Bionic nano preparation applied to acute diquat poisoning as well as preparation method and application of bionic nano preparation

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to a bionic nano preparation applied to acute diquat poisoning as well as a preparation method and application of the bionic nano preparation. The bionic nano preparation is formed by loading epigallocatechin gallate nano particles on PVA-PLGA (Polyvinyl Acetate-Poly (Lactic-co-Glycolic Acid) nano particles wrapped by an erythrocyte membrane. The bionic nano preparation with a three-layer composite structure is achieved, the purpose of nano preparation escape immune recognition is achieved, and meanwhile the anti-oxidation effect of the medicine is achieved. The preparation method is simple and easy to implement, conditions are controllable, good repeatability is achieved, and the stability of the epigallocatechin gallate can be improved. The prepared erythrocyte membrane bionic EGCG nano preparation has good stability and solubility and can be stored at-20 DEG C for a long time, and the storage life and the service life of the product are prolonged. The nano preparation shows a better diquat detoxification effect, and provides a new drug choice for diquat detoxification.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a bionic nano preparation for acute diquat poisoning, and a preparation method and application thereof. Background Art

[0002] Diquat is a non-selective herbicide. Diquat is widely used in agricultural planting, resulting in an increasing number of cases of diquat poisoning in clinical practice. Diquat poisoning is mainly manifested by extensive mucosal damage, oliguria, hematuria, renal tubular necrosis, respiratory distress, etc., with a high mortality rate, and there is currently no specific medicine. Currently, the main treatments for diquat poisoning include: gastric lavage, blood purification, symptomatic supportive treatment, etc. Drug treatment mainly uses antioxidant drugs and hormones to remove free radicals and inflammatory mediators. However, these treatments cannot fundamentally cure diquat poisoning, they can only temporarily relieve the symptoms, and they all have certain side effects and complications.

[0003] In recent years, studies have found that oxidative stress plays an important role in the pathogenesis of diquat poisoning. When diquat poisoning occurs, a large amount of reactive oxygen species is produced due to redox reactions, leading to oxidative stress, which promotes lipid peroxidation and cell death in cells. Therefore, inhibiting oxidative stress and removing reactive oxygen species may be a new strategy for treating diquat poisoning.

[0004] In recent years, researchers have begun to explore the use of antioxidants to treat diquat poisoning. For example, natural compounds from some medicinal plants have shown the effects of inhibiting oxidative stress caused by diquat and reducing organ damage in in vitro experiments and animal models. However, although these methods can inhibit oxidative stress and resist oxidation, they have failed to solve the problem of the drug's inability to continuously target and be recognized by the immune system.

[0005] Therefore, those skilled in the art construct a bionic nanoformulation that is simple in preparation, stable, and has strong antioxidant properties and bioavailability, which will provide a treatment strategy for acute diquat poisoning. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a bionic nano preparation for acute diquat poisoning and a preparation method and application thereof, so as to provide a new treatment strategy for acute diquat poisoning.

[0007] To achieve the above object, the present invention provides a bionic nano preparation for acute diquat poisoning, which is PVA-PLGA nanoparticles wrapped in red blood cell membranes and loaded with epigallocatechin gallate nanoparticles.

[0008] Figure 1 It is a schematic diagram of the structure of the bionic nano preparation provided by the present invention.

[0009] Optionally, the particle size of the bionic nanoparticle preparation is 400-600 nm.

[0010] The bionic nano preparation provided by the present invention can reduce the generation of active oxygen, reduce the release of inflammatory factors in the kidney, and thus effectively alleviate the kidney damage of patients poisoned by diquat.

[0011] The present invention provides a method for preparing the bionic nano preparation for acute diquat poisoning, comprising the following steps:

[0012] S1) PVA-PLGA nanoparticles loaded with epigallocatechin gallate were prepared by water / oil / water emulsion solvent evaporation method;

[0013] S2) The PVA-PLGA nanoparticles loaded with epigallocatechin gallate are mixed with red blood cell membranes to obtain a biomimetic nanoformulation.

