Polyethylene glycol modified bovine serum albumin doped polydopamine-selenium nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medical materials, in particular to polyethylene glycol modified bovine serum albumin doped polydopamine-selenium nanoparticles as well as a preparation method and application thereof. The nano-particle comprises a nano-selenium core and a bovine serum albumin doped polydopamine shell, the diameter of the nano-particle is about 74nm, the dispersity is good, and the preparation method is simple in process. The doping of bovine serum albumin and the modification of polyethylene glycol effectively improve the dispersibility and physiological stability of the nanoparticles. The polyethylene glycol modified bovine serum albumin doped polydopamine-selenium nanoparticles prepared by the invention have good biocompatibility and good dispersibility, can quickly and efficiently remove active oxygen, and provide a new choice for treatment of hepatic ischemia reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and more specifically to polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles, and a preparation method and application thereof. Background Art

[0002] Restoration of blood flow after liver ischemia will trigger more severe inflammatory response and oxidative stress, which is clinically known as liver ischemia-reperfusion injury. The occurrence of liver ischemia-reperfusion injury can lead to the development of liver inflammation, liver fibrosis and liver failure, directly affecting the prognosis of many patients with liver-related diseases. If timely treatment and intervention are carried out, liver ischemia-reperfusion injury can be alleviated or cured.

[0003] Selenium is an essential element for organisms and an important component of some antioxidant enzymes such as glutathione peroxidase and selenoprotein P. It plays a key role in the antioxidant defense system. Compared with inorganic selenium and organic selenium such as selenite and selenomethionine, nano-selenium has advantages such as higher antioxidant activity and low toxicity, and has gradually attracted the attention of scientific researchers.

[0004] The Chinese invention patent publication number is CN110200287A, and the name is an oyster polypeptide-nanoselenium particle complex with alcohol sobering and liver protection effects, and its preparation method and application. It discloses an oyster polypeptide nanoselenium particle complex with alcohol sobering and liver protection effects, and its preparation method and application. The method comprises: homogenizing oyster meat, adding water and active enzymes, enzymolysis, boiling water bath, centrifugation to obtain supernatant, freezing and drying the supernatant to obtain oyster polypeptide powder, adding water, mixing into a solution, adding sodium selenite solution to the oyster polypeptide solution, mixing, adding ascorbic acid solution under stirring, water bath reaction, dialysis, obtaining functionalized nanoselenium sol, ultrasound, freeze drying to obtain an oyster polypeptide nanoselenium particle complex with alcohol sobering and liver protection effects. The present invention uses oyster polypeptide to stabilize nanoselenium, significantly increases the stability of nanoselenium, can be stably stored at 4°C for 1 month, and the nanoparticles can reduce the damage of alcohol or hydrogen peroxide to the liver as detected by MTT method. If this product is transformed into a health product with liver protection function and selenium supplementation, it will produce great social and economic benefits.

[0005] The Chinese invention patent publication number is CN113082048A, and its name is a method for preparing a Se@BSA nanomedicine and its application in acute kidney injury. It discloses that the present invention relates to a method for preparing a Se@BSA nanomedicine and its application in acute kidney injury, and belongs to the field of biomaterials. The Se@BSA nanomedicine synthesized by the present invention is a nanomaterial that can be safely used for intravenous injection, which can be used to manage hypotension of ischemic kidney and damage caused by cardiovascular surgery, and is intended to allow Se@BSA release after renal I / R, thereby avoiding systemic toxicity. The specific mechanism is that Se@BSA upregulates the expression of Gpx1 mRNA and protein levels, removes ROS, inhibits the accumulation of NLRP3, prevents the assembly of inflammasomes, activates downstream inflammatory signaling pathways and NF βB phosphorylation, and slows down the progression of AKI to CKD.

