Nanometer vesicle hydrogel capable of stably loading aloe peel source as well as preparation method and application of nanometer vesicle hydrogel

Abstract

The invention relates to the technical field of biology, in particular to nano-vesicle hydrogel stably loaded with an aloe peel source as well as a preparation method and application of the nano-vesicle hydrogel. The hydrogel comprises nano vesicles (AVp-NVs) from aloe peel and a gel matrix, the mass ratio of the nano vesicles from aloe peel to the gel matrix is (2: 1)-(1: 4), and the percentage content of the gel matrix in the hydrogel is 0.25-1.5% (w / w). As a natural nano-vesicle structure, the nano-vesicle derived from the aloe peel has good biocompatibility and multiple functions including promotion of proliferation of human immortalized keratinocytes, promotion of cell migration, facilitation of elimination of inflammatory response in a wound healing process and the like. Compared with ionic carbomer gel, the non-ionic gel of hydroxypropyl methyl cellulose stearyl oxygen ether can better protect the structural integrity of the nano vesicles, the performance of the nano vesicles is reserved, and the effect of treating diabetic wound dysfunction is better achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a stable nanovesicle hydrogel loaded with aloe vera peel, its preparation method, and its application. More specifically, it relates to the stable nanovesicle hydrogel loaded with aloe vera peel and its application in the preparation of drugs for treating diabetic wound dysfunction. Background Technology

[0002] Diabetic wound dysfunction is a serious chronic complication of diabetes. Nearly 15% of the 150 million people with diabetes worldwide suffer from diabetic wounds, accounting for 50% of cases requiring amputation. Given its high morbidity and mortality, diabetic wounds impose a heavy burden on patients and society. Therefore, identifying the pathogenesis of diabetic wound diseases and improving treatment outcomes has become a key focus of diabetes research. Diabetic chronic wounds are characterized by a persistent inflammatory response, exhibiting a pathologically delayed healing process through altered angiogenesis, ultimately delaying epithelialization and inducing an imbalance in protease secretion. With the prolongation of the inflammatory phase during diabetic wound healing, inflammatory cells are universally activated, thus delaying the transition from the inflammatory phase to the proliferative phase. Large amounts of inflammatory mediators secreted by cells, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, are released to the wound site, subsequently inhibiting cell proliferation and migration required for diabetic wound healing. Furthermore, excessive activation of inflammation increases the expression of matrix metalloproteinase (MMP)-9, leading to rapid degradation of natural collagen, fibronectin, and elastin, thereby further delaying the diabetic wound healing process.

[0003] Although Western medical treatments, such as blood sugar control and local antibiotic therapy, have made some progress in the clinical treatment of diabetic wounds, they still have drawbacks, such as the easy development of drug resistance and rebound effects after discontinuation. Natural extracts, on the other hand, have good biocompatibility and biosafety and are now widely used in wound treatment.

[0004] Aloe vera (Curaçao) Aloe vera Aloe vera (L.) Burm.f. has been used in folk medicine as a natural plant wound healing agent for over 1000 years. It has both antioxidant and anti-inflammatory effects, and its efficacy in reducing oxidative stress and inflammatory responses and promoting wound healing is widely recognized. However, at present, when using aloe vera to treat wounds, people often apply aloe vera gel directly to the wound. But because aloe vera molecules are large and difficult to absorb directly, and because aloe vera gel contains sensitizing components such as calcium oxalate, this method of use lacks scientific basis, resulting in low bioavailability of aloe vera and making it difficult to exert its maximum efficacy.

[0005] Plant-derived nanovesicles (PDNVs), also known as plant-derived vesicle-like nanoparticles (PDVLNs) or plant-derived exosomes-like nanovesicles (PDENs), mainly refer to natural nanoscale membrane-structured vesicles derived from plant cells. They can be obtained through natural secretion or specific extraction and preparation techniques, and their particle size is typically distributed between 30 and 300 nm. They naturally encapsulate proteins, lipids, nucleic acids, and various plant active ingredients, and possess good biocompatibility, low immunogenicity, and a relatively long in vivo circulation time.

[0006] As naturally derived drug delivery carriers, PDNVs exhibit significant advantages. Their nanoscale and lipid membrane structures facilitate crossing complex biological barriers such as the blood-brain barrier; they can efficiently load small molecule drugs, nucleic acids, and active ingredients through physical, chemical, or biological methods, effectively protecting the loaded substances from degradation and improving delivery efficiency. Compared to traditional synthetic carriers such as liposomes, PDNVs offer several advantages, including abundant sources, ease of scalability for production, and low immunogenicity, making them a highly attractive alternative in regenerative medicine.

[0007] Currently, aloe vera peels are often discarded, resulting in resource waste. Furthermore, fresh aloe vera gel has low bioavailability and can cause sensitization and other side effects. Therefore, there is an urgent need to find a dermal preparation with high bioavailability and few side effects to meet clinical needs. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a hydrogel loaded with aloe vera peel-derived nanovesicles and its preparation method, so as to solve the problems of low bioavailability of aloe vera-related drugs and lack of highly effective and low-side-effect skin disease drugs in the prior art, and to provide the application of hydrogel loaded with aloe vera peel-derived nanovesicles in promoting the healing of diabetic wounds.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a hydrogel loaded with aloe vera peel-derived nanovesicles (AVp-NVs), comprising aloe vera peel-derived nanovesicles and a gel matrix. In the aloe vera peel-derived nanovesicle hydrogel, the mass ratio of the aloe vera peel-derived nanovesicles to the gel matrix is ​​2:1-1:4, and the gel matrix accounts for 0.25-1.5% of the hydrogel. w / w ).

[0010] Furthermore, the protein content of the aloe vera peel-derived nanovesicles is above 4 mg / mL.

[0011] Furthermore, the gel matrix is ​​hydroxypropyl methylcellulose stearyl ether (Sangelose) or carbomer.

[0012] Furthermore, the hydroxypropyl methylcellulose stearyl ether is Sangelose 60L (SGL 60L) or Sangelose 90L (SGL 90L).

[0013] Furthermore, the nanovesicle hydrogel loaded with aloe vera peel also contains glycerol, 1,3-butanediol, with or without triethanolamine.

[0014] The present invention preferably provides a nanovesicle hydrogel loaded with aloe vera peel, wherein the hydrogel comprises aloe vera peel-derived nanovesicles, Sangelose 60L or Sangelose 90L, glycerol, and 1,3-butanediol. By weight percentage, the percentages of each component in the hydrogel are as follows: aloe vera peel-derived nanovesicles 0.2-0.8%, Sangelose 60L or Sangelose 90L 0.25-1.5%, glycerol 1%-10%, 1,3-butanediol 1%-10%, and the balance being water.

[0015] Preferably, by weight percentage, the components constitute the following percentages of the hydrogel: aloe vera peel-derived nanovesicles 0.3-0.5%, Sangelose 60L 0.5-1%, glycerin 5-10%, 1,3-butanediol 5-10%, with the remainder being water.

