Cell membrane vesicle self-assembly body loaded with spirulina PDRN and preparation method of cell membrane vesicle self-assembly body

Abstract

The invention discloses a cell membrane vesicle self-assembly body loaded with spirulina PDRN and a preparation method of the cell membrane vesicle self-assembly body, and belongs to the technical field of cosmetics. The preparation method comprises the following steps: homogenizing and crushing spirulina at low temperature and high pressure, and performing graded filtration to prepare spirulina vesicle suspension; mixing with auxiliary materials such as spirulina platensis source PDRN, hydrogenated soybean phosphatidylcholine and the like to prepare a medicine-carrying vesicle premixed solution; and carrying out pre-homogenization, low-temperature fine homogenization and graded filtration to obtain the target self-assembly body. According to the invention, the spirulina natural cell membrane vesicles are taken as a core carrier, efficient encapsulation of PDRN is realized through membrane fusion recombination, the encapsulation efficiency of the obtained product can reach 55% or more, the transdermal absorption performance is excellent, UVB-induced skin light injury cells can be remarkably repaired, and the product has the effects of resisting aging, tightening, resisting wrinkles and whitening, and can be widely applied to preparation of skin care products.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, and more specifically, relates to a cell membrane vesicle self-assembly loaded with spirulina PDRN and its preparation method. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are bioactive deoxynucleotide polymers with various physiological activities, including promoting cell proliferation and repair, anti-inflammation, anti-oxidation, and promoting collagen synthesis. They have significant application value in skincare fields such as anti-aging, damage repair, and skin brightening. Among these, PDRN derived from spirulina has become a research hotspot for skincare active ingredients due to its natural origin, high biosafety, and stable activity.

[0003] However, in practical skincare applications, PDRN is a water-soluble macromolecule that is difficult to penetrate the skin's stratum corneum barrier, resulting in extremely low bioavailability. Furthermore, PDRN is easily degraded and aggregated in aqueous systems, leading to low activity retention and short-lasting effects when directly added to skincare products. Therefore, achieving efficient encapsulation and targeted transdermal delivery of PDRN is the core technological bottleneck for its large-scale application in the skincare field.

[0004] Currently, existing technologies for PDRN delivery systems mostly employ traditional liposomes and nanoemulsions as carriers. However, traditional liposomes often use synthetic phospholipids as a single matrix, resulting in drawbacks such as low encapsulation efficiency, unstable vesicle structure, easy leakage both in vivo and in vitro, and insufficient biocompatibility with the skin. At the same time, conventional liposomes have limited transdermal delivery capabilities, making it difficult to efficiently deliver active ingredients to the dermis to exert their effects.

[0005] Extracellular vesicles are naturally occurring phospholipid bilayer vesicle structures derived from cells. They possess excellent biocompatibility, targeting ability, membrane fusion capacity, and loading efficiency, making them a next-generation delivery carrier for active ingredients. Spirulina, as a single-celled prokaryote, has a simple cell membrane structure, its phospholipid composition has high affinity for human skin cell membranes, and it is widely available and inexpensive, making it a high-quality raw material for preparing natural cell membrane vesicles. However, current technologies lack specific solutions for using Spirulina cell membrane vesicles as a core carrier to load homologous Spirulina PDRN and construct vesicle self-assemblies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a delivery system using spirulina natural cell membrane vesicles as the core carrier, thereby achieving efficient encapsulation of spirulina-derived PDRN, enhancing transdermal absorption of PDRN, and improving the skin's efficient utilization of PDRN.

