Inulin gel, exosome inulin gel and preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, and discloses inulin gel, exosome inulin gel and a preparation method and application thereof.The inulin gel is prepared from inulin and a solvent, the inulin is prepared into the gel through a heating-cooling method, the inulin is dissolved through heating, the gel is formed through the crowding effect of the inulin, and the exosome inulin gel is prepared from the solvent. The exosome inulin gel relates to a solvent, inulin and an exosome, and the exosome can be a milk exosome, a plant exosome, a mesenchymal stem cell exosome, a plasma source exosome or a macrophage source exosome. According to the exosome inulin gel as well as the preparation method and the application thereof, the heating-cooling preparation method of the inulin gel and the exosome-inulin gel is simple and convenient to operate and easy to implement, meanwhile, the preparation time is short, the process stability is good, the gel uniformity is good, and meanwhile, a mouse burn wound model is used for carrying out an in-vivo experiment, so that the in-vivo experiment result is good. It is verified that the hydrogel can promote healing of wounds such as burns and scalds.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to inulin gel, exosome inulin gel, and preparation methods and applications thereof. Background Art

[0002] The skin is a key physical barrier against external stimuli, protecting the body from risks such as dehydration, ultraviolet radiation, and pathogen infection. As a protective barrier, the integrity and effective healing ability of the skin are essential for preventing infection and maintaining homeostasis. However, various physical, chemical, and biological factors may damage the integrity of the skin, leading to acute or chronic injuries or wounds. Poor wound healing is a pressing global medical problem, resulting in considerable economic losses and posing a major challenge to healthcare professionals.

[0003] Currently, the most widely used dressing in clinical practice to promote wound healing is traditional dressing, such as gauze dressing. Gauze dressing is inexpensive and has certain hygroscopic and protective effects on the treatment of general wounds. However, it absorbs little wound exudate and requires frequent dressing changes. After the dressing is soaked, bacteria easily adhere to the wound surface, causing wound infection. In addition, when changing the dressing, the gauze easily adheres to the wound and causes mechanical damage again, and has no obvious promoting effect on wound healing.

[0004] Hydrogel has good hydrophilicity and can actively replenish moisture to dry wounds, maintain moist healing conditions, and promote wound healing. It has now become a new type of wound dressing and has been used in wound care. Inulin is a natural polysaccharide found in many plants, such as onions, garlic, bananas, agave and chicory. Inulin is widely used as a substitute for fat or sugar in processed foods and has obtained generally recognized safe status from the US FDA. It has major advantages such as biodegradability, biocompatibility, non-toxicity and hydrophilicity, which makes it a promising candidate for wound dressing.

[0005] As a medium of intercellular communication and a carrier of signal transmission, exosomes have become a promising candidate in non-cellular therapy due to their unique biological properties and potential therapeutic capabilities. Compared with some delivery systems based on synthetic materials, exosomes have high circulation stability, low immunogenicity, biocompatibility and tissue homing ability due to their unique and intrinsic biological properties and particle size. Exosomes can accelerate wound healing by regulating cell function, inhibiting oxidative stress damage, inhibiting inflammatory response, promoting angiogenesis, accelerating epithelial regeneration, and promoting collagen remodeling. Milk exosomes have good biocompatibility, low immunogenicity, good gastrointestinal stability and easy large-scale extraction. They are widely used as drug delivery systems. At the same time, milk exosomes exhibit antioxidant and anti-inflammatory effects in vitro and can promote wound healing.

[0006] Combining exosomes with hydrogels can prevent the premature clearance of exosomes, allowing them to concentrate and continuously act on or near the target site, thereby achieving a more significant therapeutic effect. Therefore, the present invention applies exosomes to inulin gel to provide a method for preparing exosome inulin gel. Summary of the Invention

[0007] In view of the fact that most of the above-mentioned existing gauze dressings are made of cotton as raw material, their advantages are low price, easy availability, and ability to protect the wound surface, and they are the most widely used in clinical practice, but they absorb little wound exudate and require frequent dressing changes. After the dressing is soaked, bacteria easily adhere to the wound surface, causing wound infection, and when the dressing is changed, the gauze easily adheres to the wound and causes mechanical damage again, and has no obvious promoting effect on wound healing. Therefore, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to provide an inulin gel, an exosome inulin gel, and a preparation method and application thereof, the purpose of which is to: exhibit good biocompatibility through the hydrogel material, which is similar in nature to the extracellular matrix part, and can reduce friction and mechanical effects on surrounding tissues after absorbing water, and the hydrogel wound dressing is not easy to adhere to the wound, which makes it easy to remove without causing further trauma or damage to the wound site. At the same time, the hydrogel can provide moisture to the wound microenvironment and promote autolysis and debridement. By adding exosomes to the hydrogel, it can be given functional properties that accelerate wound healing, such as regulating cell function, inhibiting inflammatory response, promoting angiogenesis, and accelerating epithelial regeneration, thereby becoming a functional hydrogel.

