Preparation method of exosome-loaded vaseline hydrogel composite emulsion

Abstract

The invention belongs to the technical field of exosomes, and discloses a preparation method of an exosome-loaded vaseline hydrogel composite emulsion, which comprises the following specific steps: step 1, raw material preparation: preparing medical vaseline, sodium alginate, an emulsifier (Tween 80), glycerol, an antioxidant (vitamin E) and an exosome, and carrying out quality inspection on all the raw materials to ensure that no impurity or deterioration exists; and 2, melting the vaseline. According to the vaseline hydrogel composite emulsion, vaseline and hydrogel are fused, the defects of a single carrier are overcome, the vaseline hydrogel composite emulsion has good air permeability and moisturizing sealing performance, exosome can be stably released, the skin absorption efficiency is improved, wound surfaces are effectively protected, the vaseline hydrogel composite emulsion is high in stability and can be stored for a long time, damage to the exosome in the preparation process is reduced through optimization, and the vaseline hydrogel composite emulsion has good application prospects. The damage of high-temperature and high-shear to activity is avoided, and the activity of the exosome is ensured, so that the healing effect on the wound is improved, and a better scheme is provided for wound treatment.

Description

Technical Field

[0001] This invention belongs to the field of exosome technology, specifically a method for preparing an exosome-loaded petrolatum hydrogel composite emulsion. Background Technology

[0002] Exosomes are nanoscale vesicles secreted by cells, with a diameter of about 30-150 nanometers. They have a lipid bilayer membrane structure and contain bioactive substances such as proteins, lipids, RNA and DNA. They are widely present in body fluids such as blood, saliva and urine. As messengers for intercellular communication, exosomes can regulate the function of receptor cells and participate in processes such as immune response and tissue repair.

[0003] When using exosomes to heal wounds, petrolatum ointment or pure hydrogel is often used as a carrier, but there are obvious drawbacks: petrolatum ointment has poor breathability and slow penetration, making it difficult for exosomes to be released and resulting in low skin absorption efficiency; pure hydrogel has weak moisturizing and sealing properties, is prone to drying and falling off, provides insufficient protection for the wound, and exosomes are easily deactivated. In addition, the simple physical mixing of the two is prone to layering, oil separation, and clumping, resulting in poor stability and difficulty in long-term preservation. Most formulations are not optimized for exosomes, and high temperature and high shear can easily cause them to rupture and lose activity, affecting the wound treatment effect. Therefore, improvements are needed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing an exosome-loaded petrolatum hydrogel composite emulsion, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing an exosome-loaded petrolatum hydrogel composite emulsion, the specific steps of which are as follows: Step 1: Raw Material Preparation Prepare medical-grade petrolatum, sodium alginate, emulsifier (Tween 80), glycerin, antioxidant (vitamin E), and exosomes. Conduct quality checks on all raw materials to ensure they are free of impurities and spoilage. Step 2: Melt Vaseline Weigh out an appropriate amount of medical petroleum jelly according to the formula ratio and place it in a container. Then place the container in a water bath and set the water bath temperature to 80-90℃. During the heating process, use a stirrer to slowly stir the petroleum jelly to ensure that it is heated evenly until it is completely dissolved. Step 3: Prepare the hydrogel phase According to the formula ratio, weigh out the corresponding amount of sodium alginate and slowly add it to pure water while stirring to promote dissolution. After it is completely dissolved, add the specified amount of emulsifier and 5% glycerin, and continue stirring to mix it evenly. Then place the mixture in a heating device and heat it to 85°C. Maintain this temperature for 10-15 minutes to ensure that all components are fully integrated. Step 4: Emulsification and Mixing After the petroleum jelly is completely dissolved and the hydrogel phase is heated to 85°C, the hydrogel phase is slowly poured into a container containing petroleum jelly. Then, the mixture is stirred with a high-speed stirrer, and the state of the mixture is closely observed during the stirring process until it forms a uniform milky white cream. Step 5: Cooling treatment After stopping stirring, remove the container containing the emulsified mixture from the heating equipment and place it in a room temperature environment to cool naturally until the temperature drops to 40-45℃. Step Six: Add antioxidants and exosomes According to the formula ratio, accurately weigh 0.5% of antioxidant and the required concentration of exosomes. Slowly add the antioxidant to the mixture cooled to 40-45℃ and gently stir to make it initially mixed. Then, slowly add the exosomes to the mixture to avoid mechanical damage to the exosomes. Step 7: Stir gently until well combined. Use a stirrer to gently stir the mixture at low speed to ensure that the antioxidants and exosomes are evenly dispersed in the mixture to form a homogeneous system. Step 8: Filling and Solidification Choose a clean, sterile container, slowly pour the mixed compound emulsion into the container, seal the container after filling, and place it at room temperature to allow it to solidify naturally.

