Aloe vera exosome-loaded gel spray and method of making same

Abstract

The present application relates to the field of hydrogel spray, disclose a kind of gel spray of aloe exosome and its preparation method, first washing aloe, after being chopped with BPS, filtration, centrifugation, ultra-high speed centrifugation, reserve lower layer precipitate, by PBS resuspension precipitate, filtration, obtain aloe exosome suspension, store at-80 DEG C;PVA is swelled with distilled water, heated in hot water bath with stirring, obtain PVA solution;CS aqueous solution is configured;Under ice bath and slow stirring, PVA solution is slowly added to CS aqueous solution, under continuous stirring, dropwise addition β-GP solution, obtain uniform PVA / CS mixed solution;Repeat freeze-thaw 2-3 cycles, under low temperature stirring, add aloe exosome suspension, fill into light-proof spray container, obtain PVA-CS-EV hydrogel spray;The present application solves the problem that cannot realize convenient administration, cannot realize long-acting sustained-release, physical barrier and moisturizing effect in the prior art, cannot relieve inflammatory response in wound healing process, promote wound repair.

Description

Technical Field

[0001] This invention relates to the field of hydrogel sprays, and more particularly to a gel spray loaded with aloe vera exosomes and its preparation method. Background Technology

[0002] Aloe vera is a perennial evergreen succulent herb belonging to the Liliaceae family. Its gel extract has been widely proven to have moisturizing, soothing, anti-inflammatory, and wound-healing effects. However, traditional active ingredients in aloe vera gel (such as polysaccharides and anthraquinones) have large molecular weights, limited transdermal absorption efficiency, and are unstable and easily deactivated. Aloe vera exosomes, on the other hand, are novel carriers of aloe vera bioactive substances. Their nanoscale size and lipid bilayer structure make it easier to penetrate the skin barrier and precisely deliver high concentrations of aloe vera-derived active signaling molecules, theoretically possessing more efficient and stable efficacy than traditional aloe vera extracts.

[0003] However, exosomes face significant challenges in application: first, exosomes are prone to aggregation and inactivation in liquid environments, resulting in poor storage stability; second, conventional dosage forms (such as serums and creams) cannot provide long-lasting protection and sustained-release effects for exosomes, leading to low utilization rates; and third, effectively delivering exosomes to the target skin layer and maintaining their activity is a technical challenge.

[0004] Thermosensitive hydrogels are "smart" materials that exist as a flowable sol at ambient temperatures (such as room temperature). Upon contact with human skin (approximately 33-37°C), they rapidly undergo a phase transition, forming a semi-solid gel, achieving in-situ gelation. This characteristic makes them ideal as a matrix for sprays, enabling convenient drug delivery while forming a transparent protective film on the skin surface, providing long-lasting sustained release, a physical barrier, and moisturizing effects. Therefore, developing multifunctional hydrogel sprays that combine rapid film formation and long-lasting exosome release has significant clinical implications. Summary of the Invention

[0005] The present invention aims to provide a gel spray loaded with aloe vera exosomes and its preparation method, so as to achieve convenient drug delivery, form a transparent protective film on the surface of the sprayed skin, achieve long-lasting sustained release, physical barrier and moisturizing effects, relieve inflammatory response during wound healing and promote wound repair.

[0006] To achieve the above objectives, the present invention provides the following method:

[0007] The present invention provides a method for preparing a gel spray loaded with aloe vera exosomes:

[0008] S1: Wash the aloe vera with clean tap water, chop 100g of aloe vera and mix it with BPS, crush it with a blender, filter it to obtain aloe vera gel, centrifuge the aloe vera gel to remove impurities until no obvious precipitate is visible, and obtain aloe vera gel after removing impurities.

[0009] S2: The aloe vera gel after removing impurities is centrifuged at ultra-high speed, the supernatant is discarded, the lower precipitate is retained, the precipitate is resuspended in PBS and filtered to obtain an aloe vera exosome suspension, and the aloe vera exosome suspension is stored at -80℃ for preservation.

[0010] S3: Swell PVA with distilled water, place it in a hot water bath and stir and heat until completely dissolved to obtain a transparent PVA solution;

[0011] S4: Prepare a 1% w / v CS aqueous solution using 3% v / v glacial acetic acid and store it at 4°C for later use.