[0009] To solve the above technical problems, the present invention provides the following technical solution: comprising inulin and a solvent, using a heating-cooling method to prepare a gel, dissolving the inulin in water or an aqueous solution by heating, and utilizing the crowding effect of the inulin to form a gel.

[0010] As a preferred embodiment of the exosome inulin gel of the present invention, the raw materials include inulin, an aqueous solution and exosomes.

[0011] As a preferred embodiment of the exosome inulin gel of the present invention, the inulin is used as a matrix material without the need for an additional chemical cross-linking agent.

[0012] As a preferred embodiment of the exosome inulin gel of the present invention, the inulin is used as a matrix material without the need for an additional chemical cross-linking agent.

[0013] The preparation method of the inulin gel comprises the following steps:

[0014] Follow these steps:

[0015] Step 1: First, take an appropriate amount of solvent and preheat it to 70-100℃ in a 70-100℃ water bath;

[0016] Step 2: Weigh an appropriate amount of inulin, add it to the preheated aqueous solution, mix and shake well, and continue to heat in a 70-100℃ water bath for 3-60 minutes;

[0017] Step 3: Place the inulin aqueous solution at 0-25° C. and let it stand for 2-24 hours to obtain inulin gel.

[0018] As a preferred embodiment of the method for preparing the exosome inulin gel of the present invention, the solvent is water, PBS solution, physiological saline or other aqueous solutions.

[0019] As a preferred embodiment of the method for preparing the exosome inulin gel of the present invention, the inulin is used as a matrix material without the need for an additional chemical cross-linking agent.

[0020] The preparation method of the exosome inulin gel comprises the following steps:

[0021] Follow these steps:

[0022] Step 1: First, take an appropriate amount of solvent and preheat it to 70-100℃ in a 70-100℃ water bath;

[0023] Step 2: Weigh an appropriate amount of inulin, add it to the preheated aqueous solution, mix and shake well, and continue to heat in a 70-100℃ water bath for 3-60 minutes;

[0024] Step 3: After the inulin-water solution cools down, add exosomes and mix well;

[0025] Step 4: Place the raw materials mixed evenly in step 3 in a 0-15° C. environment and let it stand for 2-24 hours to obtain the exosome inulin gel.

[0026] As a preferred embodiment of the exosome inulin gel of the present invention, the standing environment in step 4 can be a refrigerator or a low-temperature incubator, or can be standing at room temperature, and the standing time can be extended to 24 hours according to actual needs.

[0027] As a preferred embodiment of the exosome inulin gel of the present invention, the preparation method may further include performing subsequent physical or chemical modification on the obtained exosome inulin gel to enhance its performance or impart specific functions to it.

[0028] As a preferred embodiment of the exosome inulin gel of the present invention, the exosomes may be milk exosomes, plant exosomes, mesenchymal stem cell exosomes, plasma-derived exosomes or macrophage-derived exosomes.

[0029] As a preferred application of the exosome inulin gel of the present invention, the inulin gel and exosome inulin gel have significant effects in promoting skin repair, wound healing, and tissue repair, and can accelerate wound inflammation relief, tissue regeneration, collagen synthesis and remodeling, and are suitable for the treatment and care of various types of wounds such as burns, scalds, cuts, diabetic ulcers, etc.

[0030] Compared with the prior art, the present invention has at least the following beneficial effects:

[0031] The heating and cooling preparation methods of inulin gel and exosome-inulin gel provided by the present invention are simple to operate and easy to execute, with short preparation time, good process stability, and good gel uniformity. In vivo experiments using a mouse burn wound model verified that the hydrogel can promote the healing of wounds such as burns and scalds, thereby enabling the exosome inulin gel of the present invention to achieve good biological activity and promote wound healing. The hydrogel of the present invention uses inulin as the only matrix material and adds exosomes as bioactive substances, which is innovative in formulation; the gel is prepared by utilizing the crowding effect of inulin through the heating-cooling method, which is innovative in the preparation method; at the same time, existing patents involving inulin gel are mainly aimed at diabetic wound healing, while the present invention broadens the scope of application and can be used for burn wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0033] Figure 1 This is a macroscopic image of milk exosome inulin gel according to the preparation method of the exosome inulin gel of the present invention.