[0007] To achieve the above objectives, the present invention discloses the following technical solutions: In a first aspect, the present invention provides a method for preparing cell membrane vesicle self-assemblies loaded with Spirulina PDRN, the method comprising the following steps: Step 1: Take Spirulina and disperse it in buffer solution at a concentration of 5-20 w / v%. Mix thoroughly to obtain algal dispersion. Place the dispersion in an environment of 4-10℃ for homogenization and disruption. Filter the homogenized mixture through 1μm and 0.22μm filter membranes step by step. Collect the filtrate to obtain Spirulina vesicle suspension. Step 2: Mix the spirulina vesicle suspension, spirulina-derived PDRN solution, hydrogenated soybean phosphatidylcholine, subtilis lipopeptide sodium, avocado oil, glycerol and solvent to obtain the drug-loaded vesicle premix. Step 3: First, pre-homogenize the drug-loaded vesicle premix by controlling the rotation speed at 8000-15000 rpm for 2-5 min. Then, perform fine homogenization at 4-15℃ by controlling the homogenization pressure at 689.5-2068.5 bar and cycle the process 3-8 times. Step 4: The homogenized vesicle emulsion is sequentially filtered through microporous membranes of 0.45 μm and 0.22 μm to obtain the cell membrane vesicle self-assembly loaded with Spirulina PDRN.

[0008] This invention is based on the high affinity principle of homologous materials. Using Spirulina cell membrane vesicles as the core carrier, it constructs a composite lipid system with hydrogenated soybean phosphatidylcholine, subtilis lipopeptide sodium, and avocado oil. Through low-temperature, high-energy, homogeneous shear force, the artificial phospholipids are fused and recombined with the natural cell membrane, simultaneously encapsulating homologous Spirulina PDRN into the aqueous chamber of the vesicles, forming structurally stable nanoscale self-assembled bodies. Utilizing the high compatibility between natural cell membrane vesicles and human skin cell membranes, the vesicles achieve transstratum corneum delivery and cell internalization, ultimately achieving targeted release and activation of PDRN.

[0009] Spirulina vesicles, as the core carriers of self-assembly, provide specific encapsulation sites and aqueous chambers for PDRN. At the same time, thanks to the phospholipid composition of the natural cell membrane, they enhance the biocompatibility and membrane fusion ability of vesicles with the skin, making them the core matrix for achieving efficient encapsulation and transdermal delivery of PDRN.

[0010] Hydrogenated soybean phosphatidylcholine, as an auxiliary phospholipid, is highly compatible with the phospholipid components of spirulina vesicles. It can optimize the compactness and stability of the vesicle bilayer, reduce PDRN leakage, and improve the compatibility of vesicles in skin care product systems.

[0011] Sodium subtilis lipopeptide, as a biosurfactant, can reduce the interfacial tension of lipid systems, improve the membrane fusion efficiency of natural vesicles and artificial phospholipids, and optimize the particle size uniformity of vesicles, thereby further improving the encapsulation efficiency of PDRN.

[0012] Avocado oil, as a lipid regulator, can embed itself in the gaps between vesicle bilayers, enhancing the flexibility and integrity of the membrane structure, reducing vesicle rupture and PDRN leakage during homogenization and storage, while also providing moisturizing and repairing effects.

[0013] Glycerin acts as a humectant and osmotic pressure regulator, maintaining the osmotic pressure balance of the vesicle system, preventing vesicles from rupturing due to osmotic pressure differences, and improving the system's moisturizing performance and skincare compatibility.

[0014] Preferably, in step 1, the spirulina is selected from at least one of spirulina wet thallus and spirulina dry powder after resuspension.

[0015] Preferably, in step 1, the buffer solution is selected from at least one of Tris-HCl buffer, PBS buffer, and sterile water, and the pH value of the buffer solution is 7.0-8.5.

[0016] Preferably, in step 1, the homogenization pressure is controlled at 800-1500 bar, and the process is repeated 3-10 times.

[0017] Preferably, in step 2, the solvent is at least one of sterile water for injection, PBS buffer, and Tris-HCl buffer.

[0018] Preferably, the amount of spirulina vesicles is 38-88% of the total lipids, the amount of hydrogenated soybean phosphatidylcholine is 10%-40 w / w of the total lipids, the amount of subtilisin sodium is 1-7 w / w of the total lipids, the amount of avocado oil is 1-15 w / w of the total lipids, and the glycerol concentration is 0.5-80 v / v.

[0019] Preferably, in step 2, the mass ratio of the spirulina-derived PDRN to the total lipids is 1:20 to 1:100.

[0020] In a second aspect, the present invention provides a cell membrane vesicle self-assembly loaded with Spirulina PDRN, wherein the cell membrane vesicle self-assembly is prepared by the preparation method described in the first aspect.

