Kaempferol liposome nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses kaempferol liposome nanoparticles and a preparation method and application thereof.The preparation method comprises the steps that firstly, liposome is prepared, kaempferol is slowly pumped into the liposome, liquid is subjected to ultrasonic treatment and stirring during pumping, water bath rotary evaporation is conducted till an organic solvent is volatilized, then deionized water is added, and the kaempferol liposome nanoparticles are obtained. And stirring until the mixture is completely hydrated to form a faint yellow emulsion, filtering, and freeze-drying to obtain powder. The prepared and synthesized kaempferol lipidosome can improve the bioavailability of kaempferol, so that the kaempferol lipidosome has a regulating effect on inflammation of traumatic ectopic ossification lesion parts and inhibits ectopic bone formation. In-vitro experiments prove that the kaempferol liposome nanoparticles can effectively inhibit LPS / IL-4 induced macrophage polarization, inhibit release of related cell factors and inhibit formation of ectopic bones. In-vivo studies show that the nanoparticles have an obvious improvement effect on traumatic ectopic ossification of rats, are good in biocompatibility and can be used for clinical treatment of traumatic ectopic ossification.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to kaempferol liposome nanoparticles and a preparation method and application thereof. Background Art

[0002] Heterotopic ossification refers to a disease in which pathological bone is formed in soft tissues outside the skeleton, such as muscles, tendons and ligaments. Heterotopic ossification often causes structural changes in the original tissue morphology, aggravates pain in the lesion area and limits the range of motion. If it forms in the spine, it will cause spinal cord compression, seriously affecting the patient's life and work.

[0003] Heterotopic ossification is usually divided into hereditary heterotopic ossification and acquired heterotopic ossification according to the cause. Hereditary heterotopic ossification is seen in some rare diseases, such as myositis ossificans progressive or fibrodysplasia ossificans progressive; acquired heterotopic ossification is more common in clinical practice and is mostly caused by trauma, burns, surgery, etc.

[0004] The mechanism of heterotopic ossification has not yet been elucidated, but it is generally believed that the three necessary conditions for heterotopic ossification are: osteogenic precursor cells, osteogenic inducers, and local microenvironment. It is believed that the formation of heterotopic ossification depends on the interaction of multiple local and systemic factors that stimulate and inhibit osteogenic formation. Histologically, heterotopic ossification is no different from callus formation, but it is subject to various stimuli during its formation. For example, after injury, burns, and surgery, a large number of perivascular lymphocytes enter the muscles and other connective tissues in the early heterotopic ossification area. With the occurrence of local inflammatory reactions, fibrosis, and angiogenesis, osteogenic factors are released, inducing the production of bone morphogenetic proteins. These factors induce mesenchymal stem cells to differentiate into osteoblasts and chondroblasts in the local microenvironment, and finally form heterotopic ossification, which is different from the normal bone tissue formation process. At the same time, there are different hypotheses about the origin of heterotopic ossification progenitor cells, including from muscles, vascular endothelial cells, adipocytes, and tendon cells.

[0005] Current treatments for heterotopic ossification include surgical resection, nonsteroidal anti-inflammatory drugs, and radiotherapy. The most effective treatment is surgery. When mature bone is found, heterotopic ossification is removed through arthroscopy or open arthrolysis, but the postoperative recurrence rate is still high, so long-term clinical results need further evaluation. For drug treatment and radiotherapy, there is no consensus on which drug should be used and when to start treatment. Therefore, the development of new drugs for the treatment of traumatic heterotopic ossification is of great significance to protecting the bone health of the people.