[0006] As a typical melanin analog, polydopamine has good biosafety and biodegradability, metal chelation ability, anti-cancer and other biological activities. At the same time, polydopamine contains a large number of phenolic hydroxyl groups, has a broad spectrum of free radical scavenging ability, and plays an important role in alleviating diseases such as oxidative stress and inflammation. Bovine serum albumin has good biocompatibility and biodegradability. Incorporating bovine serum albumin into polydopamine can further improve the dispersibility of polydopamine. At present, no research on the use of polydopamine-selenium nanoparticles for acute liver injury has been found. Therefore, the development of a bovine serum albumin-doped polydopamine-selenium nanoparticle for liver ischemia-reperfusion injury has great clinical significance. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a polyethylene glycol-modified bovine serum albumin-doped polydopamine-selenium nanoparticle, wherein the nanoparticle comprises a nano-selenium core and a bovine serum albumin-doped polydopamine shell.

[0008] Preferably, the nanoparticles have a diameter of 74 nm and good dispersibility.

[0009] Another aspect of the present invention is to provide a method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles, wherein the specific steps of the nanoparticle preparation method are as follows: S1. dissolving polyvinyl pyrrolidone and sodium selenite in deionized water, stirring to fully dissolve them to obtain a mixed solution Ⅰ; S2. dissolving ascorbic acid in deionized water, slowly adding dropwise to the mixed solution I prepared in S1 and stirring evenly to obtain a mixed solution II; S3. The mixed solution II prepared in S2 is centrifuged at high speed, and the centrifugal precipitate is washed with deionized water to obtain nano-selenium; S4. dissolving nano-selenium, dopamine hydrochloride and bovine serum albumin in Tris hydrochloric acid buffer solution and stirring slowly to obtain a mixed solution III; S5. The mixed solution III prepared in S4 is centrifuged at high speed, and the centrifugal precipitate is washed with deionized water to obtain bovine serum albumin-doped polydopamine-selenium nanoparticles; S6. dissolving bovine serum albumin-doped polydopamine-selenium nanoparticles, carboxyl polyethylene glycol thiol and tris(2-carboxyethyl)phosphine in deionized water and stirring to obtain a mixed solution IV; S7. The mixed solution IV prepared in S6 is centrifuged at high speed, and the centrifugal precipitate is washed with deionized water to obtain polyethylene glycol-modified bovine serum albumin-doped polydopamine-selenium nanoparticles.

[0010] Preferably, the molecular weight of the polyvinyl pyrrolidone in S1 is 40,000.

[0011] Preferably, the molar ratio of sodium selenite to ascorbic acid in S2 is 1:3-4, and the stirring time is 1 hour; the centrifugal speed in S3 is 13000 r / min, the time is 15 minutes, and the centrifugation and washing of S3 are repeated 3 times.

[0012] Preferably, the mass ratio of nano-selenium, dopamine hydrochloride and bovine serum albumin in S4 is 2:20:5-10, the Tris hydrochloric acid buffer solution is 0.1M, pH=8.5, and the stirring time is 12-24h.

[0013] Preferably, the centrifugal speed in S5 is 13000 r / min, the time is 15 min, and the S5 centrifugation and washing are repeated 3 times.

[0014] Preferably, the mass ratio of bovine serum albumin doped with polydopamine-selenium nanoparticles, carboxyl polyethylene glycol thiol and tri(2-carboxyethyl)phosphine in S6 is 1:9:9, the molecular weight of carboxyl polyethylene glycol thiol is 8000, the stirring time is 12h, and the pH of deionized water is adjusted to 7-8.

[0015] Preferably, the centrifugal speed in S7 is 13000 r / min, the time is 15 min, and the S7 centrifugation and washing are repeated 3 times.

[0016] Another aspect of the present invention is to provide an application of polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles, and the application of the polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles in the preparation of an agent for treating liver ischemia-reperfusion injury.

[0017] The beneficial effects of the present invention are as follows: The present invention prepares a polyethylene glycol-modified bovine serum albumin-doped polydopamine-selenium nanoparticle for liver ischemia-reperfusion injury. The nanoparticle has a core-shell structure, with nano-selenium as the core and bovine serum albumin-doped polydopamine as the shell, and the preparation method is simple. The doping of bovine serum albumin and the modification of polyethylene glycol effectively improve the dispersibility and physiological stability of the nanoparticles. The polyethylene glycol-modified bovine serum albumin-doped polydopamine-selenium nanoparticles prepared by the present invention have good biocompatibility and good dispersibility, can quickly and efficiently remove reactive oxygen, and provide a new option for the treatment of liver ischemia-reperfusion injury.