[0021] Thirdly, the present invention provides the application of the cell membrane vesicle self-assembly described in the second aspect in the preparation of skin care products with anti-aging, firming and anti-wrinkle, and whitening effects.

[0022] The beneficial effects of this invention are: 1. This invention uses natural cell membrane vesicles of Spirulina as the core carrier and a system constructed with composite lipid excipients to provide a stable encapsulation aqueous chamber for water-soluble PDRN, which greatly reduces the leakage of active ingredients during preparation and storage, and achieves efficient and stable encapsulation of Spirulina-derived PDRN. The PDRN encapsulation rate of the obtained vesicle self-assemblies is stable at over 55%. 2. This invention overcomes the skin stratum corneum barrier by leveraging the high affinity and membrane fusion ability of natural cell membrane vesicles with the skin stratum corneum, enabling efficient delivery of encapsulated PDRN to the dermis, significantly improving the bioavailability of PDRN and solving the problem of water-soluble PDRN being difficult to absorb transdermally; 3. Compared with simple PDRN solution and traditional liposome system, the self-assembled body of the present invention can more efficiently promote collagen synthesis, inhibit collagen degradation, and enhance the antioxidant capacity of cells, thus achieving excellent anti-aging, firming and wrinkle-reducing, and repairing effects. Detailed Implementation

[0023] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Unless otherwise specified, the experimental methods used in the specific embodiments are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified; and the percentages mentioned in the specific embodiments are all mass percentages unless otherwise specified.

[0025] I. In this invention Spirulina-derived PDRN: refers to polydeoxyribonucleotides extracted and purified from spirulina, with an average molecular weight range of 50-1500 kDa, possessing biological activities that promote cell repair, anti-inflammation, and antioxidant effects; Spirulina: The dried or wet thallus of various spirulina species, including Spirulina platensis (Notdst.) Geitl., belonging to the Oscillatiaceae family.

[0026] II. Self-assemblies of cell membrane vesicles loaded with Spirulina PDRN 1. Preparation of Spirulina Vesicles The core of this preparation method is to achieve the self-assembly of Spirulina cell membranes into natural vesicles through high-pressure crushing, thereby completing the preparation of the core carrier.

[0027] Step 1: Take the purified wet Spirulina algae, or the resuspended Spirulina dry powder, and disperse it in a buffer solution with pH 7.0-8.5 at a concentration of 5%-20w / v% (Tris-HCl buffer, PBS buffer, or sterile water can be selected). Mix thoroughly to obtain the algae dispersion. Step 2: Place the above dispersion in a low temperature environment of 4-10℃ and homogenize it using a high pressure homogenizer. Control the homogenization pressure to 800-1500 bar and cycle it 3-10 times. The high pressure shear force breaks the spirulina cells, causing the cell membrane fragments to spontaneously assemble into natural vesicles with uniform particle size. Step 3: Place the homogenized mixture in a 4°C environment and filter it step by step through 1μm and 0.22μm filter membranes. Collect the filtrate to obtain the Spirulina vesicle suspension. The average particle size of the spirulina vesicles in the spirulina vesicle suspension is 60-200 nm, and the PDI is less than 0.3.

[0028] 2. Preparation of drug-loaded vesicle premix The core of this preparation is to complete the system compatibility of spirulina vesicles, drugs, and lipid excipients, laying the foundation for subsequent membrane fusion and drug encapsulation.

[0029] Step 1: Dissolve the PDRN from Spirulina in sterile water for injection, PBS buffer, or Tris-HCl buffer in advance to obtain the PDRN solution for later use. Prepare hydrogenated soybean phosphatidylcholine, glycerol, subtilis lipopeptide sodium, avocado oil, and sterile water / buffer according to the prescription. Step 2: Mix the spirulina vesicle suspension, PDRN solution, hydrogenated soybean phosphatidylcholine, subtilis lipopeptide sodium, avocado oil, glycerol, and sterile water / buffer solution in a predetermined ratio to form a mixed system. Under gentle stirring conditions, the mixed system is fully hydrated and evenly dispersed to finally obtain the drug-loaded vesicle premix. In this process, the spirulina vesicles in the spirulina vesicle suspension form total lipids with hydrogenated soybean phosphatidylcholine, subtilisin sodium lipopeptide, and avocado oil. The amount of spirulina vesicles is 38-88% of the total lipids, the amount of hydrogenated soybean phosphatidylcholine is 10%-40 w / w, the amount of subtilisin sodium lipopeptide is 1-7 w / w, the amount of avocado oil is 1-15 w / w, the glycerol concentration is 0.5%-80 v / v, and the mass ratio of PDRN to total lipids is 1:20 to 1:100.