[0016] The present invention preferably provides another type of nanovesicle hydrogel loaded with aloe vera peel, wherein the hydrogel comprises aloe vera peel-derived nanovesicles, carbomer, glycerin, 1,3-butanediol, and triethanolamine. By weight percentage, the percentages of each component in the hydrogel are as follows: aloe vera peel-derived nanovesicles 0.2-0.8%, carbomer 0.25-1.5%, glycerin 5-10%, 1,3-butanediol 5-10%, triethanolamine 0.3-2%, and the balance being water.

[0017] Furthermore, in the aloe vera peel-derived nanovesicle hydrogel of the present invention, the aloe vera peel-derived nanovesicles are prepared by the following method: S1. Select fresh Aloe vera and soak it in water to remove anthraquinone components such as aloin; S2. Cut off the leaf thorns, separate the aloe vera peel from the gel, and remove any remaining gel from the leaves (scrape off with an iron spoon). Add PBS to the aloe vera peel at a ratio of 3-5:1 by weight, juice the aloe vera peel using a juicer, filter through nylon gauze, and obtain an aloe vera peel homogenate. S3. Homogenize the aloe vera peel and centrifuge at low speed to obtain the supernatant. S4. The supernatant is further homogenized using a high-pressure homogenizer. S5 is filtered sequentially through 0.45 and 0.22 μm microporous membranes; S6. The precipitate was obtained by ultra-high speed centrifugation, diluted with aqueous solution, and the particle size was measured.

[0018] In step S1, the aloe vera mentioned is Aloe vera, a plant of the Liliaceae family (Aloe vera). Aloe vera L. or Aloebarbadensis Miller).

[0019] In step S3, the speed range of the low-speed centrifugation is 1,000-5,000 × g. It is preferable to remove impurities by centrifuging at 3,000 × g for 20 min to prevent the homogenate from becoming too viscous and clogging the sample injection.

[0020] In step S4, the pressure of the high-pressure homogenization is 100-120 MPa, and the number of homogenization cycles is 10-20.

[0021] In step S6, the ultra-high speed centrifugation is performed at a speed of 100,000 × g for 30 min.

[0022] The aloe vera peel-derived nanovesicles prepared according to the above preparation method have a particle size of 50-200 nm, preferably 100-200 nm, and a zeta potential of -7.13 mV, which meets the requirements of plant extracellular vesicles with a particle size range of 50-200 nm and a slight negative charge.

[0023] The aloe vera peel-derived nanovesicles were quantified using a protein quantification method, and their protein content was ≥4 mg / mL.

[0024] The aloe vera peel-derived nanovesicles, as observed under a transmission electron microscope, exhibit a distinct elliptical or disc-shaped phospholipid bilayer morphology.

[0025] This invention provides the application of aloe vera peel-derived nanovesicles in the preparation of drugs for diabetic wound dysfunction.

[0026] Furthermore, the aforementioned diabetic wound dysfunction refers to wounds or foot ulcers caused by diabetes.

[0027] The aloe vera peel-derived nanovesicles described above can promote the healing of diabetic wounds or foot ulcers.

[0028] Verification has shown that the aloe vera peel-derived nanovesicles obtained by this invention can promote the rapid healing of diabetic wounds by promoting the proliferation and migration of human immortalized keratinocytes, regulating macrophage polarization, and inhibiting the production of inflammatory factors.

[0029] This invention also provides a method for preparing the aforementioned aloe vera peel-derived nanovesicle hydrogel, comprising the following steps: SGL 60L was weighed and dispersed evenly in an aqueous solution of aloe vera peel-derived nanovesicles at 40°C under magnetic stirring. Then, glycerol and 1,3-butanediol were added and mixed evenly. The mixture was cooled to room temperature to obtain Sangelose hydrogel loaded with aloe vera peel-derived nanovesicles.

[0030] Alternatively, under magnetic stirring, add carbomer 940 powder, glycerin, and 1,3-butanediol to an aqueous solution of aloe vera peel-derived nanovesicles, stirring continuously until the carbomer is completely dispersed (the solution is cloudy or translucent). Slowly add a neutralizing agent (triethanolamine) while stirring. Monitor the pH with a pH meter and adjust to 5.5-7.0. As neutralization proceeds, the solution gradually thickens, eventually forming a transparent or translucent carbomer gel loaded with aloe vera peel-derived nanovesicles.

[0031] This invention provides the application of loaded aloe vera peel-derived nanovesicle hydrogels in the preparation of drugs for treating diabetic wound dysfunction.

[0032] Furthermore, the aforementioned diabetic wound dysfunction refers to wounds or foot ulcers caused by diabetes.

[0033] The aforementioned aloe vera peel-derived nanovesicle hydrogel can promote the healing of diabetic wounds or foot ulcers.

[0034] This invention provides the application of nonionic hydrogels in improving the stability of plant nanovesicles.

[0035] Furthermore, The nonionic hydrogel is a hydrogel with nonionic hydroxypropyl methylcellulose stearyl ether as the matrix.

[0036] The plant nanovesicles mentioned are nanovesicles derived from aloe vera peel.

[0037] Hydrogels using nonionic hydroxypropyl methylcellulose stearyl ether as a matrix can better protect nanovesicle-like structures, thus better preserving the properties of aloe vera peel-derived nanovesicles. Their effect is significantly superior to that of ionic carbomer hydrogels.

[0038] The aloe vera peel-loaded nanovesicle hydrogel of this invention not only possesses the advantages of high stability and biocompatibility, but also exhibits a certain degree of sustained release. Furthermore, experimental results show that, compared to carbomer hydrogels, the aloe vera peel-loaded Sangelose hydrogel demonstrates the best performance in terms of porous structure, release properties, and stability.

[0039] Furthermore, the administration methods for aloe vera peel-derived nanovesicles include direct subcutaneous injection and application of aloe vera peel-derived nanovesicle hydrogel. Among these, the application of aloe vera peel-derived nanovesicle hydrogel resulted in the best wound closure rate and wound healing effect in diabetic patients.

[0040] The beneficial effects of the technical solution provided by this invention include: This invention provides a method for preparing a stable hydrogel containing aloe vera peel-derived nanovesicles and its application. Aloe vera peel-derived nanovesicles meeting the standards for plant extracellular vesicles were obtained by combining a high-pressure homogenizer with a microporous membrane, and then loaded onto a hydrogel. Aloe vera peel-derived nanovesicles, as a natural nanovesicle structure, exhibit good biocompatibility and multiple functions, including promoting the proliferation of immortalized keratinocytes, promoting cell migration, and helping to eliminate inflammatory responses during wound healing.

[0041] This invention solves the problems of low bioavailability of aloe vera, insufficient research on the preparation method of aloe vera extracellular vesicles, and the inability to directly apply fresh aloe vera gel to wounds in the prior art. It provides an effective material for drugs for skin-related diseases. The nanovesicle hydrogel loaded with aloe vera peel is highly effective in treating skin diseases, and also provides an effective material for the treatment of diabetic wounds. This invention is beneficial to the further development of research on the function of aloe vera peel-derived nanovesicles.

[0042] Furthermore, we have discovered for the first time that combining aloe vera peel-derived nanovesicles with hydrogel can significantly enhance the healing effect on diabetic wounds. Sangelose hydrogel has a more effective sustained-release platform than carbomer hydrogel, which helps prolong the duration of action of extracellular vesicles at the target site. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0044] Figure 1 This is a particle size distribution diagram of nanovesicles derived from aloe vera peel.