[0012] S5: Under ice bath and slow stirring, the cooled PVA solution is slowly added to the CS aqueous solution to mix it evenly. Then, under continuous stirring, a 4% v / v β-GP solution is added dropwise to crosslink the solution, resulting in a homogeneous PVA / CS mixed solution.

[0013] S6: Place the homogeneous PVA / CS mixed solution in a freezing environment to freeze, then take it out and let it thaw at room temperature. Repeat the freeze-thaw cycle 2-3 times to obtain PVA / CS gel.

[0014] S7: Stir the PVA / CS gel in an ice bath to make it revert to a viscous but flowable sol state; add the aloe vera exosome suspension with a concentration of 0.1% v / v under low-temperature stirring, and after ensuring uniform mixing, fill it into a pre-sterilized light-proof spray container to prepare PVA-CS-EV hydrogel spray.

[0015] Preferably, the ratio of aloe vera to BPS is 1:3.

[0016] Preferably, the aloe vera gel is centrifuged to remove impurities until no obvious precipitate is visible. The centrifugation conditions for obtaining the aloe vera gel after removing impurities are as follows: continuous centrifugation at 4°C with a centrifugal force of 1000xg→10000xg until no obvious precipitate is visible, thus obtaining the aloe vera gel after removing impurities.

[0017] Preferably, the conditions for ultra-high speed centrifugation of the aloe vera gel after impurity removal are: ultra-high speed centrifugation at 4°C with a centrifugal force of 100000xg for 90 min.

[0018] Preferably, the step of resuspending and precipitating the aloe vera exosomes with PBS, filtering to obtain an aloe vera exosome suspension, and storing the aloe vera exosome suspension at -80°C includes: resuspending and precipitating the aloe vera exosomes with PBS, filtering through a 0.22µm filter to obtain an aloe vera exosome suspension, and storing the aloe vera exosome suspension at -80°C.

[0019] Preferably, the step of swelling PVA with distilled water and then stirring and heating it in a hot water bath until it is completely dissolved to obtain a transparent PVA solution includes: swelling 8% w / v PVA with distilled water for 30 minutes, and then stirring and heating it in a 90°C water bath until it is completely dissolved to obtain a transparent PVA solution.

[0020] Preferably, the cooled PVA solution is slowly added to the CS aqueous solution under ice bath and slow stirring to mix them evenly. The ratio of PVA solution to CS aqueous solution in this step is 2:1.

[0021] Preferably, the homogeneous PVA / CS mixed solution is placed in a -20°C freezing environment for 12-16 hours.

[0022] Preferably, the homogeneous PVA / CS mixed solution is first frozen at -20°C for 12-16 hours, then removed and allowed to thaw at room temperature for 4-6 hours. This freeze-thaw cycle is repeated 2-3 times to obtain the PVA / CS gel.

[0023] This invention provides a gel spray loaded with aloe vera exosomes prepared according to the gel spray preparation method described above.

[0024] The beneficial effects of this invention are as follows: This invention synthesizes a novel PVA-CS-EV hydrogel spray, hereinafter referred to as PCE hydrogel spray, by combining polyvinyl alcohol (PVA), chitosan (CS), and aloe vera exosomes (AEVs). After preparation via a freeze-thaw cycle, the PVA hydrogel exhibits very high toughness, elasticity, and compressive strength, qualities not found in many natural polymer hydrogels. Furthermore, PVA possesses extremely high biocompatibility, excellent water solubility and film-forming properties, and good chemical stability, providing a long-lasting physical barrier and sustained-release effect. CS itself possesses unique biological functions such as antibacterial, hemostatic, wound-healing, mucosal adhesion, and biodegradability—significant advantages unmatched by synthetic materials. Moreover, the amino and hydroxyl groups on the CS molecule are easily modified, allowing for the grafting of other functional groups or polymers to customize its properties (including enhanced temperature sensitivity). In summary, the freeze-thaw cycle of PVA forms the first layer of robust network, and chitosan / β-GP is introduced as the second layer of temperature-sensitive active network. It can simultaneously achieve excellent mechanical properties and powerful biological functions, effectively protect exosome activity, realize convenient spraying and in-situ skin film formation, and achieve long-lasting, sustained-release, and highly effective skin repair effects. It has broad clinical application prospects and significance. Compared with existing hydrogel sprays, the PCE hydrogel spray constructed in this study has the following superior characteristics:

