Preparation method and application of stem cell exosome-carnosine loader

Abstract

The invention provides a preparation method and application of a stem cell exosome-carnosine loader. The preparation method comprises the following steps: firstly, treating cell supernatant of the third generation to the fifth generation of human umbilical cord mesenchymal stem cells by adopting a centrifugal method in combination with an ultra-sonar filtration method to obtain purified exosomes; then uniformly mixing the stem cell exosome with a carnosine solution, then adding a medicine, and obtaining the exosome-carnosine loading substance by adopting ultrasonic, incubation and ultrafiltration methods. By using the exosome-carnosine load, the maximum drug loading capacity of the carnosine is determined while the biological activity of the carnosine is ensured to be maintained, and more excellent anti-aging repair capacity is achieved through synergistic interaction. And the lipophilicity of barrier stratum corneum (SC) on the uppermost surface of the skin can be overcome, and compared with traditional liposome wrapping, the nano-liposome has better transdermal absorption capacity.

Description

Technical Field

[0001] The present invention relates to the technical field of skin anti-wrinkle repair, and relates to a preparation method and application of a stem cell exosome-carnosine loading material. Background Art

[0002] Exosomes are nanoscale vesicles secreted by cells, ranging in diameter from 30 to 150 nm. Rich in lipids, mRNA, microRNA, and a variety of proteins composed of growth factors and immune factors, exosomes participate in intercellular communication, alter tissue microenvironment, and regulate cellular biological functions and behavior. Due to their small size and lipid bilayer structure, exosomes can serve as a unique biological delivery system, effectively delivering bioactive molecules into cells, improving bioavailability and enabling precise treatment and repair. Compared to traditional delivery methods, exosomes offer the advantages of high efficiency, excellent biocompatibility, and synergistic effects.

[0003] Exosomes have been extensively studied for their potential applications in skin care. For example, miR-150-5P, expressed in stem cell exosomes, can accelerate the proliferation of fibroblasts and keratinocytes by regulating the PTEN-mediated PI3K / AKT pathway, replenishing aging or damaged cells and promoting skin rejuvenation. MiR-181c, expressed in stem cell exosomes, inhibits TLR4 signaling, reducing NF-κB / p65 activation and thereby alleviating inflammation. MiR-146a can inhibit the expression of pro-inflammatory genes and reduce the production of inflammatory factors, resulting in a soothing effect on the skin. Stem cell exosomes can also activate the AKT / HIF1-α pathway, promoting keratinocyte proliferation and migration, resulting in a beneficial skin repair effect.

[0004] Carnosine is a dipeptide composed of two amino acids, β-alanine and L-histidine. It is highly hydrophilic, with a partition coefficient (log P) of -2.972±0.436. As a natural antioxidant, carnosine has been shown to scavenge reactive oxygen species (ROS) and α-β unsaturated aldehydes formed by the overoxidation of fatty acids during oxidative stress, thereby delaying skin aging. Carnosine also reacts with sugars, replacing the physiological reaction between proteins and sugars, protecting proteins from glycation and achieving an anti-glycation effect. Furthermore, carnosine can enhance the skin's moisturizing ability, increase the water content of the stratum corneum, form a moisturizing barrier, and improve skin hydration.

[0005] In the field of dermatology, the efficacy of any cosmetic product containing an active ingredient is determined by two factors: the molecule's intrinsic activity and its ability to reach the site of action. To exert its function, the active ingredient must be delivered to the site of action at the appropriate concentration and for the appropriate time. To achieve effective concentrations in deeper skin tissues, the stratum corneum (SC), the skin's uppermost barrier, must be overcome. Due to the lipophilic nature of the SC, achieving skin bioavailability of hydrophilic actives is challenging.

[0006] In the prior art, despite its small molecular weight, carnosine has poor transdermal absorption. Current approaches to address this problem primarily rely on liposome encapsulation. While liposome encapsulation can improve carnosine's transdermal ability to a certain extent, the transdermal effect remains unsatisfactory. Furthermore, due to their poor biocompatibility, liposomes can easily cause skin allergic reactions after encapsulating substances such as carnosine, resulting in low safety. Therefore, the present invention proposes the use of exosomes derived from human umbilical cord mesenchymal stem cells as a carrier. Exosomes themselves have high biocompatibility and good safety. While improving carnosine's transdermal absorption, they can also protect the biological activity of carnosine, creating a synergistic effect and achieving a superior skincare effect. Summary of the Invention

[0007] In view of the deficiencies of the existing technology, the present invention provides a method for preparing a stem cell exosome-carnosine loading material and its application.

