Preparation method and application of a polypyrrole-prussian blue hybrid photothermal nanosenzyme

Abstract

The application relates to a preparation method of a poly-pyrrole-prussian blue hybrid photothermal nanolaser, polyvinylpyrrolidone is added into deionized water and stirred until completely dissolved, pyrrole and potassium ferrocyanide are added into the obtained polyvinylpyrrolidone solution, stirring is uniformly conducted, then ferric chloride hexahydrate is added, sufficient stirring and reaction are conducted, a reaction liquid is obtained, after the precipitation is removed through centrifugation, the supernatant is dialyzed and vacuum freeze-dried, and the poly-pyrrole-prussian blue hybrid photothermal nanolaser is obtained.Compared with the prior art, the method has lower requirements on equipment, mild reaction conditions, is green and environment-friendly, is suitable for large-scale production, the prepared photothermal nanolaser has good photothermal ablation effect under the irradiation of near-infrared light, can significantly inhibit the growth of tumor cells, the photothermal nanolaser also has good oxygen production performance, can decompose hydrogen peroxide to generate oxygen, and can further improve the tumor hypoxic microenvironment and enhance the in-situ thermal ablation treatment effect of the tumor, thereby providing a potential technical means for relieving the tumor hypoxic microenvironment and strengthening the in-situ thermal ablation.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to a method for preparing and applying a polypyrrole-Prussian blue hybrid photothermal nanoenzyme. Background Technology

[0002] Photothermal ablation is an emerging in situ tumor treatment method. Its principle involves irradiating a photosensitizer accumulated in the tumor area with near-infrared light, thereby generating localized high temperatures to kill tumor cells. Polypyrrole photothermal nanozymes have attracted considerable attention in the field of tumor photothermal therapy due to their good biocompatibility, excellent photostability, and good photothermal conversion effect. Under near-infrared light irradiation, they can generate sufficient heat to kill tumor cells and induce anti-tumor immunity, thus playing a therapeutic role. However, due to the hypoxic microenvironment within the tumor, photothermal nanozymes cannot effectively exert their photothermal ablation effect to kill tumor cells, significantly reducing the therapeutic effect. Furthermore, the intensity of photothermal ablation cannot be precisely controlled, posing challenges in clinical translation. Therefore, the development of photothermal nanomaterials that can improve the hypoxic microenvironment of tumors and thus enhance tumor photothermal ablation technology is particularly necessary to improve therapeutic efficacy.

[0003] Prussian blue photothermal nanozymes, with their excellent biocompatibility and unique properties, have attracted widespread attention from biomedical researchers. Existing studies have demonstrated that Prussian blue photothermal nanozymes, based on their inherent bioactivity and imaging capabilities, can serve as therapeutic agents. They can scavenge excess reactive oxygen species (ROS) in the body to treat ROS-related diseases, and through the catalytic activity of enzymes such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), they can decompose hydrogen peroxide into oxygen to improve the hypoxic state of the tumor microenvironment. Combining photothermal ablation with enzymes would undoubtedly improve the hypoxic tumor microenvironment and thus enhance the photothermal therapeutic effect on tumors.

[0004] However, there is currently a lack of technology that combines photothermal ablation with enzymes. Therefore, there is an urgent need to find a technology that can provide efficient and adjustable photothermal ablation for clinical solid tumors. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing polypyrrole-Prussian blue hybrid photothermal nanozymes. The prepared photothermal nanozymes have dual functions of photothermal and enzymatic catalytic activity. When applied to the photothermal therapy of tumors, they can decompose hydrogen peroxide to generate oxygen to improve the hypoxic microenvironment of tumors and enhance the photothermal therapeutic effect of tumors.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0007] A method for preparing a polypyrrole-Prussian blue hybrid photothermal nanozyme includes the following steps:

[0008] (1) Add polyvinylpyrrolidone to deionized water and stir until completely dissolved to obtain a polyvinylpyrrolidone solution;

[0009] (2) Add pyrrole (PY) and potassium ferrocyanide (K4Fe(CN)6) to the polyvinylpyrrolidone solution, stir evenly, then add ferric chloride hexahydrate (FeCl3·6H2O), stir the reaction thoroughly, and obtain the reaction solution.

