Flower-like manganese dioxide and preparation method and application thereof

Abstract

The invention provides flower-like manganese dioxide as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) adding a reducing agent into a potassium permanganate solution, and reducing to obtain powder A; (2) performing first mixing on the powder A obtained in the step (1), a fluorescent reagent and a first solvent to obtain powder B; (3) secondarily mixing the powder B obtained in the step (2), bovine serum albumin and a second solvent to obtain powder C; and (4) the powder C obtained in the step (3) and a targeting reagent solution are subjected to third mixing, the flower-like manganese dioxide is obtained, and the targeting reagent solution contains folic acid. The flower-like manganese dioxide prepared by the invention can provide molecular imaging and structural imaging information of joints in the treatment process of rheumatoid arthritis, has specific responsiveness, and is suitable for imaging means in early diagnosis, middle-stage detection, drug therapy, surgical treatment and the like of rheumatoid arthritis and related treatment processes.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and relates to flower-shaped manganese dioxide and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is classified as a systemic, multi-joint, chronic autoimmune arthritis that causes severe disability in patients and has a huge impact on the lives of people around the world. RA is pathologically manifested as immune cell infiltration, synovial lining hyperplasia, pannus formation, and destruction of articular cartilage and bone. It is characterized by chronic synovitis and cartilage destruction, which can cause synovial hyperplasia, pannus formation, joint inflammation, and bone erosion, ultimately leading to progressive joint dysfunction, and in severe cases, disability and death.

[0003] Magnetic resonance imaging (MRI) has become increasingly popular due to its advantages such as non-invasiveness, no radiation damage, high resolution, scanning in any orientation, and multi-nuclear multi-parameter imaging. At the same time, fluorescence imaging technology has the advantages of high detection sensitivity and no radiation hazard, and has a wide range of applications in the biomedical field.

[0004] At present, the main pathological characteristics of RA are synovitis, synovial hyperplasia, vasculitis, pannus formation and the formation of rheumatoid nodules, which lead to cartilage and bone destruction, joint deformities, etc. Without formal treatment, about 75% of patients will become disabled within 3 years. Moreover, the existing diagnostic criteria rely entirely on symptoms and strict physical examinations, which means that patients can only be diagnosed six months or a year after onset, which seriously delays the treatment time of patients. The current means cannot achieve accurate diagnosis of the early onset of RA.

[0005] CN105985962A discloses an aptamer specifically targeting rheumatoid arthritis inflammatory synovial cells and its application. Provided is an aptamer specifically targeting human rheumatoid arthritis inflammatory synovial cells, the aptamer having a nucleotide sequence of SEQID NO.1; further provided is the application of the aptamer in preparing a biological probe for identifying inflammatory synovial cells and in preparing a targeted anti-rheumatoid arthritis drug or drug carrier.

[0006] CN103403028A discloses an antibody that specifically binds to the synovial microvasculature of arthritis patients, and comprises one or more complementary determining regions (CDRs) selected from the group consisting of SEQ ID Nos. 1-4. This antigen-binding polypeptide and its conjugate can be used in the diagnosis and treatment of arthritis.

[0007] In addition to the two published patents mentioned above, there are some imaging methods applied to RA, such as musculoskeletal ultrasound and MRI. The main problem is that the current materials are not absolutely specific to RA, because non-RA symptoms such as synovitis and bone marrow edema will have signals.

[0008] Therefore, how to prepare a material with absolute specificity and high sensitivity to rheumatoid arthritis (RA) is an important research direction in this field. Summary of the invention

[0009] The purpose of the present invention is to provide a flower-shaped manganese dioxide with absolute specificity and stable structure for rheumatoid arthritis imaging, and a preparation method and application thereof.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] One of the purposes of the present invention is to provide a method for preparing flower-shaped manganese dioxide, the preparation method comprising the following steps:

[0012] (1) adding a reducing agent to a potassium permanganate solution to reduce the solution to obtain powder A;

[0013] (2) mixing the powder A, the fluorescent reagent and the first solvent described in step (1) to obtain powder B;

[0014] (3) performing a second mixing of the powder B, bovine serum albumin and a second solvent described in step (2) to obtain powder C;

[0015] (4) The powder C and the targeting agent solution of step (3) are mixed for the third time to obtain the flower-shaped manganese dioxide, wherein the targeting agent solution contains folic acid.

[0016] The present invention comprises step (1) adding oleic acid to a potassium permanganate solution for an in-situ reduction reaction to obtain powder A, wherein the potassium permanganate solution serves as an oxidant and provides a manganese source; step (2) loading a fluorescent reagent to obtain powder B loaded with the fluorescent reagent; step (3) mixing bovine serum albumin and bovine serum albumin to obtain powder C coated with bovine serum albumin; and step (4) connecting powder C with a targeting agent to finally obtain flower-shaped manganese dioxide. During the entire preparation process, the fluorescent agent can be dispersed on the surface of the flower-shaped manganese dioxide or dispersed inside the flower-shaped manganese dioxide.