[0030] 3. Homogeneous blending and molding The core of this preparation method is to achieve the fusion and recombination of phospholipids and natural vesicle membranes through high-energy shearing, and simultaneously complete the efficient encapsulation of PDRN to form stable drug-loaded composite vesicles.

[0031] Step 1: Perform high-speed shear pre-homogenization on the above premixed liquid, controlling the rotation speed at 8000-15000 rpm and the processing time at 2-5 min, so that the components are initially dispersed evenly, avoiding the problem of uneven agglomeration during subsequent fine homogenization. Step 2: Use a high-pressure micro-jet homogenizer or a high-pressure homogenizer to perform fine homogenization on the pre-homogenized system under low-temperature water bath conditions of 4-15℃, control the homogenization pressure to 689.5-2068.5 bar, and cycle the process 3-8 times. Step 3: Using high-energy fluid shear force, hydrogenated soybean phosphatidylcholine, sodium subtilis lipopeptide, avocado oil and spirulina natural vesicle membrane are fused and recombined, while PDRN is efficiently encapsulated into the water cavity inside the vesicle, finally forming a structurally stable PDRN drug-loaded composite vesicle system. Step 4: The homogenized vesicle emulsion is sequentially filtered through 0.45μm and 0.22μm microporous membranes to remove unencapsulated large PDRN aggregates and excessively large vesicle aggregates, while simultaneously performing terminal sterilization, to obtain the cell membrane vesicle self-assemblies loaded with spirulina PDRN.

[0032] III. Examples 1. Example 1 Step 1: Disperse the wet Spirulina algae in Tris-HCl buffer at a mass-to-volume ratio of 1:20 g / mL. Homogenize three times at 4℃ and a homogenization pressure of 1500 bar. After homogenization, filter the suspension through 1 μm and 0.22 μm filter membranes at 4℃ to obtain Spirulina vesicle suspension. Step 2: Take 200 mL of Spirulina vesicle suspension, 1.25 g of 20 w / w% Spirulina-derived PDRN solution, 9.5 g of hydrogenated soybean phosphatidylcholine (HSPC), 1.8 g of subtilis lipopeptide sodium, 3.7 g of avocado oil, 60 g of 2.0 v / v% glycerol, and 800 mL of Tris-HCl buffer and mix them to obtain a premix solution; Step 3: The premixed solution was pre-homogenized at 8,000 rpm for 5 min, and then homogenized 8 times using a high-pressure microfluidic homogenizer at 4°C and a homogenization pressure of 689.5 bar. The homogenized emulsion was then filtered sequentially through microporous membranes of 0.45 μm and 0.22 μm to obtain the cell membrane vesicle self-assembly of Spirulina PDRN loaded in Example 1.

[0033] 2. Example 2 Step 1: Disperse the wet Spirulina algae in Tris-HCl buffer at a mass-to-volume ratio of 1:10 g / mL. Homogenize 5 times at 6℃ and a homogenization pressure of 1000 bar. After homogenization, filter the suspension through 1μm and 0.22μm filter membranes at 6℃ to obtain Spirulina vesicle suspension. Step 2: Take 200 mL of Spirulina vesicle suspension, 3.125 g of 20 w / w% Spirulina-derived PDRN solution, 7.5 g of hydrogenated soybean phosphatidylcholine (HSPC), 1.25 g of subtilis lipopeptide sodium, 2.5 g of avocado fruit oil, 100 g of 1.0 v / v% glycerol, and 800 mL of Tris-HCl buffer and mix them to obtain a premix solution; Step 3: The premixed solution was pre-homogenized at 12000 rpm for 3 min, and then homogenized 5 times using a high-pressure microfluidic homogenizer at 10°C and a homogenization pressure of 1379 bar. The homogenized emulsion was then filtered sequentially through microporous membranes of 0.45 μm and 0.22 μm to obtain the cell membrane vesicle self-assembly of Spirulina PDRN loaded in Example 2.

