

8. HE staining

After the mice were treated, the mice were dissected, and the viscera were separated, including the heart, liver, spleen, lung, kidney and tumor, and tissue sections were made for pathological observation. Staining is to prepare dyes into solutions, immerse tissue sections in the staining agent, and after a certain period of time, the components of tissues and cells will be dyed with different colors, resulting in different refractive indices, which are convenient for observation under an optical microscope. Hematoxylin and eosin staining (HE staining) was used for staining.

The toxicity detection of embodiment 1 composite material

The sterilized PB/MnFe2O4 material was tested for cytotoxicity by detecting the co-culture of cells in different concentrations of materials (400μg/mL, 200μg/mL, 100μg/mL, 50μg/mL, 25μg/mL, no material group) Survival conditions of (material concentration is prepared by quantitatively mixing 10μL material + 90μL medium), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide) method, the cell objects selected here are HeLa cells and 4T1 cells, and the specific experimental operations are as follows:

Cell counting: Place the cells in the logarithmic growth phase in an ultra-clean workbench, remove the original medium in the culture dish (handle carefully to avoid the cells being sucked away due to excessive suction), and then add 2mL of PBS buffer solution to it Wash twice, then use a pipette to pipette 1 mL of trypsin (25%) to digest the cells. After 2 minutes, add 1 mL of medium and pipette to stop the digestion (at this time, the cells can be seen floating and not attached to the wall under the microscope), and then Use a 1.5mL centrifuge tube to collect, then centrifuge at a speed of 3000r/min for 5min. After centrifugation, remove the supernatant, add 2mL of medium, and gently blow the cells with a pipette gun (30-50 times). Take 10 μL of the cell suspension, take it to a hemocytometer for counting, and calculate the average number of the four counting areas to determine the cell amount of the total cell suspension.

Inoculation: Then inoculate 104 target cells into a 96-well plate, add a certain amount of serum-containing medium (so that the total amount of medium in each well of the cells is 100 μL), and then put it in a constant temperature incubator and incubate for 24 hours, so that Cells can grow adequately on the wall.

Adding materials: Remove the medium, add 100 μL of PBS buffer, wash twice, and then add materials of target concentration: 400 μg/mL, 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, no material Group. Each group set up 5 repeated groups and co-cultured for 24 hours in a constant temperature incubator at 37°C. After reaching the target time, the culture medium was removed and washed 3 times with PBS buffer solution (in order to remove excess residual material solution).

MTT detection: Add 20 μL of 5 mg/mL MTT solution and 80 μL of blood-free medium to each well, and then place them in a constant temperature incubator at 37°C and incubate for 4 hours in the dark. After 4 hours, carefully remove the culture medium (avoid the cells being sucked out and affect the experimental results), then add 150 μL DMSO to each well, and put the 96-well plate on a horizontal shaker at 20 r/min for 10 min under the condition of avoiding light. Then place it in a microplate reader for ultraviolet detection, and finally obtain the OD value of each well, and arrange and draw the OD value to obtain the activity data graph of HeLa and 4T1 cells.

As shown in Fig. 8, according to the above results, the magnetic Prussian blue material has good biocompatibility.

The photothermal performance of embodiment 2 material

Take the obtained sample solution, take a certain concentration of the sample in the ultraviolet dish, and then put it under the laser irradiation for 10 minutes, and use the infrared thermal imager to collect pictures at the same time, and record the change of the temperature of the material with time. Specific steps are as follows:

According to the laser power density [12] formula:

η=P/π·R ² (1)

η: laser power density, unit W/cm ² ;

P: laser power, unit W;

R: The radius of the light spot on the object irradiated by the laser, in cm.