[0006] As a preferred embodiment of the present invention, the weight percentages of medical petrolatum, sodium alginate, emulsifier, glycerin, and antioxidant in step one are as follows: medical petrolatum 70-80%, sodium alginate 10-20%, emulsifier 1-3%, glycerin 5%, and antioxidant 0.5%.

[0007] As a preferred technical solution of the present invention, after the container is placed in the water bath in step two, it is necessary to ensure that the container is completely submerged in water, but without allowing water to overflow. At the same time, the water level in the water bath should be closely observed during the heating process, and hot water should be added in a timely manner.

[0008] As a preferred technical solution of the present invention, the pure water mentioned in step three is deionized water or distilled water, and when adding sodium alginate to the pure water, it should be slowly sprinkled in and stirred continuously.

[0009] As a preferred embodiment of the present invention, when slowly pouring the hydrogel phase into the container containing petroleum jelly in step four, a dropper or funnel is used to control the pouring speed so that the hydrogel phase is evenly distributed in the petroleum jelly; when the high-speed stirrer in step four stirs the mixture, the speed is set to 2500-3500 rpm and the stirring time is 50-70 minutes.

[0010] As a preferred technical solution of the present invention, when the mixture is placed in a room temperature environment for natural cooling in step five, a thermometer is used to monitor the temperature of the mixture in real time, and the thermometer should be inserted into the center of the mixture to ensure accurate measurement.

[0011] As a preferred technical solution of the present invention, when the antioxidant is slowly added to the mixture cooled to 40-45°C in step six, it is necessary to add it while gently stirring so that the antioxidant can be quickly dispersed in the mixture.

[0012] As a preferred embodiment of the present invention, the stirring speed in step seven is controlled at 500-1000 rpm, and the stirring time is 5-10 minutes.

[0013] As a preferred technical solution of the present invention, the composite emulsion needs to undergo stability testing after preparation, specifically including: sensory and appearance stability testing, centrifugal stability testing, viscosity and rheology testing, exosome-specific testing, activity retention testing, and accelerated aging testing.

[0014] The beneficial effects of this invention are as follows: The petrolatum-hydrogel composite emulsion of this invention combines petrolatum and hydrogel, overcoming the shortcomings of a single carrier. It has both good breathability and moisturizing and occlusive properties, can stably release exosomes, improve skin absorption efficiency, effectively protect wounds, and has strong stability, allowing for long-term storage. Furthermore, the optimized preparation process reduces damage to exosomes and avoids the destruction of activity due to high temperature and high shear, ensuring the activity of exosomes and thus improving the healing effect on wounds, providing a better solution for wound treatment. Attached Figure Description

[0015] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0016] 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.

[0017] like Figure 1 As shown in the figure, this invention provides a method for preparing an exosome-loaded petrolatum hydrogel composite emulsion, the specific steps of which are as follows: Step 1: Raw Material Preparation Prepare medical-grade petrolatum, sodium alginate, emulsifier (Tween 80), glycerin, antioxidant (vitamin E), and exosomes. Conduct quality checks on all raw materials to ensure they are free of impurities and spoilage. Step 2: Melt Vaseline Weigh out an appropriate amount of medical petroleum jelly according to the formula ratio and place it in a container. Then place the container in a water bath and set the water bath temperature to 80-90℃. During the heating process, use a stirrer to slowly stir the petroleum jelly to ensure that it is heated evenly until it is completely dissolved. Step 3: Prepare the hydrogel phase According to the formula ratio, weigh out the corresponding amount of sodium alginate and slowly add it to pure water while stirring to promote dissolution. After it is completely dissolved, add the specified amount of emulsifier and 5% glycerin, and continue stirring to mix it evenly. Then place the mixture in a heating device and heat it to 85°C. Maintain this temperature for 10-15 minutes to ensure that all components are fully integrated. Step 4: Emulsification and Mixing After the petroleum jelly is completely dissolved and the hydrogel phase is heated to 85°C, the hydrogel phase is slowly poured into a container containing petroleum jelly. Then, the mixture is stirred with a high-speed stirrer, and the state of the mixture is closely observed during the stirring process until it forms a uniform milky white cream. Step 5: Cooling treatment After stopping stirring, remove the container containing the emulsified mixture from the heating equipment and place it in a room temperature environment to cool naturally until the temperature drops to 40-45℃. Step Six: Add antioxidants and exosomes According to the formula ratio, accurately weigh 0.5% of antioxidant and the required concentration of exosomes. Slowly add the antioxidant to the mixture cooled to 40-45℃ and gently stir to make it initially mixed. Then, slowly add the exosomes to the mixture to avoid mechanical damage to the exosomes. Step 7: Stir gently until well combined. Use a stirrer to gently stir the mixture at low speed to ensure that the antioxidants and exosomes are evenly dispersed in the mixture to form a homogeneous system. Step 8: Filling and Solidification Choose a clean, sterile container, slowly pour the mixed compound emulsion into the container, seal the container after filling, and place it at room temperature to allow it to solidify naturally.

