Pharmaceutical composition containing royal jelly extracellular vesicles and use thereof

Abstract

Disclosed in the present invention is a use of royal jelly extracellular vesicles (RJEVs) in the preparation of a pharmaceutical composition for treating wounds in individuals suffering from diabetes, wherein the pharmaceutical composition comprises at least one carrier, and the at least one carrier has the characteristics of adhering to the skin of the individuals.

Description

Pharmaceutical compositions containing royal jelly extracellular vesicles and their uses [Technical Field]

[0001] This invention relates to a pharmaceutical composition comprising royal jelly extracellular vesicles and its use, particularly to a pharmaceutical composition and its use for treating wounds in diabetic patients using royal jelly extracellular vesicles. [Prior Art]

[0002] Diabetic patients often experience prolonged wound healing or incomplete closure, which can easily lead to diabetic ulcers. Diabetic foot ulcers are one of the most serious problems that diabetic patients face.

[0003] Although there are many methods currently available in clinical practice to promote wound healing, including the application of appropriate antibiotic ointments and dressings to the wound, these methods are not ideal for treating wounds in diabetic patients and may cause serious side effects and adverse reactions.

[0004] Compared to wounds on normal skin, wounds in diabetic patients often heal poorly due to chronic inflammation, poor angiogenesis, reduced blood flow, neuropathy, and fluctuations in blood sugar levels, and also face a higher risk of infection. To mitigate this risk, further treatments are often required, such as growth factors, bioengineered skin regeneration templates, and hyperbaric oxygen therapy, but these treatments are not necessary for normal wounds.

[0005] Currently, the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of diabetic foot ulcers is Regranex, an ointment containing the growth factor PDGF. However, in June 2008, the FDA issued a warning that using more than three doses of Regranex may increase the risk of death in cancer patients. Therefore, there is still a very large market demand for developing safe and effective drugs to promote wound healing in diabetic patients.

[0006] Royal jelly is a known natural product with various functions, including anti-inflammatory and tissue regeneration promotion, and is widely used worldwide. Extracellular vesicles (EVs) in royal jelly retain its benefits and improve its bioavailability. To address the long-standing lack of safe and effective drugs for promoting wound healing in diabetic patients, this invention provides a pharmaceutical composition comprising royal jelly extracellular vesicles for promoting wound healing in diabetic patients. [Summary of the Invention]

[0007] To promote the healing of chronic diabetic wounds, this invention provides a pharmaceutical composition comprising royal jelly extracellular vesicles, combined with a hydrogel formulation, which can serve as a safe and cost-effective care method.

[0008] In one aspect of the present invention, a pharmaceutical composition comprising royal jelly extracellular vesicles (RJEVs) and a hydrogel is disclosed, wherein the hydrogel is selected from at least one of the group consisting of polymer Pluronic F127, poly(N-isopropyl acrylamide), polyethylene glycol-polyester copolymer, alginate, chitosan, collagen, gelatin, fibrin, hyaluronic acid, and polyethylene glycol.

[0009] In another aspect of the invention, the use of royal jelly extracellular vesicles (RJEVs) in the preparation of a pharmaceutical composition for treating wounds in individuals suffering from diabetes is disclosed, wherein the pharmaceutical composition comprises at least one carrier, and the at least one carrier has the property of adhering to the skin of the individual. [Simplified illustration]