[0018] Additional aspects and advantages of the invention will become apparent from the following description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 This is an electron microscope image of bovine serum albumin-doped polydopamine-selenium nanoparticles synthesized in Example 1 of the present invention; Figure 2 This is a diagram showing the physiological stability of polyethylene glycol-modified bovine serum albumin-doped polydopamine-selenium nanoparticles synthesized in Example 1 of the present invention; Figure 3 This is a diagram of DPPH free radical scavenging of bovine serum albumin doped with polydopamine-selenium nanoparticles modified with polyethylene glycol synthesized in Example 1 of the present invention; Figure 4 This is a cell reactive oxygen species diagram of the polydopamine-selenium nanoparticles doped with bovine serum albumin modified with polyethylene glycol synthesized in Example 1 of the present invention; Figure 5 This is a diagram showing the therapeutic effect of the bovine serum albumin-doped polydopamine-selenium nanoparticles modified with polyethylene glycol synthesized in Example 1 of the present invention on liver ischemia-reperfusion injury in vivo. DETAILED DESCRIPTION

[0020] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0022] Embodiment 1 S1. Dissolve 2 g of polyvinyl pyrrolidone and 51.9 g of sodium selenite in 30 mL of deionized water and stir to fully dissolve to obtain a mixed solution Ⅰ; S2. Dissolve 158.5 mg of ascorbic acid in 2 mL of deionized water, slowly add dropwise to the mixed solution I prepared in S1 and stir evenly to obtain a mixed solution II; S3. The mixed solution II prepared in S2 was centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate was washed with deionized water. This step was repeated 3 times to obtain nano-selenium; S4. Dissolve 6 mg of nano-selenium, 60 mg of dopamine hydrochloride and 15 mg of bovine serum albumin in 60 mL of 0.1 M, pH = 8.5 Tris hydrochloric acid buffer solution and stir slowly for 24 h to obtain a mixed solution III; S5. The mixed solution III prepared in S4 was centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate was washed with deionized water. This step was repeated 3 times to obtain bovine serum albumin-doped polydopamine-selenium nanoparticles; S6. 6 mg of bovine serum albumin doped with polydopamine-selenium nanoparticles, 54 mg of carboxyl polyethylene glycol thiol, and 54 mg of tris(2-carboxyethyl)phosphine were dissolved in 12 mL of deionized water adjusted to pH 7-8 and stirred for 12 h to obtain a mixed solution IV; S7. The mixed solution IV prepared in S6 is centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate is washed with deionized water. This step is repeated 3 times to obtain polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles.

[0023] Embodiment 2 S1. Dissolve 2 g of polyvinyl pyrrolidone and 51.9 g of sodium selenite in 30 mL of deionized water and stir to fully dissolve to obtain a mixed solution Ⅰ; S2. Dissolve 211.3 g of ascorbic acid in 2 mL of deionized water, slowly add dropwise to the mixed solution I prepared in S1 and stir evenly to obtain a mixed solution II; S3. The mixed solution II prepared in S2 was centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate was washed with deionized water. This step was repeated 3 times to obtain nano-selenium; S4. Dissolve 6 mg of nano-selenium, 60 mg of dopamine hydrochloride and 30 mg of bovine serum albumin in 60 mL of 0.1 M, pH = 8.5 Tris hydrochloric acid buffer solution and stir slowly for 12 h to obtain a mixed solution III; S5. The mixed solution III prepared in S4 was centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate was washed with deionized water. This step was repeated 3 times to obtain bovine serum albumin-doped polydopamine-selenium nanoparticles; S6. 6 mg of bovine serum albumin doped with polydopamine-selenium nanoparticles, 54 mg of carboxyl polyethylene glycol thiol, and 54 mg of tris(2-carboxyethyl)phosphine were dissolved in 12 mL of deionized water adjusted to pH 7-8 and stirred for 12 h to obtain a mixed solution IV; S7. The mixed solution IV prepared in S6 is centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate is washed with deionized water. This step is repeated 3 times to obtain polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles.