[0034] Figure 2 SEM images of inulin gel and milk exosome-inulin gel according to the preparation method of exosome inulin gel of the present invention.

[0035] Figure 3 This is a transmission electron microscope image of milk exosomes in the preparation method of the exosome inulin gel of the present invention.

[0036] Figure 4 This is a WB image of milk exosomes in the preparation method of exosome inulin gel of the present invention.

[0037] Figure 5 Images of inulin solutions prepared at different heating temperatures according to the method for preparing exosome inulin gel of the present invention.

[0038] Figure 6 This is an image of inulin clusters in an inulin solution heated at 70°C in the method for preparing exosome inulin gel of the present invention.

[0039] Figure 7 Images of the inulin solution and carboxymethyl chitosan-inulin solution used in the method for preparing exosome inulin gel of the present invention.

[0040] Figure 8 Images of inulin gels of different mass concentrations according to the preparation method of exosome inulin gel of the present invention.

[0041] Figure 9 This is an image of the wound healing of burned mice using the preparation method of the exosome inulin gel of the present invention. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0043] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0044] Reference Figure 1-9 , as an embodiment of the present invention, provides an inulin gel, an exosome inulin gel, and a preparation method and application thereof. The inulin gel includes inulin, and the inulin is prepared into a gel using a heating-cooling method, which dissolves the inulin by heating and forms a gel by utilizing the crowding effect of the inulin.

[0045] The gel is formed by using inulin as a matrix material and utilizing the crowding effect of inulin (the crowding effect refers to the situation in which, in a high-concentration solution or mixed system, the space is significantly occupied due to the presence of a large number of molecules, resulting in a limited remaining space, thereby enhancing the interaction and aggregation tendency between molecules). No additional chemical cross-linking agents are required, thus avoiding potential toxicity issues. At the same time, the crowding effect can better simulate the complex environment in the body, making it particularly suitable for biomedical applications.

[0046] Disclosed is an exosome inulin gel, the raw materials of which include a solvent, inulin and exosomes.

[0047] The preparation method of inulin gel comprises the following steps:

[0048] Step 1: First, take an appropriate amount of solvent and preheat it to 70-100℃ in a 70-100℃ water bath;

[0049] Step 2: Weigh an appropriate amount of inulin, add it to the preheated aqueous solution, mix and shake well, and continue to heat in a 70-100℃ water bath for 3-60 minutes;

[0050] Step 3: Place the inulin aqueous solution at 0-25°C for 2-24 hours to obtain inulin gel.

[0051] The exosomes may be milk exosomes, plant exosomes, mesenchymal stem cell exosomes, plasma-derived exosomes, or macrophage-derived exosomes.

[0052] The preparation method of exosome inulin gel comprises the following steps:

[0053] Step 1: First, take 2 mL of PBS and preheat it to 90°C in a 90°C water bath;

[0054] Step 2: Weigh 0.7 g of inulin and add it to the preheated PBS solution. Mix well and continue to incubate in a 90°C water bath for 5 min.

[0055] Step 3: After the inulin-PBS solution has cooled, 200 μg of milk exosomes are added and mixed evenly;

[0056] Step 4: Place the raw materials mixed evenly in step 3 at 4°C and let it stand overnight to obtain milk exosome inulin gel.

[0057] The inulin gel is prepared by using a heating and cooling method, which involves inulin and a PBS solution or water.

[0058] The standing environment in step 4 can be a refrigerator or a low-temperature incubator, or can be left standing at room temperature. The standing time can be extended to 24 hours according to actual needs.

[0059] The preparation method may further include performing subsequent physical or chemical modification on the obtained exosome inulin gel to enhance its performance or impart specific functions to it.

[0060] Application of exosome inulin gel in the preparation of medical devices with the function of promoting wound healing and skin and tissue repair.

[0061] The exosome inulin gel has good biocompatibility, biodegradability and bioactivity, and can be used in wound dressing, drug delivery and other fields.

[0062] The exosome inulin gel has a significant effect in promoting wound healing, and can accelerate the processes of wound inflammation relief, tissue regeneration, collagen synthesis and remodeling. It is suitable for the treatment and care of various types of wounds such as burns, scalds, cuts, diabetic ulcers, etc.

[0063] The hydrogel uses inulin as the sole matrix material and adds milk exosomes as bioactive substances, which is innovative in its formulation. The gel is prepared by a heating-cooling method using the crowding effect of inulin, which is innovative in its preparation method. At the same time, existing patents involving inulin gel are mainly aimed at diabetic wound healing, while the present invention broadens the scope of application and can be used for burn wound repair.