[0045] Figure 2 Zeta potential diagram of nanovesicles derived from aloe vera peel.

[0046] Figure 3 Transmission electron microscopy image of nanovesicles derived from aloe vera peel.

[0047] Figure 4 Bar graph showing the effect of different concentrations of aloe vera peel-derived nanovesicles on HaCaT cell proliferation; .

[0048] Figure 5 Flow cytometry plots showing the effect of different concentrations of aloe vera peel-derived nanovesicles on HaCaT cell apoptosis.

[0049] Figure 6 This describes the absorption and uptake of nanovesicles derived from aloe vera peel by HaCaT.

[0050] Figure 7 To demonstrate the effect of different concentrations of aloe vera peel-derived nanovesicles on the migration ability of HaCaT through scratch experiments; .

[0051] Figure 8 Transwell experiments were conducted to demonstrate the effect of different concentrations of aloe vera peel-derived nanovesicles on the migration ability of HaCaT. .

[0052] Figure 9 Bar graph showing the effect of different concentrations of aloe vera peel-derived nanovesicles on the cell viability of RAW264.7.

[0053] Figure 10 The effect of different concentrations of aloe vera peel-derived nanovesicles on the inhibition of LPS-induced NO production from RAW264.7; .

[0054] Figure 11 The effect of different concentrations of aloe vera peel-derived nanovesicles on the inhibition of LPS-induced production of inflammatory factors from RAW264.7; .

[0055] Figure 12 The effect of different concentrations of aloe vera peel-derived nanovesicles on LPS-induced RAW264.7 inflammatory marker mRNA levels; .

[0056] Figure 13 The effect of different concentrations of aloe vera peel-derived nanovesicles on LPS-induced levels of inflammatory marker proteins in RAW264.7; .

[0057] Figure 14 Macroscopic morphology diagrams of hydrogels: (A) SGL60L hydrogel without aloe vera peel nanovesicles; (B) SGL60L hydrogel with aloe vera peel nanovesicles; (C) Carbomer 940 hydrogel without aloe vera peel nanovesicles; (D) Carbomer 940 hydrogel with aloe vera peel nanovesicles.

[0058] Figure 15 The diagram shows the rheological behavior of the hydrogel.

[0059] Figure 16 This is a diagram of the microstructure of the hydrogel. A: SGL60L hydrogel loaded with aloe vera peel-derived nanovesicles under a 50x microscope; B: SGL60L hydrogel loaded with aloe vera peel-derived nanovesicles under a 200x microscope; C: Carbomer 940 hydrogel loaded with aloe vera peel-derived nanovesicles under a 50x microscope; D: Carbomer 940 hydrogel loaded with aloe vera peel-derived nanovesicles under a 200x microscope.

[0060] Figure 17 Release curves of SGL 60L hydrogel and carbomer 940 hydrogel loaded with aloe vera peel-derived nanovesicles.

[0061] Figure 18 Wound healing effect of SGL 60L hydrogel loaded with aloe vera peel-derived nanovesicles in diabetic mice. A: Representative photographs of wound closure at 0, 5, 10 and 15 days; B: Wound closure rate statistics chart; C: Schematic diagram of wound contraction; .

[0062] Figure 19 H&E staining images of wound sections from diabetic mice in each group on day 15.

[0063] Figure 20 Masson staining representations and statistical graphs of wound sections from diabetic mice in each group on day 15; .

[0064] Figure 21 Representative figures and statistical graphs of TNF-α expression in wound sections of diabetic mice in each group on day 15; .

[0065] Figure 22 Representative figures and statistical graphs of IL-1β expression in wound sections of diabetic mice in each group on day 15; . Detailed Implementation

[0066] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

[0067] Aloe vera peel-derived nanovesicles have great potential for application in the field of pharmaceutical carriers. Their small molecular structure and good biocompatibility make them an excellent drug carrier.

[0068] This invention provides a method for preparing hydrogels loaded with aloe vera peel-derived nanovesicles, addressing the issues of low bioavailability and lack of highly effective and low-side-effect skin disease medications in related technologies. This invention utilizes a combination of high-pressure homogenization and microporous membrane filtration to obtain aloe vera peel-derived nanovesicles that meet the standards for plant extracellular vesicles; it also verifies the effects of the extracted aloe vera peel-derived nanovesicles in promoting cell proliferation and migration and reducing the production of inflammatory factors, and provides an application of hydrogels loaded with aloe vera peel-derived nanovesicles in promoting wound healing in diabetic patients.

[0069] The technical solution of this patent will be further described in detail below with reference to specific embodiments.

[0070] Example 1: Extraction of nanovesicles from aloe vera peel The preparation method of aloe vera peel-derived nanovesicles includes the following steps: (1) Take fresh aloe vera. In this embodiment, Aloe vera is selected. Clean the whole aloe vera leaf and soak it in water for 3-5 days, changing the water every day to remove most of the anthraquinone components such as aloin. (2) Cut off the thorns, separate the aloe vera peel from the gel, and remove any remaining gel from the peel (scrape off with an iron spoon). Add the aloe vera peel to PBS at a mass ratio of 3:1 and juice it using a juicer. Filter the juice through nylon gauze to obtain an aloe vera peel homogenate. (3) Centrifuge the aloe vera peel homogenate at 3,000×g for 20min to remove impurities, in order to prevent the homogenate from being too viscous and clogging the sample injection. (4) The supernatant is further homogenized by a high-pressure homogenizer: the high-pressure homogenization pressure is 100MPa and the number of homogenization cycles is 10. (5) The homogenized liquid was filtered sequentially through 0.45 and 0.22 μm microporous membranes.

[0071] Figure 1 The particle size of extracellular vesicles was measured using a Malvern particle size analyzer. The average particle size of nanovesicles derived from aloe vera peel was 152.9 nm, which meets the requirements for small particle size of plant extracellular vesicles (50 nm to 250 nm).

[0072] Figure 2 The zeta potential of aloe vera peel-derived nanovesicles is -7.13 mV. Slightly negatively charged nanoparticles (NPs) are beneficial for in vivo drug delivery to reduce protein adsorption.

[0073] Figure 3 Nanovesicles derived from aloe vera peel can be observed under a transmission electron microscope to have a distinct elliptical or disc-shaped phospholipid bilayer morphology.

[0074] The protein content was 4.56 mg / mL, determined using the protein quantification method.

[0075] Example 2: Effect of aloe vera peel-derived nanovesicles on HaCaT cell proliferation rate Aloe vera peel-derived nanovesicles exhibit good biocompatibility, targeting properties, and the ability to promote cell proliferation and migration. Therefore, aloe vera peel-derived nanovesicles can be applied to the treatment of skin wound healing.

[0076] (a) Preparation of relevant solutions DMEM complete medium: Add 50 mL of premium fetal bovine serum and 5 mL of penicillin-streptomycin solution to 500 mL of DMEM medium, mix well, and store at 4°C for later use.

[0077] Aloe vera peel-derived nanovesicle stock solution: The aloe vera peel-derived nanovesicle solution extracted in Example 1 and filtered through a 0.22 μm microporous membrane was stored at 4°C for later use.