[0024] Embodiment 3 S1. Dissolve 2 g of polyvinyl pyrrolidone and 51.9 g of sodium selenite in 30 mL of deionized water and stir to fully dissolve to obtain a mixed solution Ⅰ; S2. Dissolve 158.5 mg of ascorbic acid in 2 mL of deionized water, slowly add dropwise to the mixed solution I prepared in S1 and stir evenly to obtain a mixed solution II; S3. The mixed solution II prepared in S2 was centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate was washed with deionized water. This step was repeated 3 times to obtain nano-selenium; S4. Dissolve 6 mg of nano-selenium, 60 mg of dopamine hydrochloride and 15 mg of bovine serum albumin in 60 mL of 0.1 M, pH = 8.5 Tris hydrochloric acid buffer solution and stir slowly for 12 h to obtain a mixed solution III; S5. The mixed solution III prepared in S4 was centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate was washed with deionized water. This step was repeated 3 times to obtain bovine serum albumin-doped polydopamine-selenium nanoparticles; S6. 6 mg of bovine serum albumin doped with polydopamine-selenium nanoparticles, 54 mg of carboxyl polyethylene glycol thiol, and 54 mg of tris(2-carboxyethyl)phosphine were dissolved in 12 mL of deionized water adjusted to pH 7-8 and stirred for 12 h to obtain a mixed solution IV; S7. The mixed solution IV prepared in S6 is centrifuged at 13000 r / min for 15 min, and the centrifugal precipitate is washed with deionized water. This step is repeated 3 times to obtain polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles.

[0025] Detection test The nanoparticles prepared in Example 1 were formulated into an aqueous solution for testing their physical and chemical properties.

[0026] like Figure 1The electron microscope images of the bovine serum albumin-doped polydopamine-selenium nanoparticles synthesized in Example 1 of the present invention are shown. The left image is the polydopamine-selenium nanoparticles not doped with bovine serum albumin, and the right image is the polydopamine-selenium nanoparticles doped with bovine serum albumin. Figure 1 It can be seen that compared with the polydopamine-selenium nanoparticles not doped with bovine serum albumin, the synthesized bovine serum albumin-doped polydopamine-selenium nanoparticles have significantly improved dispersibility, a core-shell structure, and a diameter of about 74 nm.

[0027] like Figure 2 The figure shows the physiological stability of the bovine serum albumin doped with polydopamine-selenium nanoparticles modified with polyethylene glycol synthesized in Example 1 of the present invention. Figure 2 It can be seen that the bovine serum albumin doped with polyethylene glycol has poor physiological stability in the culture medium and produces precipitation, while the bovine serum albumin doped with polyethylene glycol can exist stably in the culture medium.

[0028] like Figure 3 The figure shows the DPPH free radical scavenging graph of the polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles synthesized in Example 1 of the present invention. When the concentration of the polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles reaches 100 μg / mL, the scavenging rate of the DPPH free radical reaches 92%.

[0029] After AML12 cells were placed in a 96-well plate for 24 hours, 400 mM tert-butyl hydroperoxide and 200 μg / mL polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles were added, with no addition and only 400 mM tert-butyl hydroperoxide added as controls.

[0030] After incubating the cells for 3 h, they were washed with PBS, and a medium containing DCFH-DA was added to each well for a total incubation of 20 min. After washing, the level of intracellular reactive oxygen species was detected by fluorescence microscopy.

[0031] like Figure 4 As shown in the figure, the green fluorescence intensity of the cells in the tert-butyl hydrogen peroxide treatment group was significantly higher than that in the control group, indicating that a large amount of reactive oxygen species was produced. After adding polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles, the level of reactive oxygen species in the cells was significantly reduced.

[0032] Evaluation of PEG-modified bovine serum albumin doped with polydopamine-selenium nanoparticles for the treatment of hepatic ischemia-reperfusion injury in vivo: The mice were anesthetized with isoflurane and fixed on a surgical board in a supine position. After laparotomy, the left and middle hepatic vessels were blocked with a vascular clamp. After 30 minutes of ischemia, the vascular clamp was removed and the abdominal cavity was sutured.