[0018] Before preparation, the equipment needs to be cleaned and disinfected. For example, the water bath should be rinsed with clean water to remove residual impurities and dirt, and then the inner and outer walls should be wiped with 75% alcohol for disinfection. The stirrer needs to be disassembled and cleaned with neutral detergent, rinsed several times with pure water, and then wiped with alcohol for disinfection. Glassware such as measuring cylinders and beakers should be cleaned with detergent, rinsed with pure water, and then dried in a drying oven or air-dried naturally. If necessary, high-temperature and high-pressure sterilization can be performed.

[0019] In step one, the weight percentages of medical petrolatum, sodium alginate, emulsifier, glycerin, and antioxidant are as follows: medical petrolatum 70-80%, sodium alginate 10-20%, emulsifier 1-3%, glycerin 5%, and antioxidant 0.5%.

[0020] For medical-grade petroleum jelly, check its color uniformity, odor, and texture (whether it is fine and free of granules). Sodium alginate should be checked for purity; this can be done by reviewing the product inspection report or conducting a simple dissolution test to observe the dissolution rate and solution transparency. Emulsifiers should be checked to ensure they are a pale yellow, oily liquid with no layering or precipitation. Glycerin should be checked to ensure its transparency and viscosity meet standards. Antioxidants should be checked to ensure their color is pale yellow to yellow and that there are no signs of oxidation or deterioration. Exosomes require professional testing methods, such as Western blot and nanoparticle tracking analysis (NTA), to ensure their concentration and activity meet requirements.

[0021] In step two, after placing the container in the water bath, it is necessary to ensure that the container is completely submerged in water, but without causing the water to overflow. At the same time, during the heating process, the water level in the water bath should be closely monitored, and hot water should be added in a timely manner.

[0022] When melting petroleum jelly, choose a container that is heat-resistant and chemically stable, such as a stainless steel or glass beaker. The size of the container should be chosen appropriately according to the amount of medical petroleum jelly to ensure sufficient space for stirring and heating. During the heating process, be careful to prevent local overheating, which may cause the petroleum jelly to deteriorate or burn. If you find that the petroleum jelly has darkened in color or has an odor, stop heating immediately.

[0023] In step three, the pure water used is either deionized water or distilled water. When adding sodium alginate to the pure water, it should be added slowly and stirred continuously.

[0024] A magnetic stirrer can be used at a moderate speed to promote uniform dissolution. Lumps may form during dissolution; continue stirring until the lumps disappear completely. The order of adding the emulsifier and glycerin can be adjusted according to the actual situation, but be sure to stir thoroughly to ensure uniform mixing. Prevent the mixture from overflowing the container during heating.

[0025] In step four, when slowly pouring the hydrogel phase into the container containing petroleum jelly, use a dropper or funnel to control the pouring speed so that the hydrogel phase is evenly distributed in the petroleum jelly. When stirring the mixture with a high-speed stirrer in step four, the speed is set to 2500-3500 rpm and the stirring time is 50-70 minutes.

[0026] The speed and time of the high-speed mixer should be optimized according to the actual situation. Generally, the speed is set at 2500-3500 rpm and the mixing time is 50-70 minutes. However, it can be adjusted appropriately by observing the state of the mixture. If the mixture forms a uniform milky white cream in a short time, the mixing time can be shortened appropriately; otherwise, the mixing time needs to be extended.

[0027] In step five, when the mixture is placed in a room temperature environment to cool naturally, a thermometer is used to monitor the temperature of the mixture in real time. The thermometer should be inserted into the center of the mixture to ensure accurate measurement.

[0028] When cooling naturally, the container should be placed in a well-ventilated environment with a suitable room temperature. If a cooling device is used to accelerate cooling, an ice bath or a circulating water bath can be selected, but it is important to ensure that the temperature of the cooling device is uniform and to avoid localized excessively low temperatures. In addition, the cooling process should be slow and uniform to avoid sudden temperature drops that could cause the mixture to separate or crystallize.