[0006] Kaempferol (3,4',5,7-tetrahydroxyflavone), also known as kaempferol and kaempferol flavonol, is a yellow needle crystal extracted from the ginger plant kaempferol and the Santalaceae plant calendula. According to literature reports, kaempferol is distributed in fruits, vegetables and Chinese medicinal materials. For example, fruits and vegetables such as pumpkin and leek contain a certain amount of kaempferol, and kaempferol is present in many Chinese medicinal materials such as astragalus, forsythia, and ginkgo leaves. Kaempferol is slightly soluble in water and dissolves in hot ethanol, ether and dimethyl sulfoxide. From the beginning of this century to the present, domestic and foreign scholars have conducted a lot of research on the pharmacology and clinical practice of kaempferol, proving that it has multiple pharmacological effects such as antioxidant, antibacterial and anti-inflammatory, anti-cancer, liver protection, anti-depression, and prevention and treatment of photoaging, and can be used to treat cancer, infection, cardiovascular disease, etc. However, an obvious problem with kaempferol is its low bioavailability.

[0007] Liposomes are lipid bilayer vesicles with cell membrane-like structures. They are amphiphilic and can be used to deliver water-soluble and fat-soluble drugs. Their particle size is usually less than 1000nm and they have good biocompatibility. They are used to improve the therapeutic effects of active substances and provide controlled release of drugs. Currently, many liposome drugs have been approved for marketing and clinical use, but research on kaempferol liposomes is rare. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a kaempferol liposome nanoparticle and a preparation method and application thereof. The kaempferol liposome can improve the bioavailability of kaempferol, thereby producing a better therapeutic effect on traumatic heterotopic ossification.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for preparing kaempferol liposome nanoparticles comprises the following steps:

[0011] Step 1) putting egg yolk lecithin and cholesterol into a container, and then adding a dichloromethane solution to fully dissolve them; dissolving kaempferol in anhydrous ethanol solution, and then slowly pumping the kaempferol dissolved in anhydrous ethanol into the container, and performing ultrasonic and stirring on the liquid during the slow pumping; and then performing water bath rotary evaporation until the organic solvent is evaporated and a layer of oily film is observed to be formed in the container;

[0012] Step 2) Add deionized water to the container, stir until completely hydrated to form a light yellow emulsion, filter through a 0.22 μm filter membrane, and freeze-dry into powder.

[0013] Preferably, in step 1), the mass ratio of the egg yolk lecithin to cholesterol is 10:1 to 15:1, and the mass ratio of kaempferol to egg yolk lecithin is 1:10 to 1:30.

[0014] Preferably, in step 1), the volume of the dichloromethane is 6 to 15 mL, the volume of the anhydrous ethanol is 3 to 10 mL, and the volume of the deionized water is 5 to 15 mL.

[0015] Preferably, the pumping time in step 1) is 0.5 to 1 h.

[0016] Preferably, in step 1), the power of the ultrasound is 20-50W, and the stirring rate is 100-500rpm.

[0017] Preferably, the temperature of the water bath in step 1) is 30-45°C.

[0018] Preferably, the freeze-drying time in step 2) is 48 to 72 hours at a temperature of -80°C.

[0019] The kaempferol liposome nanoparticles prepared by the above preparation method.

[0020] The application of the above-mentioned kaempferol liposome nanoparticles in the preparation of drugs for treating traumatic heterotopic ossification.

[0021] A pharmaceutical composition for treating traumatic heterotopic ossification comprises the above-mentioned kaempferol liposome nanoparticles and a pharmaceutically acceptable carrier.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention characterizes the prepared kaempferol liposomes and analyzes the kaempferol release concentration. The kaempferol liposomes are co-cultured with macrophages, and the conditioned medium is collected to culture dental tendon stem cells. The effect of macrophage cytokines on the osteogenesis of tendon stem cells is analyzed. The effect on the rat traumatic heterotopic ossification model is observed by oral administration of kaempferol liposomes. Based on the expected effects of kaempferol liposomes on inflammatory response and osteogenic differentiation, the drug has broad application prospects for non-surgical treatment of traumatic heterotopic ossification. The present invention combines in vitro and in vivo experiments, and the results show that kaempferol liposomes can effectively inhibit the release of cytokines by macrophages in vivo compared with kaempferol, and have the potential to inhibit heterotopic bone formation, providing a new drug source for non-surgical treatment of traumatic heterotopic ossification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a transmission electron micrograph of the kaempferol liposome in Example 1;