[0078] (II) Cell Culture Human immortalized keratinocytes HaCaT cells (Wuhan Shangen Biotechnology Co., Ltd.) were cultured in DMEM complete medium.

[0079] Remove the cryopreserved HaCaT cells from the liquid nitrogen container and quickly place them in a 37ºC water bath to thaw within one minute. Sterilize the cryovials with 75% alcohol and open them in a clean bench. Aspirate the cryopreservation solution containing the cell suspension into a centrifuge tube, add 10 mL of the corresponding complete culture medium, and gently pipette repeatedly until the cells are evenly dispersed. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add an appropriate amount of DMEM complete culture medium, and resuspend the cells by pipetting. Transfer the cells and culture medium to a culture dish and incubate in a CO2 incubator. When the cells reach approximately 80% confluence, they are passaged. This involves aspirating the previous culture medium in a clean bench, washing with 3 mL of PBS, adding 3 mL of trypsin to digest for 2 min, and then detaching the cells from the culture dish. After stopping the digestion by adding 3 mL of fresh culture medium, the cells are centrifuged at 1000 rpm for 5 min. The supernatant is discarded to remove the trypsin. An appropriate amount of culture medium is added and the cells are resuspended. The appropriate cell suspension is then transferred to a culture dish according to the passage ratio.

[0080] (III) Cell proliferation rate determination Cells were detached from the culture dish using trypsin in a clean bench. An equal volume of DMEM complete medium was added to terminate the digestion. The cells were centrifuged, and the supernatant containing trypsin was discarded. The cells were resuspended in an appropriate amount of culture medium, counted, and diluted with fresh DMEM complete medium to a density of 2 × 10⁻⁶. 4 cells / ml. Transfer 100 μL of cell suspension to a 96-well plate, then incubate the plate at 37ºC CO2 for 4 h to allow cell adhesion. Discard the original DMEM complete medium in a cleanroom. Dilute the aloe vera peel-derived nanovesicle stock solution with DMEM complete medium to concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, and 1000 μg / mL AVp-NVs. Add 200 μL to each well, with four parallel wells for each concentration. After incubating the 96-well plate for 24 and 48 hours, remove it from the incubator, add 10 μL of CCK-8 solution to each well, and incubate for another 2 hours. Then, measure the absorbance of each well at 450 nm using a multi-mode microplate reader. Results are shown below. Figure 4 .

[0081]

[0082] The results showed that, compared with the control group, cell viability was greater than 90% at AVp-NVs concentrations of 50-1000 μg / mL after 24 h or 48 h of drug administration, indicating that aloe vera peel-derived nanovesicles had no cytotoxicity to HaCaT cells. After 24 h of drug administration, when the AVp-NVs concentration increased to 700 μg / mL, HaCaT cell proliferation significantly increased (mean ± standard deviation, n=3; compared with the control group, ...). , The proliferation of HaCaT cells showed a dose-dependent effect with increasing drug concentration, but because the proliferation of HaCaT cells did not significantly increase when the concentration of AVp-NVs was >700 μg / mL, subsequent cell experiments selected 700 μg / mL as the maximum drug dose. 48 h after drug administration, HaCaT cell viability began to increase and growth was significantly promoted at a AVp-NVs concentration of 700 μg / mL. ).

[0083] Example 3: Effect of aloe vera peel-derived nanovesicles on the apoptosis rate of HaCaT cells Immortalized HaCaT cells from humans in the logarithmic growth phase were collected, washed twice with PBS, and then digested with 1 mL of EDTA-free trypsin solution to prepare a cell suspension. The suspension was then prepared at a cell density of 8 × 10⁻⁶ cells / mL. 5 Cells were seeded per well in 6-well plates, and 2 mL of cell culture medium was added for overnight incubation. Cell samples were divided into a control group and an experimental group. After cell attachment, aloe vera peel-derived nanovesicles at concentrations of 50 μg / mL (low dose), 500 μg / mL (medium dose), and 700 μg / mL (high dose) were added for 24 h. Flow cytometry was used to analyze the effect of aloe vera peel-derived nanovesicles in the low, medium, and high dose groups on the apoptosis rate of human immortalized keratinocytes (HaCaT cells). After 24 h, cell culture medium was collected from each well of the 6-well plates, and each well was washed twice with 1 mL of culture medium. The washings were collected together in 4 mL centrifuge tubes. Add 400 μL of EDTA-free trypsin solution to each well and digest for 2 min until cells can be pipetted off. Remove the trypsin solution, add 1 mL of culture medium to each well, and pipet the cells into centrifuge tubes. Centrifuge at 800 × g for 5 min, wash twice, discard the supernatant, and retain 200 μL of cell suspension. Gently pipet the remaining cells evenly into 1 mL centrifuge tubes. Count the cells and adjust the cell density to 5 × 10⁻⁶. 5Cells were centrifuged at 800 rpm for 5 min with 500 μL PBS solution, washed once, and the supernatant was discarded, leaving approximately 20 μL of PBS. The cells were gently mixed by pipetting, and 195 μL of FITC-PI binding buffer was added to gently resuspend the cells. The cells were incubated at room temperature in the dark for 20 min. 5 μL of Annexin V-FITC was added, and the cells were gently mixed and incubated at room temperature in the dark for 20 min. 190 μL of FITC-PI binding buffer was added again, and the cells were gently resuspended and mixed. The cells were incubated at room temperature in the dark for 20 min. 10 μL of PI binding buffer was added, and the cells were incubated at room temperature in the dark for 20 min. Flow cytometry was used to analyze the effect of aloe vera peel-derived nanovesicles on the apoptosis rate of human immortalized keratinocytes (HaCaT cells). Results are shown below. Figure 5 .

[0084] The results showed that, compared with the blank control group, the apoptosis rate of human immortalized keratinocyte HaCaT cells was not significantly different after treatment with aloe vera peel-derived nanovesicles at concentrations of 50 μg / mL, 500 μg / mL, and 700 μg / mL for 24 hours. This indicates that aloe vera peel-derived nanovesicles can significantly promote apoptosis of HaCaT cells within the concentration range of 50-700 μg / mL.

[0085] Example 4: Determination of HaCaT cell uptake of aloe vera peel-derived nanovesicles Add 50 μL of PKH-26 fluorescent dye working solution to an AVp-NVs solution containing 10–200 μg of aloe vera peel-derived nanovesicle protein; vortex for 1 min; incubate for 10 min; add 7 mL of 1×PBS to the incubated extracellular vesicle-dye complex and mix; centrifuge at 100,000×g for 60 min using an ultracentrifuge to remove free PKH-26 dye from the PBS; discard the supernatant, resuspend in 200 μL of PBS, and filter through a 0.22 μm filter to obtain PKH-26-labeled aloe vera peel-derived nanovesicles. Quantify the protein concentration of PKH-26-labeled AVp-NVs using a BCA protein assay kit.