Select the laser power, then fix the laser probe, a light spot will appear after the laser is irradiated on the ground, then take a ruler to measure the diameter of the light spot and the distance from the probe to the ground, adjust the distance according to the laser power density, and then adjust the UV ratio The color dish is placed at the light spot, and the temperature change of the material is recorded, and the time-temperature curve is drawn. At the same time, the infrared thermal imager is used to collect pictures of the temperature change. Starting from 4 angles, they are different materials with the same concentration, H2O, PB, MnFe2O4 and PB/MnFe2O4; composite materials with different material concentrations under the same laser power density, 10μg/mL, 20μg/mL, 50μg/mL and 100μg /mL; composite materials with the same concentration under different laser power densities, 0, 0.5W/cm ² , 1W/cm ² , 2W/cm ² ; photothermal cycle experiments mainly verify the photothermal stability of composite materials. After 10 minutes of laser irradiation, turn off the laser, record the temperature change of the material, turn on the laser after cooling down to room temperature, irradiate for 10 minutes, then turn off the laser, and so on, repeat 3 times.

Photothermal performance test of materials

Figure 1 is the photothermal curve of the material A) Photothermal curves of composite materials with different concentrations; B) Curves of materials with the same concentration (100 μg/ml) and different power densities; C) Photothermal curves of different materials with the same concentration (100 μg/ml) Thermal curve; D) Cyclic photothermal curve of the material: E) Infrared thermal imaging pictures of materials with different concentrations.

Based on the above ultraviolet spectrum test analysis, it is found that the material has a strong absorption peak in the near infrared region, and the photothermal performance of the material is tested. First, to verify whether the composite material has photothermal properties, four material concentrations were selected, as shown in Figure 1A, which were 10 μg/mL, 20 μg/mL, 50 μg/mL and 100 μg/mL. It is found that when the laser power density is 2W/cm2, even if the material concentration is very low, the temperature difference between the front and back of the material can still reach 10°C within 10 minutes, and the temperature rises significantly, and the higher the concentration, the more obvious the temperature rise, and the highest temperature difference can reach nearly 30°C. It shows that the material has good photothermal performance.

The influence of conditions, such as laser power density, is shown in Figure 1B. Four parameters were selected, namely 0W/cm2, 0.5W/cm2, 1W/cm2, and 2W/cm2, and the concentration of the material was 100μg/ml. It was found that the higher the power density, the more obvious the temperature rise, and the temperatures were 0°C, 38°C, 48°C, and 57°C.

The effect of material recombination, as shown in Figure 1C, select H ₂ O, PB, MnFe ₂ O ₄ and PB/MnFe ₂ O ₄ materials with the same concentration, and the laser power is 2W/cm2. It is found that there is almost no temperature change before and after H ₂ O , and there is a certain increase in MnFe ₂ O ₄ , but not very obvious. However, the temperature difference between PB and PB/MnFe2O4 varies greatly before and after, and both have strong photothermal properties, but the temperature after the material is compounded is slightly higher than that of a single PB material, and there is a certain temperature superposition effect after the two are compounded.

Next is the photothermal cycle stability test of the material, mainly to test the photothermal stability of the material, and the results are shown in Figure D. The laser was turned on and off every 10 minutes, but the detection of the material temperature was not stopped. After 3 cycles, it was found that the material temperature could rise to 55°C within 10 minutes, indicating that the material had good photothermal stability.

Figure E is the infrared thermal imaging picture of the material. It mainly collects the temperature picture of the material with an infrared thermal imaging camera. The brightness and depth of the color directly reflect the temperature of the material, and it can be found that the photothermal performance of the material is better.

Cell Photothermal Experiment

See Figure 3, Figure 3A is the MTT diagram of the material and the material after being irradiated by laser light, from which it can be seen that the cytotoxicity of the material is small, even when the material concentration is very high (400 μg/mL), there are still more than 80% of the cells Survival, generally when the cell survival rate exceeds 75%, it indicates that the biotoxicity is small and the biocompatibility is high. After being irradiated by laser, the cell survival rate decreased significantly, and the cell survival rate was less than 40%. This was because the temperature of the material rose and the cells died after being irradiated by laser, which indicated that the photothermal performance of the material was good from the cell level.