[0017] The flower-shaped manganese dioxide prepared by the present invention has uniform particle size, rich folate groups on the surface, good biocompatibility, and is easy to target folate receptors. For the on-the-spot diagnosis of rheumatoid arthritis, it has the advantages of high responsiveness, high specificity, magnetic resonance capability, and biosafety.

[0018] As a preferred technical solution of the present invention, the method for preparing the potassium permanganate solution in step (1) is: stirring potassium permanganate and water to obtain a potassium permanganate solution.

[0019] Preferably, the mass ratio of potassium permanganate to water is (2-6):(1-3), wherein the mass ratio can be 2:1, 1:1, 2:3, 3:1, 3:2, 4:1, 5:1, 5:2, 5:3 or 6:1, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0020] In the present invention, if the mass ratio of potassium permanganate to water is too high, the flower-shaped manganese dioxide will be too dense, and if the mass ratio of potassium permanganate to water is too low, the flower-shaped manganese dioxide cannot be formed.

[0021] Preferably, the stirring time is 20 to 40 min, wherein the time can be 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min or 40 min, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0022] As a preferred technical solution of the present invention, the reducing agent in step (1) is oleic acid.

[0023] The present invention adopts oleic acid as a reducing agent, and the oleic acid also plays a role of a dispersant.

[0024] Preferably, the volume ratio of oleic acid to water is (1-3):(25-75), wherein the volume ratio can be 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 2:25, 2:30, 2:35, 2:40, 2:45, 2:50, 2:55, 2:60, 2:65, 2:70, 2:75, 3:25, 3:30, 3:35, 3:40, 3:45, 3:50, 3:55, 3:60, 3:65, 3:70 or 3:75, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0025] In the present invention, if the volume ratio of oleic acid to water is too high, excessive oleic acid will lead to a large amount of agglomeration of the flower-like manganese dioxide. If the volume ratio of oleic acid to water is too low, less reducing agent will be obtained and oleic acid will not provide enough active sites for the flower-like manganese dioxide.

[0026] Preferably, the reduction in step (1) comprises ultrasonic dispersion.

[0027] Preferably, the reduction time in step (1) is 10 to 14 hours, wherein the time can be 10 hours, 11 hours, 12 hours, 13 hours or 14 hours, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0028] Preferably, after the reduction in step (1), first standing, filtering, centrifugal washing, filtering and drying are sequentially performed to obtain the powder A.

[0029] Preferably, the first standing time is 20 to 30 hours, wherein the time can be 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours or 30 hours, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0030] The purpose of the first standing is to allow the reacted powder A to settle. If the first standing time is too short, the sample yield will be too low. If the first standing time is too long, the unreacted and reaction intermediate products will be mixed together, affecting the sample purity.

[0031] Preferably, the temperature of the centrifugal washing is 0-4°C, wherein the temperature can be 0°C, 1°C, 2°C, 3°C or 4°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0032] The invention sets the temperature of centrifugal washing at 0-4°C, which can prevent oleic acid from being decomposed at high temperature.

[0033] Preferably, the speed of the centrifugal washing is 14000-15000 rpm, wherein the speed may be 14000 rpm, 14100 rpm, 14200 rpm, 14300 rpm, 14400 rpm, 14500 rpm, 14600 rpm, 14700 rpm, 14800 rpm, 14900 rpm or 15000 rpm, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0034] Preferably, the centrifugal washing time is 15 to 20 min, wherein the time can be 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0035] Preferably, the washing liquid of the centrifugal washing comprises water and / or ethanol.

[0036] Preferably, the drying comprises vacuum drying.

[0037] Preferably, the drying temperature is -40 to -50°C, wherein the temperature may be -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C or -50°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0038] Low temperature drying is adopted in the present invention.

[0039] As a preferred technical solution of the present invention, the mass ratio of powder A and fluorescent reagent in step (2) is 1:(0.8-1.2), wherein the mass ratio can be 1:0.8, 1:0.9, 1:1.0, 1:1.1 or 1:1.2, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0040] In the present invention, if the mass ratio of powder A to fluorescent reagent is too large, the fluorescent reagent content will be too small and cannot effectively function. If the mass ratio of powder A to fluorescent reagent is too small, the fluorescent reagent will be too much to be loaded.

[0041] Preferably, the fluorescent reagent in step (2) includes IR780.

[0042] Preferably, in step (2), the first solvent comprises water and ethanol.

[0043] The mixed reagent of ethanol and water in the first solvent of the present invention can effectively increase the solubility of the flower-like manganese dioxide and the fluorescent reagent, and may better load the fluorescent reagent on the flower-like manganese dioxide.

[0044] Preferably, the volume ratio of water to ethanol is (1-2):1, wherein the mass ratio can be 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1 or 2:1, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0045] In the present invention, if the volume ratio of water to ethanol is too large or too small, it will lead to poor effect of fluorescent reagent loading.

[0046] As a preferred technical solution of the present invention, in step (2), the first mixing includes stirring after ultrasonic dispersion.