[0086] Place a climbing slide in a 24-well plate and seed HaCaT cells evenly at a density of 1.5 × 10⁻⁶. 5After incubating for 24 hours, the original culture medium was discarded and replaced with medium containing PKH-26-labeled AVp-NVs. Treatment was repeated for 0 hours, 0.5 hours, 4 hours, and 8 hours. After treatment, the culture medium was aspirated, and the samples were gently rinsed three times with PBS. 1 mL of 4% paraformaldehyde was added to each well for fixation for 20 minutes, the paraformaldehyde was aspirated, and the samples were washed three times with PBS. 300 mL of DAPI working solution was added to each well, and the samples were stained in the dark for 15 minutes. The stain was then removed, and the samples were washed three times with PBS. The slides were picked up with a needle and allowed to air dry. A drop of anti-quenching agent was placed on the top of a slide, and the slide was inverted to the center of the anti-quenching agent. Nail polish was applied around the edges for fixation. The slides were observed and photographed using a laser confocal microscope and stored at 4°C in the dark. Results are shown below. Figure 6 .

[0087] The results showed that aloe vera peel-derived nanovesicles could be absorbed by HaCaT cells, and after 8 hours of treatment with aloe vera peel-derived nanovesicles, a large number of PKH-26-labeled aloe vera peel-derived nanovesicles appeared in HaCaT cells.

[0088] Example 5: Scratch assay to determine cell migration rate The effects of low-dose (50 μg / mL) and high-dose (700 μg / mL) AVp-NVs on the migration ability of HaCaT cells were evaluated using a scratch assay. HaCaT cells in logarithmic growth phase were digested with trypsin and then cultured at a cell density of 2 × 10⁶ cells / mL. 5 HaCaT cells were seeded per well in 6-well plates. After confluence, the confluent HaCaT cell monolayers in the 6-well plates were scratched using a 200 μL pipette tip, and floating cells were washed with PBS. Serum-free EMDM medium (without drugs) and serum-free EMDM medium containing 50 μg / mL and 700 μg / mL AVp-NVs were added, respectively. Wound healing progress was observed at 0, 6, 12, and 24 hours using a Nikon inverted microscope. Six fields of view were randomly selected from each well, and the cell migration area was statistically analyzed using ImageJ. Results are shown below. Figure 7 .

[0089] .

[0090] The results showed that, 24 hours after administration, the migration-promoting ability of high-dose (700 μg / mL) aloe vera peel-derived nanovesicles was significantly enhanced compared with the control group.

[0091] Example 6: Transwell assay to determine cell migration rate The effects of low (50 μg / mL), medium (500 μg / mL), and high (700 μg / mL) AVp-NVs on HaCaT migration were assessed using a Transwell assay. Cells were collected, centrifuged at 800 rpm for 5 min, the supernatant was aspirated, and the cells were resuspended in FBS-free DMEM high-glucose medium and diluted to 5 × 10⁻⁶. 4 Cells / mL; 500 μL of complete culture medium was added to each well of a 24-well plate, and 100 μL of cell suspension was added to each chamber to ensure the bottom of the chamber was fully submerged in the complete culture medium. The plates were incubated for 24 h. The 24-well plate was removed, the liquid inside the chambers was aspirated, and any remaining liquid was gently wiped clean with a cotton swab. The chambers were then transferred to wells containing 500 μL of paraformaldehyde and fixed at room temperature for 10 min, followed by washing the chambers three times with PBS. The chambers were then transferred to wells containing 500 μL of 0.1% crystal violet dye and stained at room temperature for 15 min, followed by washing the chambers three times with PBS and air-drying. The chambers were then placed in a clean 24-well plate, and the lower chamber was observed and photographed using an inverted microscope. Data analysis was performed using ImageJ and GraphPad software. Results are shown below. Figure 8 .

[0092] Experimental results showed that high-dose (700 μg / mL) aloe vera peel-derived nanovesicles had the strongest ability to promote HaCaT cell migration.

[0093] Example 7: The anti-inflammatory effect of aloe vera peel-derived nanovesicles (a) Cytotoxicity assay Mouse mononuclear macrophage cell line RAW264.7 cells were cultured in complete DMEM medium. Detailed procedures were followed as described in Example 2 regarding cell proliferation assays. AVp-NVs concentrations were 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, and 700 μg / mL.

[0094] (ii) NO reagent kit for detecting NO levels RAW 264.7 cells (2 × 10⁻⁶) 5 Cells were cultured in 6-well plates for 24 hours. After removing the culture medium, various concentrations of AVp-NVs (0, 50, 100, 500, and 700 μg / mL) were mixed in preheated medium for 12 hours, followed by LPS (1 μg / mL) treatment in each well. Cells were then incubated for another 12 hours. The cell culture supernatant was collected, and nitric oxide levels were determined using a NO detection kit.

[0095] (III) ELISA kit for detecting inflammatory factor levels Follow the ELISA kit instructions. Set up standard wells and sample wells, adding 25 μL of standard diluent of different concentrations to each standard well. Set up blank control wells and sample wells. Add 25 μL of the sample to be tested to the 96-well plate (dilute 5-10 times with sample diluent depending on the sample concentration). Add 150 μL of enzyme-labeled working solution and gently shake to mix. Seal the 96-well plate with sealing film and incubate at 37°C for 2 hours. After incubation, discard the liquid in the plate, add the chromogenic reagent, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well to stop the reaction. Place the 96-well plate in a spectrophotometer and shake at medium speed for 30 seconds. Zero the instrument with the blank well and measure the absorbance of each well sequentially at different wavelengths. Calculate the levels of inflammatory factors (TNF-α, IL-1β, IL-6) in the cell supernatant based on the absorbance.

[0096] (III) Detection of mRNA levels of inflammatory factors by real-time quantitative PCR Total RNA was extracted from RAW264.7 cells using the TRIzol method and its concentration was determined. Following the reverse transcription kit instructions, the RNA was first treated to remove gDNA and then reverse transcribed to synthesize cDNA. The primers (SEQ ID No. 1-12) are designed as shown in the table below:

[0097] The SYBR Green kit was equilibrated to room temperature beforehand. cDNA and primers were appropriately diluted, and each component was added to a qPCR tube, with a total reaction volume of 20 μL. After adding the reaction solution to an 8-tube strip, the tubes were tightly capped and mixed thoroughly before RT-qPCR detection. The RT-qPCR reaction procedure is as follows:

[0098]

[0099] Using Gapdh as an internal reference, The difference between the average Ct values ​​of the target gene and the internal reference gene. Drug group and control group The difference. The values ​​represent the relative expression levels of mRNA, and statistical analysis was performed. The results are as follows: Figure 9-13 As shown.

[0100] Figure 9 The bar chart shows the effect of different concentrations of aloe vera peel-derived nanovesicles on the cell viability of RAW264.7. The results show that, compared with the control group, at 24h and 48h after drug administration, when the concentration of AVp-NVs was 50-700 μg / mL, there was no significant effect on cell growth and the cell viability was greater than 90%, indicating that the concentration of AVp-NVs at 50-700 μg / mL was not cytotoxic to RAW264.7.

[0101] Figure 10 The study investigated the inhibitory effects of different concentrations of aloe vera peel-derived nanovesicles on NO production in RAW264.7. Results showed that LPS significantly increased NO production compared to the untreated LPS group (control group). AVp-NVs at concentrations of 50-700 μg / mL significantly inhibited LPS-induced NO. Furthermore, higher doses of AVp-NVs (500 μg / mL and 700 μg / mL) significantly enhanced the ability to inhibit NO production compared to lower doses (50 μg / mL and 100 μg / mL).

