PQQ keep-alive type mitochondrial microvesicle, preparation method and application

Abstract

The invention provides PQQ keep-alive type mitochondrial microvesicles, a preparation method and application, and belongs to the technical field of cosmetics. The microvesicle is obtained through microfluidic low-oxygen denucleation, PQQ-containing buffer solution wrapping, ultrasonication, gradient ultrasound and PEG surface modification, has high stability, permeability and activity preservation rate, and can avoid inactivation of active components. The ceramide and the lecithin form a flexible liposome, and the flexible liposome wraps a plurality of components such as the PQQ keep-alive type mitochondrial microvesicles, the acetyl hexapeptide and the Ectoine, so that the transdermal absorption type anti-aging composition is formed. Under the high permeability of the flexible liposome, the nano-scale PQQ keep-alive type mitochondrial microvesicles and the like can penetrate through the stratum corneum to deeply nourish skin cells, the transdermal absorption rate is improved by 2.5 times or more, the ATP level is improved, the slow release and long-acting effects of functional components in the skin are ensured, and the effects of smoothing wrinkles and preventing and reversing skin aging are remarkable.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology and relates to a PQQ-preserving mitochondrial microvesicle, its preparation method, and its application. Background Technology

[0002] Skin aging is mainly manifested as increased wrinkles, decreased elasticity, dryness, and pigmentation. Research shows that mitochondria, as the cell's "energy factories," play a central role in the skin aging process. With age, the function of mitochondria in skin cells gradually declines, ATP synthesis decreases, and the level of reactive oxygen species (ROS) gradually increases, all of which lead to inhibited collagen synthesis and accelerated cell apoptosis. Therefore, restoring skin cell vitality by supplementing exogenous mitochondria or their active components has become a hot topic in anti-aging research.

[0003] However, mitochondria are typically 0.5-1 μm in diameter, making it difficult for them to penetrate the skin's stratum corneum barrier. Therefore, supplementing with exogenous mitochondria results in poor anti-aging effects. Furthermore, mitochondria are highly susceptible to inactivation outside the cellular environment, exhibiting poor stability in cosmetics and making it difficult to preserve their biological activity. Simultaneously, oxidative stress, mechanical damage, and environmental exposure during mitochondrial extraction can lead to a rapid loss of mitochondrial activity, resulting in a membrane potential decrease of >60%, ATP production rate of <50%, and ROS accumulation of >20%, ultimately leading to a >40% reduction in the efficacy of the final product, severely limiting its application in cosmetics.

[0004] Liposome encapsulation technology is commonly used to enhance mitochondrial activity. However, mitochondria, typically 0.5-1 μm in diameter, are large organelles, and traditional liposome encapsulation methods are inefficient. Furthermore, simple physical encapsulation cannot resolve the conflict between the size of mitochondria and transdermal absorption. Current mitochondrial extraction methods often rely on simple differential centrifugation, which easily introduces cell debris contamination and lacks strategies for preserving activity. This leads to ROS accumulation, increased membrane permeability, and a sharp decline in ATP production after extraction, resulting in poor anti-aging effects. Summary of the Invention

[0005] The purpose of this invention is to provide a PQQ-preserved mitochondrial microvesicle, its preparation method, and its application, in order to solve the problems of low mitochondrial survival rate and inability to penetrate the skin in existing mitochondria.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides a PQQ-preserved mitochondrial microvesicle, the preparation method of which includes: After mesenchymal stem cells were enucleated using a microfluidic chip, the resulting enucleated cytoplasms were added to a buffer solution and subjected to sonication, primary centrifugation, and secondary centrifugation to obtain mitochondria encapsulated by cytoplasmic membranes. The mitochondria, encapsulated by the cytoplasmic membrane, were resuspended in a resuspension solution, subjected to gradient sonication at 4°C, and then filtered to form PQQ-preserved mitochondrial microvesicles.

[0007] Secondly, this application provides a transdermal absorption type anti-aging composition, which comprises, by weight: 1-5g of PQQ-activated mitochondrial microvesicles, 0.1-0.5g of acetyl hexapeptide-8, 0.5-2g of ectoine, 1-3g of hydrolyzed collagen, 0.1-0.5g of ergothioneine, 0.5-2g of ceramide, 0.1-0.3g of lecithin, and 86-96g of deionized water.