[0010] (3) Centrifuge the reaction solution to remove the precipitate and obtain the supernatant. Dialyze the supernatant and freeze-dry it under vacuum to obtain polypyrrole-Prussian blue hybrid photothermal nanozyme (PPY@PB NEs).

[0011] In step (2), the molar ratio of pyrrole to potassium ferrocyanide is (1-15):(1-15).

[0012] Furthermore, in step (1), the concentration of the polyvinylpyrrolidone solution is 5 to 20 mg / mL, more preferably 10 mg / mL.

[0013] Furthermore, in step (2), the amount of ferric chloride hexahydrate used accounts for 25-40% of the total mass of pyrrole and potassium ferrocyanide.

[0014] Furthermore, in step (3), the centrifugation conditions are: 3000 rpm for 15 min.

[0015] Furthermore, in step (3), the dialysis uses a 100000MWCO dialysis bag and the dialysis time is 72h.

[0016] Furthermore, in step (3), the freeze-drying temperature is -80°C and the time is 48 hours.

[0017] The application of the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared by the above method in the preparation of photothermal therapy drugs for tumors.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention provides a method for preparing polypyrrole-Prussian blue hybrid photothermal nanozymes (PPY@PB NEs). These photothermal nanozymes are synthesized in one step via iron ion catalysis from polypyrrole photothermal nanozymes (PPYNEs) and Prussian blue photothermal nanozymes (PB NEs). The preparation method is simple, requires low-level equipment, operates under mild reaction conditions, and is environmentally friendly, making it suitable for large-scale industrial production. The prepared photothermal nanozymes (PPY@PB NEs) exhibit excellent photothermal properties. Under near-infrared light irradiation, PPY@PB NEs demonstrate good photothermal ablation effects, significantly inhibiting tumor cell growth. Furthermore, the prepared PPY@PB NEs exhibit good oxygen production capabilities, decomposing hydrogen peroxide to generate oxygen, thereby improving the hypoxic tumor microenvironment and enhancing the in-situ thermal ablation therapeutic effect. Attached Figure Description

[0020] Figure 1 Transmission electron microscopy image of the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared in Example 1;

[0021] Figure 2 Particle size distribution of the polypyrrole-Prussian blue hybrid photothermal nanozymes prepared in Examples 1-5;

[0022] Figure 3 The UV-Vis absorption spectra of the polypyrrole-Prussian blue hybrid photothermal nanozymes prepared in Examples 1-5;

[0023] Figure 4 The results show the photothermal performance of the polypyrrole-Prussian blue hybrid photothermal nanoenzymes prepared in Examples 1-5.

[0024] Figure 5 The photothermal performance test results of the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared in Example 1 under different conditions;

[0025] Figure 6 The oxygen production capacity test results are for the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared in Example 1.

[0026] Figure 7 The results of the blood compatibility test for the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared in Example 1;

[0027] Figure 8 The results of the biosafety test for the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared in Example 1;

[0028] Figure 9 The results show the test results of the killing ability of the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared in Example 1 against tumor cells. Detailed Implementation

[0029] The technical solution of the present invention will be described in detail below with reference to embodiments, accompanying drawings, and specific examples. These embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention.