[0008] To achieve the above objectives, one aspect of the present invention provides a method for preparing a stem cell exosome-carnosine loading material, comprising the following steps:

[0009] S1. Transfer human umbilical cord mesenchymal stem cells of passage 3 to a culture dish and culture them until passage 5 to obtain cell supernatants of passages 3 to 5;

[0010] S2. Filter through a 0.22 μm syringe filter to remove cell debris and impurities, centrifuge at 300 × g for 10 minutes to remove dead cells and large cell debris, centrifuge at 2000 × g for 10 minutes to remove cell debris, and centrifuge at 10,000 × g for 10 minutes to remove large vesicles and small cell debris. Then, 50 mL of the centrifuge fluid was added to a nano-ultrafiltration chip and enriched and purified using a negative pressure oscillation and dual-coupled ultrasonic oscillation system to obtain purified stem cell exosomes for future use.

[0011] S3. Place the purified stem cell exosomes obtained in step S2 into an ultrasonic device, and then add the carnosine solution to obtain a mixture of exosomes and carnosine;

[0012] S4. The exosome-carnosine mixture obtained in step S3 was subjected to ultrasonication under the following conditions: power 200-300 W, single working time 10 s-20 s, rest time 2 min-3 min, number of cycles 5-8 times, and system temperature 4-8°C;

[0013] S5. Incubate at 37°C for 60-180 min.

[0014] S6. Purify at a speed of 4000-5000 rpm, a time of 10-30 min, a temperature of 4-8°C, and an ultrafiltration membrane pore size of 100 kDa to obtain the stem cell exosome-carnosine load.

[0015] Furthermore, the protein concentration of the stem cell exosomes is 0.4-500 ug / mL; the carnosine concentration is 5-1000 ug / mL.

[0016] Furthermore, the exosome:carnosine drug input ratio is one of 2:1, 1:1, 1:2, 1:4, 1:8, 1:10 or 1:12.

[0017] Furthermore, the exosome concentration in the stem cell exosome-carnosine loading material is ≥50 ug / mL and still has biological activity after freezing and thawing.

[0018] Another aspect of the present invention also provides a stem cell exosome-carnosine loading material prepared by the above-mentioned method for preparing the stem cell exosome-carnosine loading material, wherein the exosome:carnosine drug input ratio is 1:8, and the cumulative permeation amount in 12 hours is increased by 310%.

[0019] Furthermore, the stem cell exosome-carnosine loading material is used to effectively promote the regenerative ability of fibroblasts and keratinocytes.

[0020] Furthermore, the exosome protein concentration is 5-500 ug / mL, the carnosine concentration is 10-200 ug / mL, and the exosome:carnosine drug input ratio is one of 1:1, 1:2, 1:4, 1:8, 1:10 or 1:12.

[0021] Furthermore, the stem cell exosome-carnosine loading material may be an aqueous solution, an emulsion or a freeze-dried powder.

[0022] Furthermore, the stem cell exosome-carnosine loading material is used in cosmetics.

[0023] Furthermore, the stem cell exosome-carnosine loading material is used in a facial mask.

[0024] The beneficial effects of the present invention compared to the prior art are:

[0025] The present invention uses ultrasound combined with co-incubation to load stem cell exosomes with carnosine. The exosomes and carnosine are mixed, and the mechanical shear force of ultrasound destroys the membrane integrity of the exosomes, allowing carnosine to enter the exosomes, thereby making carnosine loading more efficient. In addition, the co-incubation method not only restores the membrane integrity and natural activity of the exosomes, but also enables carnosine to be loaded on the surface of the exosomes, effectively improving the stratum corneum penetration. The method for preparing the stem cell exosome-carnosine loading material of the present invention achieves a drug loading capacity of 94.4 μg / mL, which is nearly double the traditional drug loading capacity of 54.4 μg / mL. The exosomes and carnosine form the stem cell exosome-carnosine loading material, and the synergistic effect of the two effectively increases the drug loading capacity and the transdermal absorption capacity. According to the "In Vitro Test Method for Skin Absorption of Chemicals" test, the cumulative penetration over 12 hours increased by 310%, which has unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A is a transmission electron microscopy image of exosomes.

[0027] Figure 1 B is the exosome particle size distribution diagram.

[0028] Figure 1 C is a nanoflow cytometry detection diagram of positive protein markers CD9, CD63, and CD81.

[0029] Figure 2 A Transmission electron microscopy image of exosome-carnosine loading.

[0030] Figure 2 B Particle size distribution of exosome-carnosine loaded materials.

[0031] Figure 2 C Western blot detection of exosome-carnosine loading.

[0032] Figure 3A This is the standard curve of carnosine.

[0033] Figure 3B The chromatograms are shown for HPLC detection of carnosine at different concentrations.