Figure 3B is the uptake rate of the material, from which it can be seen that the material can be taken up by the cells, and as time goes on, the uptake rate of the cells is higher, reaching 25%.

Figure 3C-F are pictures of cells stained by calcein AM and PI after being irradiated with laser light. It was found that HeLa cells without materials and laser were all stained with green fluorescence, indicating that the cells in the control group grew normally, while the simple materials did not. The photothermal reaction is caused, and the cells are still green fluorescent. However, when only laser light was applied, the cells did not change significantly, and all of them were still stained with green fluorescence, indicating that the cells were alive. In the absence of photothermal agents, pure 808nm laser irradiation will not produce photothermal reactions, so it will not affect cells. Under the influence of the simultaneous presence of the material and the laser, under the observation of the fluorescence microscope, red fluorescence appears at one corner. Although the range of this corner is larger than the area irradiated by the laser, it also conforms to the irradiated area. The red area is larger because the material has a photothermal effect, which converts the absorbed heat into heat to kill the cells, and due to the diffusion of heat, the activity of adjacent cells is also affected, so it is larger, but even so, outside the irradiation area There are still normal cells. In summary, this magnetic Prussian blue composite can efficiently kill cells through the near-infrared photothermal effect, and thus has great significance in the photothermal therapy of cancer.

Next, perform cell flow cytometry characterization, and take the processed cells for flow cytometry detection, and the data are shown below.

Figure 5 is the photothermal flow data diagram of cells A) Negative control group B) Positive control group C-E) Material group, the concentration of the material is 50, 100, 200 mg/ml in sequence.

It can be seen from the figure that in Figure A, which is the negative control group, there is less apoptosis, and most of the cells are in the state of living cells, while as the positive control group, due to the addition of 20mM H2O2, more than 60% of the cells are in the state of apoptosis Figures C, D, and E are the experimental groups, in which different concentrations of MPB NPs materials were added, and heat was generated under laser irradiation, which led to cell apoptosis. It can be seen that with the increase of the concentration, the living cells The proportion of apoptotic cells gradually decreased, while the proportion of apoptotic cells gradually increased, which shows that the material has a good photothermal effect, and also shows that this material has an excellent photothermal effect.

animal therapy

Next, the hyperthermia experiment on mice was carried out, as shown in FIG. 7 . Figure 7A is the real-time curve of tumor temperature during treatment, from which it can be seen that except for the treatment group (materials+MFH, materials+PTT, materials+MFH+PTT) the temperature did not change significantly, fluctuating around 36°C. In the treatment group, the changes were more obvious within 2 minutes, among which the magnetothermal treatment reached 43°C, the photothermal treatment reached 44°C, and the combined treatment could reach 55°C within 2 minutes, from which it can be seen that the material has a good effect on the animal level. , the temperature change before and after can be seen more intuitively from Figure 7B. Figure 7C graphs the change in mass of the mouse, which remains nearly balanced compared to the initial mass. Figure 7D is the volume change curve of the mouse tumors, from which it can be seen that except for the treatment group, the volume of the tumors increased significantly, and after 12 days, it was about 8 times the original size. The magneto-thermal ones are 4 times the original size, and the photothermal ones are 3 times the original size. In contrast, the tumors in the magnetothermal combined with photothermal group disappeared within 3 days, which directly reflects the therapeutic effect of the material. From the effect of the photo in Figure 7E, the changes of the tumor can be seen directly, which reflects that the material has a high therapeutic effect of combined photothermal, magnetic and thermal therapy.

pathological analysis

Figure 8 H&E stained sections of various organs and tumors of mice after 12 days, scale bar: 100 μm

Then, the tissue and cell morphology of each organ were analyzed by H&E staining, as shown in FIG. 8 . Comparing the experimental group with the control group before and after, it can be seen from the comparison that the heart, liver, spleen, lung, and kidney of the mice in the eight groups have no obvious changes in tissue morphology, indicating that the material has good biological safety and is suitable for small The mice had no obvious toxic and side effects, and the physiological status of the mice was good during the whole treatment process. However, the tissue sections of the tumor showed significant changes. Large areas of cell shrinkage, blood vessel damage and tissue necrosis appeared in the tumor tissue of the experimental group, which indicated that the tumor tissue in the treatment group had lost its physiological activity. Then have the ability to grow and increase value.

