Kaempferol biomimetic nanomaterial and preparation method and application thereof

Abstract

The present invention discloses a kaempferol mesenchymal stem cell membrane bionic material, which can more accurately target damaged tissues. The preparation method of the bionic nanomaterial is simple, has strong versatility, and is convenient for large-scale production. The present invention utilizes the mesenchymal stem cell membrane to improve the biocompatibility and targeting of the carrier and prolong the circulation time of the drug in the body; the bionic nanomaterial prepared by the present invention realizes the targeted delivery of kaempferol to the liver, providing a new treatment method for acute liver failure ALF.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to a kaempferol biomimetic nanomaterial and a preparation method and application thereof. Background Art

[0002] Acute liver failure (ALF) refers to a clinical syndrome in which liver function deteriorates rapidly in a short period of time without underlying liver disease, resulting in ascites, coagulopathy, hepatic encephalopathy and multiple organ failure. The causes of ALF are varied, the main causes include acetaminophen toxicity, drug-induced liver injury, viral infection, ischemia and autoimmune diseases. The causes of acute liver failure vary worldwide. Acute liver failure caused by APAP (paracetamol) overdose is common in Europe and other developed countries, while acute liver failure caused by viral hepatitis (type A, B and E) mainly occurs in developing countries. Patients with acute liver failure have a poor prognosis and there is no effective treatment, making the treatment of acute liver failure an important problem.

[0003] Kaempferol, also known as kaempferol-3, kaempferol, and kaempferol flavonol, is a dietary flavonoid compound. It is mainly derived from the rhizome of the ginger plant Kaempferia galanga. It is widely found in various fruits, vegetables, and natural plants. It is a very common compound in nature. Kaempferol acts on a large number of signal targets in the body and has a wide range of pharmacological activities such as anti-tumor, anti-inflammatory, antioxidant, cardioprotective, and liver protective. However, its poor water solubility and first-pass effect in the gastrointestinal tract in the body largely limit its clinical application.

[0004] Liposomes are spherical nanoshells formed by the spontaneous closure of phospholipid molecules in aqueous solution, which can simultaneously encapsulate water-soluble molecules in the lumen and fat-soluble molecules between the phospholipid bilayers, thereby improving the bioavailability of water-insoluble drugs, with high drug loading and sustained release. However, they face challenges such as rapid clearance by the mononuclear phagocyte system (MPS) and lack of selectivity between healthy and damaged liver cells.

[0005] In recent years, natural cells such as erythrocytes,12 dendritic cells (DC),13 mesenchymal stem cells (MSCs),14 macrophages have been explored as drug carriers. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the potential for self-renewal and multipotent differentiation. The application of stem cell therapy has gradually deepened and diversified, with broad prospects. MSCs can regulate the activation and proliferation of T cells, regulate the cytokines produced by immune cells, and specifically target the site of injury, thereby inhibiting inflammation and performing immune regulation. Mesenchymal stem cell membranes have the active targeting, immune escape characteristics, and long circulation of MSCs. These advantages make mesenchymal stem cell membranes promising for targeted drug delivery due to their inherent targeting ability and low immunogenicity. However, some researchers have confirmed that mesenchymal stem cells may promote tumor progression and even differentiate into tumors (F. Ma et al., Human Umbilical Cord Mesenchymal Stem Cells Promote Breast Cancer Metastasis by Interleukin-8- and Interleukin-6-Dependent Induction of CD44+ / CD24- Cells. Cell Transplantation 24, 2585-2599(2015).). In addition, some worrying results show that whole-cell drug carriers may be contaminated by biological materials and have biosafety issues. In order to overcome these shortcomings, it has been disclosed in the literature that new cell membrane-coated particles with minimal membrane protein loss have been developed (AV Kroll, RH Fang, L. Zhang, Biointerfacing and Applicationsof Cell Membrane-Coated Nanoparticles. Bioconjugate Chemistry 28, 23-32(2016).). At present, MSC membrane-modified NPs have made gratifying progress in inflammation, tissue regeneration and tumor treatment (L. Fan, A. Wei, Z. Gao, X. Mu, Current progress of mesenchymal stem cellmembrane-camouflaged nanoparticles for targeted therapy. Biomedicine &Pharmacotherapy 161, (2023).). Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a kaempferol mesenchymal stem cell membrane biomimetic material that can more accurately target damaged tissues. The preparation method of the biomimetic nanomaterial is simple, has strong versatility, and is convenient for large-scale production.