[0102] Figure 11 The study investigated the inhibitory effects of different concentrations of aloe vera peel-derived nanovesicles on LPS-induced production of inflammatory factors in RAW264.7 cells. The results showed that LPS induced an increase in the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in RAW264.7 cells, while aloe vera peel-derived nanovesicles could reduce the levels of inflammatory markers.

[0103] Figure 12 The effects of different concentrations of aloe vera peel-derived nanovesicles on LPS-induced mRNA levels of inflammatory markers in RAW264.7 were investigated. The results showed that compared with the control group, the mRNA levels of inflammatory markers in the LPS-induced group were significantly increased; while compared with the LPS-induced group, all AVp-NVs groups significantly reduced the mRNA levels of inflammatory markers.

[0104] Figure 13 The study investigated the effects of different concentrations of aloe vera peel-derived nanovesicles on LPS-induced protein levels of inflammatory markers in RAW264.7. The results showed that LPS induced an increase in the protein levels of COX-2, nitric oxide synthase, and NF-κB, while aloe vera peel-derived nanovesicles significantly reduced the protein levels of these inflammatory markers.

[0105] Example 8: Preparation of SGL 60L hydrogel (SGL 60L-NV) and carbomer 940 hydrogel (carbomer 940-NV hydrogel) loaded with aloe vera peel-derived nanovesicles (1) Preparation of Sangelose hydrogel loaded with aloe vera peel-derived nanovesicles: Weigh 60L of SGL and disperse it evenly in an AVp-NVs aqueous solution at 40℃ under magnetic stirring. Then add glycerol and 1,3-butanediol and mix evenly. Cool to room temperature and add water to make up to 100g to obtain Sangelose hydrogel loaded with aloe vera peel-derived nanovesicles. Its formula is shown in Table 4.

[0106] The protein concentration of AVp-NVs was 4 mg / mL.

[0107]

[0108] (2) Preparation of carbomer 940 hydrogel loaded with aloe vera peel nanovesicles Carbomer 940 powder, glycerol, and 1,3-butanediol were added to an AVp-NVs aqueous solution under magnetic stirring, and the mixture was stirred continuously until the carbomer was completely dispersed (the solution was turbid or translucent). A neutralizing agent (triethanolamine) was slowly added dropwise while stirring. The pH was adjusted to 5.5-7.0 using a pH meter. As neutralization proceeded, the solution gradually thickened. Water was added to bring the total volume to 100g, ultimately forming a transparent or translucent carbomer gel loaded with aloe vera peel-derived nanovesicles. The formulation is shown in Table 5.

[0109] The protein concentration of AVp-NVs was 4 mg / mL.

[0110]

[0111] Example 9: Physicochemical properties of Sangelose hydrogel and Carbomer 940 hydrogel loaded with aloe vera peel-derived nanovesicles (1) Macroscopic morphological characterization of hydrogels A certain amount of each of the following four samples were weighed and placed into sample vials: SGL 60L hydrogel without aloe vera peel nanovesicles, Sangelose hydrogel and Carbomer 940 hydrogel loaded with aloe vera peel nanovesicles (Example 8), and Carbomer 940 gel without aloe vera peel nanovesicles. The sample vials were then placed at an angle to observe the fluidity of the hydrogels and whether there was any drug precipitation or stratification. The results are shown below. Figure 14 .

[0112] The results showed that, in the group without aloe vera peel-derived nanovesicles, the SGL 60L hydrogel (Figure A) exhibited transparency, slight fluidity at a 45° tilt, and no obvious adhesion to the bottle wall. In contrast, the unloaded carbomer 940 hydrogel (Figure C) showed significantly increased viscosity under the same observation conditions, and the hydrogel as a whole did not shift when tilted.

[0113] The SGL 60L hydrogel loaded with aloe vera peel-derived nanovesicles (Figure B) changed color from transparent to light yellow. Its fluidity remained unchanged when tilted compared to the unloaded SGL 60L hydrogel (Figure A), and no particle deposition was observed at the bottom of the bottle. This indicates that the aloe vera peel-derived nanovesicles formed a stable complex with the SGL 60L hydrogel and did not induce separation. The carbomer 940 hydrogel loaded with aloe vera peel-derived nanovesicles (Figure D) also changed color from transparent to light yellow, and its fluidity when tilted was consistent with the unloaded carbomer 940 hydrogel (Figure C).

[0114] It is worth noting that both groups of hydrogel formulations loaded with aloe vera peel nanovesicles maintained physical stability after 72 hours of standing, and no stratification or flocculation was observed, confirming that the two hydrogel systems have good colloidal stability.

[0115] (2) Characterization of hydrogel rheological behavior Storage modulus (G') and loss modulus (G'') were measured using an MCR-101 rheometer. Dynamic oscillation time-scan and dynamic frequency-scan tests were performed using the rheometer, both under a fixed strain of 1% in the linear viscoelastic region (LVE). The four hydrogel samples were loaded into cylindrical parallel plate fixtures with a diameter of 40 mm and a spacing of 1000 μm. Tests were conducted at the set ambient temperature. All rheological data were collected at a fixed strain rate of 1%. Measurements were performed at 25℃ and within a scan range of 0.1–10 Hz to characterize the frequency response of the four hydrogels. Finally, the rheological properties of the hydrogel samples were plotted based on the measured data. The results are as follows: Figure 15 As shown.

[0116] Based on rheological analysis, the viscoelastic characteristics of four hydrogels were systematically evaluated. As shown in the figure, in the frequency range of 0.1-10 Hz, the storage modulus (G') of all hydrogel systems was significantly higher than the loss modulus (G''), with a difference of more than 10 times (G' / G''>10), confirming that the hydrogels possess typical elastomer properties and their three-dimensional network structure can effectively resist shear deformation. After introducing aloe vera peel-derived nanovesicles (AVp-NVs), the G' value in the hydrogel was still higher than the G'' value, indicating that the addition of NVs had almost no impact on the rheological behavior of the hydrogels. The G' value of the Carbomer 940-NV group decreased by 56.2% compared to the unloaded group (2122 Pa→929 Pa), while the G' value of the Sangelose 60L-NV group decreased by 28.6% compared to the unloaded group (479 Pa→342 Pa), indicating that the embedding of NVs structurally weakened the integrity of the gel network. This phenomenon may be related to the relatively loose network structure of SGL 60L itself. The low modulus and balanced viscoelasticity of SGL 60L hydrogel make it more suitable for flexible applications (such as skin patches and mucosal drug delivery), and its softness can improve user comfort.

[0117] (3) Characterization of hydrogel microstructure SGL 60L hydrogels and Carbomer 940 hydrogels loaded with aloe vera peel nanovesicles were poured into 60 mm diameter petri dishes. The hydrogel samples were rapidly frozen with liquid nitrogen and then placed at -80°C for 24 h. The frozen hydrogel samples were then freeze-dried in a cryogenic vacuum chamber for 6–8 hours. The dried hydrogel samples were then sputter-coated with gold under vacuum for 20 min. High-resolution microstructure images of the cross-sections of the hydrogel samples were obtained using scanning electron microscopy at 10 kV. The surface pore size and vesicle stability of the two types of hydrogels loaded with aloe vera peel nanovesicles were analyzed using SEM images to evaluate the pore structure of different hydrogel matrices and the stability of the aloe vera peel-loaded nanovesicles. Results are shown below. Figure 16 .