[0034] Figure 4A The results of HPLC detection of carnosine and exosome-carnosine loading before and after freeze-thaw in pure water and normal saline.

[0035] Figure 4B The bar graphs show the HPLC detection of carnosine and exosome-carnosine loading before and after freeze-thaw in pure water and normal saline.

[0036] Figure 5AThis is the HPLC detection chart under different drug loading methods based on co-incubation, freeze-thaw method and ultrasound method.

[0037] Figure 5B A bar chart showing the drug loading amount based on different drug loading methods including co-incubation, freeze-thaw method, and ultrasound method.

[0038] Figure 6A HPLC detection diagram under different ultrasonic conditions.

[0039] Figure 6B Download the bar graph of drug dosage for different ultrasound conditions.

[0040] Figure 7A This is the HPLC detection diagram of exosome-carnosine under different drug inputs.

[0041] Figure 7B This is a bar chart showing the download dosage of exosome-carnosine under different drug inputs.

[0042] Figure 8 Statistical bar graphs of the toxicity and proliferation of carnosine, exosomes, and exosome-carnosine-loaded substances on human fibroblasts.

[0043] Figure 9 Statistical bar graphs of the toxicity and proliferation of carnosine, exosomes, and exosome-carnosine-loaded substances on keratinocytes.

[0044] Figure 10 This is a bar graph showing the effects of carnosine, exosomes, and exosome-carnosine loading on the expression of type I collagen in human fibroblasts.

[0045] Figure 11A This is a diagram of the cell migration experiment of keratinocytes using carnosine, exosomes, and exosome-carnosine-loaded substances.

[0046] Figure 11B This is a bar graph showing statistical analysis of cell migration rate. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the following embodiments. Unless otherwise defined, technical or scientific terms used herein shall have the same meanings as those commonly understood by persons of ordinary skill in the art to which the present invention pertains. The above-mentioned features or features described in the specific examples of the present invention may be combined in any manner. These specific embodiments are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0048] Exosomes are nanoscale vesicles secreted by cells, ranging in diameter from 30 to 150 nm. Rich in lipids, mRNA, microRNA, and a variety of proteins composed of growth factors and immune factors, exosomes participate in intercellular communication, alter tissue microenvironment, and regulate cellular biological functions and behavior. Due to their small size and lipid bilayer structure, exosomes can serve as a unique biological delivery system, effectively delivering bioactive molecules into cells, improving bioavailability and enabling precise treatment and repair. Compared to traditional delivery methods, exosomes offer the advantages of high efficiency, excellent biocompatibility, and synergistic effects.

[0049] MiR-150-5P in stem cell exosomes can accelerate the proliferation of fibroblasts and keratinocytes by regulating the PTEN-mediated PI3K / AKT pathway, replenishing aging or damaged cells and promoting skin rejuvenation. MiR-181c in stem cell exosomes reduces inflammation by inhibiting the TLR4 signaling pathway and reducing NF-κB / p65 activation. MiR-146a inhibits the expression of pro-inflammatory genes and reduces the production of inflammatory factors, achieving a soothing effect on the skin. Stem cell exosomes can also activate the AKT / HIF1-α pathway, promoting keratinocyte proliferation and migration, resulting in a beneficial repair effect.

[0050] Carnosine is a dipeptide composed of two amino acids, β-alanine and L-histidine. It is highly hydrophilic, with a partition coefficient (log P) of -2.972±0.436. As a natural antioxidant, carnosine has been shown to scavenge reactive oxygen species (ROS) and α-β unsaturated aldehydes formed by the overoxidation of fatty acids during oxidative stress, thereby delaying skin aging. Carnosine also reacts with sugars, replacing the physiological reaction between proteins and sugars, protecting proteins from glycation and achieving an anti-glycation effect. Furthermore, carnosine can enhance the skin's moisturizing ability, increase the water content of the stratum corneum, form a moisturizing barrier, and improve skin hydration.

[0051] In the prior art, although carnosine has a small molecular weight, its transdermal absorption capacity is poor. The current means to solve this problem mainly relies on liposome encapsulation. Although liposome encapsulation can improve the transdermal ability of carnosine to a certain extent, the transdermal effect is still unsatisfactory. At the same time, because liposomes have poor biocompatibility, they are prone to cause skin allergic reactions after encapsulating substances such as carnosine, and their safety is not high.

[0052] This study leverages the natural biological properties of stem cell exosomes and proposes a loading strategy based on ultrasound combined with co-incubation, successfully loading carnosine into stem cell exosomes. Carnosine interacts with water molecules, increasing the hydration level of the stratum corneum, which in turn causes stratum corneum cells to swell, forming a porous structure and increasing the effective surface area of the skin, thereby promoting transdermal absorption of the exosomes. Furthermore, the exosomes encapsulate carnosine, protecting its biological activity. Furthermore, the exosomes' nanoscale size and lipid-soluble membrane enhance carnosine's bioavailability, achieving synergistic transdermal and skincare benefits.