[0014] Preferably, the step S1) specifically includes the following steps:

[0015] dissolving epigallocatechin gallate in a first solvent to obtain a first solution;

[0016] Mixing the first solution and the PLGA solution to obtain a primary emulsion;

[0017] The primary emulsion and the PVA solution are mixed to obtain a W / O / W emulsion;

[0018] The solvent is removed from the W / O / W emulsion and the emulsion is dried to obtain PVA-PLGA nanoparticles containing epigallocatechin gallate.

[0019] Preferably, the first solvent is selected from dichloromethane.

[0020] Preferably, in the first solution, the concentration of epigallocatechin gallate is 1-2 mg / mL; more preferably 1 mg / mL.

[0021] Preferably, in the PLGA solution, the solvent is dichloromethane.

[0022] Preferably, in the PLGA solution, the concentration of PLGA is 1-2 mg / mL.

[0023] Preferably, the first solution and the PLGA solution are mixed and ultrasonically treated to obtain a primary emulsion.

[0024] The power of the ultrasonic treatment is preferably 100 W, and the treatment time is preferably 3 to 7 min, more preferably 5 min.

[0025] Preferably, in the PVA solution, the solvent is dichloromethane.

[0026] Preferably, in the PVA solution, the concentration of PVA is 0.5-1 wt %, more preferably 0.7 wt %.

[0027] Preferably, the primary emulsion and the PVA solution are mixed and ultrasonically treated to obtain a W / O / W emulsion.

[0028] The power of the ultrasonic treatment is preferably 100 W, and the treatment time is preferably 2 to 5 min, more preferably 3 min.

[0029] Then, the solvent in the W / O / W emulsion is removed. The specific method for removing the solvent is not particularly limited in the present invention, and can be a method for removing the solvent well known to those skilled in the art. In some specific embodiments of the present invention, the W / O / W emulsion is volatilized stably at room temperature to remove the dichloromethane solvent by volatilization. The volatilization time is preferably 1 to 3 hours, more preferably 2 hours.

[0030] The present invention has no particular limitation on the drying method, and it may be a drying method suitable for nanoparticles well known to those skilled in the art. In some specific embodiments of the present invention, a freeze-drying method is used.

[0031] Preferably, after removing the dichloromethane solvent, the obtained emulsion from which the solvent is removed is centrifuged to collect the nanoformulation, and the PVA-PLGA nanoparticles loaded with epigallocatechin gallate are obtained by freeze-drying.

[0032] The centrifugal speed is preferably 10000-20000 rpm, more preferably 15000 rpm; the centrifugal time is preferably 10-20 min, more preferably 15 min.

[0033] Then the PVA-PLGA nanoparticles loaded with epigallocatechin gallate are mixed with red blood cell membranes to obtain a bionic nano preparation.

[0034] The present invention has no particular limitation on the source of the red blood cell membrane, which may be commercially available or prepared according to a general method known to those skilled in the art.

[0035] Preferably, the above-mentioned red blood cell membrane is obtained from an animal.

[0036] The acquisition method is preferably:

[0037] Collect blood samples, preferably orbital blood from C57BL / 6J male mice.

[0038] The blood sample was centrifuged to remove the plasma and gray-white capsule to obtain red blood cell fluid.

[0039] The centrifugal speed is preferably 800-1200 rpm, more preferably 1000 rpm; the centrifugal time is preferably 3-7 min, more preferably 5 min.

[0040] The obtained red blood cell solution was washed with cold PBS buffer until the supernatant was clear, transparent and colorless, thereby obtaining red blood cells.

[0041] The erythrocytes were induced to rupture in a PBS ice water bath, and washed with cold PBS buffer until the supernatant was colorless to obtain erythrocyte membranes.

[0042] Preferably, the red blood cells are resuspended in PBS on ice for 0.5 to 1.5 hours to induce membrane rupture. The concentration of the PBS is preferably 20% to 30%, more preferably 25%.