[0008] Thirdly, this application provides a method for preparing a transdermal absorption type anti-aging composition, the method comprising: Under nitrogen purging, PQQ-preserving mitochondrial microvesicles, acetyl hexapeptide-8, ectoine, hydrolyzed collagen, and deionized water were stirred and mixed, and then ergothioneine was added and mixed to form an active complex. Ceramide and lecithin were dissolved in anhydrous ethanol and then rotary evaporated to obtain an oil phase lipid membrane. The active complex is added to the oil phase lipid membrane, and after hydration oscillation, high-pressure homogenization, and polycarbonate membrane extrusion, a transdermal absorption anti-aging composition is obtained.

[0009] Fourthly, this application provides an application of a PQQ-preserving mitochondrial microvesicle or transdermal absorption anti-aging composition, namely, for the preparation of transdermal absorption anti-aging skin care products.

[0010] The present invention has the following beneficial effects: (1) In this application, mitochondria wrapped in cytoplasmic membrane are obtained by microfluidic hypoxia enucleation, encapsulation with PQQ buffer, and sonication. PQQ, as a core small molecule agonist, can promote the fusion and biogenesis of the fragmented nucleosomes and improve mitochondrial activity. By controlling the self-assembly of mitochondria wrapped in cytoplasmic membrane through gradient sonication and PEG surface modification, the stability and permeability of microvesicles are significantly improved, and PQQ-preserved mitochondrial microvesicles with a particle size of 50-200 nm are obtained.

[0011] (2) The activity of nanoscale PQQ-preserving mitochondrial microvesicles was monitored throughout the preparation process using JC-1, ATP detection kit and DCFH-DA to ensure that the mitochondrial activity retention rate was >95% and to avoid the inactivation of active ingredients.

[0012] (3) In this transdermal absorption type anti-aging composition, PQQ active mitochondrial microvesicles, acetyl hexapeptide, ectoin and other components are encapsulated by flexible liposomes formed by ceramide and lecithin. PQQ active mitochondrial microvesicles retain energy supply characteristics and have a small particle size. Combined with the high permeability of liposomes, they can penetrate the stratum corneum to nourish skin cells. The transdermal absorption rate is increased by more than 2.5 times, and the ATP level is increased, which synergistically exerts the anti-aging effect.

[0013] (4) This transdermal absorption anti-aging composition uses a variety of active ingredients such as PQQ-activated mitochondrial microvesicles, acetyl hexapeptide, and ectoine, which can avoid the skin irritation of traditional preparations and ensure the sustained release and long-lasting effect of the active ingredients in the skin. It has significant effects in reducing wrinkles and preventing and reversing skin aging. Attached Figure Description

[0014] Figure 1 TEM (Transmission Electron Microscope) image of PQQ-preserved mitochondrial microvesicles prepared in Example 1 of this application; Figure 2 This is a particle size analysis diagram of the PQQ-preserved mitochondrial microvesicles prepared in Example 1 of this application; Figure 3 This is a graph showing the activity of mitochondria in PQQ-preserved mitochondrial microvesicles prepared in Example 1 of this application, where the red part represents the JC-1 probe. Figure 4 This is a graph showing the detection of ATP content in skin fibroblasts in different sample groups provided in the embodiments of this application; Figure 5 This is a comparison chart showing the effects of the transdermal absorption anti-aging composition provided in Example 7 of this application. Detailed Implementation

[0015] In a first aspect, this application provides a PQQ-preserving mitochondrial microvesicle, the preparation method of which includes: S01: After mesenchymal stem cells are enucleated using a microfluidic chip, the resulting anucleate cytoplasm is added to a buffer solution and subjected to sonication, primary centrifugation, and secondary centrifugation to obtain mitochondria encapsulated by a cytoplasmic membrane.

[0016] With a density of 0.8-1×10 6Human umbilical cord mesenchymal stem cells (HMSCs) at a density of cells / mL were introduced into a microfluidic chip. Microfluidic shearing was performed for 5-10 minutes under conditions of oxygen content <3%, nitrogen atmosphere, and a flow rate of 20-50 sccm to obtain anucleate cytoplasms. A buffer solution was prepared using 200-250 mM sucrose, 8-10 mM Tris-HCl, 0.5-1 mM EGTA, 0.5-1 mM glutathione, 0.1-0.2 mM coenzyme Q10, 10-50 nM PQQ (pyrroloquinoline quinone), and 0.5-1.0 mM PMSF (benzylsulfonyl fluoride), with the Tris-HCl solution at a pH of 7.2-7.4. Enucleated cytoplasms were added to a buffer solution and sonicated for 2-3 minutes at a power of 30-40W, a frequency of 15-20kHz, and an ice bath temperature, with a 2-second pause between each 1-second sonication. During sonication, PQQ in the buffer solution, acting as a core small molecule agonist, promoted the fusion and biogenesis of the disrupted enucleated cytoplasms; simultaneously, it reduced mechanical damage to mitochondria through the cytoplasmic membrane, increasing mitochondrial survival. The sonicated enucleated cytoplasms were then initially centrifuged for 8-10 minutes at 4℃ and a centrifugation force of 800-1000g to remove broken and unfused fragments. Then, they were secondary centrifuged for 12-15 minutes at 4℃ and a centrifugation force of 10000-12000g to initially enrich the mitochondria with a cytoplasmic membrane, resulting in mitochondria with a cytoplasmic membrane thickness of 5-10 nm.