[0010] The objectives and advantages of this invention can be further understood through the following figures and detailed description: Figure 1A shows the particle size analysis results of royal jelly extracellular vesicles (RJEVs); Figure 1B shows the results of detecting extracellular vesicle (EV) marker proteins of royal jelly extracellular vesicles (RJEVs) using the Western spectrophotometric method; Figure 2A shows the cell viability test results of RJEVs on human dermal fibroblasts (HDF); Figure 2B shows the effect of RJEVs on the cell viability of human dermal fibroblasts (HDF) treated with lipopolysaccharide (LPS); Figure 3 shows the effect of RJEVs on HDF cell migration under high glucose conditions. Effects on migration rate; Figure 4A shows the cell viability test results of RJEVs on mouse macrophages (Raw264.7); Figure 4B shows the cell viability test results of RJEVs on lipopolysaccharide (LPS)-induced mouse macrophages (Raw264.7); Figure 4C shows the effect of RJEVs on reducing the activation of LPS-induced inflammation-related transcription factors; Figure 5A shows the cell viability test results of RJEVs on human leukemia monocytokine cell line (THP-1); Figure 5B shows the effect of RJEVs on the cell viability test results of LPS-induced human leukemia monocytokine cell line (THP-1). The results of cell viability assays for RJEVs are shown in Figures 5C to 5F, which show the effects of RJEVs on the inflammatory factors IL-6, IL-8, IL-1β, and TNF-α induced by lipopolysaccharide (LPS) in human leukemia monocytogenes cell line (THP-1), respectively. Figure 5G shows the effect of RJEVs on the expression of phosphorylated NF-κB protein induced by LPS in THP-1 cells. Figure 6A shows the cell viability assay results of RJEVs on human umbilical vein endothelial cells (HUVECs). Figure 6B shows the cell viability assay results of RJEVs on human umbilical vein endothelial cells (HUVECs) affected by a high glucose environment. Results; Figure 6C shows the effect of RJEVs on the cell migration rate of human umbilical vein endothelial cells (HUVECs) under high glucose conditions; Figure 6D shows the effect of RJEVs on angiogenesis of human umbilical vein endothelial cells (HUVECs) under high glucose conditions; Figure 7A shows the state changes of PF127 at different temperatures; Figure 7B shows the effect of RJEVs and its hydrogel formulation on the healing of diabetic wounds; Figure 7C shows the effect of RJEVs and its hydrogel formulation on the wound healing rate of diabetic wounds; and Figure 7D shows the tissue staining results of diabetic wounds treated with RJEVs compared to the control group. [Implementation Method]

[0011] The present invention will be described more clearly with reference to the following embodiments. It should be noted that the following description of preferred embodiments of the invention is for illustration and description only, and is not intended to be exhaustive or to limit the precise forms disclosed.

[0012] To promote the healing of chronic diabetic wounds, this invention provides a pharmaceutical composition comprising royal jelly extracellular vesicles and a hydrogel. The hydrogel serves as a scaffold for the delivery of royal jelly extracellular vesicles and prolongs the duration of action of the vesicles. The hydrogel used in this invention is selected from one or more combinations of the group consisting of polymer Pluronic F127, poly(N-isopropyl acrylamide), polyethylene glycol-polyester copolymer, alginate, chitosan, collagen, gelatin, fibrin, hyaluronic acid, and polyethylene glycol. In one embodiment, the pharmaceutical composition comprises one of the above-mentioned hydrogels, for example, polymer Pluronic F127. In other embodiments, the pharmaceutical composition comprises two or more hydrogels, for example, a combination of polymer Pluronic F127 and hyaluronic acid, or a combination of polymer Pluronic F127 and sodium alginate.

[0013] For wound dressings, hydrogels with a mechanical strength (elastic modulus) in the range of 1 to 100 kPa are generally considered suitable because they can mimic the softness of human skin and achieve a good balance between flexibility and durability. Compared to the mechanical strength of gauze or cotton (1 MPa to 1 GPa), this range of mechanical strength of hydrogels is more suitable for human skin and has better compatibility with human skin.

[0014] In the embodiments of this invention, the hydrogel used in the pharmaceutical composition comprises a thermosensitive hydrogel. A thermosensitive hydrogel is a temperature-sensitive biomedical material that is a flowing liquid at room temperature and rapidly solidifies into a gel at physiological temperatures. Thermosensitive hydrogels can further bind active ingredients such as antibiotics, drugs, or growth factors to increase the time for these active ingredients to remain at the affected area for slow release, absorption, and metabolism. Due to its high stability and biocompatibility, thermosensitive hydrogels can be widely used in medical applications such as wound dressings and artificial skin.

[0015] Table 1 lists several thermosensitive hydrogels suitable for the pharmaceutical compositions used in this case. These thermosensitive hydrogels have good elastic modulus, typically between 1 kPa and 100 kPa. Among these thermosensitive hydrogels, PF-127 and chitosan have received FDA approval, while certain polyethylene glycol-based formulations have been approved for use in wound care products. Table 1

[0016] In addition to temperature-sensitive hydrogels, the pharmaceutical compositions of the present invention may also contain other types of hydrogels (as shown in Table 2), which are also biocompatible and suitable for use in wound dressings. Therefore, the hydrogels in Tables 1 and 2 can be combined arbitrarily to form the carrier for delivering royal jelly extracellular vesicles in the pharmaceutical compositions of this invention. When temperature-sensitive hydrogels are combined with other types of hydrogels, the other types of hydrogels account for approximately 0.01% to 1% of the total weight of the hydrogels. However, not all biocompatible hydrogels are suitable for use in wound dressings. Considering factors such as environmental pollution or skin adhesion, some non-biodegradable hydrogels (e.g., polyacrylamide hydrogels) or highly hydrophilic hydrogels (e.g., polyvinyl alcohol hydrogels) are not suitable for use in wound dressings. Table 2