[0053] Example 1

[0054] A method for preparing a stem cell exosome-carnosine loading material comprises the following steps:

[0055] S1. Transfer human umbilical cord mesenchymal stem cells of passage 3 to a culture dish and culture them until passage 5 to obtain cell supernatants of passages 3 to 5;

[0056] S2. Filter through a 0.22 μm syringe filter to remove cell debris and impurities, centrifuge at 300 × g for 10 minutes to remove dead cells and large cell debris, centrifuge at 2000 × g for 10 minutes to remove cell debris, and centrifuge at 10,000 × g for 10 minutes to remove large vesicles and small cell debris. Then, 50 mL of the centrifuge fluid was added to a nano-ultrafiltration chip and enriched and purified using a negative pressure oscillation and dual-coupled ultrasonic oscillation system to obtain purified stem cell exosomes for future use.

[0057] S3. Place the purified stem cell exosomes obtained in step S2 into an ultrasonic device, and then add the carnosine solution to obtain a mixture of exosomes and carnosine;

[0058] S4. The exosome-carnosine mixture obtained in step S3 was subjected to ultrasonication under the following conditions: power 200, single working time 10 s, rest time 2 min, number of cycles 5, and system temperature 4°C;

[0059] S5. Incubate at 37°C for 60 min.

[0060] S6. Purify at a speed of 4000 rpm, a time of 10, a temperature of 4°C, and an ultrafiltration membrane pore size of 100 kDa to obtain the stem cell exosome-carnosine load.

[0061] Example 2

[0062] A method for preparing a stem cell exosome-carnosine loading material comprises the following steps:

[0063] S1. Transfer human umbilical cord mesenchymal stem cells of passage 3 to a culture dish and culture them until passage 5 to obtain cell supernatants of passages 3 to 5;

[0064] S2. Filter through a 0.22 μm syringe filter to remove cell debris and impurities, centrifuge at 300 × g for 10 minutes to remove dead cells and large cell debris, centrifuge at 2000 × g for 10 minutes to remove cell debris, and centrifuge at 10,000 × g for 10 minutes to remove large vesicles and small cell debris. Then, 50 mL of the centrifuge fluid was added to a nano-ultrafiltration chip and enriched and purified using a negative pressure oscillation and dual-coupled ultrasonic oscillation system to obtain purified stem cell exosomes for future use.

[0065] S3. Place the purified stem cell exosomes obtained in step S2 into an ultrasonic device, and then add the carnosine solution to obtain a mixture of exosomes and carnosine;

[0066] S4. The exosome-carnosine mixture obtained in step S3 was subjected to ultrasonication under the following conditions: power 300 W, single working time 20 s, rest time 3 min, 8 cycles, and system temperature 8°C;

[0067] S5. Incubate at 37°C for 180 min.

[0068] S6. Purify at a speed of 5000 rpm, a time of 30 min, a temperature of 4-8°C, and an ultrafiltration membrane pore size of 100 kDa to obtain the stem cell exosome-carnosine load.

[0069] In the optimal embodiment of the present invention, the ultrasonic conditions are: ultrasonic power 280W, single working time 15s, rest time 2min, cycle number 8 times, system temperature 4°C, incubation conditions, 37°C, 120min.

[0070] Exemplarily, the protein concentration of the stem cell exosomes is 0.4-500 ug / mL; the carnosine concentration is 5-1000 ug / mL.

[0071] Exemplarily, the exosome:carnosine drug input ratio is one of 2:1, 1:1, 1:2, 1:4, 1:8, 1:10 or 1:12.

[0072] Exemplarily, the exosome concentration in the stem cell exosome-carnosine loading material is ≥50 ug / mL and still has biological activity after freezing and thawing.

[0073] Exemplarily, the stem cell exosome-carnosine loading material is used to increase the transdermal absorption capacity of carnosine and improve the bioavailability of carnosine.

[0074] Another aspect of the present invention also provides a stem cell exosome-carnosine loading material prepared by the above-mentioned method for preparing the stem cell exosome-carnosine loading material, wherein the exosome:carnosine drug input ratio is 1:8, and the cumulative permeation amount in 12 hours is increased by 310%.

[0075] Another aspect of the present invention provides a stem cell exosome-carnosine loading material, produced by the aforementioned method, which exhibits a greater ability to promote fibroblast proliferation than carnosine alone, exosomes alone, or a mixture of carnosine and exosomes. Therefore, the stem cell exosome-carnosine loading material can synergistically promote fibroblast proliferation, exhibiting excellent anti-aging potential while also reducing economic costs.