[0017] During the process of mitochondria being enveloped by the cytoplasmic membrane, parameters were monitored in real time using the JC-1 assay, the ATP (adenosine triphosphate) assay kit, and the DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) assay kit to detect mitochondrial activity. In this application, the JC-1, DCFH-DA, and ATP assay kits all employ commonly used mitochondrial fluorescent probes and assay kits in the art.

[0018] S02: The mitochondria wrapped in the cytoplasmic membrane are resuspended in a resuspension solution, subjected to gradient sonication at 4°C, and filtered to form PQQ-preserved mitochondrial microvesicles.

[0019] Mitochondria encapsulated in the cytoplasmic membrane were resuspended in PBS solution at pH 7.4 containing 10-50 nM PQQ and 0.2-0.5 mM glutathione, forming a mixture with a concentration of 0.8-1 mg / mL. Gradient sonication was then performed at 4°C. The first stage of sonication used a power of 40-50 W, a frequency of 15-20 kHz, and a duration of 4-5 min, with a 1.5-2 s interval followed by a 3-4 s interval. The second stage of sonication used a power of 80-100 W, a frequency of 35-40 kHz, and a duration of 4-5 min, with a 1.5-2 s interval followed by a 3-4 s interval. The first stage of sonication disrupted the outer cytoplasmic membrane, while the second stage promoted the self-assembly of the cytoplasmic membrane.

[0020] After sonication, PEG-2000 with a molecular weight of 2000 Da and a mass of 0.2-0.3% of the mitochondrial membrane-encapsulated cytoplasm was added. The mixture was shaken at 120-150 rpm for 25-30 min to modify the surface of the mitochondrial membrane-encapsulated cytoplasm, resulting in microvesicles with a particle size of 50-200 nm. Dynamic light scattering (DLS) analysis confirmed that the microvesicles exhibited a particle size distribution (PDI) < 0.15, PQQ-induced ATP production > 92%, ROS < 3%, and mitochondrial viability retention > 90%.

[0021] Microvesicles were sequentially filtered through filters with pore sizes of 0.8 μm, 0.45 μm, and 0.22 μm to remove large, broken particles and impurities, yielding liquid PQQ-preserved mitochondrial microvesicles. The PQQ-preserved mitochondrial microvesicles were analyzed to have a particle size of 50-200 nm, encapsulation efficiency >85%, yield >75%, and zeta potential of -20 to -30 mV. Activity was verified using a JC-1, ATP, and ROS assay kit, showing a membrane potential >85%, ATP yield >92%, and ROS <5%.

[0022] Secondly, this application provides a transdermal absorption type anti-aging composition, which comprises, by weight: 1-5g of PQQ-activated mitochondrial microvesicles, 0.1-0.5g of acetyl hexapeptide-8, 0.5-2g of ectoine, 1-3g of hydrolyzed collagen, 0.1-0.5g of ergothioneine, 0.5-2g of ceramide, 0.1-0.3g of lecithin, and 86-96g of deionized water.

[0023] In this application, the PQQ-preserving mitochondrial microvesicles are prepared using the method described in the first aspect. They retain key proteins such as the TOM / TIM complex of the mitochondrial membrane and some genetic material such as mtDNA, thus mimicking mitochondrial function and promoting energy metabolism in recipient cells. Furthermore, the particle size of these PQQ-preserving mitochondrial microvesicles is 50-200 nm, significantly smaller than the diameter of the original mitochondria, which facilitates transdermal absorption.