[0033] Polydopamine-selenium nanoparticles doped with polyethylene glycol-modified bovine serum albumin were injected into the tail vein. The mice were killed 24 hours later and the livers were obtained for hematoxylin-eosin staining.

[0034] like Figure 5 As shown in the figure, the liver ischemia-reperfusion injury group showed necrotic foci of varying sizes and shapes, and disordered tissue structure. The mice injected with polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles through the tail vein had significantly fewer necrotic foci and a significantly reduced degree of damage, proving that the nanoparticles can effectively improve liver tissue damage caused by ischemia-reperfusion stimulation.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may also have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles, characterized in that: The nanoparticles include a nano-selenium core and a bovine serum albumin-doped polydopamine shell.

2. The polyethylene glycol-modified bovine serum albumin-doped polydopamine-selenium nanoparticles according to claim 1, characterized in that: The diameter of the nanoparticles is 74 nm.

3. The method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 1, characterized in that: The specific steps of the nanoparticle preparation method are as follows: S1. dissolving polyvinyl pyrrolidone and sodium selenite in deionized water, stirring to fully dissolve them to obtain a mixed solution Ⅰ; S2. dissolving ascorbic acid in deionized water, slowly adding dropwise to the mixed solution I prepared in S1 and stirring evenly to obtain a mixed solution II; S3. The mixed solution II prepared in S2 is centrifuged at high speed, and the centrifugal precipitate is washed with deionized water to obtain nano-selenium; S4. dissolving nano-selenium, dopamine hydrochloride and bovine serum albumin in Tris hydrochloric acid buffer solution and stirring slowly to obtain a mixed solution III; S5. The mixed solution III prepared in S4 is centrifuged at high speed, and the centrifugal precipitate is washed with deionized water to obtain bovine serum albumin-doped polydopamine-selenium nanoparticles; S6. dissolving bovine serum albumin-doped polydopamine-selenium nanoparticles, carboxyl polyethylene glycol thiol and tris(2-carboxyethyl)phosphine in deionized water and stirring to obtain a mixed solution IV; S7. The mixed solution IV prepared in S6 is centrifuged at high speed, and the centrifugal precipitate is washed with deionized water to obtain polyethylene glycol-modified bovine serum albumin-doped polydopamine-selenium nanoparticles.

4. The method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 3, characterized in that: The molecular weight of polyvinyl pyrrolidone in S1 is 40,000.

5. The method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 3, characterized in that: The molar ratio of sodium selenite to ascorbic acid in S2 is 1:3-4, and the stirring time is 1 hour; the centrifugal speed in S3 is 13000r / min, the time is 15 minutes, and the centrifugation and washing of S3 are repeated 3 times.

6. The method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 3, characterized in that: The mass ratio of nano-selenium, dopamine hydrochloride and bovine serum albumin in S4 is 2:20:5-10, the Tris hydrochloric acid buffer solution is 0.1M, pH=8.5, and the stirring time is 12-24h.

7. The method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 3, characterized in that: The centrifugal speed in S5 is 13000 r / min, the time is 15 min, and the S5 centrifugation and washing are repeated 3 times.

8. The method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 3, characterized in that: The mass ratio of bovine serum albumin doped with polydopamine-selenium nanoparticles, carboxyl polyethylene glycol thiol and tri(2-carboxyethyl)phosphine in S6 is 1:9:9, the molecular weight of carboxyl polyethylene glycol thiol is 8000, the stirring time is 12 hours, and the pH of deionized water is adjusted to 7-8.

9. The method for preparing polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 3, characterized in that: The centrifugal speed in S7 is 13000 r / min, the time is 15 min, and the S7 centrifugation and washing are repeated 3 times.

10. The use of polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles according to claim 1, characterized in that: The application of the polyethylene glycol-modified bovine serum albumin doped with polydopamine-selenium nanoparticles in preparing a reagent for treating liver ischemia-reperfusion injury.

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