[0017] In the pharmaceutical composition of this case, the royal jelly extracellular vesicles have a concentration of 10 μg / ml to 500 μg / ml (including but not limited to 10, 20, 50, 100, and 500 μg / ml), and the hydrogel has an elastic modulus of 1 kPa to 100 kPa, such as 5 kPa to 100 kPa, 5 kPa to 50 kPa, or 15 kPa to 50 kPa. When the pharmaceutical composition of this case contains multiple hydrogels, the overall elastic modulus of the multiple hydrogels should be between 1 and 100 kPa to meet the viscosity and elastic modulus required for wound dressings. Overall, the royal jelly extracellular vesicles in the pharmaceutical composition account for 0.05% to 5% of the total weight, and the hydrogel in the pharmaceutical composition accounts for 95% to 99.95% of the total weight.

[0018] Any value falling within the numerical range described herein can be used as a maximum or minimum value to derive sub-ranges; for example, the numerical range of "1 kPa to 100 kPa" should be understood to include any sub-range between the minimum value of 1 kPa and the maximum value of 100 kPa, such as: 5 kPa to 100 kPa, 1 kPa to 10 kPa, and 20 kPa to 50 kPa, etc. Furthermore, the various numerical endpoints described herein can be arbitrarily chosen as maximum or minimum values ​​to derive numerical ranges; for example, 10, 20, 50, 100, or 500 μg / ml can derive numerical ranges of 10 to 500 μg / ml, 20 to 100 μg / ml, or 50 to 100 μg / ml.

[0019] According to embodiments of the present invention, the pharmaceutical composition can be formulated into a dosage form for topical administration. Specifically, the pharmaceutical composition can be formulated into the form of ointment, gel, paste, patch, film, oral ointment, or wound dressing. Preferably, the pharmaceutical composition can be formulated into the form of wound dressing, ointment, oral ointment, or gel. More preferably, the pharmaceutical composition can be formulated into the form of wound dressing or gel.

[0020] Optionally, in addition to the hydrogel, the pharmaceutical composition of this application may also include other pharmaceutically acceptable components such as carriers, additives, adjuvants, and / or excipients. For example, the pharmaceutical composition may include, but is not limited to, any combination of one or more of the group consisting of solvents, solubilizers, stabilizers, tension enhancers, penetration enhancers, pH adjusters, wetting agents, surfactants, buffers, preservatives, viscosity modifiers, emulsifiers, suspending agents, flavoring agents, colorants, and antibacterial agents.

[0021] On the other hand, the present invention provides the use of royal jelly extracellular vesicles (RJEVs) in the preparation of a pharmaceutical composition for treating wounds in individuals with diabetes, the pharmaceutical composition comprising at least one carrier having the property of adhering to the individual's skin.

[0022] The term "diabetes" as used in this article includes, but is not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, cystic fibrosis-related diabetes (CFRD), latent autoimmune diabetes in adults (LADA), or monogenic diabetes.

[0023] As used in this article, the term "wound" refers to an open lesion on the body surface that is not healing or heals slowly due to damage or rupture of the mucous membranes or skin, including wounds on the body surface such as the mouth, cornea, or skin. As used in this article, the terms "diabetic ulcer" or "diabetic wound" refer to an ulcer or wound that is a complication of diabetes and typically does not heal within 1 to 3 months.

[0024] When the pharmaceutical composition of the present invention is used to treat diabetic wounds, the pharmaceutical composition contains about 10 μg / ml to 500 μg / ml of royal jelly extracellular vesicles (including but not limited to 10, 20, 50, 100 and 500 μg / ml) and a hydrogel having an elastic modulus of 1 kPa to 100 kPa.

[0025] In embodiments of the invention, the individual suffering from diabetes can be a human or an animal. Preferably, the individual is a mammal, such as a human or a non-human mammal (including primates, pigs, rodents, and rabbits). In one embodiment of the invention, the individual is a diabetic pig.

[0026] In the pharmaceutical compositions of the present invention, the carrier has the property of adhering to the individual's skin. Suitable carriers having the above-mentioned properties include: films, breathable adhesive tapes, hydrophilic dressings (artificial skin), foams, hydrophilic fiber dressings, alginate dressings, and gels, etc. In embodiments of the present invention, the carrier is one or more hydrogels. As mentioned above, the hydrogel used in the pharmaceutical compositions of this invention has an elastic modulus of 1 to 100 kPa and is compatible with the individual's skin.

