Tripterygium wilfordii exosome-like nano-vesicle modified prussian blue nano-material and application thereof
Through the Prussian blue nanomaterial modified by Tribute Vine exosome-like nanovesicles, the problems of adverse reactions and insufficient biosafety of existing rheumatoid arthritis treatment drugs have been solved, and nanomaterial preparation with excellent photothermal treatment capabilities and high biosafety is achieved, which is suitable for photothermal treatment of rheumatoid arthritis.
Patent Information
- Application Number
- CN202411967038.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-25
AI Technical Summary
The existing rheumatoid arthritis treatment drugs have adverse reactions and insufficient biosafety, especially the hepatotoxicity caused by chemotherapy drugs prepared by the medicinal active ingredients of Triton Vine limits its widespread use.
Prussian blue nanomaterial modified with tripod vine exosome-like nanovesicles is formed by coating tripod vine exosome-like nanovesicles outside block-shaped Prussian blue nanoparticles. This material is prepared by physical extrusion method and treated with a polycarbonate film to obtain nanomaterials with excellent photothermal conversion and oxidation resistance.
This material has excellent photothermal treatment capabilities, which can effectively reduce inflammatory macrophages in rheumatoid arthritis disease sites, remove reactive oxygen species in disease sites, coordinate immune regulation, and achieve improvement of the immune microenvironment of rheumatoid arthritis. At the same time, the material has good biosafety, low hemolysis rate and low cytotoxicity, and is suitable for nanobiomedicine applications.
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Abstract
Description
Technical Field
[0001] The present application belongs to the field of nanomaterial synthesis and also to the field of biomedical technology, and specifically relates to a Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles and its application in the preparation of drugs for the treatment of rheumatoid arthritis and a preparation method thereof. Background Art
[0002] Rheumatoid arthritis (RA) is an autoimmune disease with erosive arthritis as the main clinical manifestation. At present, the treatment of rheumatoid arthritis mainly adopts chemical drugs such as non-steroidal anti-inflammatory drugs, immunosuppressants, etc., and there are also some herbal medicines, such as Tripterygium wilfordii polyglycoside tablets prepared with medicinal active ingredients extracted from Tripterygium wilfordii. The prior art reports that some of these drugs have some adverse reactions. For example, the hepatotoxicity caused by chemotherapy drugs prepared with medicinal active ingredients of Tripterygium wilfordii limits their wide application, such as Tripterygium wilfordii polyglycoside tablets.
[0003] Research on safe, efficient and biosafe treatment of rheumatoid arthritis is of great significance and has broad prospects. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles, and its application in drugs for treating rheumatoid arthritis and a preparation method thereof in view of the deficiencies in the prior art. The Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles of the present invention has excellent photothermal conversion performance and antioxidant properties, and the hemolysis rate remains at an extremely low level, and the cytotoxicity is low. It is used for photothermal treatment of rheumatoid arthritis, has excellent photothermal treatment ability, and has good biosafety.
[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:
[0006] The first aspect of the present invention provides a Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles, which is a nanoscale particle with a block structure, including block-shaped Prussian blue nanoparticles and Tripterygium wilfordii exosome-like nanovesicles coated outside the Prussian blue nanoparticles.
[0007] According to the above scheme, the Prussian blue nanomaterials modified with Tripterygium wilfordii exosome-like nanovesicles have uniform size, with a particle size of 130-160nm.
[0008] According to the above scheme, the encapsulation thickness of Tripterygium wilfordii exosome-like nanovesicles is in the range of 3-7.5 nm.
[0009] According to the above scheme, a method for preparing Prussian blue nanomaterials modified with Tripterygium wilfordii exosome-like nanovesicles is as follows: Tripterygium wilfordii exosome nanovesicles PELNVs are added to a Prussian blue nanoparticle (PB NPs) solution and mixed, and the mixed system is repeatedly passed through a polycarbonate membrane by physical extrusion to obtain Prussian blue nanomaterials modified with Tripterygium wilfordii exosome-like nanovesicles; the Prussian blue nanomaterials modified with Tripterygium wilfordii exosome-like nanovesicles include Prussian blue nanoparticles and Tripterygium wilfordii exosome-like nanovesicles coated outside the Prussian blue nanoparticles.