[0024] Acetyl hexapeptide-8 is a neurotransmitter inhibitory peptide with a botulinum toxin-like mechanism. It can block neuromuscular transmission, relax facial muscles, and thus reduce the formation of dynamic wrinkles. Ectoin has excellent cytoprotective effects and can stabilize the structure of biological macromolecules under extreme conditions. Hydrolyzed collagen can replenish collagen lost from the skin and improve moisturizing ability. Ergothioneine, as a natural antioxidant, can build an antioxidant defense line, protect mitochondria from ROS damage, and synergize with PQQ to maintain the mitochondrial reducing environment during the preservation of its activity.

[0025] Ceramides and lecithin serve as building blocks for lipid carriers. Ceramides can repair the skin barrier and enhance the adhesion of the stratum corneum; lecithin has good amphiphilicity and can form a stable bilayer structure to encapsulate water-soluble active ingredients. The ceramide used in this application is ceramide NP.

[0026] According to the test results, the transdermal absorption type anti-aging composition of this application has a stability of >97% at 4°C; according to the Franz diffusion cell test, the transdermal absorption rate is >65% within 24 hours, and the PQQ-mediated mitochondrial activity preservation rate is >90%.

[0027] Thirdly, this application provides a method for preparing a transdermal absorption type anti-aging composition, the method comprising: S01: Under conditions of nitrogen purging, temperature of 2-8℃, and stirring speed of 80-150 rpm, PQQ-preserving mitochondrial microvesicles, acetyl hexapeptide-8, ectoine, hydrolyzed collagen, and deionized water are stirred and mixed to form an aqueous mixture. Nitrogen purging maintains a low-oxygen environment, thus preserving the activity of PQQ-preserving mitochondrial microvesicles. Ergothioneine is then added to the aqueous mixture, and the mixture is stirred and mixed under conditions of 2-8℃ and stirring speed of 80-150 rpm to form an active complex. The addition of ergothioneine constructs the first line of antioxidant defense in the aqueous mixture, synergistically protecting the activity of PQQ-preserving mitochondrial microvesicles with a >95% activity retention rate.

[0028] S02: Ceramide and lecithin were added to anhydrous ethanol and stirred until completely dissolved. The anhydrous ethanol was then evaporated using a rotary evaporator at a temperature of 40-55℃ and a rotation speed of 40-60 rpm to obtain an oil phase lipid membrane. The oil phase lipid membrane was immediately resuspended in a resuspension containing PQQ to ensure its activity.

[0029] S03: Add the active complex to the oil phase lipid membrane and hydrate and shake at 35-37℃ for 45-60 min to form a primary liposome suspension. During the hydration and shaking process, the dried oil phase lipid membrane swells upon contact with water, spontaneously detaches and closes, encapsulating the active complex containing PQQ-preserving mitochondrial microvesicles and other active ingredients in a hydrophilic core, thereby ensuring the uniform distribution and preservation of activity of the PQQ-preserving mitochondrial microvesicles. After cyclic high-pressure homogenization at 500-1000 bar for 2-5 cycles, extrude through a 0.1 μm polycarbonate membrane 8-15 times to obtain a transdermal anti-aging composition.

[0030] The high-pressure homogenization and polycarbonate film extrusion process ensures that the transdermal anti-aging composition has a uniform particle size at the nanoscale, which greatly enhances its transdermal performance.

[0031] Fourthly, this application provides an application of a PQQ-retaining mitochondrial microvesicle or transdermal absorption anti-aging composition, specifically for the preparation of transdermal absorption anti-aging skincare products. These skincare products can be serums, lotions, or masks, used to improve skin wrinkles, enhance skin elasticity, and promote collagen synthesis, while simultaneously achieving long-lasting energy replenishment through PQQ-retaining mitochondrial microvesicles.

[0032] When the transdermal absorption anti-aging composition of this application is used in the preparation of skin care products, the amount added is 1-5% of the total mass of the skin care products.

[0033] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0034] Example 1 This application provides a PQQ-preserving mitochondrial microvesicle, the preparation method of which includes: S101: A density of 1×10 6Human umbilical cord mesenchymal stem cells (HMSCs) at a density of cells / mL were introduced into a microfluidic chip and microfluidically sheared for 8 min under nitrogen-filled conditions with an oxygen content of <3% to obtain anucleated cytoplasms. A buffer solution was prepared using 220 mM sucrose, 9 mM Tris-HCl, 0.8 mM EGTA, 0.6 mM glutathione, 0.15 mM coenzyme Q10, 20 nM PQQ, and 0.8 mM PMSF, with the Tris-HCl at pH 7.3. The anucleated cytoplasms were added to the buffer solution and sonicated for 2.5 min at 35 W, 15 kHz, and an ice bath, with a 2-s interval between sonications. The sonicated anucleated cytoplasms were then initially centrifuged at 4 °C and 900 g for 9 min, followed by a secondary centrifugation at 4 °C and 11000 g for 12 min to obtain mitochondria encapsulated in a 7 nm thick cytoplasmic membrane.