[0027] Depending on the condition of the diabetic wound, those skilled in the art can adjust the frequency and / or timing of administration of the pharmaceutical composition. For example, the frequency and / or timing of administration of the pharmaceutical composition can be adjusted from three to four times daily to once every three to five days. According to an embodiment of the invention, the pharmaceutical composition of the invention is administered to the wound of diabetic pigs once every three to four days.

[0028] The technical effects of the present invention will be further illustrated in the following specific examples with reference to experimental data. However, it should be understood that the scope of the present invention is not limited to the exemplary examples.

[0029] Example 1: Preparation of Royal Jelly Extracellular Vesicles (RJEVs)

[0030] [Amended according to Rule 26, 10.09.2025] Royal jelly from Western honeybees (Apis mellifera) was purchased from a beekeeping farm in Taiwan, China. After diluting the royal jelly with PBS, the sample was centrifuged at 3,000g for 30 minutes to obtain the supernatant. The supernatant was centrifuged at 16,000g for 30 minutes, and then filtered through a 0.22μm filter membrane to remove residual debris. The extracellular vesicles of royal jelly were then purified by filtration.

[0031] To further confirm whether the product obtained by the above method is royal jelly extracellular vesicle, the particle size or marker proteins of the extracellular vesicles (EVs) can be used for verification. Generally, the particle size of extracellular vesicles is between 30 and 200 nm, and royal jelly extracellular vesicles contain marker proteins such as transmembrane protein CD63, connexin syntenin, major royal jelly protein 1 (MRJP1), MRJP2, MRJP3, MRJP5, defensin-1, and the antimicrobial peptide jellein-1. The royal jelly extracellular vesicles purified by the procedure in Example 1 were analyzed using a nanoparticle size analyzer (…). Particle Metrix (Germany) analysis showed a size distribution peak of 121 nm (as shown in Figure 1A), consistent with the particle size of extracellular vesicles. Western spectrophotometry analysis of the royal jelly extracellular vesicles purified using the procedure of Example 1 also revealed the presence of the marker proteins CD63 and syntenin (as shown in Figure 1B).

[0032] Example 2: Analysis of the toxicity and activation of royal jelly extracellular vesicles on human dermal fibroblasts

[0033] [Amended according to Rule 26, 10.09.2025] Human dermal fibroblast cell line (HDF) was purchased from the Taiwan Center for Biological Resources Conservation and Research and cultured in minimal basal medium (MEM) supplemented with 1.5 g / L sodium bicarbonate, 0.1 mM non-essential amino acids (NEAA), 100 U / mL penicillin / streptomycin, 1 mM sodium pyruvate, and 10% fetal bovine serum. Extracellular vesicles obtained in Example 1 were labeled with the ExoSpark exosome extracellular membrane fluorescent labeling kit - green stain (Dongjin Chemical Research Institute, Japan), and then the labeled extracellular vesicles were reacted with human dermal fibroblasts for 24 hours. Cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI) stain and observed using a conjugate focal microscope LSM 700 (Zeiss, Germany). Overlaying images stained with ExoSpark and DAPI confirmed that human dermal fibroblasts can take up extracellular vesicles of royal jelly (not shown).

[0034] On the other hand, the royal jelly extracellular vesicles obtained in Example 1 were serially diluted to obtain royal jelly extracellular vesicles at concentrations of 10, 20, 50, 100, and 500 μg / ml. Human dermal fibroblasts were treated with royal jelly extracellular vesicles at concentrations of 0, 10, 20, 50, 100, and 500 μg / ml, and cell viability was measured. As shown in Figure 2A, royal jelly extracellular vesicles at concentrations from 10 μg / ml to 500 μg / ml were non-toxic to human dermal fibroblasts. As shown in Figure 2B, lipopolysaccharide (LPS) treatment reduced cell viability, while treatment with royal jelly extracellular vesicles at concentrations of 10 μg / mL and 20 μg / mL restored cell viability, showing a significant difference compared to cells treated with LPS alone.

[0035] Example 3: Effects of royal jelly extracellular vesicles on fibroblast aging, cell migration rate and collagen expression in a high-glucose environment.

[0036] A cellular senescence β-galactosidase (SA-β-gal) staining kit (purchased from Cell Signaling Technology, USA) was used to assess SA-β-gal expression in fibroblasts. HDF cells were stimulated with 25.5 mM glucose to simulate a high glucose (HG) environment. In another group, HDF cells were pretreated with 20 μg / mL royal jelly extracellular vesicles for one hour before high glucose stimulation, 24 hours prior. Cells were then observed under an optical microscope and analyzed using ImageJ software. The results of SA-β-gal activity analysis showed that a high glucose environment induced cellular senescence and increased SA-β-gal expression, while royal jelly extracellular vesicles reversed this cellular senescence state, reducing SA-β-gal expression and activation (not shown).