[0029] In step six, when slowly adding the antioxidant to the mixture cooled to 40-45°C, it is necessary to gently stir while adding so that the antioxidant can be quickly dispersed in the mixture.

[0030] Exosomes are usually in the form of lyophilized powder or solution. If it is lyophilized powder, it should be reconstituted with an appropriate amount of buffer or physiological saline first. During the reconstitution process, be careful to gently shake or centrifuge to ensure that the exosomes are fully dissolved. Then, slowly add the reconstituted exosome solution to the mixture. Avoid generating bubbles and mechanical stirring during the addition process.

[0031] In step seven, the mixer speed is controlled at 500-1000 rpm, and the mixing time is 5-10 minutes.

[0032] Stirring at a low speed can avoid generating excessive shear force that could damage the exosomes. During stirring, it is necessary to closely observe the state of the mixture. When the mixture is homogeneous and there are no local concentrations that are too high or too low, stirring can be stopped. During stirring, care should be taken to ensure that the impeller blades do not touch the bottom or walls of the container to avoid friction and heat, which could affect the activity of the exosomes. If bubbles are found in the mixture, the stirring speed should be reduced or stirring should be stopped. Stirring can be resumed only after the bubbles disappear.

[0033] Among them, the composite emulsion needs to undergo stability testing after preparation, including: sensory and appearance stability testing, centrifugal stability testing, viscosity and rheology testing, exosome-specific testing, activity retention testing, and accelerated aging testing.

[0034] Sensory and appearance stability tests Test method: Take the prepared samples and store them in an environment of 25℃ / 60%RH and 40℃ / 75%RH for 1-3 months respectively. Observe the color, odor and uniformity of the samples visually every week. Judgment criteria: During storage, the sample should not change color or produce any odor, and should remain in a uniform state without any suspended matter or oil-water separation. If, during the observation process, the sample is found to have a significant color change, an odor, or unevenness such as layering or sedimentation, the sample is deemed to be unstable. Centrifugal stability test Test method: Take an appropriate amount of sample and put it into a centrifuge tube. Place the centrifuge tube in a centrifuge, set the centrifugation speed to 3000 rpm and the centrifugation time to 30 minutes. After centrifugation, observe whether the sample in the centrifuge tube shows stratification. Another method is to vacuum emulsify and disperse the sample and then cool it, and then check whether the sample shows discoloration or precipitation. Judgment criteria: If the sample does not show stratification after centrifugation, or if the sample does not change color or produce precipitation after vacuum emulsification, dispersion and cooling, it indicates that the sample has good centrifugal stability under the test conditions; if stratification, discoloration or precipitation occurs, it indicates that the sample has poor centrifugal stability. Viscosity and rheological testing Test method: The Brookfield viscometer was used to test the samples, and the storage modulus (G') and loss modulus (G'') of the samples were measured. The rheological curves were plotted based on the test data to analyze the rheological properties of the samples. Judgment criteria: The storage modulus (G') of the sample must be greater than the loss modulus (G''). This indicates that the sample can maintain a certain structural strength after coating and will not easily liquefy. If G'≤G'', it means that the sample may not be able to maintain a stable shape after coating and is prone to liquefaction, indicating poor stability. Exosome-specific assays, namely particle size and zeta potential assays Test methods: The size and polydispersity index (PDI) of exosomes were measured using a nanoparticle tracking analyzer (NTA) or a dynamic light scattering analyzer (DLS). Measurements were taken at the beginning of sample preparation and after a period of storage to compare the changes in exosome particle size and polydispersity index before and after storage. Judgment criteria: The exosome particle size should be less than 200 nm and remain stable, the polydispersity index (PDI) should be less than 0.3, and the changes in exosome particle size and polydispersity index after storage should be less than 10%. If these ranges are exceeded, it indicates that the exosomes may have aggregated or degraded during storage, and their stability has been affected. Activity retention test Test methods: The content of CD63 / CD81 protein in the sample was detected by enzyme-linked immunosorbent assay (ELISA), or the content of miRNA was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The tests were performed at the beginning of sample preparation and after a period of storage, and the retention rate of exosome activity was calculated. Judgment criteria: The exosome activity retention rate should be greater than 85%. If the retention rate is less than 85%, it indicates that the exosome activity has been lost during storage and the stability of the sample does not meet the requirements. Accelerated aging test Test method: The sample was stored at 40℃ / 75%RH for 3 months. According to the principle of accelerated aging test, this condition is equivalent to storage at room temperature (around 25℃) for 2 years. During the storage period, samples were taken periodically and high performance liquid chromatography (HPLC) was used to detect whether degradation products were generated in the sample. Judgment criteria: If no obvious degradation products are found during accelerated aging, or the content of degradation products is within an acceptable range, and other stability test indicators mentioned above are met, it indicates that the sample has good stability under accelerated aging conditions, and it can be inferred that it can remain relatively stable even after being stored at room temperature for 2 years; if a large amount of degradation products are detected, or other stability test indicators are not met, it indicates that the sample has poor stability and is not suitable for long-term storage.