[0035] S102: Mitochondria encapsulated in cytoplasmic membrane were resuspended in PBS solution at pH 7.4 containing 30 nM PQQ and 0.4 mM glutathione to form a mixture with a concentration of 0.9 mg / mL. Gradient sonication was performed at 4°C. The first stage of sonication was performed at a power of 45 W, a frequency of 18 kHz, and a duration of 4.5 min, with a 3-second pause between every 1.5 s sonication sessions. The second stage of sonication was performed at a power of 90 W, a frequency of 38 kHz, and a duration of 4.5 min, with a 3-second pause between every 1.5 s sonication sessions. After sonication, PEG-2000 (2000 Da, 0.25% of the mass of the mitochondria encapsulated in cytoplasmic membrane) was added, and the mixture was shaken at 140 rpm for 28 min to obtain microvesicles with a particle size of 80 nm. The microvesicles were then filtered sequentially through filters with pore sizes of 0.8 μm, 0.45 μm, and 0.22 μm to obtain liquid PQQ-preserved mitochondrial microvesicles.

[0036] Example 2 This application provides a PQQ-preserving mitochondrial microvesicle, the preparation method of which includes: S201: A material with a density of 0.8 × 10⁻⁶ 6Human umbilical cord mesenchymal stem cells (HMSCs) at a density of cells / mL were introduced into a microfluidic chip and microfluidically sheared for 5 min under nitrogen-filled conditions with oxygen content <3% to obtain anucleated cytoplasms. A buffer solution was prepared using 200 mM sucrose, 10 mM Tris-HCl, 0.5 mM EGTA, 0.5 mM glutathione, 0.2 mM coenzyme Q10, 50 nM PQQ, and 0.5 mM PMSF, with the Tris-HCl at pH 7.2. The anucleated cytoplasms were added to the buffer solution and sonicated for 2 min at 30 W, 15 kHz, and an ice bath, with a 2-s interval between sonications. The sonicated anucleated cytoplasms were then initially centrifuged at 4 °C and 1000 g for 8 min, followed by a secondary centrifugation at 4 °C and 10000 g for 15 min to obtain mitochondria encapsulated in a 10 nm thick cytoplasmic membrane.

[0037] S202: Mitochondria encapsulated in cytoplasmic membrane were resuspended in PBS solution at pH 7.4 containing 50 nM PQQ and 0.2 mM glutathione to form a mixture with a concentration of 0.8 mg / mL. Gradient sonication was performed at 4°C. The first stage of sonication was performed at a power of 40 W, a frequency of 15 kHz, and a duration of 5 min, with a 2-second sonication interval followed by a 3-second pause. The second stage of sonication was performed at a power of 80 W, a frequency of 35 kHz, and a duration of 4 min, with a 2-second sonication interval followed by a 3-second pause. After sonication, PEG-2000 (molecular weight 2000 Da, 0.2% of the mass of the mitochondria encapsulated in cytoplasmic membrane) was added, and the mixture was shaken at 120 rpm for 30 min to obtain microvesicles with a particle size of 50 nm. The microvesicles were then filtered sequentially through filters with pore sizes of 0.8 μm, 0.45 μm, and 0.22 μm to obtain liquid PQQ-preserved mitochondrial microvesicles.

[0038] Example 3 This application provides a PQQ-preserving mitochondrial microvesicle, the preparation method of which includes: S301: A material with a density of 0.9 × 10⁻⁶ 6Human umbilical cord mesenchymal stem cells (HMSCs) at a density of cells / mL were introduced into a microfluidic chip and microfluidically sheared for 10 min under nitrogen-filled conditions with oxygen content <3% to obtain enucleated cytoplasms. A buffer solution was prepared using 250 mM sucrose, 8 mM Tris-HCl, 1 mM EGTA, 1 mM glutathione, 0.1 mM coenzyme Q10, 10 nM PQQ, and 0.8 mM PMSF, with the Tris-HCl at pH 7.4. The enucleated cytoplasms were added to the buffer solution and sonicated for 3 min at 40 W, 20 kHz, and an ice bath, with a 2-s interval between sonications. The sonicated enucleated cytoplasms were then initially centrifuged at 4 °C and 800 g for 10 min, followed by a secondary centrifugation at 4 °C and 12000 g for 12 min to obtain mitochondria encapsulated in a 5 nm thick cytoplasmic membrane.