[0030] Example 1

[0031] A method for preparing a polypyrrole-Prussian blue hybrid photothermal nanozyme includes the following steps:

[0032] (1) Add 0.1g of polyvinylpyrrolidone to 10mL of deionized water and stir until completely dissolved to obtain a polyvinylpyrrolidone solution;

[0033] (2) Add 1 mmol (69.2 μL) of pyrrole (PY) and 0.1333 mmol (49.11 mg) of potassium ferrocyanide (K4Fe(CN)6) to the polyvinylpyrrolidone solution, stir well, then add 30 mg of ferric chloride hexahydrate (FeCl3·6H2O), stir for 2 h to obtain the reaction solution;

[0034] (3) Centrifuge the reaction solution at 3000 rpm for 15 min to remove the precipitate and obtain the supernatant. Dialyze the supernatant using a 100000MWCO dialysis bag for 72 h and then freeze-dry it under vacuum at -80℃ for 48 h to obtain a blue-black powder, which is the polypyrrole-Prussian blue hybrid photothermal nanozyme (PPY@PB NEs).

[0035] Example 2

[0036] In step (2), the amount of pyrrole used is 2 mmol, and the amount of potassium ferrocyanide used is 0.1333 mmol. The rest is the same as in Example 1.

[0037] Example 3

[0038] In step (2), the amount of pyrrole used is 1 mmol, and the amount of potassium ferrocyanide used is 1 mmol. The rest is the same as in Example 1.

[0039] Example 4

[0040] In step (2), the amount of pyrrole used is 0.1333 mmol, and the amount of potassium ferrocyanide used is 1 mmol. The rest is the same as in Example 1.

[0041] Example 5

[0042] In step (2), the amount of pyrrole used is 0.1333 mmol, and the amount of potassium ferrocyanide used is 2 mmol. The rest is the same as in Example 1.

[0043] Comparative Example 1

[0044] Preparation of polypyrrole photothermal nanozymes (PPYNEs):

[0045] In step (2), potassium ferrocyanide (K4Fe(CN)6) is not added, and the rest is the same as in Example 1.

[0046] Comparative Example 2

[0047] Preparation of Prussian blue photothermal nanozymes (PB NEs):

[0048] In step (2), pyrrole is not added, and the rest is the same as in Example 1.

[0049] Figure 1 The image shows a transmission electron microscope (TEM) image of the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared in Example 1. The image shows that the PPY@PB NEs are circular in shape.

[0050] Figure 2 The particle size distributions of the polypyrrole-Prussian blue hybrid photothermal nanozymes prepared in Examples 1-5 show that the PPY@PB NEs photothermal nanozymes prepared in these examples exhibit a normal particle size distribution, are appropriately sized, and possess good passive targeting ability against tumor tissue. In different preparation ratios across the examples, the particle size of the nanozymes increases with increasing pyrrole content.

[0051] Figure 3 The UV-Vis absorption spectra of the polypyrrole-Prussian blue hybrid photothermal nanozymes prepared in Examples 1-5 show that the PPY@PB NEs photothermal nanozymes prepared in the examples of this invention all have obvious characteristic absorption peaks at 700 nm, and the intensity of the absorption peaks is positively correlated with the amount of potassium ferrocyanide added. These are characteristic absorption peaks of PB, proving that PB has been successfully hybridized into the photothermal nanozymes.

[0052] 1. Test the photothermal properties of the polypyrrole-Prussian blue hybrid photothermal nanozymes prepared in Examples 1-5.

[0053] Test method: The polypyrrole-Prussian blue hybrid photothermal nanozymes prepared in Examples 1-5 were each prepared into 500 μg / mL aqueous solutions, with deionized water used as a control group. A power density of 3.0 W / cm² was used. 2 The PPY@PB NEs solution was irradiated with an 808nm laser for 5 minutes. After that, the 808nm laser was turned off to allow the temperature to drop naturally. Every 30 seconds, a handheld thermal imager was used to take thermal images and record the temperature, and a temperature curve was plotted.

[0054] Test results are available Figure 4 It can be seen that the photothermal performance of PPY@PB NEs is mainly affected by the amount of pyrrole added. The greater the amount of pyrrole added, the stronger the photothermal performance of the nanoparticles. The temperature of the deionized water control group did not change significantly throughout the process.

[0055] 2. Testing the photothermal performance of polypyrrole-Prussian blue hybrid photothermal nanozymes under different conditions.