[0064] In the prepared milk exosome-inulin gel, there are 100 μg of milk exosomes in each 1 mL of gel system (excluding the increase in gel volume caused by the dissolution of inulin). Figure 1 Shown are macroscopic images of milk exosome-inulin gel, from left to right: inulin solution with added milk exosomes, milk exosome-inulin gel, and inverted image of milk exosome-inulin gel;

[0065] like Figure 2 Shown are SEM images of inulin gel and milk exosome-inulin gel from left to right, with a scale of 50 μm.

[0066] Experiment 1: Characterization of milk exosomes

[0067] like Figure 3 Shown is a transmission electron microscopy image of milk exosomes, with a scale bar of 100 nm;

[0068] like Figure 4 Shown are Western blot images of milk exosomes.

[0069] Experiment 2: Effect of temperature on gel preparation

[0070] 1) Prepare four 2 mL aliquots of PBS and preheat in a water bath at 30°C, 50°C, 70°C, and 90°C.

[0071] 2) Weigh four 0.7g portions of inulin and add them to the preheated PBS solution. Mix and shake well. Continue to incubate in a water bath at 30°C, 50°C, 70°C, and 90°C for 5 minutes.

[0072] 3) Observe the dissolution of inulin;

[0073] like Figure 5 The figure shows the inulin solutions prepared at different heating temperatures. The experiment was conducted using a 35% ratio. It was found that the solubility of inulin was significantly different when the preparation temperature was 30°C, 50°C, 70°C, and 90°C. When the preparation temperature was no higher than 70°C, it was found that a large amount of inulin clumps could not be dissolved after heating (such as Figure 6 The image shown is of inulin clusters in an inulin solution heated at 70°C). This will result in an uneven texture of the gel formed during the subsequent cooling process. Therefore, matching the appropriate temperature and feed ratio is crucial for preparing inulin gel with good uniformity.

[0074] Experiment 3: Effect of raw materials on gel preparation

[0075] 1) Preheat 2 mL of PBS in a 90°C water bath.

[0076] 2) Weigh 0.07 g of carboxymethyl chitosan and 0.63 g of inulin, add to the preheated PBS solution, shake well, and continue to incubate in a 90°C water bath for 5 min;

[0077] 3) Observe the dissolution of carboxymethyl chitosan and inulin and compare with the inulin solution prepared at 90°C in Test Example 2;

[0078] like Figure 7 The images shown are of an inulin solution and a carboxymethyl chitosan-inulin solution. The carboxymethyl chitosan-inulin solution was not fully dissolved, which resulted in an uneven texture in the gel formed during the cooling process. Gels such as carboxymethyl chitosan and hyaluronic acid typically require thorough stirring after adding water. Uneven stirring can lead to uneven dispersion, resulting in localized gel formation while other areas remain in solution. This suggests that heating-cooling gel preparation requires ensuring that all raw material components are substantially dissolved during the heating process to produce a uniform gel. The use of multi-component materials to prepare gels requires consideration of the solubility of each component and mixing conditions, which can increase raw material costs and process complexity.

[0079] Experiment 4: Effect of inulin concentration on gel preparation

[0080] 1) Take 2 mL of PBS and preheat to 90°C in a 90°C water bath;

[0081] 2) Weigh 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 g of inulin and add them to the preheated PBS solution. Mix well and continue to incubate at 90°C in a water bath for 5 min.

[0082] 3) Place the obtained inulin-PBS solution in a 4°C refrigerator and let it stand overnight to obtain inulin gel;

[0083] In Experiment 3, the mass concentrations of inulin in the prepared inulin gel were 20%, 25%, 30%, 35%, 40% and 45% (w / v) in sequence. When the mass concentration of inulin was low, no gel state could be formed or only a partial gel was formed. When the mass concentration of inulin was high, heating at 90°C could not completely dissolve the inulin, which may make the texture of the formed inulin gel uneven, such as Figure 8 Shown are images of inulin gels at different mass concentrations.

[0084] Experiment 5: Promoting wound healing in burned mice

[0085] 1) Six-week-old female Balb / c mice were placed in a standard environment (20–26°C, 12 / 12 h light / dark cycle) for one week;

[0086] 2) Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate solution (injection dose: 100 μL / 10 g mouse body weight). After ensuring that the mice were fully anesthetized, a circular burn wound was created on the back of the mice using a copper hammer under standardized conditions (100°C, 15 s).

[0087] 3) 24 hours later, the burned skin was excised using a 10 mm skin punch and scalpel to complete the dorsal burn wound model. The mice with the completed dorsal burn wound model were evenly divided into three groups: a control group, an inulin gel group, and a milk exosome-inulin gel group, and each group received corresponding treatment.