[0039] S302: Mitochondria encapsulated in cytoplasmic membrane were resuspended in PBS solution at pH 7.4 containing 10 nM PQQ and 0.5 mM glutathione to form a 1 mg / mL mixture. Gradient sonication was performed at 4 °C. The first stage of sonication was performed at 50 W power, 20 kHz frequency, and 4 min time, with a 1.5 s interval followed by a 4 s pause. The second stage of sonication was performed at 100 W power, 40 kHz frequency, and 5 min time, with a 2 s interval followed by a 4 s pause. After sonication, PEG-2000 (2000 Da, 0.3% of the mass of the mitochondria encapsulated in cytoplasmic membrane) was added, and the mixture was shaken at 150 rpm for 25 min to obtain microvesicles with a particle size of 150 nm. The microvesicles were then filtered sequentially through filters with pore sizes of 0.8 μm, 0.45 μm, and 0.22 μm to obtain liquid PQQ-preserved mitochondrial microvesicles.

[0040] Example 4 This application provides a PQQ-preserving mitochondrial microvesicle, the preparation method of which includes: S401: A material with a density of 0.85 × 10⁻⁶ 6Human umbilical cord mesenchymal stem cells (HMSCs) at a density of cells / mL were introduced into a microfluidic chip and microfluidically sheared for 8 min under nitrogen-filled conditions with oxygen content <3% to obtain anucleated cytoplasms. A buffer solution was prepared using 230 mM sucrose, 8.5 mM Tris-HCl, 0.8 mM EGTA, 0.7 mM glutathione, 0.16 mM coenzyme Q10, 30 nM PQQ, and 1.0 mM MPMSF, with the Tris-HCl at pH 7.3. The anucleated cytoplasms were added to the buffer solution and sonicated for 2 min at 35 W, 16 kHz, and an ice bath, with a 2-s interval between sonications. The sonicated anucleated cytoplasms were then initially centrifuged at 4 °C and 950 g for 8 min, followed by a secondary centrifugation at 4 °C and 11500 g for 13 min to obtain mitochondria encapsulated in a cytoplasmic membrane with a thickness of 8 nm.

[0041] S402: Mitochondria encapsulated in cytoplasmic membrane were resuspended in PBS solution at pH 7.4 containing 40 nM PQQ and 0.4 mM glutathione to form a mixture with a concentration of 0.9 mg / mL. Gradient sonication was performed at 4°C. The first stage of sonication was performed at 45 W power, 16 kHz frequency, and 5 min time, with a 2 s sonication interval followed by a 4 s interval. The second stage of sonication was performed at 85 W power, 36 kHz frequency, and 4 min time, with a 2 s sonication interval followed by a 4 s interval. After sonication, PEG-2000 with a molecular weight of 2000 Da and a mass of 0.2% of the mitochondrial membrane encapsulation mass was added, and the mixture was shaken at 140 rpm for 28 min to obtain microvesicles with a particle size of 70 nm. The microvesicles were then filtered sequentially through filters with pore sizes of 0.8 μm, 0.45 μm, and 0.22 μm to obtain liquid PQQ-preserved mitochondrial microvesicles.

[0042] Example 5 This application provides a transdermal anti-aging composition, which comprises, by weight: 2g of PQQ-activated mitochondrial microvesicles, 0.2g of acetyl hexapeptide-8, 1g of ectoine, 2g of hydrolyzed collagen, 0.2g of ergothioneine, 1g of ceramide, 0.2g of lecithin, and 93.46g of deionized water as described in Example 1.

[0043] This application also provides a method for preparing the transdermal anti-aging composition, the method comprising: S501: Under nitrogen purging, at a temperature of 4°C and a stirring speed of 100 rpm, PQQ-preserving mitochondrial microvesicles, acetyl hexapeptide-8, ectoine, hydrolyzed collagen, and deionized water were stirred and mixed to form an aqueous mixture. Ergothioneine was added to the aqueous mixture, and the mixture was stirred and mixed at 4°C and a stirring speed of 100 rpm to form an active complex.

[0044] S502: Ceramide and lecithin were added to anhydrous ethanol and stirred until completely dissolved. The anhydrous ethanol was then evaporated using a rotary evaporator at 50°C and 55 rpm to obtain an oil-phase lipid membrane. The oil-phase lipid membrane was immediately resuspended in a resuspension containing PQQ to ensure its activity.