[0025] Figure 2 The results of the in vitro experimental evaluation of kaempferol liposomes in Example 1 are as follows: A is a particle size diagram of blank liposomes, kaempferol liposomes and kaempferol liposome lyophilized powder; B is a potential diagram of blank liposomes, kaempferol liposomes and kaempferol liposome lyophilized powder; C is a drug release curve;

[0026] Figure 3 The effect of 8 μM and 50 μM kaempferol liposomes on the polarization of NR8383 cells detected by flow cytometry in Example 2: A is M1 type; B is M2 type;

[0027] Figure 4 The effect of 8 μM and 50 μM kaempferol liposomes on the expression of related cytokines in NR8383 cells detected by enzyme-linked immunosorbent assay (ELISA) in Example 2: A is IL-1β; B is IL-6; C is IL-10; D is TNF-α; E is TGF-β1; wherein, ** P<0.01, *** P < 0.001, **** P < 0.0001;

[0028] Figure 5 The effect of kaempferol liposomes on the proliferation of tendon stem cells (A) and macrophages (B) in Example 2; wherein, **** P < 0.0001;

[0029] Figure 6 The osteogenesis effect of macrophages treated with different concentrations of kaempferol liposomes on tendon stem cells in Example 2: A is alkaline phosphatase staining; B is alizarin red staining;

[0030] Figure 7 The 3D reconstruction of CT of the ectopic bones of rats in each group at 6 and 12 weeks after the treatment of the rats with traumatic heterotopic ossification model in Example 3;

[0031] Figure 8 The figures are statistical graphs of ectopic bone surface area and volume of rats in each group at 6 and 12 weeks after the treatment of the traumatic heterotopic ossification model rats in Example 3: A is a statistical graph of ectopic bone volume of rats at 6 weeks; B is a statistical graph of ectopic bone surface area of ​​rats at 6 weeks; C is a statistical graph of ectopic bone volume of rats at 12 weeks; D is a statistical graph of ectopic bone surface area of ​​rats at 12 weeks; wherein, * P<0.05, ** P<0.01, **** P < 0.0001, ns means no significant difference;

[0032] Fig. 9 The expression of cytokines IL-1β, IL-6, IL-10, TNF-α and TGF-β1 in the lesion site in Example 3;

[0033] Fig.10 This is the SOFG staining of rats in each group treated with drugs in Example 3 after 6 and 12 weeks. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0036] The experimental animals used in the following examples were SD male rats from the Experimental Animal Center of Xuzhou Medical University.

[0037] Example 1 Preparation and Characterization of Kaempferol Liposomes

[0038] 1. Preparation of Kaempferol Liposomes

[0039] 60mg of egg yolk lecithin and 5mg of cholesterol were placed in an eggplant-shaped bottle, and then 5mL of dichloromethane solution was added to fully dissolve to obtain blank liposomes. 3mg of kaempferol was weighed and dissolved in 5mL of anhydrous ethanol solution. The kaempferol dissolved in anhydrous ethanol was pumped into the blank liposomes in the eggplant-shaped bottle by a micropump for 0.5h. During the slow pumping, the liquid was ultrasonicated (ultrasonic power 20W) and stirred (100rpm). Rotary evaporation was performed in a 45℃ water bath until the organic solvent was evaporated, and an oily film was observed to form in the eggplant-shaped bottle. 10mL of deionized water was added to the eggplant-shaped bottle, and stirred (200rpm) until it was completely hydrated to form a light yellow emulsion, and then filtered through a 0.22μm filter membrane to obtain kaempferol liposomes, which were then lyophilized into powder to obtain kaempferol liposome lyophilized powder (kaempferol liposome nanoparticles).