[0047] Preferably, the ultrasonic dispersion time is 20 to 30 min, wherein the time can be 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min or 30 min, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0048] Preferably, the stirring time is 5 to 7 hours, wherein the time may be 5 hours, 6 hours or 7 hours, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0049] Preferably, after the first mixing in step (2), a second standing, centrifugal washing, filtering and drying are sequentially performed to obtain the powder B.

[0050] Preferably, the second standing time is 6 to 12 hours, wherein the time may be 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0051] Preferably, the temperature of the centrifugal washing is 0-4°C, wherein the temperature can be 0°C, 1°C, 2°C, 3°C or 4°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0052] Preferably, the speed of the centrifugal washing is 14000-15000 rpm, wherein the speed may be 14000 rpm, 14100 rpm, 14200 rpm, 14300 rpm, 14400 rpm, 14500 rpm, 14600 rpm, 14700 rpm, 14800 rpm, 14900 rpm or 15000 rpm, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0053] Preferably, the centrifugal washing time is 15 to 20 min, wherein the time can be 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0054] Preferably, the drying comprises vacuum drying.

[0055] Preferably, the drying temperature is -40 to -50°C, wherein the temperature may be -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C or -50°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0056] As a preferred technical solution of the present invention, the second solvent in step (3) includes water.

[0057] Preferably, the mass ratio of the powder B to bovine serum albumin in step (3) is 1:(3-24), wherein the mass ratio can be 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23 or 1:24, but is not limited to the listed values. Other unlisted values ​​within the numerical range are also applicable, preferably 1:(7-9).

[0058] In the present invention, if the mass ratio of powder B to bovine serum albumin is too large, the water solubility of the synthesized sample may be poor, and if the mass ratio of powder B to bovine serum albumin is too small, the bovine serum albumin may be excessive and deteriorate.

[0059] Preferably, the mass ratio of the powder B and the second solvent in step (3) is 1:(3-7), wherein the mass ratio can be 1:3, 1:4, 1:5, 1:6 or 1:7, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0060] Preferably, after the second mixing in step (3), centrifugal washing, filtration and drying are sequentially performed to obtain the powder C.

[0061] Preferably, the temperature of the centrifugal washing is 0-4°C, wherein the temperature can be 0°C, 1°C, 2°C, 3°C or 4°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0062] Preferably, the speed of the centrifugal washing is 14000-15000 rpm, wherein the speed may be 14000 rpm, 14100 rpm, 14200 rpm, 14300 rpm, 14400 rpm, 14500 rpm, 14600 rpm, 14700 rpm, 14800 rpm, 14900 rpm or 15000 rpm, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0063] Preferably, the centrifugal washing time is 15 to 20 min, wherein the time can be 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0064] Preferably, the drying comprises vacuum drying.

[0065] Preferably, the drying temperature is -40 to -50°C, wherein the temperature may be -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C or -50°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0066] As a preferred technical solution of the present invention, the method for preparing the targeting reagent solution in step (4) comprises: dissolving the targeting reagent in a phosphate buffer to obtain the targeting reagent solution.

[0067] Preferably, the targeting agent is folic acid-polyethylene glycol-N-hydroxysuccinimide active ester.

[0068] The targeting agent of the present invention contains folic acid components, and the folic acid can target macrophages in the microenvironment of rheumatoid arthritis.

[0069] Preferably, the molecular weight of polyethylene glycol in the folic acid-polyethylene glycol-N-hydroxysuccinimide active ester is 1800-2200Da, wherein the molecular weight can be 1800Da, 1850Da, 1900Da, 1950Da, 2000Da, 2050Da, 2100Da, 2150Da or 2200Da, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0070] The folic acid in the folic acid-polyethylene glycol-N-hydroxysuccinimide active ester of the present invention has targeting properties and contains an amino group that can be connected with the powder C. If the molecular weight of the polyethylene glycol in the folic acid-polyethylene glycol-N-hydroxysuccinimide active ester of the present invention is too large, the particle size of the synthesized sample will be too large, and if the molecular weight is too small, it will be difficult to control the quality of the synthesized sample.

[0071] Preferably, the pH of the phosphate buffer is 7.0-8.0, wherein the pH may be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0072] Preferably, the mass ratio of the folic acid-polyethylene glycol-N-hydroxysuccinimide active ester to powder C is (1-3):(10-30), wherein the mass ratio can be 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, 1:30, 2:10, 2:12, 2:14, 2:16, 2:18, 2:20, 2:22, 2:24, 2:26, ​​2:28, 3:10, 3:12, 3:14, 3:16, 3:18, 3:20, 3:22, 3:24, 3:26, 3:28 or 3:30, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0073] As a preferred technical solution of the present invention, the time for the third mixing in step (4) is 4 to 6 hours, wherein the time can be 4h, 4.2h, 4.4h, 4.6h, 4.8h, 5h, 5.2h, 5.4h, 5.6h, 5.8h or 6h, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0074] Preferably, after the third mixing in step (4), centrifugal washing, filtration and drying are sequentially performed to obtain the flower-like manganese dioxide.

