Kaempferol biomimetic nanomaterial, preparation method therefor, and use thereof

Abstract

Disclosed is a kaempferol mesenchymal stem cell membrane biomimetic material capable of more accurately targeting a damaged tissue. The biomimetic nanomaterial is simple to prepare, high in universality, and suitable for large-scale production. The present invention uses mesenchymal stem cell membranes to improve the biocompatibility and targeting property of the carrier and prolong the circulation time of the drug in vivo. The biomimetic nanomaterial prepared by the present invention enables targeted delivery of kaempferol to the liver, providing a novel treatment method for acute liver failure ALF.

Description

A kaempferol biomimetic nanomaterial, a preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a kaempferol biomimetic nanomaterial, a preparation method and application thereof. BACKGROUND

[0002] Acute liver failure (ALF) is a clinical syndrome characterized by rapid deterioration of liver function in a short period of time, resulting in ascites, coagulopathy, hepatic encephalopathy and multiple organ failure. The causes of ALF are diverse, and the main causes include acetaminophen toxicity, drug-induced liver injury, viral infection, ischemia and autoimmune disease. The causes of acute liver failure vary worldwide, and APAP (acetaminophen) overdose-induced acute liver failure is common in Europe and other developed countries, while viral hepatitis (type A, B and E) -induced acute liver failure mainly occurs in developing countries. The prognosis of patients with acute liver failure is poor and there is no effective treatment, making how to treat acute liver failure an important problem.

[0003] Kaempferol, also known as kaempferol-3, kaempferol and kaempferol, belongs to dietary flavonoid compounds, mainly derived from the rhizomes of Zingiberaceae plants, and widely exists in various fruits, vegetables and natural plants, and 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, heart protection and liver protection, but its poor water solubility and first-pass effect in the gastrointestinal tract greatly 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 lipid-soluble molecules between the phospholipid bilayers, thereby improving the bioavailability of water-insoluble drugs, and have 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 red blood cells, 12 dendritic cells (DCs), 13 mesenchymal stem cells (MSCs), 14 macrophages have been explored as drug carriers. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with self-renewal and pluripotent differentiation potential. The application of stem cell therapy is gradually deepening and diversifying, with broad prospects. MSCs can regulate the activation and proliferation of T cells, regulate the cytokines produced by immune cells, and specifically target the injury site, thereby inhibiting inflammation and immune regulation. Mesenchymal stem cell membranes have the active targeting of MSCs, immune escape characteristics, and long circulation, and these advantages make mesenchymal stem cell membranes have the prospect of 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 biomaterials and have biological safety problems. In order to overcome these shortcomings, there are documents that have developed new cell membrane-coated particles with minimal loss of membrane proteins (A. V. Kroll, R. H. Fang, L. Zhang, Biointerfacing and Applications of Cell Membrane-Coated Nanoparticles. Bioconjugate Chemistry 28, 23-32 (2016).). Currently, MSC membrane-modified NPs have made gratifying progress in inflammation, tissue regeneration, and tumor therapy (L. Fan, A. Wei, Z. Gao, X. Mu, Current progress of mesenchymal stem cell membrane-camouflaged nanoparticles for targeted therapy. Biomedicine & Pharmacotherapy 161, (2023).). SUMMARY

[0006] Invention purposes: The purpose of the present application is to provide a kaempferol mesenchymal stem cell membrane biomimetic material, which can more accurately target the damaged tissue. The preparation method of the biomimetic nanomaterial is simple, universal, and convenient for large-scale production.

[0007] Technical scheme:

[0008] The present application provides a kind of kaempferol biomimetic nanomaterial, which is composed of mesenchymal stem cell membrane, liposome and kaempferol liposome. The average particle size of the nanomaterial is 135-140 nm.