[0040] Take 10 mg of the product to make a suspension, take 1 μL of the suspension and drop it on a copper mesh, and observe the morphology of the product under an electron microscope. Figure 1 As shown, the kaempferol liposome nanoparticles are oval in shape and uniform in size, with black opaque kaempferol visible in the middle, indicating that kaempferol is successfully encapsulated in the liposomes.

[0041] 2. Characterization and determination of kaempferol liposomes

[0042] Ultraviolet wavelength scanning was performed on kaempferol, the kaempferol liposomes prepared as above and the methanol solution of blank liposomes respectively. Kaempferol had a characteristic absorption value at 360nm, while the liposomes had no absorption value within the wavelength range, indicating that there was no interference with the blank liposomes. After the carrier was destroyed by methanol and the drug was dissolved, the kaempferol liposomes still had the absorption value of kaempferol at 360nm, indicating that kaempferol could be successfully encapsulated into the preparation, and the encapsulation efficiency was measured to be 90.6%.

[0043] The particle size and potential of kaempferol liposomes were measured using a multi-angle particle size and potential analyzer nanoparticle size tracking analyzer. Figure 2 As shown in A, kaempferol liposomes did not change much even after freeze-drying, and Figure 2 As shown in B, the potential increases after freeze-drying and has better stability.

[0044] Kaempferol liposomes were added into dialysis bags with molecular cutoff of 8000-140000D, and the drug release rate was measured at different times. Figure 2 As shown in C, kaempferol liposomes have a higher release rate and a sustained release effect.

[0045] Example 2 Verification of the in vitro function of kaempferol liposomes

[0046] 1. Effects of kaempferol liposomes on NR8383 cell behavior

[0047] (1) Polarization of NR8383 cells

[0048] NR8383 cells were cultured at 1×10 6 Inoculate in 6-well plates, wait for cells to grow to 80%, starve for 6 hours, then add corresponding reagents. Perform the following grouping experiments:

[0049] ①Group 1 (M1 type):

[0050] Blank group (Ctrl): no substance was added;

[0051] M1 model group (Model): LPS (100 μg / mL);

[0052] Experimental group (8 kaempferol): LPS (100 μg / mL) + 8 μM kaempferol liposomes;

[0053] Experimental group (50 μM kaempferol): LPS (100 μg / mL) + 50 μM kaempferol liposomes.

[0054] ②Group 2 (M2 type):

[0055] Blank group (Ctrl): no substance was added;

[0056] M2 model group (Model): IL-4 (10 μg / mL);

[0057] Experimental group (8 kaempferol): IL-4 (10 μg / mL) + 8 μM kaempferol liposomes;

[0058] Experimental group (50 μM kaempferol liposomes): IL-4 (10 μg / mL) + 50 μM kaempferol liposomes.

[0059] After 48 h, the cells were digested with trypsin without EDTA, then the digestion was terminated by centrifugation and the cells were washed three times with PBS. The cells were stained with CD206, CD86, and F4 / 80 antibodies for 30 min in the dark.

[0060] The effects of kaempferol liposomes on the polarization of rat macrophages NR8383 were investigated by detecting the expressions of F 4 / 80, CD86 and CD206 on the surface of macrophages after treatment in each group.

[0061] The results are as follows Figure 3 As shown in the figure, compared with the blank group, the expression of the model group in M1 and M2 macrophages increased, indicating that the model was successfully established. Compared with the model group, the kaempferol liposomes in the experimental group inhibited the polarization of macrophages in a dose-dependent manner.

[0062] (2) Expression of NR8383 cell-related cytokines

[0063] The grouping experiment was carried out in the same manner as above, and the cell culture supernatant was collected after 48 h.

[0064] The expression of cytokines in NR8383 cells treated with kaempferol liposomes was detected by ELISA. High expression of cytokines was induced by LPS / IL-4 stimulation of NR8383 cells.