[0118] Based on scanning electron microscopy (SEM) characterization results, this study revealed the microstructural differences between two hydrogel systems loaded with aloe vera peel-derived nanovesicles. As shown in the figure, under low magnification (×50), the SGL 60L-NV hydrogel (Figure A) and the carbomer 940-NV hydrogel (Figure C) exhibit typical three-dimensional interconnected porous structures. The high specific surface area and high porosity not only create superior conditions for deep cell growth and transmembrane transport of substances, but also significantly improve the penetration efficiency of drugs in wounds, while accelerating the drainage of exudate.

[0119] Under high magnification (×200), spherical nanoparticles (180-220 nm in diameter) were clearly visible on the pore wall surface of the SGL 60L-NV hydrogel (red arrow in Figure B), and their morphological integrity was completely consistent with the NV characteristics characterized by previous transmission electron microscopy. However, no complete NV structure was observed on the pore wall surface of the Carbomer 940-NV hydrogel (Figure D) under high magnification. This lack of vesicles may be related to the fact that Carbomer, as an ionic gel, disrupts vesicle stability. The analysis of these microstructural features provides key morphological evidence for comparing the functional differences between the two types of hydrogel-supported vesicle systems.

[0120] Example 10: Determination of the release rate of SGL 60L hydrogel loaded with aloe vera peel-derived nanovesicles SGL 60L hydrogels loaded with aloe vera peel-derived nanovesicles were immersed in PBS and placed in a shaker at 37°C and 100 rpm. The release medium was periodically extracted and fresh buffer solution was added. The absorbance of the release medium at 595 nm was measured using the BCA protein quantification method. The content of released aloe vera peel-derived nanovesicles was determined using a predetermined calibration curve. The cumulative release per unit E was calculated using the following formula. r .

[0121]

[0122] Er: Cumulative drug release; Ve: Replacement volume of PBS; V0: Total volume of release medium; C: Concentration of release solution at the time of the first replacement sampling; m NV : Total mass of drug carried by nanoparticles; i, n: Number of times PBS was replaced. The experiment was repeated three times, and the average value of the results was taken.

[0123] The cumulative drug release at each sampling point was calculated using the above formula. Nonlinear fitting was performed using plotting software; the vertical axis represents the standardized cumulative release percentage, and the horizontal axis represents the time node. The release rate curve is shown below. Figure 17 .

[0124] Based on the release performance results, this study reveals that the SGL 60L hydrogel loaded with aloe vera peel-derived nanovesicles has an effective sustained release capability.

[0125] like Figure 17 As shown, AVp-NVs were successfully released within 72 h, and in its SGL 60L hydrogel, AVp-NVs achieved a release rate of 74% at 48 h, and continued to be released at 60 h and 72 h.

[0126] Within 72 h, AVp-NVs in its carbomer 940 hydrogel achieved a 60% release rate at 48 h and continued to be released at 60 h and 72 h.

[0127] The results showed that both the SGL 60L hydrogel and the carbomer 940 hydrogel loaded with vesicles possessed effective sustained-release platforms, which helped prolong the duration of action of the vesicles at the target site. However, the vesicles exhibited poor stability in carbomer and their release was less effective than that of the SGL 60L hydrogel.

[0128] Therefore, the following wound healing experiment was conducted in diabetic mice using SGL 60L hydrogel loaded with aloe vera peel-derived nanovesicles.

[0129] Example 11: Wound healing effect of SGL 60L hydrogel loaded with aloe vera peel-derived nanovesicles on diabetic mice. (1) Establishment of a diabetic wound mouse model Experimental animals: 36 male C57 mice, 6-8 weeks old, weighing 20 (±2) g, provided by Liaoning Changsheng Biotechnology Co., Ltd., animal qualification certificate number: SYXK(Liaoning)2020-0001. Housing environment: Standard SPF-grade animal room, temperature: (23±2)℃, relative humidity: 50%~60%, with alternating 12-hour light and dark cycles. Mice had free access to water and feed. All animals in this experiment were housed in the animal room of Shenyang Pharmaceutical University according to the animal husbandry regulations of Shenyang Pharmaceutical University, and animal experimental procedures were conducted under the guidance of the Experimental Animal Protection Association.

[0130] Male C57 mice were acclimatized for 2 weeks and then fed a 45% high-fat diet (HFD) for 5 weeks. After a 12-hour fast, HFD-fed mice were induced to have diabetes (DM) by intravenous injection of streptozotocin (50 mg / kg in 0.1 M citrate buffered saline, pH 4.5) via the tail vein. Blood glucose levels were measured using Roche test strips after 6 days, and a level above 16.7 mM was considered indicative of DM. The diabetic mice were maintained on a normal diet for another 2 months to establish a stable diabetic animal model. The diabetic animal model and blood glucose levels were reconfirmed before wound formation. After anesthesia with sodium barbital (Sigma-Aldrich) (1%, 50 mg / kg), a full-thickness wound with a depth of 2 mm was made on the back of each mouse through a sterile disposable 8 mm diameter dermal biopsy puncture.

[0131] (2) Grouping of mice Mice were randomly divided into six groups of six each. Mice were kept alone with normal diet and water. To observe the wound healing process, the wounds were photographed using a macro camera on days 0, 5, 10, and 15 post-surgery.

[0132]

[0133] (3) Mouse sampling Animals were euthanized, and wound tissue was collected. The tissue was then fixed in 10% formalin neutral buffer (Sigma-Aldrich) and further embedded in paraffin. Sections were then subjected to H&E, Masson trichrome staining, and immunohistochemistry according to the manufacturer's manual. Histological images were obtained under a microscope. Results are shown in […]. Figure 18-22 .

[0134] Figure 18Representative photographs (A), wound closure rate statistics (B), and wound contraction diagrams (C) are shown for each group at 0, 5, 10, and 15 days. A and C represent the macroscopic healing process of the wound. Compared with the control group (NC), the model group (DW) showed obvious ulceration and suppuration on day 5, and the wound scab formation was slower than other groups. The SGL 60 blank hydrogel group (HG) showed better healing than the DW group, but was inferior to the low-dose AVp-NVs injection group (NV-L) and the high-dose injection group (NV-H). Compared with the NV-H group, the hydrogel group loaded with aloe vera peel-derived nanovesicles (HG-NV) promoted faster wound healing, achieving near-perfect re-epithelialization on day 15 without redness or swelling.

[0135] Quantitative analysis shows that ( Figure 18 (B) Compared to the DW group, the HG group showed better wound healing promotion, which may be due to its appropriate moisturizing ability and unique biocompatibility, which can accelerate the wound healing process by promoting re-epithelialization, indicating the potential of SGL 60L hydrogel as a wound covering material. Compared to the HG group, although both the NV-L and NV-H groups showed good effects in promoting diabetic wound healing, the wound healing rate of the HG-NV group was consistently the highest among all treatment groups at all time points. It is worth emphasizing that the closure rate of the HG-NV group reached 97.16±1.3% on day 15, which highlights the positive effect of local hydrogel administration on diabetic wound healing, indicating that the administration method of aloe vera peel-loaded nanovesicle hydrogel is more conducive to wound healing than direct subcutaneous injection.