[0088] 4) Each wound was then fixed with a 1622W Tegaderm (3M) transparent dressing and gauze. The initial burn was defined as day -1, and the completion of the back burn wound model was defined as day 0. Dressings were changed at each time point (day 0, day 3, day 6, day 9, day 12, and day 15) for each group, and the wound closure status of each group of mice was recorded.

[0089] The results are as follows Figure 9 As shown in the figures (from top to bottom: control group, inulin gel group, milk exosome-inulin gel group), the healing speed of mouse wounds was significantly accelerated after being treated with inulin gel and milk exosome-inulin gel, proving that the inulin gel and milk exosome-inulin gel prepared by the present invention can significantly promote the healing of burn wounds in mice.

[0090] The provided heating-cooling preparation method of inulin gel and milk exosome-inulin gel is simple to operate and easy to execute, and has a short preparation time, good process stability, and good gel uniformity. The raw materials of inulin and milk exosomes are cheap and have high economic benefits. The prepared inulin gel and milk exosome-inulin gel can significantly promote the healing of wounds in burned mice.

[0091] The hydrogel of the present invention uses inulin as the sole matrix material and adds milk exosomes as bioactive substances, which is innovative in formulation. The gel is prepared by a heating-cooling method using the crowding effect of inulin, which is innovative in preparation method. At the same time, existing patents involving inulin gel are mainly aimed at diabetic wound healing, while the present invention broadens the scope of application and can be used for burn wound repair. In addition, the high economic benefits and safety of inulin and milk exosomes make them ideal choices for wound treatment. The hydrogel using these as raw materials is easy to prepare and low-cost. In vivo experiments using a mouse burn wound model verified that the hydrogel can promote the healing of burn wounds.

[0092] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An inulin gel, characterized in that The raw materials include inulin and a solvent. The gel is prepared by a heating-cooling method. The inulin is dissolved in water or an aqueous solution by heating, and the crowding effect of the inulin is used to form a gel.

2. An exosome inulin gel, characterized in that The raw materials include inulin, aqueous solution and exosomes.

3. The inulin gel according to claim 1, wherein: The inulin is used as a matrix material and no additional chemical cross-linking agent is required.

4. The method for preparing inulin gel according to claim 1, characterized in that: The following steps are involved: Step 1: First, take an appropriate amount of solvent and preheat it to 70-100℃ in a 70-100℃ water bath; Step 2: Weigh an appropriate amount of inulin, add it to the preheated aqueous solution, mix and shake well, and continue to heat in a 70-100℃ water bath for 3-60 minutes; Step 3: Place the inulin aqueous solution at 0-25° C. and let it stand for 2-24 hours to obtain inulin gel.

5. The inulin gel according to claim 1, wherein: The solvent is water, PBS solution, physiological saline or other aqueous solutions.

6. The exosome inulin gel according to claim 2, characterized in that: The inulin is used as a matrix material and no additional chemical cross-linking agent is required.

7. The method for preparing the exosome inulin gel according to claim 2, wherein: The following steps are involved: Step 1: First, take an appropriate amount of solvent and preheat it to 70-100℃ in a 70-100℃ water bath; Step 2: Weigh an appropriate amount of inulin, add it to the preheated aqueous solution, mix and shake well, and continue to heat in a 70-100℃ water bath for 3-60 minutes; Step 3: After the inulin-water solution cools down, add exosomes and mix well; Step 4: Place the raw materials mixed evenly in step 3 in a 0-15° C. environment and let it stand for 2-24 hours to obtain the exosome inulin gel.

8. The method for preparing exosome inulin gel according to claim 5, characterized in that: The standing environment in step 4 can be a refrigerator or a low-temperature incubator, or can be left standing at room temperature. The standing time can be extended to 24 hours according to actual needs.

9. The method for preparing the exosome inulin gel according to claim 6, wherein: The preparation method may further include performing subsequent physical or chemical modification on the obtained exosome inulin gel to enhance its performance or impart specific functions to it.

10. The exosome inulin gel according to claim 3, characterized in that: The exosomes may be milk exosomes, plant exosomes, mesenchymal stem cell exosomes, plasma-derived exosomes, or macrophage-derived exosomes.

11. The use of the inulin gel or exosome inulin gel according to claims 1-2, characterized in that: Inulin gel and exosome inulin gel have significant effects in promoting skin repair, wound healing, and tissue repair. They can accelerate wound inflammation relief, tissue regeneration, collagen synthesis and remodeling, and are suitable for the treatment and care of various types of wounds such as burns, scalds, cuts, diabetic ulcers, etc.

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