[0025] (1) Aloe vera exosomes have good antibacterial and anti-inflammatory effects;

[0026] (2) It can be easily sprayed to achieve in-situ film formation and is suitable for defects of different shapes;

[0027] (3) It can release aloe vera exosomes in a slow-release manner to achieve long-lasting, slow-release and highly effective skin repair effects. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0029] Figure 1 A schematic flowchart illustrating a method for preparing a gel spray loaded with aloe vera exosomes according to an embodiment of the present invention;

[0030] Figure 2 Provided for embodiments of the present invention: (A) Representative transmission electron microscopy (TEM) images of AEVs; (B) Nanoparticle tracking analysis (NTA) images of AEVs;

[0031] Figure 3Provided for embodiments of the present invention: (AB) Scanning electron microscope (SEM) images of PCE hydrogel at different scales; (CD) Scanning electron microscope (SEM) images of PCE hydrogel loaded with AEVs;

[0032] Figure 4 Provided for embodiments of the present invention: (A) Overall views of the wounds of SD mice at various days (days 0, 4, 8, and 12); (B) Statistical analysis chart of wound closure area;

[0033] Figure 5 The following images are provided for embodiments of the present invention: (A) representative H&E staining images of different treatment groups on day 12; (B) representative Masson staining images of different treatment groups on day 12. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this invention are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or end that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or ends.

[0036] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0037] Aloe exosomes are novel carriers of aloe bioactive substances. Their nanoscale size and lipid bilayer structure facilitate penetration of the skin barrier, precisely delivering high concentrations of aloe-derived active signaling molecules, theoretically offering higher efficacy and greater stability than traditional aloe extracts. However, exosomes face significant challenges in application: firstly, exosomes are prone to aggregation and inactivation in liquid environments, resulting in poor storage stability; secondly, conventional dosage forms (such as serums and creams) cannot provide long-lasting protection and sustained-release effects for exosomes, leading to low utilization rates; and thirdly, effectively delivering exosomes to the target skin layer and maintaining their activity remains a technical challenge.

[0038] Thermosensitive hydrogels are "smart" materials that exist as a flowable sol at ambient temperatures (such as room temperature). Upon contact with human skin (approximately 33-37°C), they rapidly undergo a phase transition, forming a semi-solid gel, achieving in-situ gelation. This characteristic makes them ideal as a matrix for sprays, enabling convenient drug delivery while forming a transparent protective film on the skin surface, providing long-lasting sustained release, a physical barrier, and moisturizing effects. Therefore, developing multifunctional hydrogel sprays that combine rapid film formation and long-lasting exosome release has significant clinical implications.

[0039] The present invention aims to provide a gel spray loaded with aloe vera exosomes and its preparation method, so as to achieve convenient drug delivery, form a transparent protective film on the surface of the sprayed skin, achieve long-lasting sustained release, physical barrier and moisturizing effects, relieve inflammatory response during wound healing and promote wound repair.

[0040] like Figure 1 As shown in the figure, a specific embodiment of the present invention provides a method for preparing a gel spray loaded with aloe vera exosomes, comprising the following steps:

[0041] S1: Wash the aloe vera with clean tap water, chop 100g of aloe vera and mix it with BPS, crush it with a blender, filter it to obtain aloe vera gel, centrifuge the aloe vera gel to remove impurities until no obvious sediment is visible, and obtain aloe vera gel after removing impurities.

[0042] In this embodiment of the invention, the ratio of aloe vera to BPS is 1:3; the aloe vera gel is centrifuged to remove impurities until no obvious precipitate is visible, and the centrifugation conditions for obtaining the aloe vera gel after removing impurities are: continuous centrifugation at 4°C with a centrifugal force of 1000xg→10000xg until no obvious precipitate is visible, and the aloe vera gel after removing impurities is obtained.

[0043] S2: After removing impurities, the aloe vera gel is centrifuged at ultra-high speed, the supernatant is discarded, and the lower precipitate is retained. The precipitate is resuspended in PBS and filtered to obtain an aloe vera exosome suspension. The aloe vera exosome suspension is stored at -80℃.