[0076] Another aspect of the present invention provides a stem cell exosome-carnosine loading material, produced by the aforementioned method for preparing the stem cell exosome-carnosine loading material, which exhibits a greater ability to increase keratinocyte proliferation than carnosine alone, exosomes alone, or a mixture of carnosine and exosomes. Therefore, the stem cell exosome-carnosine loading material can achieve a synergistic effect in promoting keratinocyte proliferation, demonstrating excellent efficacy in promoting epidermal cell renewal while reducing economic benefits.

[0077] Another aspect of the present invention provides a stem cell exosome-carnosine-loaded material produced by the aforementioned method for preparing a stem cell exosome-carnosine-loaded material. This material increases collagen expression in fibroblasts compared to pure carnosine, pure exosomes, or a mixture of carnosine and exosomes. Thus, the stem cell exosome-carnosine-loaded material can achieve a synergistic effect in promoting collagen expression, demonstrating excellent anti-aging capabilities while also reducing economic benefits. Another aspect of the invention also provides a stem cell exosome-carnosine-loaded material produced by the aforementioned method for preparing a stem cell exosome-carnosine-loaded material. This material can increase the migration rate of keratinocytes, achieving a synergistic effect in promoting keratinocyte migration and demonstrating excellent repair efficacy.

[0078] Exemplarily, the exosome protein concentration is 5-500 ug / mL, the carnosine concentration is 10-200 ug / mL; and the exosome:carnosine drug input ratio is one of 1:1, 1:2, 1:4, 1:8, 1:10 or 1:12.

[0079] The preferred exosome:carnosine drug input ratio is 1:2.

[0080] Exemplarily, the stem cell exosome-carnosine loading material may be an aqueous solution, an emulsion, or a lyophilized powder.

[0081] Exemplarily, the stem cell exosome-carnosine loading substance is used in cosmetics. By applying the stem cell exosome-carnosine loading substance to cosmetics, it has a better skin-beautifying effect due to its high drug loading capacity and high transdermal property.

[0082] Exemplarily, the stem cell exosome-carnosine loading material is used in a facial mask. Exosomes have the effect of protecting the biological activity of carnosine, so the two act synergistically to make the stem cell exosome-carnosine loading material have better moisturizing properties.

[0083] Figure 1 A is a transmission electron microscopy image of exosomes. Figure 1 B is the particle size distribution diagram, Figure 1 C is the nanoflow cytometry detection diagram of positive protein markers CD9, CD63, and CD81.

[0084] The experimental results show that:

[0085] Figure 1 A is a transmission electron microscopy image of exosomes, which have a typical "saucer-like" morphology.

[0086] Figure 1 B shows that the overall particle size of exosomes is 40-150 nm, with an average particle size of 73.3 nm, which is consistent with the typical size of exosomes.

[0087] Figure 1 C is a nanoflow cytometry detection image, in which CD9, CD63, and CD81 are all positively expressed, which is consistent with the positive protein expression characteristics on the surface of exosomes.

[0088] Figure 2 A. Figure 2 B. Figure 2 The results of the C test showed that Figure 2 A is a transmission electron microscopy image of the exosome-carnosine loading. It can be seen that the morphology of the exosomes did not change after loading.

[0089] Figure 2 B is the particle size distribution diagram of the exosome-carnosine loaded material. It can be seen that after loading with carnosine, the overall particle size is slightly larger, with an average particle size of 82.7 nm.

[0090] Figure 2 C is the WB detection image of exosome-carnosine loading, in which all exosome positive proteins are expressed and all negative proteins are not expressed.

[0091] Figure 2 The overall results showed that carnosine was successfully loaded into exosomes without affecting the properties of the exosomes themselves.

[0092] Figure 3A Based on Figure 3BThe standard curve is drawn with the horizontal axis representing the concentration of carnosine and the vertical axis representing the peak area.

[0093] Figure 3B This is the chromatogram of HPLC detection of different concentrations of carnosine (12.5μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 400μg / mL). Figure 3B This is the HPLC peak diagram for detecting carnosine at different concentrations.

[0094] Figure 4A The chromatograms of carnosine and exosome-carnosine loading before and after freezing and thawing in pure water and normal saline. Figure 4B Based on Figure 4A The chromatogram shows a bar chart of carnosine content. The results show that the stem cell exosomes (MSC-EXO) themselves have no peak. A 200 μg / mL carnosine solution was prepared and frozen and thawed in PBS or saline, and then HPLC detection was performed. It was found that the carnosine content in PBS was 173.3 μg / mL, and the carnosine content in saline was 170.8 μg / mL. When stem cell exosomes were added and frozen and thawed, the carnosine content in PBS was 205.1 μg / mL, and the carnosine content in saline was 203.04 μg / mL. The results show that stem cell exosomes can effectively protect the biological activity of carnosine.