[0010] According to the above scheme, the pore particle size of the polycarbonate membrane is preferably 300-500 nm. In some specific embodiments, a polycarbonate membrane with a pore particle size of 400 nm can be selected.
[0011] Provided is an application of the above-mentioned Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles in the preparation of rheumatoid arthritis drugs. The Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles is a nanoscale particle with a block structure, including block-shaped Prussian blue nanoparticles and tripterygium wilfordii exosome-like nanovesicles coated outside the Prussian blue nanoparticles.
[0012] Provided is a method for preparing a Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles, comprising the following steps:
[0013] 1) Wash the tubers of Tripterygium wilfordii and soak them in water overnight, break them and add PBS buffer solution to dilute them to obtain the original pulp;
[0014] 2) centrifuging the puree obtained in step 1) to obtain the supernatant, removing tuber debris, dead cells, and cell debris;
[0015] 3) subjecting the supernatant collected after the last centrifugation in step 2) to ultracentrifugation, discarding the supernatant, and obtaining a homogenate suspension;
[0016] 4) subjecting the homogenate suspension obtained in step 3) to sucrose density gradient ultracentrifugation, collecting the Tripterygium wilfordii exosome-like nanovesicles (PELNVs) and performing vacuum freeze-drying to obtain PELNVs powder;
[0017] 5) adding the PELNVs powder obtained in step 4) into the Prussian blue nanoparticle (PB NPs) solution and stirring the solution to react, using physical extrusion to repeatedly pass the reaction solution through a polycarbonate membrane, and centrifuging to obtain PELNVs-modified PB NPs (PELNVs@PBNPs).
[0018] According to the above scheme, in step 1), the mass volume ratio of Tripterygium wilfordii tuber and PBS buffer solution is: 300g: 200-400mL.
[0019] According to the above scheme, in step 2), at 0-4°C, centrifuge at 2000-4000g for 20-40min and take the supernatant; then centrifuge at 8000-12000g for 50-70min and take the supernatant. The first centrifugation mainly removes tuber debris and dead cells, and the second centrifugation mainly removes cell debris.
[0020] According to the above scheme, in step 3), the ultracentrifugation parameters are: 0-4°C, 140000-160000g centrifugation for 80-100 min.
[0021] According to the above scheme, in step 4), in the sucrose density gradient ultracentrifugation, the mass fractions of the sucrose gradient are 8%, 15%, 30%, 45% and 60%, respectively, and the centrifugation is carried out at 140000-160000g at 0-4°C for 100-150min.
[0022] Preferably, the sucrose gradient of the 30% / 45% layer is the main source of plant exosome-like nanovesicles (PELNVs). The 30% / 45% layer is mainly collected for vacuum freeze-drying to obtain PELNVs powder for standby use.
[0023] According to the above scheme, in step 5), the preparation of Prussian blue nanoparticles is as follows: polyvinyl pyrrolidone and potassium ferrocyanide are dissolved in a dilute hydrochloric acid solution, stirred to completely dissolve, reacted at 70-90° C. for 20-28 hours, washed and centrifuged to obtain Prussian blue nanoparticles (PB NPs).
[0024] Preferably, the cleaning comprises: washing with deionized water and ethanol for 2-3 times respectively; and the centrifugation comprises: centrifugation at 13000-16000g for 12-18min.
[0025] Preferably, the mass ratio of potassium ferrocyanide to polyvinyl pyrrolidone is 1:25-30.
[0026] Preferably, the concentration of the dilute hydrochloric acid solution is 0.01-0.02 mol / L.
[0027] According to the above scheme, in step 5), the mass ratio of Prussian blue nanoparticles to PELNVs powder is 1:1-3.