[0007] Technical solution:

[0008] The invention provides a kaempferol biomimetic nano material, which is composed of liposomes for mesenchymal stem cell membranes and kaempferol liposomes. The average particle size of the nano material is 135-140nm.

[0009] The present invention also provides kaempferol bionic nanomaterials, and the preparation method is as follows:

[0010] 1) Preparation of mesenchymal stem cell membrane: After culturing mesenchymal stem cells, the cells are collected, centrifuged, resuspended once, centrifuged, resuspended twice, frozen and thawed, and centrifuged. Finally, the precipitate is collected to obtain the mesenchymal stem cell membrane;

[0011] 2) Preparation of kaempferol liposomes: dissolve lecithin, cholesterol and kaempferol in anhydrous ethanol, add them into ultrapure water and stir them after mixing, and pass them through a membrane using a liposome extruder to obtain kaempferol liposomes;

[0012] 3) The mesenchymal stem cell membrane obtained in step 1) is extruded by a liposome extruder, mixed with kaempferol liposomes, ultrasonicated in an ice bath, and extruded again by the liposome extruder to obtain kaempferol biomimetic nanomaterials.

[0013] Furthermore, in step 1), the cells are resuspended once with a phosphate buffered saline solution, and the cells are resuspended twice with a hypotonic solution.

[0014] Furthermore, the hypotonic solution is an aqueous solution containing 10mmol / L Tris-HCL, 1mmol / L KCL, 1.5mmol / L MgCl2 and 1mmol / L PMSF in concentrations thereof.

[0015] Furthermore, in the step 2), the mass ratio of lecithin, cholesterol and kaempferol is 3:1:1.

[0016] Furthermore, in the step 3), the mass ratio of the mesenchymal stem cell membrane to the kaempferol liposome is 1:1-1:10.

[0017] Furthermore, the temperature of the centrifugation step in step 1) is 4°C.

[0018] Furthermore, in the step 2), the kaempferol liposomes are obtained by sequentially extruding through polycarbonate membranes of 800 nm, 400 nm and 200 nm on a liposome extruder.

[0019] The present invention also provides application of kaempferol bionic nanomaterial in preparing medicine for treating acute liver failure.

[0020] Beneficial effects:

[0021] (1) The nano bionic material provided by the present invention is obtained by physical extrusion, i.e., thin film extrusion, and is simple to prepare and easy to mass produce.

[0022] (2) The present invention designs a nano-bionic material that utilizes mesenchymal stem cell membrane to improve the biocompatibility and targeting of the carrier and prolong the circulation time of the drug in the body.

[0023] (3) The bionic nanomaterials prepared in the present invention achieve targeted delivery of kaempferol to the liver, providing a new treatment for acute liver failure (ALF). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the preparation process of bionic nanomaterial KAE@ML.

[0025] Figure 2 Schematic diagram of characterization of biomimetic nanomaterials: Figure 2 a is the transmission electron microscope (TEM) image of biomimetic nanomaterials L and ML, with a scale of 100 nm; Figure 2 b is the dynamic light scattering (DLS) measurement image of biomimetic nanomaterials L and ML; Figure 2 c is the Zeta potential of biomimetic nanomaterials L and ML; Figure 2 d is the encapsulation efficiency and drug loading at three different ratios of M and L; Figure 2 e is the in vitro cumulative drug release of biomimetic nanomaterials KAE, KAE@L, and KAE@ML in PBS at pH 7.4.

[0026] Figure 3 Live and dead images of cells in the bionic nanomaterials KAE, KAE@L and KAE@ML.

[0027] Figure 4 Targeting of biomimetic nanomaterials: Figure 4 a is the fluorescence intensity of the Control group, PBS group, L group, and ML group detected by IndiGO imaging system 1, 3, and 7 days after injection. Figure 4 b is a typical in vitro image of the main organs of each group 24 hours after injection.

[0028] Figure 5 The therapeutic effects of biomimetic nanomaterials: Figure 5 a is the determination of ALT and AST levels of liver function in the Control, ALF and treated groups of mice. Figure 5b is the survival curve of the mice in the Control group, ALF group and each group after treatment. Figure 5 c is the HE images of rats in each group. DETAILED DESCRIPTION

[0029] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments and drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0030] The sources of raw materials used in the following examples are as follows:

[0031] Mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC).