[0043] In the present invention, after the obtained red blood cell membrane is ultrasonically treated, a micro extruder is used to sequentially extrude polycarbonate porous membranes of 400 nm and 200 nm. Preferably, each membrane with each pore size is extruded at least 9 times to obtain the red blood cell membrane.

[0044] Preferably, the PVA-PLGA nanoparticles loaded with epigallocatechin gallate are mixed with erythrocyte membranes, magnetically stirred, and then ultrasonically treated to obtain a bionic nano-preparation.

[0045] The magnetic stirring time is preferably 30 to 60 min, more preferably 45 min.

[0046] The power of the ultrasonic treatment is preferably 50-200 W, more preferably 100 W; the time of the ultrasonic treatment is preferably 3-7 min, more preferably 5 min.

[0047] Preferably, after ultrasonic treatment, the suspension is repeatedly extruded through a 200 nm porous polycarbonate membrane using a micro extruder to obtain a biomimetic nanoformulation.

[0048] The present invention provides the use of the bionic nano preparation for acute diquat poisoning in the preparation of a medicine for treating or alleviating acute diquat poisoning.

[0049] Specifically, the treating or alleviating acute diquat poisoning is treating or alleviating kidney damage caused by acute diquat poisoning.

[0050] Compared with the prior art, the present invention provides a bionic nano preparation for acute diquat poisoning, which is PVA-PLGA nanoparticles wrapped in red blood cell membranes and loaded with epigallocatechin gallate nanoparticles.

[0051] The present invention achieves the following beneficial effects:

[0052] ① The present invention realizes a bionic nano-preparation with a three-layer composite structure, which not only achieves the purpose of nano-preparation escaping immune recognition, but also exerts the antioxidant effect of the drug;

[0053] ② The preparation method provided by the present invention is simple and easy, the conditions are controllable, and it has good repeatability, and can improve the stability of epigallocatechin gallate. The erythrocyte membrane biomimetic EGCG nano preparation prepared by the present invention has good stability and solubility, and can be stored for a long time at -20°C, which increases the storage and service life of the product;

[0054] ③ Compared with the epigallocatechin gallate raw material drug, the erythrocyte membrane biomimetic EGCG nano preparation of the present invention exhibits a better detoxification effect of diquat, providing a new drug option for diquat detoxification. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a schematic diagram of the structure of the bionic nano preparation provided by the present invention;

[0056] Figure 2 It is a Coomassie brilliant blue staining band diagram of the bionic nano preparation provided by the present invention;

[0057] Figure 3 It is a transmission electron microscope image of the bionic nano preparation provided by the present invention;

[0058] Figure 4 is a particle size distribution diagram of the bionic nano preparation provided by the present invention;

[0059] Figure 5 It is a potential distribution diagram of the bionic nano preparation provided by the present invention;

[0060] Figure 6 It is a graph of the relative cell viability of the bionic nanoformulation provided by the present invention;

[0061] Figure 7 It is a graph showing the results of creatinine detection and urea nitrogen detection of the bionic nano preparation provided by the present invention. DETAILED DESCRIPTION

[0062] In order to further illustrate the present invention, the following is a detailed description in conjunction with the embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0063] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0064] Example 1.

[0065] 1.1 Extraction of red blood cell membrane

[0066] 0.5-1 mL of orbital blood was collected from C57BL / 6J male mice, placed in an EDTA spray tube, and centrifuged at 1000 rpm for 5 min to remove plasma and gray-white capsule. The obtained erythrocytes were washed three times with cold 1×PBS buffer (5000 rpm, 10 min) until the supernatant was clear and colorless to obtain erythrocytes. Subsequently, the erythrocytes were resuspended in a 25% PBS ice bath for 1 hour to induce membrane rupture. Subsequently, the erythrocytes were repeatedly washed with cold 0.25×PBS solution at 20000 rpm, 4°C for 20 min / time until the supernatant was colorless, and the light pink particles were collected to obtain the erythrocyte membrane. The collected erythrocyte membranes were sonicated for 2 minutes using a probe sonicator (100 W), and then 400 and 200 nm polycarbonate porous membranes were extruded in sequence using a micro extruder. Each membrane with a pore size was extruded at least 9 times to obtain the erythrocyte membrane, and the obtained erythrocyte membrane was suspended in a PBS solution and stored at 4°C for later use.