[0044] In this embodiment of the invention, the conditions for ultra-high speed centrifugation of the aloe vera gel after removing impurities are as follows: ultra-high speed centrifugation at 4°C with a centrifugal force of 100000xg for 90 min; resuspending the precipitate with PBS, filtering through a 0.22um filter to obtain an aloe vera exosome suspension, and storing the aloe vera exosome suspension at -80°C for preservation.

[0045] S3: Swell PVA with distilled water, place it in a hot water bath and stir and heat until completely dissolved to obtain a transparent PVA solution.

[0046] In this embodiment of the invention, PVA with a concentration of 8% w / v was swollen in distilled water for 30 minutes, and then stirred and heated in a 90°C water bath until completely dissolved to obtain a transparent PVA solution.

[0047] S4: Prepare a 1% w / v aqueous solution of CS using 3% v / v glacial acetic acid and store it at 4°C for later use.

[0048] S5: Under ice bath and slow stirring, the cooled PVA solution is slowly added to the CS aqueous solution to mix evenly. Then, under continuous stirring, a 4% v / v β-GP solution is added dropwise to crosslink the solution, resulting in a homogeneous PVA / CS mixed solution.

[0049] In this embodiment of the invention, the cooled PVA solution is slowly added to the CS aqueous solution under ice bath and slow stirring to mix it evenly. The ratio of PVA solution to CS aqueous solution in this step is 2:1.

[0050] S6: First, freeze the homogeneous PVA / CS mixture in a freezing environment, then remove it and allow it to thaw at room temperature. Repeat the freeze-thaw cycle 2-3 times to obtain the PVA / CS gel.

[0051] In this embodiment of the invention, a homogeneous PVA / CS mixed solution is placed in a -20°C freezing environment for 12-16 hours; then it is taken out and allowed to thaw at room temperature for 4-6 hours, and the freeze-thaw cycle is repeated 2-3 times to obtain a PVA / CS gel.

[0052] S7: Stir the PVA / CS gel in an ice bath until it returns to a viscous but flowable sol state; add aloe vera exosome suspension at a concentration of 0.1% v / v while stirring at low temperature, and after ensuring uniform mixing, fill it into a pre-sterilized light-proof spray container to prepare PVA-CS-EV hydrogel spray.

[0053] This invention provides a gel spray loaded with aloe vera exosomes prepared according to the gel spray preparation method described above.

[0054] like Figure 2 As shown, this is a representative transmission electron microscope (TEM) image of AEVs provided in an embodiment of the present invention; the AEVs exhibit a uniform group of nanovesicles with vesicle shapes;

[0055] (B) AEVs nanoparticle tracking analysis (NTA) figure: The average diameter of aloe vera exosomes was 122.03±6.28nm, and the total particle concentration was 5.0E+10 Particles / mL, proving the successful extraction of AEVs.

[0056] like Figure 3 The images shown are scanning electron microscope (SEM) images of the PCE hydrogel at different scales, provided in embodiments of the present invention; the microporous morphology of the PCE hydrogel.

[0057] (CD) Scanning electron microscope (SEM) image of AEVs loaded on PCE hydrogel: Successful synthesis of PCE hydrogel, with AEVs uniformly distributed in the hydrogel.

[0058] like Figure 4 As shown, this invention provides: (A) Overall view of the wounds of SD mice at various days (days 0, 4, 8, and 12); the PVE-gel (experimental gel group) showed the fastest wound healing speed and the largest healing area, demonstrating a good healing-promoting effect;

[0059] (B) Statistical analysis of wound closure area; PVE-gel (experimental gel group) had the highest wound closure rate, especially on DAY8 and DAY12, which was significantly different from the control group and commercial gel group.

[0060] like Figure 5 As shown, the following are representative H&E staining images of different treatment groups on day 12 provided by the embodiments of the present invention: (A) Yellow arrows indicate newly formed epidermis and dermis, and red arrows indicate newly formed hair follicles; newly formed capillaries can be seen in the PVE-gel group, the epidermis is thicker than other experimental groups, and the hair follicles and sebaceous glands are arranged in a clear and regular manner.

[0061] (B) Representative Masson staining images of different treatment groups on day 12; the PVE-gel group had the highest collagen fiber content (blue area), the thickest epidermis, and the collagen and muscle fibers were neatly arranged, with a tissue structure close to that of normal skin.