[0045] S503: An active complex was added to the oil phase lipid membrane and hydrated and shaken at 37°C for 60 min to form a primary liposome suspension. After cyclic high-pressure homogenization three times at 800 bar using a high-pressure homogenizer, the mixture was extruded ten times through a polycarbonate membrane with a pore size of 0.1 μm to obtain a transdermal absorption anti-aging composition.

[0046] Example 6 This application provides a transdermal absorption type anti-aging composition, which comprises, by weight: 1g of PQQ-preserving mitochondrial microvesicles, 0.1g of acetyl hexapeptide-8, 0.5g of ectoine, 1g of hydrolyzed collagen, 0.1g of ergothioneine, 0.5g of ceramide, 0.1g of lecithin, and 96g of deionized water as described in Example 1.

[0047] This application also provides a method for preparing the transdermal anti-aging composition, which is the same as in Example 5, except that the high-pressure homogenization pressure is 600 bar.

[0048] Example 7 This application provides a transdermal anti-aging composition, which comprises, by weight: 5g of PQQ-activated mitochondrial microvesicles, 0.5g of acetyl hexapeptide-8, 1g of ectoine, 2g of hydrolyzed collagen, 0.5g of ergothioneine, 1.5g of ceramide, 0.15g of lecithin, and 86g of deionized water as described in Example 1.

[0049] This application also provides a method for preparing the transdermal anti-aging composition, which is the same as in Example 5, except that the high-pressure homogenization pressure is 1000 bar.

[0050] In this application, the PQQ-preserving mitochondrial microvesicles prepared in Example 1 were subjected to TEM, particle size distribution, and mitochondrial activity assays, respectively, to obtain the attached... Figure 1-3 .

[0051] From the appendix Figure 1 As can be seen, the PQQ-preserving mitochondrial microvesicles prepared in Example 1 of this application contain nanoscale mitochondrial contents with a generally uniform particle size ranging from 50 to 200 nm.

[0052] From the appendix Figure 2 As can be seen, the particle size of the PQQ-preserving mitochondrial microvesicles prepared in Example 1 of this application is mainly 50-200nm. The particle size is small and can penetrate the stratum corneum and enter the skin cells, thereby allowing the mitochondria in the PQQ-preserving mitochondrial microvesicles to enter the skin cells, achieving the effects of reducing wrinkles, preventing and reversing skin aging.

[0053] From the appendix Figure 3 As can be seen, the mitochondrial membrane potential of the PQQ-preserved mitochondrial microvesicles prepared in Example 1 of this application was well maintained after JC-1 RED mitochondrial membrane potential staining.

[0054] This application also tested the cellular energy metabolism and anti-wrinkle efficacy of the transdermal absorption anti-aging compositions prepared in Examples 5-7, as detailed below: 1. Cellular energy metabolism detection Cellular energy metabolism (ATP) detection of intracellular ATP levels is an important indicator for measuring cell viability and anti-aging ability. In this application, human skin fibroblasts (HDF) were seeded in 96-well plates and divided into a blank control group, a positive control group, and three sample groups. The blank control group received no sample, the positive control group received 10 μM coenzyme Q10, and the three sample groups received 1% of the transdermal anti-aging composition prepared in Examples 5-7, respectively. After culturing the human skin fibroblasts for 24 hours, the intracellular ATP content of each group was measured using an ATP detection kit, as shown in Table 1 and Appendix. Figure 4 .

[0055] Table 1: ATP content in HDF From Table 1 and Appendix Figure 4 As can be seen, compared with the blank control, the transdermal absorption anti-aging compositions prepared in Examples 5-7 can significantly increase the ATP level in fibroblasts, and the effect is better than that of the common anti-aging ingredient coenzyme Q10. This indicates that the transdermal absorption anti-aging compositions provided in this application effectively deliver mitochondrial active components through PQQ-preserving mitochondrial microvesicles, restarting cellular energy metabolism.

[0056] 2. Anti-wrinkle efficacy test Thirty female volunteers aged 40-55 with noticeable crow's feet around their eyes were recruited and randomly divided into three groups. Each group used a transdermal anti-aging composition prepared in Examples 5-7. Testing method: Volunteers applied the sample around their eyes morning and evening for 28 consecutive days. Facial images were taken using a VISIA skin analyzer on days 0 and 28, and the changes in the area and depth of crow's feet were analyzed. The results are shown in Table 2 and Appendix. Figure 5 .