[0028] According to the above scheme, in step 5), the concentration of the PBNPs solution is 1-3 mg / mL.
[0029] According to the above scheme, in step 5), the stirring reaction time is 10-30 minutes.
[0030] According to the above scheme, in step 5), the centrifugation parameters are: 13000-16000 g for 12-18 min. Centrifuge and purify, and collect the lower precipitate, namely PELNVs-modified PB NPs (PELNVs@PB NPs).
[0031] Prussian blue nanoparticles (PB NPs), as a typical metal organic framework, have strong light absorption ability and high photothermal conversion efficiency in the near infrared region, and have received extensive attention in the biomedical field. In addition, it has been reported that PB NPs have multiple enzyme activities that can be used to remove multiple reactive oxygen species (ROS) and alleviate multiple inflammatory diseases, but at the same time, they have problems such as low catalytic efficiency, high cytotoxicity, and poor therapeutic effect due to large-scale removal in the blood circulation.
[0032] Beneficial effects of the present invention:
[0033] 1. The present invention constructs a Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles by combining Tripterygium wilfordii exosome-like nanovesicles with Prussian blue. The Tripterygium wilfordii exosome-like nanovesicles are coated on the outside of the block-shaped Prussian blue nanoparticles. The obtained Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles is in the form of block-shaped nanoscale particles with uniform size and good biocompatibility. It can avoid being cleared in the blood circulation, has excellent photothermal conversion performance and antioxidant performance, and the hemolysis rate remains at an extremely low level, with low cytotoxicity and good biosafety.
[0034] 2. The Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles constructed by the present invention is used for photothermal therapy of rheumatoid arthritis, which has excellent photothermal therapy ability. Through photothermal therapy, it can effectively reduce inflammatory macrophages in the disease site of rheumatoid arthritis, eliminate reactive oxygen in the disease site, and coordinate immune regulation to achieve improvement of the immune microenvironment of rheumatoid arthritis. It has excellent effect and good biosafety.
[0035] 3. The preparation method of the present invention is simple to operate, low in cost, and easy to prepare in large quantities, and has broad prospects in nanobiomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is the TEM image of the Tripterygium wilfordii PELNVs prepared in Example 1.
[0037] Figure 2 This is a physical picture of the reaction of the Tripterygium wilfordii PELNVs prepared in Example 1 with 1M H2O2 for 1 hour.
[0038] Figure 3 The overall antioxidant capacity of the Tripterygium wilfordii PELNVs prepared in Example 1 at different concentrations was monitored by the ABTS method.
[0039] Figure 4 This is the transmission electron microscopy (TEM) image of the PB NPs prepared in Example 1.
[0040] Figure 5 TEM image of PELNVs@PB NPs prepared in Example 1, where (b) is a partial enlarged image of (a).
[0041] Figure 6 The hydrated particle sizes of PB NPs, PELNVs, and PELNVs@PB NPs prepared in Example 1.
[0042] Figure 7 The potentials of PB NPs, PELNVs, and PELNVs@PB NPs prepared in Example 1.
[0043] Figure 8 The overall antioxidant capacity of PB NPs and PELNVs@PB NPs prepared in Example 1 at different concentrations was monitored by the ABTS method.
[0044] Fig. 9 The temperature rise of PELNVs@PB NPs prepared in Example 1 under near-infrared excitation light of 808 nm at different powers.
[0045] Fig.10 Hemolysis test of PELNVs@PB NPs prepared in Example 1 at different concentrations.
[0046] Fig.11 The toxicity experiment of PB NPs and PELNVs@PB NPs prepared in Example 1 at different concentrations on normal macrophages was performed.
[0047] Fig.12 The PELNVs@PB NPs prepared in Example 1 at different concentrations were 2 The therapeutic effect of 808nm near-infrared light on inflammatory macrophages. DETAILED DESCRIPTION
[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0049] Example 1
[0050] Provided is a method for synthesizing a Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles, comprising the following steps:
[0051] 1) Preparation of plant exosome-like nanovesicles (PELNVs)
[0052] ① 300 g of Tripterygium wilfordii tubers were washed with deionized water and soaked in deionized water overnight. The next day, they were broken with a wall-breaking machine and diluted with 300 mL of PBS buffer solution.