[0032] Lecithin: Shanghai Aladdin Biochemical Technology Co., Ltd., 8002-43-5

[0033] Cholesterol: Hubei Dingxintong Pharmaceutical Co., Ltd., D162

[0034] Kaempferol: Shanghai Aladdin Biochemical Technology Co., Ltd., K107144

[0035] Tris-HCL: Shanghai Aladdin Biochemical Technology Co., Ltd., T301502

[0036] KCL: Shanghai Aladdin Biochemical Technology Co., Ltd., P433492

[0037] MgCl2: Shanghai Bio-Tech Biotechnology Co., Ltd., ST269

[0038] PMSF phenylmethylsulfonyl fluoride: Jiangsu KeyGen Biotech Co., Ltd., KGB5105-10

[0039] Example 1 Preparation of Kaempferol Biomimetic Nanomaterial KAE@ML

[0040] 1) Preparation of mesenchymal stem cell membrane

[0041] 1-1) Cultivate mesenchymal stem cells in a 10 cm dish according to the existing technology (Mesenchymal Stem Cell Membrane-Camouflaged Liposomesfor Biomimetic Delivery of Cyclosporine A for Hepatic Ischemia-ReperfusionInjury Prevention), and collect the cells after the dish is full of mesenchymal stem cells;

[0042] 1-2) Centrifuge the cells in step 1-1) at low speed (1000 rpm, 5 min) at 4°C, collect the precipitate, resuspend the cells in pre-cooled PBS (phosphate buffered saline), centrifuge again (4°C, 1000 rpm, 5 min) to collect the precipitate, and repeat this centrifugation step three times;

[0043] 1-3) In an ice bath, suspend the precipitated cells (about 108 cells) obtained in step 1-2) in 2 mL of hypotonic solution (water as solvent, concentrations of each component are 10 mmol / L Tris-HCL, 1 mmol / L KCL, 1.5 mmol / LMgCl2 and 1 mmol / L PMSF) and resuspend the cells for 6-8 hours.

[0044] 1-4) Repeatedly freeze and thaw the cell suspension obtained in step 1-3) 5-7 times under liquid nitrogen and room temperature conditions to fully rupture the cells. After low-speed centrifugation (850g, 15min) at 4°C, collect the supernatant and centrifuge it again (15000g, 30min). The precipitate is the stem cell membrane fragment (M), which is quantified by Bicinchonininc acid (BCA) and stored at -80°C;

[0045] 2) Preparation of Kaempferol Liposomes

[0046] Take lecithin, cholesterol and kaempferol in anhydrous ethanol at a mass ratio of 3:1:1, dissolve them by oscillation and ultrasonication, and after they are completely dissolved, slowly inject them into ultrapure water with a 1 ml syringe (stirring while adding), stir with a magnetic stirrer until there is no ethanol smell, ultrasonicate for 5 min in an ice bath, 40% amplitude, ultrasonicate for 10 s and stop for 10 s, and pass them through 800 nm, 400 nm and 200 nm polycarbonate membranes in sequence with a liposome extruder to obtain kaempferol liposomes (KAE@L);

[0047] 3) Preparation of kaempferol biomimetic nanomaterials

[0048] The mesenchymal stem cell membrane (M) obtained in step 1) is extruded with a 200 nm liposome extruder, and then mixed with kaempferol liposomes, wherein the volume ratio of the mesenchymal stem cell membrane (M) to kaempferol liposomes is 1:1, and then ultrasonicated in an ice bath for 5 minutes, with an amplitude of 40%, and an interval of 10 seconds for 10 seconds, and then extruded again with a 200 nm liposome extruder to obtain the nanomaterial (KAE@ML).

[0049] Comparative Example 2 Preparation of biomimetic material ML without kaempferol

[0050] The difference from Example 1 is that the kaempferol in step 2) can be removed, and the other steps are the same. The nanomaterial finally obtained is a mixed material ML of mesenchymal stem cell membrane (M) and empty liposome (L).