The magnetocaloric experiment of embodiment 3 materials

This example mainly investigates the magneto-caloric properties of the material. During the test, a fluorescent optical fiber thermometer is used to record the temperature change of the material solution, mainly from three angles: 1. The concentration of the material, 200μg/mL, 400μg/mL, 800μg /mL; 2. Experimental test conditions, such as current, field strength, frequency, etc., and here mainly refers to the size of the magnetic induction coil current. When the material concentration is constant, change the current size, 5A, 10A, 20A, 30A and 40A; 3. The same experimental conditions, the same concentration but different materials, H2O, PB, MnFe2O4 and PB/MnFe2O4. Draw the time-temperature curve according to the measured temperature, and calculate the heat production rate (Specific Absorption Rate, SAR) of the material according to the curve [13], which is the amount of energy converted into heat energy per unit mass of the sample. The higher the heat production rate, The better the magnetocaloric performance:

In the formula: C is the specific heat capacity of the material solution, J/g K;

ΔT/Δt is the slope of the temperature-time curve;

m _Fe is the content of iron element contained in a unit mass of material.

Magnetic-caloric performance test of materials

Figure 2 The magnetocaloric heating curve of materials A) Materials with different concentrations; B) Different current sizes; C) Materials with different concentrations at the same concentration; D) Infrared thermal imaging pictures of materials with different concentrations; E) SAR values of materials and corresponding 3 minutes temperature

Through the VSM test, it is found that the material has high magnetic properties, so the next step is to test the magnetocaloric properties of the material. Mainly from three perspectives, namely the concentration of the material, the conditions of the experimental test, the SAR value of the magnetic heating, and the influence of the material before and after compounding.

Figure 2A is to explore the influence of material concentration. Under the experimental conditions of 500KHz and 12A/H, three concentrations were selected, 200μg/mL, 400μg/mL and 800μg/mL. It is found that the higher the concentration, the better the temperature rise performance of the material, which means the better the magnetocaloric performance of the material. Even at a very low concentration (200μg/mL), the temperature can still reach 42°C.

Next is the effect of experimental conditions, as shown in Fig. 2B. It is found that the larger the current, the faster the temperature rise, especially when the experimental current is 40A, the current rises rapidly to 70°C within 3 minutes, which is very obvious.

Figure 2C is to explore whether material compounding has an effect on the magnetocaloric properties. It was found that the temperature of H2O and PB hardly changed, but the temperature of MnFe2O4 and PB/MnFe2O4 increased significantly, and both could reach above 42°C within 10 minutes, which could achieve the therapeutic effect. And after compounding, the temperature of the material is slightly higher than that of single MnFe2O4, mainly because the particle size of the material becomes larger, and the magnetocaloric properties are affected by the particle size of the material. Within a certain range, the larger the particle size, the higher the temperature.

Figure 2D is an infrared thermal imaging picture of the material, which mainly reflects the temperature change of the material from a macroscopic perspective through the infrared thermal imaging camera. The higher the temperature, the brighter the color. It is found that before and after the magnetic treatment, the material change is more obvious.

Figure 2E is the SAR value of the material, corresponding to 3 minutes, it directly reflects the magnetocaloric properties of the material, the higher the SAR, the better the magnetocaloric properties, which can reach nearly 1000W/g, compared with some related literature reports, the value is higher .

Magnetocaloric Effect of Cells

It can be seen from Figure 4 that the magnetocaloric effect is similar to the photothermal effect in Figure 3. After magnetothermic treatment, the activity of cells and the survival rate decreased, indicating that the material has a magnetocaloric effect, which converts the absorbed magnetic energy into heat energy, thereby converting Cells are killed, and this can be seen from the fluorescent staining effect. After the material is magnetically heated, the activity of the cells decreases or even dies. Therefore, the cells are not stained with fluorescence. Black color indirectly illustrates the magnetocaloric effect of the material, indicating that this material can be used for magnetothermic therapy of cancer.