[0037] To analyze the effect of royal jelly extracellular vesicles on cell migration rate, culture inserts were used... The cells (HDF cells) were attached to 12-well plates. After approximately 95% confluence, the culture media were removed, and the cells were washed with Hank's balanced salt solution (HBSS). Then, 20 μg / ml royal jelly extracellular vesicles were added, and cell migration was observed using an optical microscope at 0, 8, and 24 hours. Adding 30 mM glucose to the culture medium served as a high-glucose induction condition, as shown in Figure 3. Compared to the control group without glucose, the high-glucose environment inhibited cell migration rate at both 8 and 24 hours. Compared to the high-glucose environment (HG) group, the treatment with royal jelly extracellular vesicles (HG+RJEVs) improved cell migration rate in the 24-hour cell migration test, indicating that royal jelly extracellular vesicles help increase wound healing rate under high-glucose conditions.

[0038] Type I collagen is a major component of the extracellular matrix of the dermis. In a high-glucose environment, the inhibitory effect of the high-glucose environment on collagen expression can be observed, while treatment with royal jelly extracellular vesicles can restore collagen expression that is reduced due to the high-glucose environment (not shown).

[0039] The results of Examples 1-3 show that royal jelly extracellular vesicles have nanoscale particle size and can be internalized into human dermal fibroblasts. Royal jelly extracellular vesicles are not only non-toxic to cells, but can also reverse high-glucose-induced cell aging, inhibited collagen expression, and slowed cell migration.

[0040] Example 4: Analysis of the toxicity and activation of extracellular vesicles of royal jelly on mouse macrophages

[0041] Mouse macrophages RAW-Blue TMCells were purchased from InvivoGen (USA) as reporter cells for the activation of inflammation-related transcription factors NF-κB and AP-1. RAW-Blue cells were then used. TM Cell cultures were prepared with 200 μg / mL zeopycin, 2 mM L-glutamic acid, 10% fetal bovine serum (FBS), and 100 μg / mL nomomycin. TM (Normocin TM The cells were cultured in DMEM medium containing 100 U / mL penicillin / streptomycin and placed in a cell culture incubator at 37°C with 5% carbon dioxide. The extracellular vesicles obtained in Example 1 were labeled with the ExoSpark exosome extracellular membrane fluorescent labeling kit – green stain (Dongjin Chemical Research Institute, Japan). The labeled extracellular vesicles were then reacted with mouse macrophages for 24 hours. The cell nuclei were labeled with DAPI stain and observed using a conjugate focal microscope LSM 700 (Zeiss, Germany). Overlaying the images stained with ExoSpark and DAPI confirmed that mouse macrophages could take up royal jelly extracellular vesicles (not shown).

[0042] Mouse macrophages were treated with extracellular vesicles of royal jelly at concentrations of 0, 10, 20, 50, 100, and 500 μg / ml, and cell viability was measured. As shown in Figure 4A, extracellular vesicles of royal jelly from 10 μg / ml to 500 μg / ml showed no toxicity to mouse macrophages. On the other hand, mouse macrophages were treated with extracellular vesicles of royal jelly at concentrations of 0, 10, or 20 μg / ml for 1 hour, followed by treatment with 1 μg / ml LPS for 24 hours. As shown in Figure 4B, extracellular vesicles of royal jelly at concentrations of 10 μg / mL and 20 μg / mL reduced the inhibitory effect of lipopolysaccharide on cell viability, showing a significant difference compared to cells treated with LPS alone.

[0043] Since the activation of NF-κB and AP-1 can induce cells to produce secretory embryonic alkaline phosphatase (SEAP), it can be transmitted through QUANTI-Blue. TM The amount of secreted SEAP was detected using the InvivoGen reagent, which can monitor the activation of NF-κB and AP-1. To analyze the effect of royal jelly extracellular vesicles on the activation of inflammation-related transcription factors NF-κB and AP-1, mouse macrophages were seeded into 96-well plates, followed by treatment with 1 μg / ml LPS and the addition of 10 μg / ml or 20 μg / ml royal jelly extracellular vesicles. After 18 hours of reaction, the cell supernatant was collected, and QUANTI-Blue was applied. TMThe (QB) solution was added to the supernatant, mixed thoroughly, and reacted for 2 hours. The absorbance at 640 nm was measured using a spectrophotometer to determine the SEAP expression level. Royal jelly extracellular vesicles at concentrations of 10 μg / ml and 20 μg / ml reduced the activation of lipopolysaccharide-induced inflammation-related transcription factors NF-κB and AP-1, with a particularly significant difference at 20 μg / ml compared to cells treated with LPS alone (as shown in Figure 4C). Cell morphology observation under an optical microscope revealed that LPS-induced macrophages exhibited a dendritic activation state. Treatment of LPS-induced macrophages with 20 μg / ml royal jelly extracellular vesicles reduced the number of dendritic cells, indicating that royal jelly extracellular vesicles can reduce LPS-induced macrophage activation (not shown).