[0034] 3. Example 3 Step 1: Disperse the wet Spirulina algae in Tris-HCl buffer at a mass-to-volume ratio of 1:5 g / mL. Homogenize three times at 10℃ and a homogenization pressure of 1500 bar. After homogenization, filter the suspension through 1 μm and 0.22 μm filter membranes at 10℃ to obtain Spirulina vesicle suspension. Step 2: Take 200 mL of Spirulina vesicle suspension, 11.38 g of 20 w / w% Spirulina-derived PDRN solution, 4.6 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.46 g of subtilis lipopeptide sodium, 0.46 g of avocado oil, 100 g of 0.5 v / v% glycerol, and 800 mL of Tris-HCl buffer and mix them to obtain a premix solution; Step 3: The premixed solution was pre-homogenized at 15000 rpm for 2 min, and then homogenized three times using a high-pressure microfluidic homogenizer at 15°C and a homogenization pressure of 2068.5 bar. The homogenized emulsion was then filtered sequentially through microporous membranes of 0.45 μm and 0.22 μm to obtain the cell membrane vesicle self-assembly of Spirulina PDRN loaded in Example 3.

[0035] 4. Example 4 The preparation method in Example 2 was adjusted as follows: Step 1: Take 3.125g of 20w / w% Spirulina-derived PDRN solution, 27.5g of hydrogenated soybean phosphatidylcholine (HSPC), 1.25g of subtilis lipopeptide sodium, 2.5g of avocado oil, 100g of 1.0v / v% glycerol, and 1000mL of Tris-HCl buffer and mix them to obtain a premixed solution; Step 2: The premixed liquid was pre-homogenized at 12000 rpm for 3 min, and then homogenized 5 times using a high-pressure micro-jet homogenizer at 10°C and a homogenization pressure of 1379 bar. The homogenized emulsion was then filtered sequentially through microporous membranes of 0.45 μm and 0.22 μm to obtain Example 4. The difference from Example 2 is that Example 4 lacks spirulina vesicle suspension, and the missing amount is made up with hydrogenated soybean phosphatidylcholine and buffer solution.

[0036] 5. Example 5 The preparation method in Example 2 was adjusted as follows: Step 1 is the same as Step 1 in Example 2; Step 2: Take 200 mL of Spirulina vesicle suspension, 3.125 g of 20 w / w% Spirulina-derived PDRN solution, 8.75 g of hydrogenated soybean phosphatidylcholine (HSPC), 2.5 g of avocado oil, 100 g of 1.0 v / v% glycerol, and 800 mL of Tris-HCl buffer and mix them to obtain a premixed solution; Step 3: Same as step 3 in Example 2, to obtain Example 5; The difference from Example 2 is that Example 5 lacks sodium subtilis lipopeptide, and the missing amount is made up with hydrogenated soybean phosphatidylcholine.

[0037] 6. Example 6 The preparation method in Example 2 was adjusted as follows: Step 1 is the same as Step 1 in Example 2; Step 2: Take 200 mL of Spirulina vesicle suspension, 3.125 g of 20 w / w% Spirulina-derived PDRN solution, 10 g of hydrogenated soybean phosphatidylcholine (HSPC), 1.25 g of subtilis lipopeptide sodium, 100 g of 1.0 v / v% glycerol, and 800 mL of Tris-HCl buffer and mix them to obtain a premixed solution; Step 3: Same as step 3 in Example 2, to obtain Example 6; The difference from Example 2 is that Example 6 lacks avocado oil, and the missing amount is made up with hydrogenated soybean phosphatidylcholine.