Magnetothermal-optical-thermal superimposed use of the material of embodiment 4

According to the photothermal and magnetothermal results of the above materials, a concentration of 200mg/ml was selected for detection (at this temperature, all materials have a heating effect, especially photothermal), a magnetothermal photothermal platform was built, and then the photothermal and magnetic The same time is measured under thermal experimental conditions, and infrared thermal imaging is used to take pictures to record the temperature change, and then the time-temperature curve is drawn.

Figure 6 is the combined magneto-thermal-photothermal curve of the material. The experimental condition in Figure 6A is 500KHz, 30A. It is found that under this condition, the temperature rises by about 10°C within 10 minutes, and under the same concentration conditions, the temperature rise of PTT is relatively more obvious, about 16°C, as shown in Figure 6B. After superposition, the temperature difference is about 20°C, and the temperature rise is more obvious, which shows that the superposition effect of MFH and PTT is better, and further shows the feasibility and effect of superimposed use. Next, we chose 200μg/mL material to try magnetothermal plus photothermal. As shown in Figure 6D, we found that the temperature difference is more obvious, the temperature can reach more than 70°C, and the temperature difference exceeds 30°C, the effect is very good. Finally, it was found that after the combined use of the two, the temperature rise was more obvious, the rising speed was faster, and the temperature was higher, which confirmed the possibility of the combined use of magneto-thermal and photothermal, both of which are hyperthermia, and indirectly explained the role of this composite material in cancer hyperthermia. The field has great potential.

The above is only a preferred embodiment of the present invention, so the scope of implementation of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention within range.

Claims (10)

1. Prussian blue-purposes of the Manganese Ferrite composite nano materials as physiotherapy agent, wherein the Prussian blue-iron Sour manganese composite nano materials are PB-MnFe₂O₄。

2. purposes as described in claim 1, which is characterized in that the physiotherapy agent includes that photo-thermal therapy agent or magnetic heat are controlled Treat agent.

3. purposes as described in claim 1, which is characterized in that the physiotherapy agent is photo-thermal/magnetic heat integration therapeutic agent.

4. the purposes as described in claims 1 or 2 or 3, which is characterized in that the Prussian blue-Manganese Ferrite composite Nano material In material, PB and MnFe₂O₄Mass ratio is 1:1-5.

5. the purposes as described in claims 1 or 2 or 3, which is characterized in that the Prussian blue-Manganese Ferrite composite Nano material In material, PB and MnFe₂O₄Mass ratio is 1:2-3.

6. the purposes as described in claims 1 or 2 or 3, which is characterized in that the described Prussian blue preparation method of alkalinity includes： Take PB and MnFe₂O₄Sample, according to mass concentration 1：The ratio of 1-5 mixes, and it is 1-4 then to adjust solution ph, is stirred overnight Afterwards, repeatedly Magnetic Isolation, cleaning until solution is clarified.

7. the purposes as described in claims 1 or 2 or 3, which is characterized in that the described Prussian blue preparation method of alkalinity includes： Take PB and MnFe₂O₄Sample, according to mass concentration 1：The ratio of 2-3 mixes, and it is 1.5-2.5 then to adjust solution ph, stirred After night, Magnetic Isolation, cleaning are until solution is clarified repeatedly.

8. a kind of physiotherapy agent contains Prussian blue-Manganese Ferrite composite nano materials.

9. a kind of physiotherapy agent as claimed in claim 8, which is characterized in that the physiotherapy agent includes photo-thermal therapy Agent or magnetic heat cure agent.

10. a kind of physiotherapy agent as claimed in claim 8, which is characterized in that the physiotherapy agent is photo-thermal/magnetic heat Combination therapy agent.