[0035] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an exosome-loaded petrolatum hydrogel composite emulsion, characterized in that, The specific steps are as follows: Step 1: Raw material preparation Prepare medical-grade petrolatum, sodium alginate, emulsifier (Tween 80), glycerin, antioxidant (vitamin E), and exosomes. Conduct quality checks on all raw materials to ensure they are free of impurities and spoilage. Step 2: Melt Vaseline Weigh out an appropriate amount of medical petroleum jelly according to the formula ratio and place it in a container. Then place the container in a water bath and set the water bath temperature to 80-90℃. During the heating process, use a stirrer to slowly stir the petroleum jelly to ensure that it is heated evenly until it is completely dissolved. Step 3: Prepare the hydrogel phase According to the formula ratio, weigh out the corresponding amount of sodium alginate and slowly add it to pure water while stirring to promote dissolution. After it is completely dissolved, add the specified amount of emulsifier and 5% glycerin, and continue stirring to mix it evenly. Then place the mixture in a heating device and heat it to 85°C. Maintain this temperature for 10-15 minutes to ensure that all components are fully integrated. Step 4: Emulsification and Mixing After the petroleum jelly is completely dissolved and the hydrogel phase is heated to 85°C, the hydrogel phase is slowly poured into a container containing petroleum jelly. Then, the mixture is stirred with a high-speed stirrer, and the state of the mixture is closely observed during the stirring process until it forms a uniform milky white cream. Step 5: Cooling Treatment After stopping stirring, remove the container containing the emulsified mixture from the heating equipment and place it in a room temperature environment to cool naturally until the temperature drops to 40-45℃. Step Six: Add antioxidants and exosomes According to the formula ratio, accurately weigh 0.5% of antioxidant and the required concentration of exosomes. Slowly add the antioxidant to the mixture cooled to 40-45℃ and gently stir to make it initially mixed. Then, slowly add the exosomes to the mixture to avoid mechanical damage to the exosomes. Step 7: Gently stir until well combined. Use a stirrer to gently stir the mixture at low speed to ensure that the antioxidants and exosomes are evenly dispersed in the mixture to form a homogeneous system. Step 8: Filling and Solidification Choose a clean, sterile container, slowly pour the mixed compound emulsion into the container, seal the container after filling, and place it at room temperature to allow it to solidify naturally.

2. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: The weight percentages of the medical petrolatum, sodium alginate, emulsifier, glycerin, and antioxidant mentioned in step one are as follows: medical petrolatum 70-80%, sodium alginate 10-20%, emulsifier 1-3%, glycerin 5%, and antioxidant 0.5%.

3. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: After placing the container in the water bath as described in step two, it is necessary to ensure that the container is completely submerged in water, but without causing the water to overflow. At the same time, during the heating process, the water level in the water bath should be closely monitored, and hot water should be added in a timely manner.

4. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: The pure water mentioned in step three is deionized water or distilled water. When adding sodium alginate to the pure water, it should be added slowly and stirred continuously.

5. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: When slowly pouring the hydrogel phase into the container containing petroleum jelly in step four, use a dropper or funnel to control the pouring speed so that the hydrogel phase is evenly distributed in the petroleum jelly; when stirring the mixture with a high-speed stirrer in step four, the speed should be set to 2500-3500 rpm and the stirring time should be 50-70 minutes.

6. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: When the mixture is placed in a room temperature environment to cool naturally as described in step five, the temperature of the mixture should be monitored in real time using a thermometer. The thermometer should be inserted into the center of the mixture to ensure accurate measurement.

7. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: When adding the antioxidant slowly to the mixture cooled to 40-45°C as described in step six, it is necessary to stir gently while adding so that the antioxidant can be quickly dispersed in the mixture.

8. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: The stirring speed mentioned in step seven is controlled at 500-1000 rpm, and the stirring time is 5-10 minutes.

9. The method for preparing an exosome-loaded petrolatum hydrogel composite emulsion according to claim 1, characterized in that: The composite emulsion needs to undergo stability testing after preparation, specifically including: sensory and appearance stability testing, centrifugal stability testing, viscosity and rheology testing, exosome-specific testing, activity retention testing, and accelerated aging testing.

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