[0056] Test method: (1) The PPY@PB NEs prepared in Example 1 were prepared into aqueous solutions of 100, 300, and 500 μg / mL, respectively, with deionized water as the control group. The power density of the 808nm laser was set to 1.0 W / cm². 2 Each group of solutions was irradiated with laser for 5 minutes, and thermal images were taken and the temperature was recorded every 30 seconds using a handheld thermal imager to test the photothermal performance of PPY@PB NEs at different concentrations.

[0057] (2) The PPY@PB NEs prepared in Example 1 were prepared into an aqueous solution of 500 μg / mL, with deionized water as the control group. Power densities of 1.0, 2.0, and 3.0 W / cm³ were used respectively. 2 The PPY@PB NEs solution was irradiated with an 808nm laser for 5 minutes. Thermal images were taken and the temperature was recorded every 30 seconds using a handheld thermal imager to test the photothermal performance of PPY@PB NEs under different laser power densities.

[0058] Test results are available Figure 5 ,in Figure 5 A represents the photothermal properties of PPY@PB NEs at different concentrations. Figure 5 B represents the photothermal properties of PPY@PBNEs under different laser power densities. It can be seen that PPY@PBNEs exhibit photothermal properties under 808nm laser power densities (1.0W / cm²). 2 The heat generation effect after irradiation was significant, and the higher the concentration of photothermal nanozyme, the better the heat generation effect. PPY@PB NEs reached 46.1℃ at a concentration of 500 μg / mL; while the temperature of the deionized water control group did not change significantly after 5 minutes of irradiation. Figure 5 As can be seen from B, the heat generation effect of PPY@PB NEs increases with increasing laser power density. The solution temperature of PPY@PB NEs is at 3.0 W / cm². 2 The laser irradiation at high power density can reach 68.6℃, while the temperature of the deionized water control group did not change significantly after 5 minutes of irradiation.

[0059] 3. The oxygen production capacity of the polypyrrole-Prussian blue hybrid photothermal nanozymes PPY@PB NEs prepared in Example 1, PPYNEs prepared in Comparative Example 1, and PB NEs prepared in Comparative Example 2 was tested.

[0060] Test method: Prepare PPY@PB NEs, PPYNEs and PB NEs into 500 μg / mL aqueous solutions, take 1 mL of each, and add 200 μL H2O2 to each. Use deionized water as a control group. Use a handheld oxygen detector to detect the oxygen production of each group every 2 minutes.

[0061] Test results are available Figure 6 As can be seen, the oxygen content in the PPYNEs group and the deionized water group did not change significantly, while the oxygen content in PB NEs and PPY@PB NEs increased significantly, indicating that PPY@PB NEs has the ability to decompose H2O2 to produce oxygen.

[0062] 4. Detection of blood compatibility of polypyrrole-Prussian blue hybrid photothermal nanozymes

[0063] Test method: The PPY@PB NEs prepared in Example 1 were prepared into aqueous solutions of 25, 50, 100, and 200 μg / mL as experimental group samples. Deionized water and PBS were used as positive and negative controls, respectively. Then, the aqueous solutions of PPY@PB NEs at different concentrations (25, 50, 100, and 200 μg / mL) and the control group samples were co-incubated with 10% mouse red blood cell suspension for 2 hours and centrifuged. The absorbance of the supernatant at 541 nm (the characteristic UV absorption peak of hemoglobin) was detected. The hemolysis rate of each group of samples was calculated by the absorbance ratio to determine the blood compatibility of the nanoparticles.

[0064] The results are as follows Figure 7 As shown, deionized water and PBS were used as positive and negative controls, respectively. Calculations showed that when the highest concentration of PPY@PB NEs reached 200 μg / mL, the hemolysis rate was less than 5%, indicating that PPY@PB NEs has good blood compatibility.