[0053] ② Centrifuge at 3000g for 30 min at 4°C to remove tuber debris and dead cells, and take the supernatant.
[0054] ③ Centrifuge at 10,000 g for 60 min at 4°C to remove cell debris and take the supernatant.
[0055] ④ Centrifuge at 150,000 g for 90 min at 4°C, discard the supernatant, and obtain a homogenate suspension.
[0056] ⑤ For further purification, the homogenate suspension was subjected to sucrose gradient ultracentrifugation (SGC) (sucrose gradient mass fraction was 8%, 15%, 30%, 45% and 60%), and centrifuged at 150000g for 120 min at 4°C. The 30% / 45% layer of sucrose gradient was used as the main source of plant exosome-like nanovesicles (PELNVs), and the collected PELNVs were vacuum-lyophilized to obtain PELNVs powder.
[0057] 2) Preparation of Prussian blue nanoparticles (PB NPs)
[0058] ① Dissolve polyvinyl pyrrolidone and potassium ferrocyanide in 0.01M dilute hydrochloric acid solution and stir until completely dissolved to obtain solution A, in which the concentration of polyvinyl pyrrolidone is 75 mg / mL and the concentration of potassium ferrocyanide is 2.75 mg / mL.
[0059] ② After heating solution A in an oil bath at 80°C for 24 hours, wash it with deionized water and ethanol three times, centrifuge it at 15000g for 15 minutes to obtain Prussian blue nanoparticles (PB NPs). Prepare a 2 mg / mL PB NPs solution for later use.
[0060] 3) Preparation of PELNVs-modified PB NPs
[0061] Take 1 mL of the 2 mg / mL PB NPs solution obtained in step 2) and add 4 mg of PELNVs powder obtained in step 1) and stir for 20 min to obtain solution B. Use physical extrusion to make solution B repeatedly pass through a polycarbonate membrane with a pore size of 400 nm, centrifuge at 15000 g for 15 min, discard the supernatant, and obtain PELNVs-modified PB NPs (PELNVs@PB NPs).
[0062] Figure 1 This is a TEM image of the Tripterygium wilfordii PELNVs prepared in this example. The image shows that the size of the Tripterygium wilfordii PELNVs is 122.78±19.29 nm.
[0063] Figure 2 This is a physical picture of the reaction of the prepared Tripterygium wilfordii PELNVs with 1M H2O2 for 1 hour. The picture shows that after the reaction of Tripterygium wilfordii PELNVs with 1M H2O2 for 1 hour, a large number of bubbles appeared in the solution, indicating that the Tripterygium wilfordii PELNVs have good CAT activity.
[0064] Figure 3 The ABTS method is used to monitor the overall antioxidant capacity of the Tripterygium wilfordii PELNVs prepared in this example at different concentrations. The figure shows that the Tripterygium wilfordii PELNVs have overall antioxidant capacity, and the higher the concentration of PELNVs, the stronger the overall antioxidant capacity.
[0065] Figure 4 The transmission electron microscope (TEM) image of the PB NPs prepared in this example shows that the PB NPs are evenly distributed, have clear edges and corners, and have a size of 107.14±6.98nm.
[0066] Figure 5 The TEM image of the PELNVs@PB NPs prepared in this example shows that the PELNVs@PB NPs have rounded edges and corners, and their size is 143.23±16.16nm. The image shows that there is an obvious film-like coating on the surface of the PB NPs.
[0067] Figure 6 The hydrated particle sizes of PB NPs, PELNVs, and PELNVs@PB NPs prepared in this example are shown in the figure: the hydrated particle sizes of PB NPs, PELNVs, and PELNVs@PB NPs are 200.03±2.95nm, 135.9±1.13nm, and 304.78±1.27nm, respectively.