[0044] Example 5: Analysis of the toxicity of royal jelly extracellular vesicles to human leukemia monocytic cell lines and the expression of inflammatory factors.

[0045] [Amended according to Rule 26, 10.09.2025] The THP-1 cell line (purchased from the Taiwan Center for Biological Resources Conservation and Research) is a human leukemia monocytic cell line widely used in immunological research. THP-1 cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamic acid, 1.5 g / L sodium bicarbonate, 4.5 g / L glucose, 10 mM HEPES buffer, 1.0 mM sodium pyruvate, 10% FBS, and 100 U / mL penicillin-streptomycin. To activate the signal transduction enzyme protein kinase C and induce the monocytic cell line to differentiate into macrophages, cells were cultured for 24 hours with 100 ng / mL phorbol 12-myristate 13-acetate (PMA).

[0046] THP-1 cells were treated with extracellular vesicles of royal jelly at concentrations of 0, 10, 20, 50, 100, and 500 μg / ml, and cell viability was measured. As shown in Figure 5A, extracellular vesicles of royal jelly from 10 μg / ml to 500 μg / ml were not toxic to THP-1 cells. THP-1 cells were pretreated with extracellular vesicles of royal jelly at concentrations of 0, 10, and 20 μg / ml for 1 hour, followed by stimulation with 1 μg / ml LPS for 24 hours, and cell viability was measured. As shown in Figure 5B, LPS treatment did not decrease the viability of THP-1 cells.

[0047] LPS induces the release of inflammatory factors from THP-1 cells, including IL-1β, IL-6, IL-8, and TNF-α. To further confirm the effect of royal jelly extracellular vesicles on the release of inflammatory factors from THP-1 cells, this invention used the Luminex Discovery Assay kit (purchased from R&D Systems, USA) to study cytokines, and analyzed the data using the Luminex 200 multifunctional fluorescent biomolecule multiplex analysis system (purchased from Luminex Corporation, USA). As shown in Figures 5C-5F, treatment with royal jelly extracellular vesicles can reduce the expression of LPS-induced inflammatory factors. Western spectrophotometry was used to detect the expression of the inflammation-related transcription factor NF-κB, revealing that royal jelly extracellular vesicles can reduce LPS-induced NF-κB activation in THP-1 cells (as shown in Figure 5G). Furthermore, microscopic observation of THP-1 cells revealed that royal jelly extracellular vesicles can reduce LPS-induced activation of M1 macrophages (not shown). These results demonstrate that royal jelly extracellular vesicles have the activity of reducing the expression of induced inflammatory factors in THP-1 cells.

[0048] Example 6: Effects of royal jelly extracellular vesicles on migration rate and angiogenesis of human umbilical vein endothelial cells in a high-glucose environment

[0049] Human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell (USA) and cultured on 2 μg / cm² plates. 2 Cells were cultured in fibronectin culture dishes. The cells were maintained in the manufacturer's provided medium, with the medium changed every 1-2 days. Cells at passage numbers 3-12 were collected for subsequent experiments. Exosparkler staining confirmed that human umbilical vein endothelial cells could take up royal jelly extracellular vesicles (not shown). Human umbilical vein endothelial cells were treated with royal jelly extracellular vesicles at concentrations of 0, 10, 20, 50, 100, and 500 μg / ml, and cell viability was measured. As shown in Figure 6A, royal jelly extracellular vesicles at concentrations from 10 μg / ml to 500 μg / ml were non-toxic to human umbilical vein endothelial cells. Human umbilical vein endothelial cells were pretreated with royal jelly extracellular vesicles at concentrations of 0, 10, and 20 μg / ml, followed by a reaction with 30 mM glucose for 24 hours, and cell viability was measured. As shown in Figure 6B, high glucose treatment was also non-toxic to human umbilical vein endothelial cells.