[0009] The present application also provides a kaempferol biomimetic nanomaterial, and the preparation method is as follows:

[0010] 1) Preparation of mesenchymal stem cell membrane: after culturing mesenchymal stem cells, collect the cells, and sequentially centrifuge, resuspend once, centrifuge, resuspend twice, freeze-thaw, centrifuge, and finally collect the precipitate as the mesenchymal stem cell membrane;

[0011] 2) Preparation of kaempferol liposome: take egg phospholipid, cholesterol and kaempferol in anhydrous ethanol, mix and dissolve, then add to ultrapure water and stir, pass through the membrane with a liposome extrusion machine to obtain kaempferol liposome;

[0012] 3) The mesenchymal stem cell membrane obtained in step 1) is extruded with a liposome extrusion machine, mixed with kaempferol liposome, ice-bath ultrasonic, and extruded again with a liposome extrusion machine to obtain kaempferol biomimetic nanomaterial.

[0013] Further, the resuspension in step 1) uses phosphate buffered saline solution to resuspend the cells, and the resuspension in step 2) uses hypotonic solution to resuspend the cells.

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

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

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

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

[0018] Further, the kaempferol liposome in step 2) is extruded through polycarbonate membranes of 800 nm, 400 nm and 200 nm in sequence with a liposome extrusion machine.

[0019] The application also provides application of the kaempferol biomimetic nanomaterial in preparation of a medicine for treating acute liver failure.

[0020] Advantages:

[0021] (1) The nanobiomimetic material provided by the application is obtained through physical extrusion, that is, film extrusion, and is simple to prepare and easy to mass-produce.

[0022] (2) The application designs a nanobiomimetic material, and the mesenchymal stem cell membrane is used to improve the biocompatibility, targeting property and circulation time of the carrier in the body.

[0023] (3) The biomimetic nanomaterial prepared by the application realizes targeted delivery of kaempferol to the liver, and provides a new treatment method for acute liver failure ALF. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the preparation process of the biomimetic nanomaterial KAE@ML.

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

[0026] Figure 3 is a cell live and dead diagram of the biomimetic nanomaterial KAE, KAE@L and KAE@ML.

[0027] Figure 4 is the targeting property of the biomimetic nanomaterial: Figure 4a is the fluorescence intensity of the Control group, the PBS group, the L group and the ML group after being injected for 1, 3 and 7 days, which is detected by using the IndiGO imaging system, and Figure 4b is a typical ex vivo image of the main organs of each group after being injected for 24 h.

[0028] Figure 5 is the therapeutic effect of the biomimetic nanomaterial: Figure 5a is the determination of the liver function ALT and AST levels of the Control group, the ALF group and each group of mice after treatment, Figure 5b is a survival curve diagram of the mice in the Control group, the ALF group and each group of mice after treatment, and Figure 5c is an HE image of each group of rats. DETAILED DESCRIPTION

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

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

[0031] Mesenchymal stem cells: purchased from American Type Culture Collection.

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

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

[0034] Kaempferol: Shanghai Aladdin Bio-Chem Technology Co., Ltd., K107144

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

[0036] KCL: Shanghai Aladdin Bio-Chem Technology Co., Ltd., P433492

[0037] MgCl2: Shanghai Biyun Tian Biological Technology Co., Ltd., ST269

[0038] PMSF benzyl sulfonyl fluoride: Jiangsu Kaikai Biological Technology 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) According to the prior art (Mesenchymal Stem Cell Membrane-Camouflaged Liposomes for Biomimetic Delivery of Cyclosporine A for Hepatic Ischemia-Reperfusion Injury Prevention), mesenchymal stem cells were cultured in a 10 cm dish, and when they were fully grown, the cells were collected;

[0042] 1-2) The cells of step 1-1) were centrifuged at low speed (1000 rpm, 5 min) at 4°C, and the precipitate was resuspended with pre-cooled PBS (phosphate buffered saline) and centrifuged again (4°C, 1000 rpm, 5 min). The centrifugation step was repeated three times.

[0043] 1-3) Under ice bath conditions, the precipitated cells (about 10 8The cells were suspended in 2 mL of a hypotonic solution (10 mmol / L Tris-HCL, 1 mmol / L KCL, 1.5 mmol / L MgCl2 and 1 mmol / L PMSF in water) and the cells were resuspended for 6-8 h.

[0044] 1-4) The cell suspension obtained in step 1-3) was repeatedly frozen and thawed 5-7 times under liquid nitrogen and room temperature conditions to fully rupture the cells. After low-speed centrifugation (850 g, 15 min) at 4°C, the supernatant was centrifuged again (15000 g, 30 min) and the precipitate was the stem cell membrane fragments (M), which were quantified by Bicinchonininc acid (BCA) and stored at -80°C.