[0075] Preferably, the temperature of the centrifugal washing is 0-4°C, wherein the temperature can be 0°C, 1°C, 2°C, 3°C or 4°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0076] Preferably, the speed of the centrifugal washing is 14000-15000 rpm, wherein the speed may be 14000 rpm, 14100 rpm, 14200 rpm, 14300 rpm, 14400 rpm, 14500 rpm, 14600 rpm, 14700 rpm, 14800 rpm, 14900 rpm or 15000 rpm, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0077] Preferably, the centrifugal washing time is 15 to 20 min, wherein the time can be 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0078] Preferably, the drying comprises vacuum drying.

[0079] Preferably, the drying temperature is -40 to -50°C, wherein the temperature may be -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C or -50°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0080] The second object of the present invention is to provide a flower-shaped manganese dioxide, wherein the flower-shaped manganese dioxide is prepared by the preparation method of the flower-shaped manganese dioxide as described in the first object.

[0081] The flower-shaped manganese dioxide prepared by the invention has uniform particle size, rich folate groups on the surface, good biocompatibility, and is easy to target folate receptors.

[0082] The third object of the present invention is to provide an application of the flower-shaped manganese dioxide as described in the second object, wherein the flower-shaped manganese dioxide is applied in the field of biomedical material technology.

[0083] The flower-shaped manganese dioxide prepared by the invention can be applied to magnetic resonance imaging and fluorescence multimodal imaging of rheumatoid arthritis.

[0084] Compared with the prior art, the present invention has the following beneficial effects:

[0085] (1) The preparation method of the flower-shaped manganese dioxide of the present invention has the advantages of simple reaction steps, low cost, and green environmental protection. The prepared functionalized flower-shaped manganese dioxide has uniform particle size, stable structure, good biosafety, magnetic resonance performance, and multimodal imaging function;

[0086] (2) The flower-shaped manganese dioxide prepared by the present invention can provide molecular imaging and structural imaging information of joints during the treatment of rheumatoid arthritis, has specific responsiveness, and is suitable for imaging methods in the early diagnosis, mid-term detection, drug treatment, surgical treatment, etc. of rheumatoid arthritis and related treatment processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 It is a transmission electron microscope image of the flower-shaped manganese dioxide in Example 1 of the present invention.

[0088] Figure 2 This is the Zeta potential diagram of flower-shaped manganese dioxide in ultrapure water according to Example 1 of the present invention.

[0089] Figure 3 This is a graph of water and particle size of flower-shaped manganese dioxide in ultrapure water according to Example 1 of the present invention.

[0090] Figure 4 It is the ultraviolet absorption spectrum of the flower-shaped manganese dioxide in Example 1 of the present invention at different times in a microenvironment solution simulating rheumatoid arthritis.

[0091] Figure 5 This is a graph of the r1 relaxation curves of the flower-shaped manganese dioxide in Example 1 of the present invention in a microenvironment solution simulating rheumatoid arthritis and under normal conditions.

[0092] Figure 6 This is a graph showing the cell viability after co-culturing HUVEC cells with flower-shaped manganese dioxide of different concentrations in Example 1 of the present invention for 24 hours.

[0093] Figure 7 This is a graph of cell viability after co-culturing RAW254.7 cells with flower-shaped manganese dioxide of different concentrations in Example 1 of the present invention for 24 hours.

[0094] Figure 8 This is a fluorescent imaging diagram of the flower-shaped manganese dioxide obtained in Application Example 1 of the present invention in CIA model mice.

[0095] Fig. 9 This is a magnetic resonance imaging image of the flower-shaped manganese dioxide obtained in Application Example 1 of the present invention in CIA model mice. DETAILED DESCRIPTION

[0096] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0097] Example 1

[0098] This embodiment provides a method for preparing flower-shaped manganese dioxide, and the preparation method comprises the following steps:

[0099] (1) Weigh 100 mg of potassium permanganate and 50 mL of ultrapure water into a beaker, stir vigorously for 30 min to obtain a potassium permanganate solution, add 2 mL of oleic acid to the potassium permanganate solution, and disperse by ultrasonication to form a stable emulsion. After the reduction reaction for 12 h, the solution is allowed to stand overnight, filtered, the residue is collected, the residue is dispersed in ultrapure water for centrifugal washing, the residue is collected, and vacuum dried at -45°C to obtain powder A, wherein the centrifugal washing temperature is 2°C, the speed is 14500 rpm, and the time is 18 min;

[0100] (2) Ultrapure water and anhydrous ethanol were mixed and stirred in a ratio of 3:2 to obtain 100 mL of a mixed solution, 20 mg of powder A and 20 mg of IR780 were added, and the mixture was dispersed by ultrasonication for 25 min, stirred for 6 h, and allowed to stand for 9 h. The mixture was centrifugally washed with anhydrous ethanol and ultrapure water, the residue was collected, and vacuum dried at -45 °C to obtain powder B, wherein the centrifugal washing temperature was 2 °C, the speed was 14500 rpm, and the time was 18 min;