[0050] On the other hand, cell migration assays were performed using human umbilical vein endothelial cells, and the experimental method for the cell migration assays was the same as in Example 3. As shown in Figure 6C, compared with the control group without added glucose, the high glucose environment slowed down the cell migration rate, while the cell migration rate of cells treated with 20 μg / ml royal jelly extracellular vesicles increased under the high glucose environment.

[0051] To analyze the effect of extracellular vesicles of royal jelly on angiogenesis, the basement membrane matrix was... (Corning, USA) Pre-coated into 96-well plates (50 μl / well), and incubated at 37°C for 30 minutes to promote reaction. Gelatinize and inoculate with 2×10 5 Human umbilical vein endothelial cells / pores. The high-glucose environment group (HG) was treated with 30 mM glucose for 4 hours. The group treated with royal jelly extracellular vesicles was pre-treated with 20 μg / ml royal jelly extracellular vesicles for 1 hour, followed by 30 mM glucose and 4 hours of reaction. Finally, the formation of tubular structures was photographed using an optical microscope. As shown in Figure 6D, the high-glucose environment inhibited angiogenesis in human umbilical vein endothelial cells, while treatment with 20 μg / ml royal jelly extracellular vesicles promoted angiogenesis in the high-glucose environment. In the angiogenesis assay, vascular endothelial growth factor A (VEGFA) and platelet-endothelial cell adhesion molecule CD31 are biomarkers used to identify endothelial cells. The results showed that the high-glucose environment inhibited the expression of VEGFA and CD31 in human umbilical vein endothelial cells, while royal jelly extracellular vesicles promoted the VEGFA and CD31 expression reduced by the high-glucose environment (not shown). These results indicate that royal jelly extracellular vesicles can improve angiogenesis affected by the high-glucose environment.

[0052] Example 7: Preparation of the pharmaceutical composition of the present invention

[0053] The hydrogel (95% to 99.95% by weight) is mixed with royal jelly extracellular vesicles (0.05% to 5% by weight). The mixture is then continuously stirred at 4-8°C for at least 30 minutes to ensure uniform dispersion of the royal jelly extracellular vesicles within the hydrogel matrix. After stirring, the final product is defoamed to remove entrained air bubbles, thereby obtaining a homogeneous and stable material. Those skilled in the art will understand that the steps for forming the final product of the hydrogel and royal jelly extracellular vesicle mixture may differ depending on the characteristics of the hydrogel used. The above steps are merely illustrative examples and are not intended to limit the preparation method of the pharmaceutical composition of this invention.

[0054] The pharmaceutical composition of the present invention may comprise at least one hydrogel, such as any combination of one or more of the following: polymer Pluronic F127, poly(N-isopropyl acrylamide), polyethylene glycol-polyester copolymer, alginate, chitosan, collagen, gelatin, fibrin, hyaluronic acid, and polyethylene glycol. In one embodiment, 20% Pluronic F127 is used as the hydrogel of the pharmaceutical composition of the present invention. Since Pluronic F127 is a thermosensitive hydrogel, it can rapidly solidify from a solution state to a gel state at physiological temperatures. As shown in Figure 7A, 20% PF127 is in a solution state at 4°C and in a gel state at 37°C.

[0055] Table 3 provides the viscosity and elastic modulus (i.e., storage modulus) for several different hydrogel types. The elastic modulus (kPa) was measured using a DHR-2 rheometer (TA Instruments-Waters LLC., USA), with a measurement frequency range of [missing information]. Viscosity was measured using a ROTAAVISC me-vi rotary viscometer (IKA, Germany) with a rotor SP-11 at a speed of 60 rpm. The hydrogel compositions consisted of 20% Pluronic F-127 mixed with different proportions of hyaluronic acid (molecular weight 1,200–2,200 kDa) or mixed with different proportions of sodium alginate (molecular weight 12 kDa–40 kDa). Table 3

[0056] Based on the viscosity and elasticity coefficients of the hydrogels in the table above, the suitable addition range for hyaluronic acid mixed with 20% Pluronic F-127 is approximately 0.01% to 0.1% of the total weight of the hydrogel, while the suitable addition range for sodium alginate mixed with 20% Pluronic F-127 is approximately 0.1% to 1% of the total weight of the hydrogel. Hydrogel combinations within these ranges exhibit ideal viscosity and elasticity coefficients, making them suitable as dressings for skin wounds.