[0068] Figure 7 The potentials of PB NPs, PELNVs, and PELNVs@PB NPs prepared in this example are shown in the figure: the potentials of PBNPs, PELNVs, and PELNVs@PB NPs are -27.9±5.95mV, -9.33±0.97mV, and -38.9±0.47mV, respectively.
[0069] Figure 8The ABTS method was used to monitor the overall antioxidant capacity of PB NPs and PELNVs@PB NPs prepared in this example at different concentrations. The figure shows that the antioxidant capacity of PELNVs@PB NPs is significantly improved compared with that of PB NPs. When the concentration is 62.5μg / mL, the significant difference in antioxidant capacity between the two reaches *; when the concentration is 125μg / mL and 200μg / mL, the significant difference in antioxidant capacity between the two reaches ***. The statistical one-way analysis of variance calculated the significant difference, *p<0.05, **p<0.01, ***p<0.001.
[0070] Fig. 9 The temperature rise of the PELNVs@PB NPs prepared in this example under the 808nm near-infrared excitation light of different powers. The figure shows that the same concentration (all 200μg / mL) of PELNVs@PB NPs under the 808nm near-infrared excitation light, with the increase of laser power, the heating rate increases, and the temperature reaches the highest after 8 minutes.
[0071] Fig.10 The hemolysis test of the PELNVs@PB NPs prepared in this example at different concentrations is shown in the figure: even after adding 400 μg / mL high concentration of PELNVs@PB NPs and incubating for 4 hours, the hemolysis rate remains at a low level, indicating that PELNVs@PB NPs have high biosafety.
[0072] Fig.11 The figure shows the cytotoxicity test of different concentrations of PB NPs, PELNVs@PB NPs and normal macrophages RAW264.7 incubated for 8 hours. The figure shows that under the same concentration conditions, the survival rate of cells incubated with PELNVs@PB NPs is higher than that of cells incubated with PB NPs for 8 hours, and there is a significant difference between the two under the concentration conditions of 200μg / mL and 100μg / mL, indicating that the Prussian blue nanomaterial PELNVs@PB NP modified with Tripterygium wilfordii exosome-like nanovesicles has low cytotoxicity and better biosafety.
[0073] Fig.12 Different concentrations of PELNVs@PB NPs were incubated with lipopolysaccharide-stimulated inflammatory macrophages RAW264.7 for 4 h and then illuminated with 0.5 W / cm 2 The survival rate of inflammatory macrophages after 5 minutes of 808nm near-infrared light irradiation. The figure shows that with the increase of PELNVs@PB NP concentration, the cell survival rate dropped sharply. When the PELNVs@PB NP concentration was 200μg / mL, the cell survival rate was 13.83%, indicating that PELNVs@PB NP has excellent photothermal therapy ability and can effectively kill inflammatory macrophages.
[0074] The present invention uses tripterygium wilfordii exosome-like nanovesicles and Prussian blue nanoparticles to construct a tripterygium wilfordii exosome-like nanovesicle-modified Prussian blue nanomaterial, wherein the tripterygium wilfordii exosome-like nanovesicles are coated on the outside of the block-shaped Prussian blue nanoparticles, and the obtained tripterygium wilfordii exosome-like nanovesicle-modified Prussian blue nanomaterial is in the form of block-shaped nanoscale particles with uniform size and good biocompatibility, can avoid being cleared in the blood circulation, and has excellent antioxidant performance and photothermal conversion performance ( Figure 8 and Fig. 9 ). And the hemolysis rate remains at a very low level (such as Fig.10 ), low cytotoxicity, and the survival rate of normal macrophage RAW264.7 before composite modification can be significantly improved (such as Fig.11 The Prussian blue nanomaterial modified with the Tripterygium wilfordii exosome-like nanovesicles constructed by the present invention is used for photothermal therapy of rheumatoid arthritis, which has excellent photothermal therapy ability and can effectively kill inflammatory macrophages ( Fig.12 ), with excellent effect and good biosafety.