[0038] 7. Example 7 The preparation method in Example 2 was adjusted as follows: Step 1 is the same as Step 1 in Example 2; Step 2: Take 200 mL of Spirulina vesicle suspension, 3.125 g of 20 w / w% Spirulina-derived PDRN solution, 11.25 g of hydrogenated soybean phosphatidylcholine (HSPC), 100 g of 1.0 v / v% glycerol, and 800 mL of Tris-HCl buffer and mix them to obtain a premixed solution. Step 3: Same as step 3 in Example 2, to obtain Example 7; The difference from Example 2 is that Example 7 lacks sodium subtilis lipopeptide and avocado oil, and the missing amounts are made up with hydrogenated soybean phosphatidylcholine.

[0039] IV. Performance Testing 1. Encapsulation efficiency test 1.1 Test Method The encapsulation efficiency (EE) of Spirulina PDRN in cell membrane vesicle self-assemblies loaded with Spirulina PDRN was determined by ultrafiltration centrifugation. 1 mL of each vesicle self-assembly sample from Examples 1-7 was placed in an ultrafiltration centrifuge tube, centrifuged at 10000 r / min for 10 min, and the filtrate was collected. The concentration of free Spirulina PDRN (W1) was determined by HPLC. Separately, 1 mL of each of the above samples was subjected to ultrasonic demulsification for 10 min, filtered through a 0.45 μm organic filter membrane, and the concentration of total Spirulina PDRN after demulsification was determined by HPLC (W2).

[0040] The encapsulation efficiency of Spirulina PDRN was calculated using the following formula. All samples were measured in triplicate, and the average value was taken as the final result: .

[0041] 1.2 Results and Analysis 1.2.1 Test Results The results are shown in Table 1; Table 1 Encapsulation efficiency test results Group Encapsulation rate / % Example 1 67.36 Example 2 70.28 Example 3 65.15 Example 4 32.47 Example 5 58.19 Example 6 59.63 Example 7 55.71 1.2.2 Results Analysis The encapsulation rates of Examples 1-3 were all stable within the range of 65.15% to 70.28%, with Example 2 showing the highest encapsulation rate. This indicates that in the preparation process of the present invention, Spirulina vesicles serve as the core carrier, and the combination of hydrogenated soybean phosphatidylcholine, subtilis lipopeptide sodium, and avocado oil can effectively encapsulate Spirulina PDRN, forming structurally complete vesicle self-assemblies.

[0042] Example 4 showed that the encapsulation rate dropped sharply to 32.47% due to the absence of Spirulina vesicles, proving that Spirulina natural vesicles are the core matrix for effective PDRN encapsulation. Their cell membrane structure can provide specific encapsulation sites and aqueous chambers for PDRN, and the encapsulation effect is far superior to that of liposome systems constructed from phospholipids alone.

[0043] The encapsulation efficiency of both Example 5 (lacking sodium subtilisin) and Example 6 (lacking avocado oil) decreased significantly. This indicates that sodium subtilisin can improve the fusion efficiency of phospholipids with the natural vesicle membrane, and avocado oil can enhance the flexibility and integrity of the vesicle bilayer. Both can reduce PDRN leakage during encapsulation and homogenization, making them synergistic excipients for improving vesicle encapsulation efficiency.

[0044] 2. Transdermal performance test 2.1 Test Objective Using an in vitro Bama miniature pig skin permeation model combined with laser confocal fluorescence microscopy imaging technology, the vertical penetration depth of PDRN-loaded cell membrane vesicle self-assemblies in skin tissue was qualitatively observed and quantitatively determined by fluorescently labeled Spirulina PDRN, thus evaluating its transdermal delivery performance.

[0045] 2.2 Preparation of Fluorescently Labeled Samples FITC-PDRN was covalently fluorescently labeled with fluorescein isothiocyanate (FITC). After dialysis purification to remove free fluorescein, FITC-PDRN was prepared. Following the formulations and preparation processes of Examples 2 and 4-7, the original spirulina-derived PDRN in the system was replaced with FITC-PDRN in equal molar amounts to prepare the corresponding fluorescently labeled test samples. At the same time, a FITC-PDRN aqueous solution with a concentration of 2000 μg / mL was prepared as a control solution.