[0065] 5. Determining the biosafety of polypyrrole-Prussian blue hybrid photothermal nanozymes

[0066] Test method: The PPY@PB NEs prepared in Example 1 were prepared into aqueous solutions of 50, 100, 200, 400, and 800 μg / mL, respectively. The PPY@PB NEs solutions of different concentrations (50, 100, 200, 400, and 800 μg / mL) were co-incubated with MEF (SCRC-1040, ATCC) and 4T1 cells (CRL-2539, ATCC) for 24 h, respectively. Cell viability was detected by CCK-8 assay.

[0067] Test results are as follows Figure 8As shown, different concentrations of PPY@PB NEs did not exhibit significant toxicity to MEF and 4T1 cells, and cell viability remained above 85% in all groups. This indicates that PPY@PB NEs did not cause significant cell damage within this concentration range.

[0068] 6. Detection of the killing ability of polypyrrole-Prussian blue hybrid photothermal nanozymes against tumor cells.

[0069] Test method: PPY@PB NEs prepared in Example 1 were prepared into aqueous solutions of 25, 50, 100, 200, and 400 μg / mL, respectively. Different concentrations (25, 50, 100, 200, and 400 μg / mL) of PPY@PB NEs were co-incubated with 4T1 cells (CRL-2539, ATCC) for 2 h, followed by irradiation with 808 nm near-infrared light. The photothermal killing effect of PPY@PB NEs was studied by detecting cell viability.

[0070] The results are as follows Figure 9 As shown, the activity of 4T1 cells gradually decreased with increasing concentration of PPY@PB NEs. When the nanoparticle concentration reached 400 μg / mL, the activity of 4T1 cells was only 15%. These results demonstrate that PPY@PB NEs possesses excellent photothermal killing ability against tumor cells and can significantly inhibit their growth.

[0071] Although the present invention has been described in detail in the general description and specific embodiments above, those skilled in the art will still have room for modification based on the present invention. Therefore, all simple variations, modifications, or substitutions made without departing from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a polypyrrole-Prussian blue hybrid photothermal nanozyme, characterized in that, Includes the following steps: (1) Add polyvinylpyrrolidone to deionized water and stir until completely dissolved to obtain a polyvinylpyrrolidone solution; (2) Add pyrrole and potassium ferrocyanide to the polyvinylpyrrolidone solution, stir evenly, then add ferric chloride hexahydrate, stir the reaction thoroughly, and obtain the reaction solution. (3) Centrifuge the reaction solution to remove the precipitate and obtain the supernatant. Dialyze the supernatant and freeze-dry it under vacuum to obtain the polypyrrole-Prussian blue hybrid photothermal nanozyme. In step (2), the molar ratio of pyrrole to potassium ferrocyanide is (1-15):(1-15).

2. The method for preparing polypyrrole-Prussian blue hybrid photothermal nanozyme as described in claim 1, characterized in that, In step (1), the concentration of the polyvinylpyrrolidone solution is 5–20 mg / mL.

3. The method for preparing polypyrrole-Prussian blue hybrid photothermal nanozyme as described in claim 1, characterized in that, In step (2), the amount of ferric chloride hexahydrate used accounts for 25-40% of the total mass of pyrrole and potassium ferrocyanide.

4. The method for preparing polypyrrole-Prussian blue hybrid photothermal nanozyme as described in claim 1, characterized in that, In step (3), the centrifugation conditions are: 3000 rpm for 15 min.

5. The method for preparing polypyrrole-Prussian blue hybrid photothermal nanozyme as described in claim 1, characterized in that, In step (3), the dialysis uses a 100000MWCO dialysis bag and the dialysis time is 72h.

6. The method for preparing polypyrrole-Prussian blue hybrid photothermal nanozyme according to any one of claims 1 to 5, characterized in that, In step (3), the freeze-drying temperature is -80℃ and the time is 48h.

7. The use of the polypyrrole-Prussian blue hybrid photothermal nanozyme prepared by the method of any one of claims 1-6 in the preparation of photothermal therapeutic drugs for tumors.

Images