[0101] (3) Dissolve 20 mg of the powder B obtained in step (2) in 100 mL of ultrapure water, add 160 mg of bovine serum albumin, stir for 4 h, wash by centrifugation with ultrapure water, collect the residue, and dry it in vacuo at -45 °C to obtain powder C, wherein the centrifugal washing temperature is 2 °C, the speed is 14500 rpm, and the time is 18 min;

[0102] (4) Dissolve 10 mg of folic acid-polyethylene glycol-N-hydroxysuccinimide active ester in 10 mL of phosphate buffer solution with a pH of 7.5, add 100 mg of powder C from step (3), stir for 5 h, wash with ultrapure water by centrifugation, collect the filter residue, and dry in vacuo at -45 ° C to obtain the flower-like manganese dioxide, wherein the centrifugal washing temperature is 2 ° C, the speed is 14500 rpm, and the time is 18 min.

[0103] The transmission electron microscope image of the flower-like manganese dioxide prepared in this example is as follows: Figure 1 As shown, it can be seen that the morphology of the energetic flower-like manganese dioxide is a typical honeycomb-like monodispersed nanosphere with uniform particle size, uniform dispersion and a large specific surface area.

[0104] The Zeta potential diagram of the flower-shaped manganese dioxide prepared in this example in ultrapure water is as follows: Figure 2 As shown, Figure 1 The Zeta potential of powder A (hMnO2), powder B (hMnO2-IR), powder C (hMnO2-IR@BSA), and powder D (flower-like manganese dioxide, hMnO2-IR@BSA-PEG-FA) dissolved in ultrapure water showed that the Zeta potential of hMnO2 began to increase after loading with IR780, and the Zeta potential gradually decreased with the addition of bovine serum albumin and folic acid-polyethylene glycol-N-hydroxysuccinimide active ester.

[0105] The water content and particle size of the flower-shaped manganese dioxide prepared in this example in ultrapure water are shown in the figure below. Figure 3 As shown, the water content and particle size of powder A (hMnO2), powder B (hMnO2-IR), powder C (hMnO2-IR@BSA), and powder D (flower-like manganese dioxide, hMnO2-IR@BSA-PEG-FA) dissolved in ultrapure water. With the addition of IR780, bovine serum albumin and folic acid-polyethylene glycol-N-hydroxysuccinimide active ester, the water content and particle size of the samples continued to increase, proving that the surface of the flower-like manganese dioxide was successfully modified.

[0106] The flower-shaped manganese dioxide prepared in Example 1 of the present invention was subjected to responsiveness test, magnetic resonance ability test and biosafety test. The test methods and test results are as follows:

[0107] 1. Responsiveness Test

[0108] A microenvironment solution simulating rheumatoid arthritis was prepared, which was a mixed solution of phosphate buffer (pH=6.5) and hydrogen peroxide (concentration of 1 mmol / L), and 100 μL of 200 mg / mL flower-shaped manganese dioxide was added. The absorption at different times was recorded by UV-visible absorption spectroscopy.

[0109] The response experiment of flower-like manganese dioxide was carried out by simulating the microenvironment of rheumatoid arthritis. Figure 4 The ultraviolet absorption spectra of the flower-shaped manganese dioxide prepared by the present invention at different times in a microenvironment solution simulating rheumatoid arthritis can clearly be seen that the absorption peak of manganese dioxide at 380nm disappears after 3 minutes, proving that the flower-shaped manganese dioxide can be responsive in a microenvironment solution simulating rheumatoid arthritis.

[0110] 2. Magnetic Resonance Ability Test

[0111] The tests were carried out in a microenvironment solution simulating rheumatoid arthritis and under normal conditions (phosphate buffer solution pH=7.4), and 100 μL of flower-like manganese dioxide of different concentrations was added and allowed to react for 10 minutes before testing its magnetic resonance relaxation rate.

[0112] Figure 5 The r1 relaxation curve of the flower-shaped manganese dioxide prepared by the present invention in the microenvironment solution simulating rheumatoid arthritis and under normal conditions. As can be seen from the figure, the r1 value of the flower-shaped manganese dioxide under normal conditions is 0.006955mM -1 s -1 The r1 value of flower-like manganese dioxide in the microenvironment solution simulating rheumatoid arthritis is 5.9460 mM -1 s -1 , which is 85.49 times higher than the r1 value under normal conditions, proving that flower-like manganese dioxide can have good magnetic resonance imaging ability in the microenvironment solution simulating rheumatoid arthritis.

[0113] 3. Biosafety Testing

[0114] The CCK-8 method was used to detect the cytotoxicity of the prepared flower-shaped manganese dioxide. The CCK-8 cytotoxicity test results showed that after HUVEC cells and RAW254.7 cells were co-cultured with different concentrations of flower-shaped manganese dioxide for 24 hours, the results were as follows Figure 6 and Figure 7 When the concentration of flower-shaped manganese dioxide was 0.125 mmol / L, the cell survival rate was still maintained above 80%, indicating that the prepared flower-shaped manganese dioxide had good biological safety.