[0045] 2) Preparation of kaempferol liposomes

[0046] Take the egg lecithin, cholesterol and kaempferol, dissolve them in anhydrous ethanol according to the mass ratio of 3:1:1, shake and ultrasonically dissolve, then slowly inject 1 ml of the solution into ultrapure water (stir while adding), stir with a magnetic stirrer until there is no ethanol smell, ice bath ultrasonication for 5 min, 40% amplitude, ultrasonication for 10 s and stop for 10 s, pass through 800 nm, 400 nm and 200 nm polycarbonate membranes in sequence using a liposome extruder, and kaempferol liposomes (KAE@L) are obtained.

[0047] 3) Preparation of kaempferol biomimetic nanomaterials

[0048] The mesenchymal stem cell membrane (M) obtained in step 1) was extruded using a 200 nm liposome extruder, mixed with kaempferol liposomes, and the volume ratio of the mesenchymal stem cell membrane (M) to kaempferol liposomes was 1:1. Ice bath ultrasonication was performed for 5 min at 40% amplitude, ultrasonication for 10 s and stop for 10 s, and the nanomaterial (KAE@ML) was obtained by extruding the mixture using a 200 nm liposome extruder.

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

[0050] The difference between Example 1 and Comparative Example 2 is that the kaempferol in step 2) is removed, and the other steps are the same. The final nanomaterial is a mixture of mesenchymal stem cell membrane (M) and empty liposomes (L), which is ML.

[0051] The comparative example 2 was characterized, and the transmission electron microscope (TEM) image confirmed the nanoscale structure of the liposome, and the characteristic core-shell structure could be observed on the surface of ML (Figure 2a). The Malvern particle size instrument test showed that the average particle size of L was 106.1 nm, and the average particle size of ML was 138.7 nm. The particle size of the ML group increased by 32.6 nm compared with the particle size of the L group (Figure 2b). The average Zeta potential of L was -24.909 mV, and the average Zeta potential of ML was -31.52 mV. ML showed more negative Zeta potential than L, indicating that the cell membrane was successfully encapsulated on the surface of the liposome (Figure 2c).

[0052] Example 3 Preparation of kaempferol mesenchymal stem cell membrane biomimetic material KAE@ML

[0053] The difference between its preparation method and the embodiment is that in step 3) preparation of kaempferol biomimetic nanomaterial, 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 its preparation method and the embodiment is that in step 3) preparation of kaempferol biomimetic nanomaterial, the mass ratio of mesenchymal stem cell membrane (M) to kaempferol liposome is 1:5.

[0056] Example 5 Encapsulation efficiency and drug loading rate

[0057] 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, as shown in Figure 2d. Different ratios of M and KAE@L have different encapsulation efficiency and drug loading capacity. When the ratio is 1:1, the encapsulation efficiency of KAE in ML is 88.54%, and the drug loading capacity is 7.43%. When the ratio is 1:5, the encapsulation efficiency of KAE in ML is 37.13%, and the drug loading capacity is 2.16%. When the ratio is 1:10, the encapsulation efficiency of KAE in ML is 29.58%, and the drug loading capacity is 3.01%. Therefore, when the mass ratio of M and KAE@L is 1:1, the encapsulation efficiency and drug loading capacity are both high.

[0058] Example 6 In vitro drug release kinetics

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

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

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

[0062] Example 8 Targeting of biomimetic material ML not containing kaempferol prepared in Comparative Example 2

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

[0064] In order to evaluate the in vivo targeting behavior of the biomimetic nanomaterial, the Cy5.5-loaded biomimetic nanomaterial was injected intravenously into the rat body, and the distribution of the biomimetic nanomaterial L and ML was monitored at predetermined time points using the IndiGO imaging system. The results showed that 24h after injection, the fluorescence intensity of the ML group was the strongest. In contrast, the PBS group had almost no observable fluorescence (Figure 4a). 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 4b). Therefore, it is proved that the mesenchymal stem cell membrane wrapped liposome (ML) has good active targeting ability.