[0095] Figure 5A The chromatograms of carnosine detected by HPLC were obtained based on co-incubation with three drug loading methods, freeze-thaw method, and ultrasound method.

[0096] Figure 5B Based on Figure 5A The bar chart of the carnosine drug loading in the chromatogram shows that the drug loading by co-incubation is 54.4 μg / mL, the drug loading by freeze-thaw method is 51.8 μg / mL, and the drug loading by ultrasound method is 94.4 μg / mL.

[0097] The results showed that the drug loading capacity of the ultrasound method was the highest, followed by co-incubation. Therefore, the ultrasound method combined with co-incubation was selected as the drug loading method.

[0098] Figure 6A This is the chromatogram of HPLC detection of carnosine under different input ratios and different ultrasonic conditions. Figure 6B Based on Figure 6A Histogram of the chromatogram.

[0099] The experimental results showed that stem cell exosomes (MSC-EXO) themselves had no peak. When the ultrasonic process was not optimized, when the carnosine: exosome (Carnosine: EXO) ratio was 1:2, the drug loading capacity was 50.6μg / mL, when the carnosine: exosome (Carnosine: EXO) ratio was 1:1, the drug loading capacity was 69.02μg / mL, and when the carnosine: exosome (Carnosine: EXO) ratio was 2:1, the drug loading capacity was 97.8μg / mL. After optimizing the ultrasonic process, the drug loading capacity was 123.5 μg / mL when the carnosine:exosome ratio was 1:2, 165.1 μg / mL when the carnosine:exosome ratio was 1:1, and 216.9 μg / mL when the carnosine:exosome ratio was 2:1. In summary, under the same ultrasonic process, the drug loading capacity increased with increasing carnosine input; after optimizing the ultrasonic power, single working time, and number of cycles, the drug loading capacity was increased at the same carnosine:exosome ratio.

[0100] Figure 7A This is the HPLC chromatogram of carnosine and exosomes at different input ratios. Figure 7B Based on Figure 7A Histogram of the chromatogram.

[0101] The experimental results showed that when the carnosine: exosome ratio was 1:2, the drug loading capacity was 123.9 μg / mL; when the carnosine: exosome ratio was 1:1, the drug loading capacity was 164.7 μg / mL; when the carnosine: exosome ratio was 2:1, the drug loading capacity was 261.1 μg / mL; when the carnosine: exosome ratio was 4:1, the drug loading capacity was 329.4 μg / mL, when the carnosine: exosome ratio was 8:1, the drug loading capacity was 499.7 μg / mL, when the carnosine: exosome ratio was 10:1, the drug loading capacity was 368.2 μg / mL, and when the carnosine: exosome ratio was 12:1, the drug loading capacity was 358.8 μg / mL. In summary, when the carnosine: exosome ratio was 8:1, it had the highest drug loading capacity of 499.7 μg / mL.

[0102] Figure 8The experimental results showed that DMEM was the blank control, Carnosine was a simple carnosine solution with a concentration of 500 μg / mL, MSC-EXO was a stem cell exosome with a particle concentration of 1×10*11particles / mL, Carnosine+MSC-EXO was a simple mixture of stem cell exosomes (1×10*11particles / mL) and carnosine (500 μg / mL), Car-EXO was an exosome-carnosine load prepared by the method described in the present invention by co-incubation with carnosine loaded into stem cell exosomes by ultrasound, Car-EXO (2×) was a Car-EXO solution diluted 2 times, and Car-EXO (5×) was a Car-EXO solution diluted 5 times.

[0103] The results showed that the cell activity of fibroblasts treated with Carnosine was 105.6%, the cell activity of fibroblasts treated with MSC-EXO was 110.9%, the cell activity of fibroblasts treated with Carnosine+MSC-EXO was 116.9%, the cell activity of fibroblasts treated with Car-EXO was 130.1%, the cell activity of fibroblasts treated with Car-EXO (2×) was 115.4%, and the cell activity of fibroblasts treated with Car-EXO (5×) was 106.5%.

[0104] In summary, carnosine alone, exosomes alone, a mixture of carnosine and exosomes, and carnosine loaded onto exosomes showed no cytotoxicity to fibroblasts. Car-EXO exhibited the highest cell proliferation capacity, and even after two-fold dilution, its cell proliferation capacity remained higher than that of carnosine alone, exosomes alone, and a mixture of carnosine and exosomes alone. Therefore, loading carnosine into exosomes can achieve a synergistic effect in promoting fibroblast proliferation, possessing excellent anti-aging potential and reducing economic benefits.