[0067] 1.2 Preparation of EGCG nanoparticles

[0068] Epigallocatechin gallate nanoparticles (EGCG-NPs) were prepared using a water / oil / water emulsion solvent evaporation method. 1 mg EGCG and 10 mg PLGA were dissolved in dichloromethane for 60 min at room temperature. Then, 0.7 wt% polyvinyl alcohol was added, and the resulting solution was treated with an ultrasonic homogenizer for 5 min to obtain EGCG-loaded nanoparticles. The nanoparticles were then rinsed three times in distilled water, freeze-dried for 48 h, and stored at -80 °C.

[0069] 1.3 Preparation of erythrocyte membrane biomimetic EGCG nanoparticles

[0070] 2 mL of 1 mg / mL EGCG nanoparticle suspension was mixed with erythrocyte membranes in 500 µL whole blood under magnetic stirring for 45 min. The mixture was then sonicated at 100 W for 5 min. Finally, the suspension was repeatedly extruded through a 200 nm porous polycarbonate membrane using a micro-extruder to obtain erythrocyte membrane biomimetic EGCG nanoparticles.

[0071] 1.4 Characterization and morphology of erythrocyte membrane-mimetic EGCG nanoparticles

[0072] 1.4.1 Nanoparticle size and potential

[0073] The prepared EGCG-RBCm / NPs were dispersed in PBS buffer and ultrasonically mixed, and the particle size and Zeta potential were measured using a dynamic light scattering particle size analyzer.

[0074] 1.4.2 Nanoparticle morphology

[0075] The prepared EGCG-RBCm / NPs were dispersed in PBS buffer and then ultrasonically mixed. An appropriate amount of the mixed solution was dropped on a copper mesh, stained with phosphotungstic acid, and then dried at room temperature. The morphology of EGCG-RBCm / NPs was observed by transmission electron microscopy (TEM).

[0076] 1.4.3 Characterization of membrane proteins of nanoparticles

[0077] The extracted red blood cell lysate, red blood cell membrane, PLGA and EGCG-RBCm / NPs were placed in a centrifuge tube, and a quantitative amount of RIPA (strong) lysis buffer containing 1% protease inhibitor was added, and then continuously blown to mix evenly, lysed on ice for 30 minutes, centrifuged at 4°C and 12000 r / min for 20 minutes, the precipitate was discarded, and the red blood cell membrane protein concentration in each sample was measured by the BCA method. An equal amount of supernatant was taken from each group, and after adding buffer, the mixture was vortexed and heated for 10 min to denature the protein (100°C). A 12% SDS-PAGE gel was used and the samples were loaded in order. Then, protein gel electrophoresis was performed, first at 80 V for 60 min, and then at 120 V for 60 min. After the gel electrophoresis, the gel was completely taken out and immersed in Coomassie brilliant blue staining solution. The protein bands on the gel were shaken on a shaker for 30 min, and Coomassie brilliant blue staining was performed on the gel. After staining, the gel was washed with double distilled water, and the destaining solution was changed several times for destaining, and photographed and recorded.

[0078] like Figure 2 As shown, the biomimetic hybrid membrane prepared in this example was subjected to Coomassie Brilliant Blue staining protein analysis, and the results showed that the biomimetic hybrid membrane was successfully fused and prepared.

[0079] like Figure 3 As shown, the erythrocyte membrane bionic EGCG nanoparticles bionic nanoformulation prepared in this example was subjected to transmission electron microscopy image analysis. The results showed that uniformly dispersed erythrocyte membrane bionic EGCG nanoparticles were successfully prepared, and the bionic hybrid membrane was wrapped around the outer layer of the EGCG nanoparticles.