[0115] HUVEC cell culture conditions: Add 3mL of DMEM culture medium containing 10% fetal bovine serum to a 60nm cell culture dish and culture it in a constant temperature incubator at 37℃ and 5% CO2. HUVEC is an adherent cell. When the cells grow to 80%, digest them with 1mL of 0.25% trypsin solution for 2min, terminate the trypsin effect with 1mL of culture medium containing 10% fetal bovine serum, and repeatedly blow the cells at the bottom of the dish to fully disperse them. Centrifuge at 100rpm for 5min, discard the supernatant, add fresh culture medium to the cell pellet, blow evenly and transfer to a new culture dish at a ratio of 1:4 to continue culturing for later use.

[0116] Raw264.7 cell culture conditions: Add 3mL of DMEM culture medium containing 10% fetal bovine serum to a 60nm cell culture dish and culture it in a constant temperature incubator at 37℃ and 5% CO2. When the cells grow to 80%, repeatedly blow the cells on the bottom of the dish to fully disperse them. Centrifuge at 100rpm for 5min, discard the supernatant, add fresh culture medium to the cell pellet, blow evenly and transfer to a new culture dish at a ratio of 1:4 to continue culturing for later use.

[0117] It can be seen from the above test conditions that the flower-shaped manganese dioxide prepared in Example 1 of the present invention has excellent responsiveness, magnetic resonance capability and biosafety.

[0118] Example 2

[0119] This embodiment provides a method for preparing flower-shaped manganese dioxide, and the preparation method comprises the following steps:

[0120] (1) Weigh 150 mg of potassium permanganate and 75 mL of ultrapure water into a beaker, stir vigorously for 40 min to obtain a potassium permanganate solution, add 3 mL of oleic acid to the potassium permanganate solution, and disperse by ultrasonication to form a stable emulsion. After the reduction reaction for 14 h, the solution is allowed to stand overnight, filtered, the residue is collected, the residue is dispersed in ultrapure water for centrifugal washing, the residue is collected, and vacuum dried at -40°C to obtain powder A, wherein the centrifugal washing temperature is 0°C, the speed is 14000 rpm, and the time is 15 min;

[0121] (2) Ultrapure water and anhydrous ethanol were mixed and stirred in a ratio of 3:2 to obtain 100 mL of a mixed solution, 20 mg of powder A and 20 mg of IR780 were added, and the mixture was dispersed by ultrasonication for 30 min, and then stirred for 7 h, and allowed to stand for 6 h. The mixture was centrifugally washed with anhydrous ethanol and ultrapure water, the residue was collected, and vacuum dried at -40°C to obtain powder B, wherein the centrifugal washing temperature was 0°C, the speed was 14000 rpm, and the time was 15 min;

[0122] (3) 30 mg of the powder B prepared in step (2) was dissolved in 100 mL of ultrapure water, and 240 mg of bovine serum albumin was added. After stirring for 5 h, the mixture was centrifuged and washed with ultrapure water. The residue was collected and dried under vacuum at -40 °C to obtain powder C, wherein the centrifugal washing temperature was 0 °C, the speed was 14000 rpm, and the time was 15 min;

[0123] (4) Dissolve 15 mg of folic acid-polyethylene glycol-N-hydroxysuccinimide active ester in 10 mL of phosphate buffer solution with a pH of 7.0, add 150 mg of powder C from step (3), stir for 5 h, wash by centrifugation with ultrapure water, collect the filter residue, and dry in vacuo at -40°C to obtain the flower-like manganese dioxide, wherein the centrifugal washing temperature is 0°C, the speed is 14000 rpm, and the time is 15 min.

[0124] Example 3

[0125] This embodiment provides a method for preparing flower-shaped manganese dioxide, and the preparation method comprises the following steps:

[0126] (1) Weigh 50 mg of potassium permanganate and 25 mL of ultrapure water into a beaker, stir vigorously for 20 min to obtain a potassium permanganate solution, add 1 mL of oleic acid to the potassium permanganate solution, and disperse by ultrasonication to form a stable emulsion. After the reduction reaction for 12 h, the solution is allowed to stand overnight, filtered, the residue is collected, the residue is dispersed in ultrapure water for centrifugal washing, the residue is collected, and vacuum dried at -50°C to obtain powder A, wherein the centrifugal washing temperature is 4°C, the speed is 15000 rpm, and the time is 20 min;

[0127] (2) Ultrapure water and anhydrous ethanol were mixed and stirred in a ratio of 3:2 to obtain 100 mL of a mixed solution, 20 mg of powder A and 20 mg of IR780 were added, and the mixture was dispersed by ultrasonication for 20 min, and then stirred for 5 h, and allowed to stand for 12 h. The mixture was centrifugally washed with anhydrous ethanol and ultrapure water, the residue was collected, and vacuum dried at -50 °C to obtain powder B, wherein the centrifugal washing temperature was 4 °C, the speed was 15000 rpm, and the time was 20 min;