[0105] Figure 9 Statistical bar graphs of the toxicity and proliferation of carnosine, exosomes, and exosome-carnosine-loaded substances on keratinocytes.

[0106] Interpretation of experimental results: The results showed that the cell activity of keratinocytes after treatment with Carnosine was 105.06%, the cell activity of keratinocytes after treatment with MSC-EXO was 110.4%, the cell activity of keratinocytes after treatment with Carnosine+MSC-EXO was 115.7%, the cell activity of keratinocytes after treatment with Car-EXO was 125.9%, the cell activity of keratinocytes after treatment with Car-EXO (2×) was 115.8%, and the cell activity of keratinocytes after treatment with Car-EXO (5×) was 107.15%.

[0107] In summary, carnosine alone, exosomes alone, a mixture of carnosine and exosomes, and carnosine loaded onto exosomes all showed no cytotoxicity to keratinocytes. Car-EXO exhibited the highest cell proliferation capacity, and even after two-fold dilution, its cell proliferation capacity remained higher than that of carnosine alone, exosomes alone, and a mixture of carnosine and exosomes alone. Therefore, loading carnosine into exosomes can achieve a synergistic effect in promoting keratinocyte proliferation, demonstrating excellent efficacy in promoting epidermal cell renewal while also reducing economic benefits.

[0108] Figure 10 This is a bar graph showing the effects of carnosine, exosomes, and exosome-carnosine loading on the expression of type I collagen in human fibroblasts.

[0109] Interpretation of experimental results: After Carnosine treatment of fibroblasts, the collagen expression level was 95.4 ng / mL, after MSC-EXO treatment of fibroblasts, the collagen expression level was 104.2 ng / mL, after Carnosine+EXO treatment of fibroblasts, the collagen expression level was 110.3 ng / mL, after Car-EXO treatment of fibroblasts, the collagen expression level was 127.7 ng / mL, after Car-EXO (2×) treatment of fibroblasts, the collagen expression level was 109.3 ng / mL, and after Car-EXO (5×) treatment of fibroblasts, the collagen expression level was 101.9 ng / mL. In summary, after Car-EXO treated fibroblasts, the collagen expression level was the highest. After 2-fold dilution, the collagen expression level was still higher than that of pure carnosine, pure exosomes, and a mixture of carnosine and exosomes. Therefore, loading carnosine into exosomes can achieve the effect of synergistically promoting collagen expression, showing excellent anti-aging ability and reducing economic benefits.

[0110] Figure 11A A is a graph showing the cell migration experiment of keratinocytes using carnosine, exosomes, and exosome-carnosine loading. B is a bar graph showing the statistical analysis of cell migration rate.

[0111] Interpretation of experimental results: After Carnosine treatment of keratinocytes, the cell migration rate was 26.3% after 6 hours, after MSC-EXO treatment of keratinocytes, the cell migration rate was 32.03% after 6 hours, after Carnosine+EXO treatment of keratinocytes, the cell migration rate was 52.2% after 6 hours, after Car-EXO treatment of keratinocytes, the cell migration rate was 71.9% after 6 hours, after Car-EXO (2×) treatment of keratinocytes, the cell migration rate was 43.1% after 6 hours, and after Car-EXO (5×) treatment of keratinocytes, the cell migration rate was 22.3% after 6 hours. In summary, after Car-EXO treatment of keratinocytes, the cell migration rate was the highest after 6 hours, which can achieve the ability of synergistically promoting keratinocyte migration and show excellent repair efficacy.

[0112] Table 1 shows the cumulative permeation results of carnosine, liposome-encapsulated carnosine, and exosome-carnosine loaded products.

[0113]

[0114] Interpretation of experimental results: The Franz diffusion cell method was used to quantitatively analyze the permeation of pig skin at three time points: 1 hour, 6 hours, and 12 hours. The results showed that when carnosine was loaded into exosomes, the permeation rate was the highest at all three time points. This was higher than the permeation rates of carnosine, exosomes, a mixture of carnosine and exosomes, and liposome-encapsulated carnosine. This confirms that carnosine can interact with water molecules, increasing the hydration level of the stratum corneum, causing the stratum corneum cells to swell and form a porous structure, thereby promoting the transdermal absorption of exosomes. Furthermore, the nanoscale size and lipid-soluble membrane of exosomes enhance the bioavailability of carnosine, achieving a synergistic transdermal effect.