[0051] Comparative Example 2 was characterized, and transmission electron microscopy (TEM) images confirmed the nanoscale structure of the liposomes. A characteristic core-shell structure ( Figure 2 a). Malvern particle size analyzer test shows that the average particle size of L is 106.1nm, and the average particle size of ML is 138.7nm. The particle size of ML group increased by 32.6nm compared with that of L group ( Figure 2 b). The average Zeta potential of L is -24.909 mV, and the average Zeta potential of ML is -31.52 mV. ML exhibits a more negative Zeta potential than L, indicating that the cell membrane is successfully encapsulated on the surface of the liposome ( Figure 2 c).

[0052] Example 3 Preparation of Kaempferol Mesenchymal Stem Cell Membrane Biomimetic Material KAE@ML

[0053] The difference between the preparation method and the example is that in step 3) in the preparation of kaempferol biomimetic nanomaterials, the mass ratio of mesenchymal stem cell membrane (M) to kaempferol liposome is 1:10.

[0054] Example 4 Preparation of Kaempferol Mesenchymal Stem Cell Membrane Biomimetic Material KAE@ML

[0055] The difference between the preparation method and the example is that in step 3) of preparing the kaempferol biomimetic nanomaterial, the mass ratio of the mesenchymal stem cell membrane (M) to the kaempferol liposome is 1:5.

[0056] Example 5 Encapsulation efficiency and drug loading rate

[0057] The KAE@ML obtained in Examples 1, 3, and 4 respectively represent different mass ratios of mesenchymal stem cell membrane M and kaempferol liposome KAE@L, such as Figure 2 As shown in Figure d, the encapsulation efficiency and drug loading of M and KAE@L are different in different ratios. When the ratio is 1:1, the encapsulation efficiency of KAE in ML is 88.54%, and the drug loading is 7.43%. When the ratio is 1:5, the encapsulation efficiency of KAE in ML is 37.13%, and the drug loading is 2.16%. When the ratio is 1:10, the encapsulation efficiency of KAE in ML is 29.58%, and the drug loading is 3.01%. Therefore, when the mass ratio of M and KAE@L is 1:1, the encapsulation efficiency and drug loading are both high.

[0058] Example 6 In vitro drug release kinetics

[0059] Taking KAE@ML prepared in Example 1 as an example, 2% Tween-80 / PBS in which KAE is well soluble was used as the drug release medium. The KAE drug content outside the dialysis bag was detected at a fixed time point and the drug release curve was drawn. The results showed that the cumulative release of free KAE reached 54.88% in 6 hours, and 75.37% was released in about 12 hours, and then the release behavior tended to be stable; KAE@L only released 42.67% in 6 hours and only 47.33% in 12 hours; KAE@ML released 32.21% and 38.17% in 6 hours and 12 hours, respectively ( Figure 2 e), indicating that KAE@ML has a sustained-release effect on drugs and can prolong the circulation time of drugs in the body.

[0060] Example 7 Biocompatibility of KAE@ML biomimetic nanomaterials

[0061] Figure 3 Kaempferol (KAE), KAE@L, and KAE@ML were co-cultured with hepatocytes (LO2) for 24 h, and then live-dead staining was performed. Live cells were green and dead cells were red. The cell status was observed under a fluorescence microscope ( Figure 3 ). The results showed that KAE, KAE@L, and KAE@ML had low toxicity to LO2 and good biocompatibility.

[0062] Example 8 Targeting of the biomimetic material ML prepared in Comparative Example 2 without kaempferol

[0063] The control group was normal SD rats, the PBS group was injected with free dye Cy5.5 through the tail vein into the SD rats, the L group was injected with blank liposomes dyed with Cy5.5 through the tail vein into the SD rats. The ML group was injected with blank mesenchymal stem cell membrane-wrapped liposomes dyed with Cy5.5 through the tail vein into the SD rats.

[0064] To evaluate the in vivo targeting behavior of biomimetic nanomaterials, we injected Cy5.5-loaded biomimetic nanomaterials intravenously into rats and used the IndiGO imaging system to monitor the distribution of biomimetic nanomaterials L and ML at predetermined time points. The results showed that 24 hours after injection, the fluorescence intensity of the ML group was the strongest. In contrast, there was almost no observable fluorescence in the PBS group ( Figure 4 a). Then the rat tissues were dissected and the main organs were imaged to monitor the distribution behavior of L and ML in vivo. The results showed that the fluorescence was mainly distributed in the liver tissue, which was consistent with the results of in vivo imaging, and the fluorescence intensity of the ML group was the highest ( Figure 4 b). Therefore, it is proved that the MSC membrane-encapsulated liposomes (ML) have good active targeting ability.