[0065] The results are as follows Figure 4 As shown in the figure, compared with the blank group, the expression of IL-1β, IL-6, IL-10, TNF-α and TGF-β1 in the model group (LPS / IL-4) increased (P < 0.0001), indicating that the model was successfully established. Compared with the model group, the kaempferol liposomes in the experimental group reduced the release of cytokines in a dose-dependent manner.

[0066] 2. Effects of macrophages treated with different concentrations on osteogenic differentiation of tendon stem cells

[0067] (1) Effects of kaempferol liposomes on the proliferation of tendon stem cells

[0068] Tendon stem cells 5 Cells were seeded in 96-well plates and cultured overnight. NR8383 cells were plated at 2×10 5 Cells were seeded in a 96-well plate and cultured overnight. On the second day, gradient concentrations (mM) of kaempferol and kaempferol liposome solution were added, and the cells were further incubated for 24 h. At the end of the experiment, the corresponding experimental reagent solution was added to each well, and the CCK8 experiment was checked for absorbance of each well at 450 nm by SynergyTM H1 microplate reader.

[0069] The results of cell viability experiments were as follows Figure 5 As shown, at 48h of culture, there was no significant difference in proliferation ability between concentrations less than 100 μM (P>0.05), indicating that kaempferol liposomes have a promoting effect on the proliferation of tendon stem cells.

[0070] (2) Effects of macrophages treated with different concentrations of kaempferol liposomes on the osteogenic activity of tendon stem cells

[0071] For the co-culture assay, a transwell system was used and the pore size of the porous membrane was selected to be 0.4 μm. NR8383 cells were pretreated and seeded in the upper chamber of the transwell system, while TSPCs were cultured in the lower chamber at a ratio of 4:1. After 7 days of osteogenic induction, alkaline phosphatase staining was performed. After 14 days of osteogenic induction, alizarin red staining was performed. The following grouping experiments were performed:

[0072] Blank group (Control): untreated macrophages;

[0073] Model group: macrophages treated with LPS (100 μg / mL) + IL-4 (10 μg / mL);

[0074] Experimental group (8 kaempferol): macrophages were treated with LPS (100 μg / mL) + IL-4 (10 μg / mL) + 8 μM kaempferol liposomes;

[0075] Experimental group (50 μM liposomes): macrophages were treated with LPS (100 μg / mL) + IL-4 (10 μg / mL) + 50 μM kaempferol liposomes.

[0076] The experimental results are as follows Figure 6 As shown in the figure, compared with the blank group, the alizarin red staining and alkaline phosphatase staining in the model group increased significantly, indicating that macrophages can effectively promote the osteogenic activity of tendon stem cells. The alizarin red staining and alkaline phosphatase staining in the macrophage groups treated with different concentrations of kaempferol liposomes were significantly reduced, indicating that after treatment with kaempferol liposomes, macrophages can significantly reduce the osteogenic differentiation ability of tendon stem cells.

[0077] Example 3 In vivo functional study of G10@HAP / MSN@ZnO@COS

[0078] 1. Effect of kaempferol liposomes in the treatment of ectopic bone in rats

[0079] (1) Experimental procedures

[0080] After anesthesia with 1% sodium pentobarbital, a 0.5 cm longitudinal incision was made along the medial aspect of the distal hindlimb. The Achilles tendon was located and transected at the midpoint without suturing, and the skin incision was then sutured with 4-0 Vicryl. To evaluate the effects of kaempferol and liposome kaempferol on heterotopic ossification, the animals were randomly divided into the following groups:

[0081] Blank group (Ctrl): Achilles tendon was not transected;

[0082] Model group: Achilles tendon transection;

[0083] Treatment group (50 mt): Achilles tendon transection + 50 mg / kg / d kaempferol gavage;

[0084] Treatment group (50 shanfa): Achilles tendon transection + oral administration of 50 mg / kg / d kaempferol liposomes.

[0085] The mice were gavaged for 1 week, and CT scans were performed at 6 and 12 weeks.

[0086] (2) Experimental results

[0087] like Figure 7 As shown, at 6 and 12 weeks, the ectopic bone in the treatment group was significantly reduced, and the same concentration of kaempferol liposomes had a better therapeutic effect than kaempferol.