[0115] The present invention uses ultrasound combined with co-incubation to load stem cell exosomes-carnosine. The exosomes and carnosine are mixed, and the mechanical shear force of ultrasound destroys the membrane integrity of the exosomes, allowing carnosine to enter the exosomes, thereby making carnosine loading more efficient. In addition, the co-incubation method not only restores the membrane integrity and natural activity of the exosomes, but also enables carnosine to be loaded on the surface of the exosomes, effectively improving the stratum corneum penetration. The method for preparing the stem cell exosome-carnosine loading material of the present invention achieves a drug loading capacity of 94.4 μg / mL, which is nearly double the traditional drug loading capacity of 54.4 μg / mL. The exosomes and carnosine form the stem cell exosome-carnosine loading material, and the synergistic effect of the two effectively increases the drug loading capacity while effectively improving the transdermal absorption capacity. According to the "In Vitro Test Method for Skin Absorption of Chemicals" test, the cumulative penetration over 12 hours increased by 310%.

Claims

1. A method for preparing a stem cell exosome-carnosine loading material, characterized by: The following steps are involved: S1. Transfer human umbilical cord mesenchymal stem cells of passage 3 to a culture dish and culture them until passage 5 to obtain cell supernatants of passages 3 to 5; S2. Filter through a 0.22 μm syringe filter to remove cell debris and impurities, centrifuge at 300 × g for 10 minutes to remove dead cells and large cell debris, centrifuge at 2000 × g for 10 minutes to remove cell debris, and centrifuge at 10,000 × g for 10 minutes to remove large vesicles and small cell debris. Then, 50 mL of the centrifuge fluid was added to a nano-ultrafiltration chip and enriched and purified using a negative pressure oscillation and dual-coupled ultrasonic oscillation system to obtain purified stem cell exosomes for future use. S3. Place the purified stem cell exosomes obtained in step S2 into an ultrasonic device, and then add the carnosine solution to obtain a mixture of exosomes and carnosine; S4. The exosome-carnosine mixture obtained in step S3 was subjected to ultrasonication under the following conditions: power 200-300 W, single working time 10 s-20 s, rest time 2 min-3 min, number of cycles 5-8 times, and system temperature 4-8°C; S5. Incubate at 37°C for 60-180 min. S6. Purify at a speed of 4000-5000 rpm, a time of 10-30 min, a temperature of 4-8°C, and an ultrafiltration membrane pore size of 100 kDa to obtain the stem cell exosome-carnosine load.

2. The method for preparing the stem cell exosome-carnosine-loaded material according to claim 1, characterized in that: The protein concentration of the stem cell exosomes is 0.4-500 ug / mL; the carnosine concentration is 5-1000 ug / mL.

3. The method for preparing the stem cell exosome-carnosine-loaded material according to claim 1, characterized in that: The exosome:carnosine drug input ratio is one of 2:1, 1:1, 1:2, 1:4, 1:8, 1:10 or 1:

12.

4. The stem cell exosome-carnosine-loaded material prepared according to the method for preparing a stem cell exosome-carnosine-loaded material according to any one of claims 1 to 3, characterized in that: The stem cell exosome-carnosine loading material is used to increase the transdermal absorption capacity of carnosine and improve the bioavailability of carnosine.

5. The stem cell exosome-carnosine-loaded material prepared by the method for preparing the stem cell exosome-carnosine-loaded material according to claim 4, characterized in that: The stem cell exosome-carnosine loading is used to effectively promote the regenerative capacity of fibroblasts and keratinocytes.

6. The stem cell exosome-carnosine-loaded material prepared by the method for preparing the stem cell exosome-carnosine-loaded material according to claim 4, characterized in that: The exosome:carnosine drug input ratio was 1:8, and the cumulative permeation volume increased by 310% in 12 hours.

7. The stem cell exosome-carnosine-loaded material prepared by the method for preparing the stem cell exosome-carnosine-loaded material according to claim 4, characterized in that: The exosome protein concentration is 5-500ug / mL, the carnosine concentration is 10-200ug / mL, and the exosome:carnosine drug input ratio is one of 1:1, 1:2, 1:4, 1:8, 1:10 or 1:

12.

8. The stem cell exosome-carnosine-loaded material prepared by the method for preparing a stem cell exosome-carnosine-loaded material according to claim 4, characterized in that: The stem cell exosome-carnosine loading material may be an aqueous solution, an emulsion or a lyophilized powder.

9. The stem cell exosome-carnosine-loaded material prepared by the method for preparing a stem cell exosome-carnosine-loaded material according to claim 4, characterized in that: The stem cell exosome-carnosine loading material is used in cosmetics.

10. The stem cell exosome-carnosine-loaded material prepared by the method for preparing a stem cell exosome-carnosine-loaded material according to claim 4, characterized in that: The stem cell exosome-carnosine loading material is used in a facial mask.

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