[0065] Example 9 The therapeutic effect of biomimetic nanomaterials on acute liver failure

[0066] The Control group was normal SD rats, the ALF group was SD rats with acute liver failure, the KAE group was SD rats with acute liver failure administered with kaempferol by oral gavage, the KAE@L group was SD rats with acute liver failure administered with kaempferol liposomes via tail vein, and the KAE@ML group was SD rats with acute liver failure administered with kaempferol liposomes coated with mesenchymal stem cell membranes via tail vein injection.

[0067] We used an automatic analyzer to further detect the liver function levels of SD rats in each group after treatment, and the results showed that the liver function levels of rats increased after treatment. The aspartate aminotransferase (AST) value of the blank group rats was 213.15±4.00 (U / L), the AST value of the ALF group rats was 1492.33±21.01 (U / L), the AST value of the KAE group rats was 824.24±23.54 (U / L), the AST value of the KAE@L group rats was 786.02±12.79 (U / L), and the AST value of the KAE@ML group was 579.83±12.27 (U / L). In the detection of alanine aminotransferase (ALT), the values ​​of the blank control group, ALF group, KAE group, KAE@L group, KAE group and KAE@ML group were 47.25±3.40 (U / L), 1058.34±18 (U / L), 791.71±14.03 (U / L), 627.70±13.8 (U / L), 444.38±19.41 (U / L), respectively. Figure 5 a). Therefore, compared with the ALF group, the AST and ALT levels of rats in the KAE, KAE@L and KAE@ML groups were all reduced, but the decrease in the KAE@ML group was more obvious, and liver inflammation was alleviated. The survival curve showed that the KAE@ML group significantly improved the survival rate of rats compared with the other groups ( Figure 5 b) HE (hematoxylin-eosin staining) results also showed that after KAE@ML treatment, the liver damage of rats was significantly reduced compared with other groups ( Figure 5 c). This indicates that KAE@ML has a good therapeutic effect on ALF rats.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kaempferol biomimetic nanomaterial, characterized in that: The method is composed of mesenchymal stem cell membrane and kaempferol liposome, wherein the mass ratio of the mesenchymal stem cell membrane to the kaempferol liposome is 1:1-1:10; The preparation method of the kaempferol biomimetic nanomaterial is as follows: 1) Preparation of mesenchymal stem cell membrane: After culturing mesenchymal stem cells, the cells are collected, centrifuged, resuspended once, centrifuged, resuspended twice, frozen and thawed, and centrifuged. Finally, the precipitate is collected to obtain the mesenchymal stem cell membrane; 2) Preparation of kaempferol liposomes: dissolve lecithin, cholesterol and kaempferol in anhydrous ethanol, add them into ultrapure water and stir them after mixing, and pass them through a membrane using a liposome extruder to obtain kaempferol liposomes; 3) extruding the mesenchymal stem cell membrane obtained in step 1) using a liposome extruder, mixing it with kaempferol liposomes, ultrasonicating it in an ice bath, and extruding it again using the liposome extruder to obtain kaempferol biomimetic nanomaterials; In step 1), the cells are resuspended once with a phosphate buffered saline solution, and the cells are resuspended twice with a hypotonic solution.

2. The kaempferol biomimetic nanomaterial according to claim 1, characterized in that: The average particle size of the nano material is 135-140 nm.

3. The kaempferol biomimetic nanomaterial according to claim 1, characterized in that: The hypotonic solution is an aqueous solution containing 10mmol / L Tris-HCL, 1mmol / L KCL, 1.5mmol / L MgCl2 and 1mmol / L PMSF in concentrations thereof.

4. The kaempferol biomimetic nanomaterial according to claim 1, characterized in that: In the step 2), the mass ratio of lecithin, cholesterol and kaempferol is 3:1:

1.

5. The kaempferol biomimetic nanomaterial according to claim 1, characterized in that: The temperature of the centrifugation step in step 1) is 4°C.

6. The kaempferol biomimetic nanomaterial according to claim 1, characterized in that: In the step 2), the kaempferol liposomes are obtained by sequentially extruding through polycarbonate membranes of 800 nm, 400 nm and 200 nm on a liposome extruder.

7. Use of the kaempferol biomimetic nanomaterial according to any one of claims 1 to 6 in the preparation of a drug for treating acute liver failure.

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