[0062] The beneficial effects of this invention are as follows: This invention synthesizes a novel PVA-CS-EV hydrogel spray, hereinafter referred to as PCE hydrogel spray, by combining polyvinyl alcohol (PVA), chitosan (CS), and aloe vera exosomes (AEVs). After preparation via a freeze-thaw cycle, the PVA hydrogel exhibits very high toughness, elasticity, and compressive strength, qualities not found in many natural polymer hydrogels. Furthermore, PVA possesses extremely high biocompatibility, excellent water solubility and film-forming properties, and good chemical stability, providing a long-lasting physical barrier and sustained-release effect. CS itself possesses unique biological functions such as antibacterial, hemostatic, wound-healing, mucosal adhesion, and biodegradability—significant advantages unmatched by synthetic materials. Moreover, the amino and hydroxyl groups on the CS molecule are easily modified, allowing for the grafting of other functional groups or polymers to customize its properties (including enhanced temperature sensitivity). In summary, the freeze-thaw cycle of PVA forms the first layer of robust network, and chitosan / β-GP is introduced as the second layer of temperature-sensitive active network. It can simultaneously achieve excellent mechanical properties and powerful biological functions, effectively protect exosome activity, realize convenient spraying and in-situ skin film formation, and achieve long-lasting, sustained-release, and highly effective skin repair effects. It has broad clinical application prospects and significance. Compared with existing hydrogel sprays, the PCE hydrogel spray constructed in this study has the following superior characteristics:

[0063] (1) Aloe vera exosomes have good antibacterial and anti-inflammatory effects;

[0064] (2) It can be easily sprayed to achieve in-situ film formation and is suitable for defects of different shapes;

[0065] (3) It can release aloe vera exosomes in a slow-release manner to achieve long-lasting, slow-release and highly effective skin repair effects.

[0066] The above descriptions are merely embodiments of the present invention. Commonly known technical solutions or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a gel spray loaded with aloe vera exosomes, characterized in that, The method includes: S1: Wash the aloe vera with clean tap water, chop 100g of aloe vera and mix it with PBS. The ratio of aloe vera to PBS is 1:

3. Crush the aloe vera with a mixer, filter it, and obtain aloe vera gel. Centrifuge the aloe vera gel to remove impurities. Centrifuge continuously at 4°C with a centrifugal force of 1000×g→10000×g until no obvious precipitate is visible. The aloe vera gel after removing impurities is obtained. S2: The aloe vera gel after removing impurities is subjected to ultra-high speed centrifugation at 4°C and a centrifugal force of 100000×g for 90 min. The supernatant is discarded, and the lower precipitate is retained. The precipitate is resuspended with PBS and filtered through a 0.22μm filter to obtain an aloe vera exosome suspension. The aloe vera exosome suspension is stored at -80°C for preservation. S3: After swelling PVA with a concentration of 8% w / v in distilled water for 30 min, stir and heat in a 90℃ water bath until completely dissolved to obtain a transparent PVA solution; S4: Prepare a 1% w / v CS aqueous solution using 3% v / v glacial acetic acid and store it at 4°C for later use. S5: Under ice bath and slow stirring, the cooled PVA solution is slowly added to the CS aqueous solution. The ratio of the amount of PVA solution to the amount of CS aqueous solution is 2:

1. After mixing evenly, under continuous stirring, a cold 4% v / v β-GP solution is added dropwise to crosslink the solution, resulting in a homogeneous PVA / CS mixed solution. S6: Place the homogeneous PVA / CS mixed solution in a -20℃ freezing environment for 12-16 hours, then take it out and let it thaw at room temperature for 4-6 hours. Repeat the freeze-thaw cycle 2-3 times to obtain PVA / CS gel. S7: Stir the PVA / CS gel in an ice bath to make it revert to a viscous but flowable sol state; add the aloe vera exosome suspension with a concentration of 0.1% v / v under low-temperature stirring, and after ensuring uniform mixing, fill it into a pre-sterilized light-proof spray container to prepare PVA-CS-EV hydrogel spray.

2. A gel spray loaded with aloe vera exosomes prepared according to the method for preparing a gel spray loaded with aloe vera exosomes as described in claim 1.

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