[0057] Table 2: Improvement in wrinkles As shown in Table 2, with prolonged use, the area and depth of wrinkles around the eyes in volunteers showed significant improvement, with the high-concentration groups in Examples 5 and 7 showing more pronounced effects; additionally, [the following text is missing]. Figure 5 The actual usage diagrams clearly show that the area and depth of wrinkles around the eyes are significantly improved, indicating that the transdermal absorption anti-aging composition provided in this application has excellent anti-wrinkle and firming effects.

[0058] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing PQQ-preserved mitochondrial microvesicles, characterized in that, include: After mesenchymal stem cells were enucleated using a microfluidic chip, the resulting enucleated cytoplasms were added to a buffer solution and subjected to sonication, primary centrifugation, and secondary centrifugation to obtain mitochondria encapsulated by cytoplasmic membranes. The mitochondria, encapsulated by the cytoplasmic membrane, were resuspended in a resuspension solution, subjected to gradient sonication at 4°C, and then filtered to form PQQ-preserved mitochondrial microvesicles.

2. The method for preparing PQQ-preserving mitochondrial microvesicles according to claim 1, characterized in that, Nitrogen gas is continuously introduced into the microfluidic chip, and the oxygen content is <3%; the flow rate of the mesenchymal stem cells is 20-50 sccm.

3. The method for preparing PQQ-preserving mitochondrial microvesicles according to claim 1, characterized in that, The buffer solution comprises 200-250 mM sucrose, 8-10 mM Tris-HCl, 0.5-1 mM EGTA, 0.5-1 mM glutathione, 0.1-0.2 mM coenzyme Q10, 10-50 nM PQQ, and 0.5-1.0 mM PMSF, wherein the pH of Tris-HCl is 7.2-7.

4.

4. The method for preparing PQQ-preserving mitochondrial microvesicles according to claim 1, characterized in that, The ultrasonic disruption is performed at a power of 30-40W, a frequency of 15-20kHz, an ice bath temperature, and a time of 2-3 minutes, with a 2-second pause between each 1-second ultrasonic cycle; the primary centrifugation is performed at a temperature of 4℃, a centrifugal force of 800-1000g, and a time of 8-10 minutes; the secondary centrifugation is performed at a temperature of 4℃, a centrifugal force of 10000-12000g, and a time of 12-15 minutes.

5. The method for preparing PQQ-preserving mitochondrial microvesicles according to claim 1, characterized in that, The resuspension is a PBS solution with pH 7.4 containing 10-50 nM PQQ and 0.2-0.5 mM glutathione.

6. The method for preparing PQQ-preserving mitochondrial microvesicles according to claim 1, characterized in that, The gradient ultrasound includes: The first stage of ultrasound: power of 40-50W, frequency of 15-20kHz, duration of 4-5min, with 1.5-2s intervals and 3-4s intervals between each ultrasound. The second stage of ultrasound: power of 80-100W, frequency of 35-40kHz, duration of 4-5min, with 1.5-2s intervals and 3-4s breaks between each ultrasound.

7. PQQ-preserved mitochondrial microvesicles prepared by the preparation method according to any one of claims 1-6.

8. A transdermal absorption type anti-aging composition, characterized in that, The composition by weight includes: 1-5g of PQQ-preserving mitochondrial microvesicles as described in claim 7, 0.1-0.5g of acetyl hexapeptide-8, 0.5-2g of ectoine, 1-3g of hydrolyzed collagen, 0.1-0.5g of ergothioneine, 0.5-2g of ceramide, 0.1-0.3g of lecithin, and 86-96g of deionized water.

9. A method for preparing a transdermal absorption type anti-aging composition, characterized in that, include: Under nitrogen purging, PQQ-preserving mitochondrial microvesicles, acetyl hexapeptide-8, ectoine, hydrolyzed collagen, and deionized water were stirred and mixed, and then ergothioneine was added and mixed to form an active complex. Ceramide and lecithin were dissolved in anhydrous ethanol and then rotary evaporated to obtain an oil phase lipid membrane. The active complex is added to the oil phase lipid membrane, and after hydration oscillation, high-pressure homogenization, and polycarbonate membrane extrusion, a transdermal absorption anti-aging composition is obtained.

10. The PQQ-preserving mitochondrial microvesicles of claim 7 or the transdermal absorption anti-aging composition of claim 8 are used to prepare transdermal absorption anti-aging skin care products.

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