[0128] (3) Dissolve 10 mg of the powder B obtained in step (2) in 100 mL of ultrapure water, add 80 mg of bovine serum albumin, stir for 3 h, wash by centrifugation with ultrapure water, collect the residue, and dry it in vacuo at -50 °C to obtain powder C, wherein the centrifugal washing temperature is 4 °C, the speed is 15000 rpm, and the time is 20 min;

[0129] (4) Dissolve 5 mg of folic acid-polyethylene glycol-N-hydroxysuccinimide active ester in 10 mL of phosphate buffer solution with a pH of 8.0, add 50 mg of powder C from step (3), stir for 5 h, wash with ultrapure water by centrifugation, collect the filter residue, and dry in vacuo at -50°C to obtain the flower-like manganese dioxide, wherein the centrifugal washing temperature is 4°C, the speed is 15000 rpm, and the time is 20 min.

[0130] Example 4

[0131] In this example, except that oleic acid in step (1) is replaced by sulfurous acid, other conditions are the same as those in Example 1.

[0132] Comparative Example 1

[0133] In this comparative example, except that step (3) is not performed and the powder B prepared in step (2) is directly mixed with the targeting agent solution in step (4), other conditions are the same as those in Example 1.

[0134] Application Example 1

[0135] The flower-shaped manganese dioxide prepared in Example 1 of the present invention is used as a multimodal imaging agent in the treatment of rheumatoid arthritis. Through magnetic resonance and fluorescence imaging, it provides molecular imaging and structural imaging information of joints during the treatment of rheumatoid arthritis. It can be used in the early diagnosis, mid-term detection, drug treatment, surgical treatment, etc. of rheumatoid arthritis and imaging methods in related treatment processes.

[0136] The flower-shaped manganese dioxide prepared in Example 1 was applied to fluorescence imaging and magnetic resonance imaging:

[0137] (1) Establishment of CIA mouse model: 5-6 week old female SPF grade DBA / 1J mice and 7-8 week old female Wistar rats were selected and the model was established after 1 week of feeding. Primary immunization: bovine type II collagen (2 mg mL -1 ) and Freund's complete adjuvant (1 mg mL -1 ) were fully mixed and emulsified, and then injected subcutaneously at the base of the mouse tail; boosting immunization: 2 weeks after modeling, bovine type II collagen (2 mg mL -1 ) and Freund's incomplete adjuvant (1 mg mL -1 ) were mixed thoroughly into emulsified form and injected subcutaneously at the base of the mouse tail.

[0138] (2) Fluorescence imaging: One week after the booster immunization, the DBA / 1J mice in step (1) were injected with 100 μL of 0.125 mM (manganese ion concentration) flower-shaped manganese dioxide via the tail vein. At different times after the injection, in vivo near-infrared fluorescence imaging was performed using an IVIS Lumina XR SeriesIII Spectrum system (PerkinElmer, Waltham, MA) at an excitation wavelength of 780 nm and an emission wavelength of 810 nm.

[0139] Figure 8 For this application example, a fluorescence imaging diagram of flower-like manganese dioxide in CIA model mice was prepared. As can be seen from the figure, bright fluorescence can be seen in the joints of the mice 24 hours after the tail vein injection of flower-like manganese dioxide, proving that flower-like manganese dioxide can responsively achieve fluorescence imaging in rheumatoid arthritis.

[0140] (3) Magnetic resonance imaging: One week after the booster immunization, the Wistar rats in step (1) were injected with 150 μL of 0.125 mM (manganese ion concentration) flower-shaped manganese dioxide through the tail vein. At different times after the injection, T1-weighted magnetic resonance imaging images of the Wistar rats were obtained on a 3.0T MRI (Umr770 3.0T) imaging system;

[0141] Fig. 9 For this application example, a magnetic resonance imaging image of flower-like manganese dioxide in CIA model mice was prepared. As shown in the figure, 24 hours after the tail vein injection of flower-like manganese dioxide, the synovium in the joints of the mice became brighter, and the synovial hyperplasia of rheumatoid arthritis can be seen, proving that flower-like manganese dioxide can responsively achieve magnetic resonance imaging in rheumatoid arthritis.

[0142] As can be seen from the above application examples, the flower-shaped manganese dioxide prepared by the present invention has specific response ability under the microenvironment conditions simulating rheumatoid arthritis, and is an imaging agent used in the treatment process of rheumatoid arthritis. It is suitable for early diagnosis, mid-term detection, drug treatment, surgical treatment, etc. in the treatment process of rheumatoid arthritis and imaging means in related treatment processes. It is also suitable for multimodal imaging agents for related diseases with weak acid and high hydrogen peroxide microenvironment, such as cancer, osteoarthritis, etc., and has broad application prospects.