[0080] like Figure 4 As shown, the particle size analysis of the erythrocyte membrane biomimetic EGCG nanoparticles biomimetic nano preparation prepared in this example was performed. The results showed that the erythrocyte membrane biomimetic EGCG nanoparticles were successfully prepared.

[0081] like Figure 5 As shown, the erythrocyte membrane biomimetic EGCG nanoparticles biomimetic nano preparation prepared in this example was subjected to potential analysis. The results showed that the erythrocyte membrane biomimetic EGCG nanoparticles were successfully prepared.

[0082] Example 2. Application of biomimetic nanoparticles in human renal tubular epithelial cells

[0083] 2.1 Cytotoxicity studies

[0084] 2.1.1 Purchase biologically identified HK-2 cells from the Cell Bank of the Chinese Academy of Sciences and subculture them in a cell culture incubator containing 90% DMEM-F12 medium and 10% FBS at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for cell experiments. DQ and EGCG-RBCm / NPs were stored in a certain concentration of liquid using sterile PBS. During the experiment, they were diluted to the corresponding concentration using 100% DMEM-F12 medium for subsequent experiments.

[0085] 2.1.2 Preparation of main solution

[0086] Diquat (DQ) solution (for cells): Dissolve 50 mg of DQ powder in 5 mL of sterile PBS, filter with a 0.22 μm mesh, and store at 4°C. It is valid within 1 month and can be diluted immediately before use.

[0087] EGCG-RBCm / NPs solution (for cells): Dilute the previously prepared 10 mM EGCG-RBCm / NPs solution with DMEM-F12 medium, filter through a 0.22 μm mesh, and store at 4°C. It is valid for 1 week.

[0088] 2.1.3 Cell viability CCK-8 assay

[0089] The cells are divided into 1*10 4 Each well was inoculated into a 96-well culture plate, and according to the experimental requirements, the concentrations of EGCG-RBCm / NPs were 1, 5, 10, 25, and 50 μM, respectively. Each group had 6 sub-wells. After 48 hours of inoculation, the serum-free medium containing the above-mentioned drugs was replaced and cultured for another 12 hours. After 12 hours, the culture medium was replaced with a serum-free medium containing 10% CCK-8 and incubated for 2 hours. After the incubation, the absorbance was measured at a wavelength of 450 nm. The relative cell viability was calculated. The experimental results are shown in Figure 6 As shown, Figure 6 The results showed that EGCG-loaded biomimetic nanoparticles had no toxic effect on human renal tubular epithelial cells. Figure 6 The vertical axis Cellviability represents cell viability.

[0090] Example 3. Application of bionic nanoformulation in treating renal injury in mice poisoned by diquat

[0091] 3.1 Experimental animals

[0092] Sixty SPF healthy male C57BL / 6J mice, 6 weeks old and weighing 20-25 g, were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., with a production license number of SCXK (Zhejiang) 2019-0001. They were kept in the Experimental Animal Center of the First Affiliated Hospital of Wenzhou Medical University.

[0093] 3.2 Experimental Grouping

[0094] Control group: After 0.1 mL of PBS was injected intraperitoneally, 0.1 mL of PBS was injected intraperitoneally on the other side after an interval of 30 minutes. Diquat poisoning group: The DQ solution prepared above was injected intraperitoneally at 50 mg / kg, and 0.1 mL of PBS was injected intraperitoneally on the other side after an interval of 30 minutes. EGCG administration group: DQ solution was injected intraperitoneally at 50 mg / kg, and EGCG solution was injected intraperitoneally at a dose of 20 mg / kg on the other side after an interval of 30 minutes. The intraperitoneal injection of EGCG continued for 3 days. EGCG-RBCm / Nps administration group: The DQ solution prepared above was injected intraperitoneally at 50 mg / kg, and EGCG-RBCm / Nps solution was injected intraperitoneally at a dose of 20 mg / kg on the other side after an interval of 30 minutes. The intraperitoneal injection of EGCG-RBCm / Nps continued for three days. Three days after DQ injection, the mice were anesthetized with 1% sodium pentobarbital and blood was collected from the eyeballs.