[0136] Figure 19 The images show H&E staining of wound sections from diabetic mice in each group on day 15. Histopathological analysis (H&E staining) revealed that, compared to the NC group, the DW group had a significantly thicker epidermis with granular layer hyperplasia and tightly packed, disorganized collagen fiber bundles. While the HG group had a relatively smooth epidermis, its thickness was greater than that of the NV-L group. Both the NV-H and HG-NV groups showed relatively thinner epidermis with a clear structure and looser, more regularly arranged collagen fibers. In the HG-NV group, the formation of new skin appendages, hair follicles, was clearly observed.

[0137] Figure 20 The images show representative Masson staining images and statistical diagrams of wound sections from diabetic mice in each group on day 15. Masson staining results showed that the DW group had the lowest collagen deposition compared to the NC group. While the HG group had higher collagen deposition than the DW group, it was still significantly lower than the NV-L and NV-H groups. The HG-NV group had the highest collagen deposition, the best arrangement of newly formed collagen fibers, and enhanced extracellular matrix reconstruction and tissue remodeling.

[0138] Figure 21-22TNF-α (inflammation-related biomarkers) was collected from wound sections of diabetic mice in each group on day 15. Figure 21 ) and IL-1β ( Figure 22 The histochemical analysis of the expression of AVp-NVs is presented in the figure and statistical diagram. Immunohistochemical results showed no significant difference in expression levels between the HG and DW groups, indicating that the hydrogel had almost no anti-inflammatory effect. In contrast, compared with the DW group, the expression levels of TNF-α and IL-1β in the NV-L and NV-H groups were significantly reduced (**P<0.01), indicating that AVp-NVs have an anti-inflammatory effect. Compared with the NV-H group, the HG-NV group showed enhanced inhibition of TNF-α (*P<0.05) and a greater trend of reduced IL-1β expression. This indicates that the hydrogel loaded with aloe vera peel-derived nanovesicles can inhibit the inflammatory response during the healing process.

[0139] The above experimental results indicate that the HG group has a positive effect on promoting epidermal repair and collagen deposition, making it a hydrogel dressing suitable for wound repair. Subcutaneous injection of AVp-NVs has a good effect on promoting the healing of diabetic wounds, manifested in promoting epidermal repair, collagen deposition, and inhibiting the expression of inflammatory factors. The HG-NV group is significantly superior to subcutaneous injection of AVp-NVs around the wound in promoting the healing of diabetic wounds.

[0140] In summary, this invention provides a method for preparing aloe vera peel-derived nanovesicle hydrogel. Aloe vera peel-derived nanovesicles meeting the standards for plant extracellular vesicles were obtained through a combination of high-pressure homogenization and microporous filtration. Unexpectedly, cell experiments revealed that these aloe vera peel-derived nanovesicles not only promote the proliferation and migration of immortalized human keratinocytes but also inhibit LPS-induced inflammatory responses without significantly affecting macrophage proliferation. Furthermore, using SGL 60L to load aloe vera peel-derived nanovesicle hydrogel can better promote the healing of diabetic wounds, exhibiting repair and regeneration effects on skin wounds.

Claims

1. Nanovesicles derived from aloe vera peel, characterized in that, The aloe vera peel-derived nanovesicles were prepared by the following method: S1. Select fresh Aloe vera and soak it in water to remove anthraquinone components; S2. Cut off the leaf thorns, separate the aloe vera peel from the gel, and remove any residual gel from the leaves. Add PBS at a ratio of 3-5:1 (mass ratio of PBS to aloe vera peel), juice, filter, and obtain aloe vera peel homogenate. S3. Homogenize the aloe vera peel and centrifuge at low speed to obtain the supernatant. S4. The supernatant is further homogenized using a high-pressure homogenizer. S5 is filtered sequentially through 0.45 and 0.22 μm microporous membranes; S6. The precipitate was obtained by ultra-high speed centrifugation, diluted with aqueous solution, and the particle size was measured.

2. A nanovesicle hydrogel loaded with aloe vera peel, characterized in that, The hydrogel comprises aloe vera peel-derived nanovesicles as described in claim 1 and a gel matrix, wherein the mass ratio of aloe vera peel-derived nanovesicles to gel matrix is ​​2:1 to 1:4, and the gel matrix accounts for 0.25 to 1.5% of the weight of the hydrogel.

3. The aloe vera peel-derived nanovesicle hydrogel according to claim 2, characterized in that, The gel matrix is ​​hydroxypropyl methylcellulose stearyl ether or carbomer, and the hydroxypropyl methylcellulose stearyl ether is preferably Sangelose 60L or Sangelose 90L.

4. The aloe vera peel-derived nanovesicle hydrogel according to claim 2 or 3, characterized in that, It also contains glycerol, 1,3-butanediol, with or without triethanolamine.

5. The aloe vera peel-derived nanovesicle hydrogel according to claim 2 or 3, characterized in that, The hydrogel comprises aloe vera peel-derived nanovesicles, Sangelose 60L or Sangelose 90L, glycerin, and 1,3-butanediol. By weight percentage, each component constitutes the following percentage of the hydrogel: aloe vera peel-derived nanovesicles 0.2-0.8%, Sangelose 60L or Sangelose 90L 0.25-1.5%, glycerin 1%-10%, 1,3-butanediol 1%-10%, with the balance being water. Preferably, the composition is: aloe vera peel-derived nanovesicles 0.3-0.5%, Sangelose 60L 0.5-1%, glycerin 5-10%, 1,3-butanediol 5-10%, with the balance being water.

6. The aloe vera peel-derived nanovesicle hydrogel according to claim 2 or 3, characterized in that, The hydrogel contains aloe vera peel-derived nanovesicles, carbomer, glycerin, 1,3-butanediol, and triethanolamine. By weight percentage, the content of each component in the hydrogel is as follows: aloe vera peel-derived nanovesicles 0.2-0.8%, carbomer 0.25-1.5%, glycerin 5-10%, 1,3-butanediol 5-10%, triethanolamine 0.3-2%, and the balance being water.

7. The use of the aloe vera peel-derived nanovesicles of claim 1 or the aloe vera peel-loaded nanovesicle hydrogels of any one of claims 2-6 in the preparation of a medicament for treating diabetic wound dysfunction.

8. The application according to claim 7, characterized in that, The aforementioned diabetic wound dysfunction refers to wounds or foot ulcers caused by diabetes.

9. The application according to claim 7 or 8, characterized in that, The aloe vera peel-derived nanovesicles or hydrogels loaded with aloe vera peel-derived nanovesicles exert therapeutic effects on diabetic wound dysfunction by promoting the proliferation and migration of human immortalized keratinocytes, regulating macrophage polarization, and inhibiting the production of inflammatory factors.

10. The application of nonionic hydrogels in the preparation of drugs with improved stability of plant nanovesicles, wherein the nonionic hydrogel is preferably a hydrogel with nonionic hydroxypropyl methylcellulose stearyl ether as a matrix; and the plant nanovesicles are preferably nanovesicles derived from aloe vera peel.

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