Claims
1. A Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles, characterized in that: The Prussian blue nanomaterial modified with the tripterygium wilfordii exosome-like nanovesicles is a nanoscale particle with a block-like structure, including block-like Prussian blue nanoparticles and the tripterygium wilfordii exosome-like nanovesicles coated outside the Prussian blue nanoparticles.
2. The nanomaterial according to claim 1, characterized in that The Prussian blue nanomaterial modified with the Tripterygium wilfordii exosome-like nanovesicles has a uniform size, with a particle size of 130-160 nm; the encapsulation thickness of the Tripterygium wilfordii exosome-like nanovesicles is in the range of 3-7.5 nm.
3. The nanomaterial according to claim 1, characterized in that The preparation method of the Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles comprises: adding tripterygium wilfordii exosome nanovesicles PELNVs to a Prussian blue nanoparticle solution and mixing, and repeatedly passing the mixed system through a polycarbonate membrane by physical extrusion to obtain the Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles; the Prussian blue nanomaterial modified with tripterygium wilfordii exosome-like nanovesicles comprises Prussian blue nanoparticles and tripterygium wilfordii exosome-like nanovesicles coated outside the Prussian blue nanoparticles.
4. The nanomaterial according to claim 3, characterized in that: The pore size of the polycarbonate membrane is 300-500nm.
5. Use of the Prussian blue nanomaterial modified with the Tripterygium wilfordii exosome-like nanovesicles according to any one of claims 1 to 4 in the preparation of a drug for rheumatoid arthritis.
6. A method for preparing the Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles according to any one of claims 1 to 4, characterized in that: The steps include: 1) Wash the tubers of Tripterygium wilfordii and soak them in water overnight, break them and add PBS buffer solution to dilute them to obtain the original pulp; 2) centrifuging the puree obtained in step 1) to obtain the supernatant, removing tuber debris, dead cells, and cell debris; 3) subjecting the supernatant collected after the last centrifugation in step 2) to ultracentrifugation, discarding the supernatant, and obtaining a homogenate suspension; 4) subjecting the homogenate suspension obtained in step 3) to sucrose density gradient ultracentrifugation, collecting the Tripterygium wilfordii exosome-like nanovesicles and performing vacuum freeze-drying to obtain PELNVs powder; 5) adding the PELNVs powder obtained in step 4) into the Prussian blue nanoparticle solution and stirring for reaction, using physical extrusion to repeatedly pass the reaction solution through a polycarbonate membrane, and centrifuging to obtain the Prussian blue nanomaterial modified with Tripterygium wilfordii exosome-like nanovesicles.
7. The preparation method according to claim 6, characterized in that: In the step 1), the mass volume ratio of the tuber of Tripterygium wilfordii and the PBS buffer solution is: 300 g: 200-400 mL.
8. The preparation method according to claim 6, characterized in that: In the step 2), centrifuge at 2000-4000 g for 20-40 min at 0-4° C. to obtain the supernatant; then centrifuge at 8000-12000 g for 50-70 min to obtain the supernatant; In the step 3), the ultracentrifugation parameters are: 0-4°C, 140000-160000g centrifugation for 80-100 min; In the step 4), in the sucrose density gradient ultracentrifugation, the mass fractions of the sucrose gradient are 8%, 15%, 30%, 45% and 60% respectively, and the centrifugation is carried out at 140000-160000g for 100-150min at 0-4°C.
9. The preparation method according to claim 6, characterized in that: In the step 5), the preparation of Prussian blue nanoparticles is as follows: polyvinyl pyrrolidone and potassium ferrocyanide are dissolved in a dilute hydrochloric acid solution, stirred to completely dissolve, reacted at 70-90° C. for 20-28 hours, washed and centrifuged to obtain Prussian blue nanoparticles.
10. The preparation method according to claim 6, characterized in that: In the step 5), the mass ratio of Prussian blue nanoparticles to PELNVs powder is 1:1-3; and the stirring reaction time is 10-30 min.