[0143] The flower-shaped manganese dioxide prepared in Examples 1-4 of the present invention and Comparative Example 1 was subjected to a responsiveness test, and the test results are shown in Table 1.

[0144] The method of responsiveness test is as follows: a microenvironment solution simulating rheumatoid arthritis is prepared, the solution is a mixed solution of phosphate buffer (pH=6.5) and hydrogen peroxide (concentration is 1 mmol / L), and 100 μL of 200 mg / mL of flower-shaped manganese dioxide of Examples 1-4 and Comparative Example 1 is added separately. The absorption at different times is recorded by UV-visible absorption spectroscopy.

[0145] Table 1

[0146] Respond Response time Water Solubility Example 1 yes 3 minutes excellent Example 2 yes 3 minutes excellent Example 3 yes 3 minutes excellent Example 4 no - good Comparative Example 1 yes 3 minutes Difference

[0147] It can be seen from the above table that the flower-shaped manganese dioxide prepared in Examples 1-3 of the present invention has excellent responsiveness; in Example 4, oleic acid in step (1) is replaced with sulfurous acid, and flower-shaped manganese dioxide cannot be generated; in Comparative Example 1, bovine serum albumin is not coated. The water solubility of the flower-shaped manganese dioxide finally prepared is poor.

[0148] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing flower-shaped manganese dioxide, characterized in that: The preparation method comprises the following steps: (1) adding a reducing agent to a potassium permanganate solution to reduce the solution to obtain powder A; (2) mixing the powder A, the fluorescent reagent and the first solvent described in step (1) to obtain powder B; (3) performing a second mixing of the powder B, bovine serum albumin and a second solvent described in step (2) to obtain powder C; (4) The powder C and the targeting agent solution of step (3) are mixed for the third time to obtain the flower-shaped manganese dioxide, wherein the targeting agent solution contains folic acid.

2. The preparation method according to claim 1, characterized in that: The preparation method of the potassium permanganate solution in step (1) is: stirring potassium permanganate and water to obtain a potassium permanganate solution; Preferably, the mass ratio of potassium permanganate to water is (2-6):(1-3); Preferably, the stirring time is 20 to 40 minutes.

3. The preparation method according to claim 2, characterized in that: The reducing agent in step (1) is oleic acid; Preferably, the volume ratio of oleic acid to water is (1-3):(25-75); Preferably, the reduction in step (1) comprises ultrasonic dispersion; Preferably, the reduction time in step (1) is 10 to 14 hours; Preferably, after the reduction in step (1), first standing, filtering, centrifugal washing, filtering and drying are sequentially performed to obtain the powder A; Preferably, the first standing time is 20 to 30 hours.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The mass ratio of the powder A to the fluorescent reagent in step (2) is 1:(0.8-1.2); Preferably, the fluorescent reagent in step (2) comprises IR780; Preferably, in step (2), the first solvent comprises water and ethanol; Preferably, the volume ratio of water to ethanol is (1-2):

1.

5. The preparation method according to any one of claims 1 to 4, characterized in that: Step (2) the first mixing includes stirring after ultrasonic dispersion; Preferably, the ultrasonic dispersion time is 20 to 30 minutes; Preferably, the stirring time is 5 to 7 hours; Preferably, after the first mixing in step (2), second standing, centrifugal washing, filtering and drying are sequentially performed to obtain the powder B; Preferably, the second standing time is 6 to 12 hours.

6. The preparation method according to any one of claims 1 to 5, characterized in that: Step (3) the second solvent comprises water; Preferably, the mass ratio of the powder B to bovine serum albumin in step (3) is 1:(3-24), preferably 1:(7-9); Preferably, the mass ratio of the powder B to the second solvent in step (3) is 1:(3-7); Preferably, after the second mixing in step (3), centrifugal washing, filtration and drying are sequentially performed to obtain the powder C.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The method for preparing the targeting agent solution in step (4) comprises: dissolving the targeting agent in a phosphate buffer to obtain the targeting agent solution; Preferably, the targeting agent is folic acid-polyethylene glycol-N-hydroxysuccinimide active ester; Preferably, the molecular weight of polyethylene glycol in the folic acid-polyethylene glycol-N-hydroxysuccinimide active ester is 1800-2200 Da; Preferably, the pH of the phosphate buffer is 7.0 to 8.0; Preferably, the mass ratio of the folic acid-polyethylene glycol-N-hydroxysuccinimide active ester to powder C is (1-3):(10-30).

8. The preparation method according to any one of claims 1 to 7, characterized in that: The third mixing time in step (4) is 4 to 6 hours; Preferably, after the third mixing in step (4), centrifugal washing, filtration and drying are sequentially performed to obtain the flower-like manganese dioxide.

9. A flower-shaped manganese dioxide, characterized in that: The flower-shaped manganese dioxide is prepared by the preparation method of flower-shaped manganese dioxide according to any one of claims 1 to 8.

10. A use of the flower-shaped manganese dioxide as claimed in claim 9, characterized in that: The flower-shaped manganese dioxide is applied in the technical field of biomedical materials.

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