[0088] like Figure 8 As shown in the figure, the surface area and volume of bone tissue decreased in each group after 6 or 12 weeks of medication, and the surface area and volume of the kaempferol liposome group were lower than those of the kaempferol group (P < 0.05). This shows that kaempferol liposome has a good anti-ossification effect on traumatic heterotopic ossification.

[0089] 2. Cytokine expression in lesion sites after drug treatment

[0090] The grouping experiment was the same as the above steps, and the mice were gavaged for 1 week. After 7 days, the Achilles tendon was sampled and immunohistochemical staining was performed.

[0091] The experimental results are as follows Fig. 9 As shown in the figure, compared with the blank group, the expressions of IL-1β, IL-6, IL-10, TNF-α and TGF-β1 in the model group were increased, while those in the kaempferol group and kaempferol liposome group were significantly decreased compared with the model group. This indicates that kaempferol reduces macrophage-induced osteogenesis by reducing the release of IL-1β, IL-6, IL-10, TNF-α and TGF-β1 in macrophages.

[0092] 3. Safranin fast green staining to further observe the ectopic bone condition of rats

[0093] The grouping experiment was the same as the above steps, and the mice were gavaged for 1 week. At 6 and 12 weeks, the Achilles tendon was sampled, embedded in paraffin, and stained with safranin fast green.

[0094] The experimental results are as follows Fig.10 As shown, tissue sections showed that the cells in the model group were dense and there were a large number of cartilage and bone cells. The treatment group had more chondrocytes than the model group, indicating that the treatment group did effectively inhibit the progression of ectopic bone.

[0095] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing kaempferol liposome nanoparticles, characterized in that: The following steps are involved: Step 1) putting egg yolk lecithin and cholesterol into a container, and then adding a dichloromethane solution to fully dissolve them; dissolving kaempferol in anhydrous ethanol solution, and then slowly pumping the kaempferol dissolved in anhydrous ethanol into the container, and performing ultrasonic and stirring on the liquid during the slow pumping; and then performing water bath rotary evaporation until the organic solvent is evaporated and a layer of oily film is observed to be formed in the container; Step 2) Add deionized water to the container, stir until completely hydrated to form a light yellow emulsion, filter through a 0.22 μm filter membrane, and freeze-dry into powder.

2. The method for preparing kaempferol liposome nanoparticles according to claim 1, wherein: In step 1), the mass ratio of the egg yolk lecithin to cholesterol is 10:1 to 15:1, and the mass ratio of kaempferol to egg yolk lecithin is 1:10 to 1:

30.

3. The method for preparing kaempferol liposome nanoparticles according to claim 1, characterized in that: Step 1) The volume of the dichloromethane is 6-15 mL, the volume of the anhydrous ethanol is 3-10 mL, and the volume of the deionized water is 5-15 mL.

4. The method for preparing kaempferol liposome nanoparticles according to claim 1, wherein: The pumping time in step 1) is 0.5 to 1 h.

5. The method for preparing kaempferol liposome nanoparticles according to claim 1, characterized in that: Step 1) The power of the ultrasound is 20-50W, and the stirring rate is 100-500rpm.

6. The method for preparing kaempferol liposome nanoparticles according to claim 1, characterized in that: Step 1) The temperature of the water bath is 30-45°C.

7. The method for preparing kaempferol liposome nanoparticles according to claim 1, characterized in that: Step 2) The freeze-drying time is 48 to 72 hours at a temperature of -80°C.

8. Kaempferol liposome nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the kaempferol liposome nanoparticles according to claim 8 in the preparation of a medicament for treating traumatic heterotopic ossification.

10. A pharmaceutical composition for treating traumatic heterotopic ossification, characterized in that: The method comprises the kaempferol liposome nanoparticles as claimed in claim 8, and a pharmaceutically acceptable carrier.

Images