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

[0066] Control group is normal SD rats, ALF group is acute liver failure of SD rats, KAE group is acute liver failure of SD rats with intragastric administration of kaempferol, KAE@L group is acute liver failure of SD rats with intravenous administration of kaempferol liposome, KAE@ML group is acute liver failure of SD rats with intravenous administration of mesenchymal stem cell membrane-encapsulated kaempferol liposome.

[0067] We further detected the liver function of SD rats in each group after treatment using an automatic analyzer, and the results showed that the liver function of rats increased after treatment. The aspartate aminotransferase (AST) value of the blank group was 213.15±4.00 (U / L), the AST value of the ALF group was 1492.33±21.01 (U / L), the AST value of the KAE group was 824.24±23.54 (U / L), the AST value of the KAE@L group 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 alanine aminotransferase (ALT) test, the values of the blank control group, the ALF group, the KAE group, the KAE@L group, and the 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), and 444.38±19.41 (U / L), respectively (Figure 5a). Therefore, compared with the ALF group, the AST and ALT levels of the KAE group, the KAE@L group, and the KAE@ML group were decreased, but the decrease in the KAE@ML group was more obvious, and the liver inflammation was relieved. The survival curve showed that the KAE@ML group significantly improved the survival rate of rats compared with other groups (Figure 5b). The HE (hematoxylin-eosin staining) results also showed that the liver damage of rats was significantly reduced after KAE@ML treatment compared with other groups (Figure 5c). It is proved that KAE@ML has good therapeutic effect on ALF rats.

[0068] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A kaempferol biomimetic nanomaterial, characterized in that, The mesenchymal stem cell membrane is composed of liposomes and kaempferol liposomes.

2. The biomimetic nanomaterial of kaempferol according to claim 1, characterized by the fact that, The average particle size of the nanomaterial is 135-140 nm.

3. The kaempferol biomimetic nanomaterial according to claim 1 or 2, characterized by the fact that, The preparation method is as follows: 1) Preparation of mesenchymal stem cell membrane: after culturing mesenchymal stem cells, the cells are collected, and the cells are sequentially subjected to centrifugation, primary resuspension, centrifugation, secondary resuspension, freeze-thawing, centrifugation, and finally the precipitate is collected as the mesenchymal stem cell membrane; 2) Preparation of kaempferol liposomes: take lecithin, cholesterol and kaempferol in anhydrous ethanol, mix and dissolve, then add to ultrapure water and stir, pass through the membrane with a liposome extrusion machine, and obtain kaempferol liposomes; 3) The mesenchymal stem cell membrane obtained in step 1) is extruded with a liposome extrusion machine, mixed with kaempferol liposomes, ice-bath ultrasonic, and extruded again with a liposome extrusion machine to obtain kaempferol biomimetic nanomaterial.

4. The biomimetic nano-material of kaempferol according to claim 3, characterized by the fact that, The primary resuspension in step 1) uses phosphate buffered saline solution to resuspend the cells, and the secondary resuspension uses a hypotonic solution to resuspend the cells.

5. The biomimetic nano-material of kaempferol according to claim 4, characterized by the fact that, The hypotonic solution is an aqueous solution containing 10 mmol / L Tris-HCL, 1 mmol / L KCL, 1.5 mmol / L MgCl2 and 1 mmol / L PMSF.

6. The biomimetic nano-material of kaempferol according to claim 3, characterized by the fact that, The mass ratio of lecithin, cholesterol and kaempferol in step 2) is 3:1:

1.

7. The biomimetic nano-material of kaempferol according to claim 3, characterized by the fact that, The mass ratio of mesenchymal stem cell membrane to kaempferol liposomes in step 3) is 1:1-1:

10.

8. The biomimetic nano-material of kaempferol according to claim 3, characterized by the fact that, The temperature of the centrifugation step in step 1) is 4℃.

9. The biomimetic nano-material of kaempferol according to claim 3, characterized by the fact that, The kaempferol liposomes in step 2) are sequentially extruded through polycarbonate membranes of 800 nm, 400 nm and 200 nm in the liposome extrusion machine.

10. Use of the kaempferol biomimetic nanomaterial of any one of claims 1-9 in the preparation of a drug for treating acute liver failure.

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