[0057] Example 8: Evaluation of the effect of royal jelly extracellular vesicles on diabetic wound healing in an animal model

[0058] The disease progression and induction patterns in pigs are similar to those in humans. Landmark pigs were selected as experimental animals and injected with 40 mg / kg streptozotocin (STZ) to induce a diabetic model. Wound healing experiments were conducted once the pigs' blood glucose levels exceeded 100 mg / dL. After shaving the pigs, the wounds were disinfected with 75% alcohol and 7.5% iodine. A 4 cm wound was then made on the skin surface, and the wound depth was recorded. No materials were added to the wounds of the control group. In the RJEV group, 50 μg / ml royal jelly extracellular vesicles dispersed in phosphate buffer solution were added to the wounds. In the PF-127 / RJEV group, 50 μg / ml royal jelly extracellular vesicles were mixed with 20% PF-127 and applied to the wound surface, followed by coverage with a 3M waterproof and breathable dressing. Samples were administered repeatedly every three to four days, and the appearance of the wounds was photographed on days 0, 10, 21, and 31. The healing area of ​​each wound compared to day 0 was calculated using ImageJ image processing software. Wound healing rate (%) = [1 - (remaining wound area / original wound area)] × 100%. The experimental results showed that the addition of royal jelly extracellular vesicles and the application of PF-127 containing royal jelly extracellular vesicles significantly accelerated the wound healing rate, and the wounds remained moist with intact edges, indicating that royal jelly extracellular vesicles significantly promoted tissue granulation and re-epithelialization (as shown in Figures 7B to 7C).

[0059] On day 42 of the animal model, tissue sections and staining were performed on both the control and RJEV groups. As shown in Figure 7D, compared to intact skin, the control group showed incomplete epidermal coverage and poor collagen deposition on day 42. Consistent with the healing process, the RJEV group showed complete reepithelialization, fully developed dermal matrix, and well-arranged collagen fibers on day 42. The results from the animal model demonstrate that both the use of royal jelly extracellular vesicles alone and the combination with royal jelly extracellular vesicle hydrogel promote the healing of diabetic wounds.

[0060] By using the pharmaceutical composition of the present invention, the healing of diabetic wounds can be effectively promoted, and it is non-cytotoxic. It can be applied to the development of wound dressings and to improve wound care.

[0061] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims. Furthermore, no embodiment or claim of the present invention needs to achieve all the objectives, advantages, or features disclosed in the present invention. In addition, the abstract and headings are merely for assisting in patent document searches and are not intended to limit the scope of the invention. [Symbol Explanation]

Claims

1. A pharmaceutical composition comprising: Royal jelly extracellular vesicles (RJEVs); as well as The hydrogel is selected from at least one of the group consisting of polymer Pluronic F127, poly(N-isopropyl acrylamide), polyethylene glycol-polyester copolymer, alginate, chitosan, collagen, gelatin, fibrin, hyaluronic acid and polyethylene glycol.

2. The pharmaceutical composition according to claim 1, wherein the royal jelly extracellular vesicles have a concentration of 10 μg / ml to 500 μg / ml, the hydrogel has an elastic modulus of 1 kPa to 100 kPa, the royal jelly extracellular vesicles in the pharmaceutical composition account for 0.05% to 5% of the total weight, and the hydrogel in the pharmaceutical composition accounts for 95% to 99.95% of the total weight.

3. The pharmaceutical composition of claim 1, wherein the hydrogel comprises a temperature-sensitive hydrogel.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a wound dressing or wound gel.

5. Use of royal jelly extracellular vesicles (RJEVs) in the preparation of a pharmaceutical composition for treating wounds in an individual with diabetes, the pharmaceutical composition comprising at least one carrier, wherein the at least one carrier has the property of adhering to the skin of the individual.

6. The use according to claim 5, wherein the royal jelly extracellular vesicles have a concentration of 10 μg / ml to 500 μg / ml, the royal jelly extracellular vesicles in the pharmaceutical composition account for 0.05% to 5% of the total weight, and the at least one carrier in the pharmaceutical composition accounts for 95% to 99.95% of the total weight.

7. The use according to claim 5, wherein the pharmaceutical composition is a wound dressing or wound gel, and the individual is a human or animal.

8. The use according to claim 5, wherein the at least one carrier is a hydrogel, the hydrogel being selected from the group consisting of polymer Pluronic F127, poly(N-isopropyl acrylamide), polyethylene glycol-polyester copolymer, alginate, chitosan, collagen, gelatin, fibrin, hyaluronic acid, and polyethylene glycol.

9. The use according to claim 8, wherein the hydrogel comprises a temperature-sensitive hydrogel.

10. The use according to claim 8, wherein the hydrogel has an elastic modulus of 1 kPa to 100 kPa.

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