[0095] 3.3 Detection of serum creatinine and blood urea nitrogen levels in each group of mice

[0096] 3.3.1 Creatinine test:

[0097]

[0098] Calculate the creatinine content in the sample according to the formula in the instruction manual. Note: K is the dilution factor, the value is 186 / 246

[0099] Creatinine content (μmol / L) = [(Assay A2-K*Assay A1)-(Blank A2-K*Blank A1)] / [(Standard A2-K*Standard A1)-(Blank A2-K*Blank)]*Standard concentration (442μmol / L)

[0100] 3.3.2 Urea nitrogen detection:

[0101]

[0102] Calculate the urea nitrogen content in the sample according to the formula in the instruction manual. Urea nitrogen content (mmol / L) = (measured OD value - blank measured value) / (standard OD value - blank OD value) * standard concentration (10mmol / L) * sample dilution multiple before testing.

[0103] The results are as follows Figure 7 As shown, it can be seen that the red blood cell membrane biomimetic EGCG nanoparticle group can effectively alleviate the renal damage caused by diquat poisoning. Figure 7 The vertical axis Serum Cr represents serum creatinine; Serum BUN represents blood urea nitrogen.

[0104] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A bionic nano preparation for acute diquat poisoning, characterized in that: Epigallocatechin gallate nanoparticles are encapsulated in PVA-PLGA nanoparticles wrapped in red blood cell membranes.

2. The bionic nano preparation for acute diquat poisoning according to claim 1, characterized in that: The particle size of the bionic nano preparation is 400-600 nm.

3. The method for preparing the bionic nano preparation for acute diquat poisoning according to any one of claims 1 to 2, comprising the following steps: S1) PVA-PLGA nanoparticles loaded with epigallocatechin gallate were prepared by water / oil / water emulsion solvent evaporation method; S2) The PVA-PLGA nanoparticles loaded with epigallocatechin gallate are mixed with red blood cell membranes to obtain a biomimetic nanoformulation.

4. The preparation method according to claim 3, characterized in that: The step S1) specifically includes the following steps: dissolving epigallocatechin gallate in a first solvent to obtain a first solution; Mixing the first solution and the PLGA solution to obtain a primary emulsion; The primary emulsion and the PVA solution are mixed to obtain a W / O / W emulsion; The solvent is removed from the W / O / W emulsion and the emulsion is dried to obtain PVA-PLGA nanoparticles containing epigallocatechin gallate.

5. The preparation method according to claim 4, characterized in that: The first solvent is selected from dichloromethane; In the first solution, the concentration of epigallocatechin gallate is 1-2 mg / mL.

6. The preparation method according to claim 4, characterized in that: In the PLGA solution, the solvent is dichloromethane; In the PLGA solution, the concentration of PLGA is 1-2 mg / mL.

7. The preparation method according to claim 4, characterized in that: In the PVA solution, the solvent is dichloromethane; In the PVA solution, the concentration of PVA is 0.5-1wt%.

8. The preparation method according to claim 3, characterized in that: The erythrocyte membrane is prepared according to the following method: Collect blood samples; The blood sample is centrifuged to remove the plasma and gray-white capsule to obtain red blood cell fluid; The obtained red blood cell solution was washed with cold PBS buffer until the supernatant was clear, transparent and colorless to obtain red blood cells; The erythrocytes were induced to rupture in a PBS ice water bath, and washed with cold PBS buffer until the supernatant was colorless to obtain erythrocyte membranes.

9. Use of the bionic nanoformulation for acute diquat poisoning according to any one of claims 1 to 2 in the preparation of a drug for treating or alleviating acute diquat poisoning.

10. The use according to claim 9, characterized in that: The treating or alleviating acute diquat poisoning is treating or alleviating kidney damage caused by acute diquat poisoning.

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