[0046] 2.3 Test Method The processed Bama miniature pig skin was fixed between the supply and receiving pools of a Franz diffusion cell, with the stratum corneum facing the supply pool and the dermis facing the receiving pool, ensuring a tight fit between the skin and the diffusion cell, free of air bubbles and leakage. A constant temperature water bath was set at 32±0.5℃, and the magnetic stirring speed was 200 r / min. After the system equilibrated for 30 min, the test samples from Examples 2, 4-7, and the control solution were added to the supply pool, with three replicates for each experiment. After administration, the samples were incubated in the dark for 5 h. The skin was then removed, rinsed, thoroughly cleaned, and dried. The samples were frozen and sectioned, and the sections were observed using a laser confocal microscope. The results are shown in Table 2.

[0047] 2.4 Results and Analysis 2.4.1 Test Results The results of the 5-hour skin penetration depth test for each group of samples are shown in Table 2; Table 2 Results of skin penetration depth test for each group of samples after 5 hours Group Average penetration depth / μm Example 2 192.36±18.74 Example 4 86.52±9.13 Example 5 112.47±15.25 Example 6 125.83±14.09 Example 7 98.15±7.82 FITC-PDRN control solution 62.74±5.36 2.4.2 Results Analysis As shown in Table 2, the self-assembled body of Example 2 can penetrate the stratum corneum and epidermis of the skin and reach the dermis, with an average penetration depth of 192.36 μm, which is better than that of the groups of Examples 4-7 and the control solution group, demonstrating excellent transdermal barrier penetration ability and deep delivery performance.

[0048] The results of Example 4 indicate that Spirulina vesicles are the core matrix for achieving efficient transdermal delivery. They are highly similar to human skin cell membranes and can carry encapsulated PDRN through the stratum corneum lipid membrane and intercellular penetration pathways to achieve deep delivery. Their transdermal ability is far superior to that of liposome systems constructed from artificial phospholipids alone.

[0049] The results of Examples 5-7 show that sodium subtilis lipopeptide can optimize the particle size uniformity and membrane fusion efficiency of vesicles, making it easier for nanoscale vesicles to penetrate deep into the skin through the intercellular spaces; avocado oil can enhance the flexibility and structural integrity of the vesicle bilayer, reducing vesicle rupture and leakage of contents during transdermal delivery. Both are key synergistic excipients for improving the transdermal delivery capability of vesicles.

[0050] In summary, this invention uses natural spirulina cell membrane vesicles as the core and combines them with composite lipid excipients to construct a vesicle self-assembly, which can significantly break through the skin stratum corneum barrier and efficiently deliver encapsulated PDRN to the dermis, solving the problem that water-soluble PDRN is difficult to absorb through the skin and cannot reach the target layer to exert its activity.

[0051] 3. Effects of PDRN-loaded self-assemblies on the repair of damaged cells 3.1 HaCat cell SLS damage test (1) Experimental materials: HaCat cells (modeling induction concentration: 1% SLS).

[0052] (2) Experimental method: CCK-8 method.

[0053] (3) Test samples: Example 2, Example 4-7, Spirulina-derived PDRN, Ectoin, Vitamin C.

[0054] (4) Grouping: Blank control group (no induction and no drug administration), negative control group (induction only and no drug administration), positive control group 1 (induction + 100 ng / mL ectoine co-incubation), positive control group 2 (induction + 100 μg / mL vitamin C co-incubation), Example 2 group (induction + 0.25% Example 2 co-incubation), Example 4, 5, 6, 7 groups (induction + 0.25% Example 4, 5, 6, 7 co-incubation), PDRN control (induction + 1% spirulina-derived PDRN co-incubation).

[0055] (5) Results: See Table 3 for details.

[0056] Table 3 Results of SLS damage test in HaCat cells Group Relative cell viability Improvement rate (compared to negative control) Blank control group 100 / negative control group 40.93 / 100 ng / mL ectoine 101.06 146.91% 100 μg / mL Vitamin C 98.32 140.22% Example 2 (concentration 0.25wt%) 93.74 129.03% Example 4 (concentration 0.25wt%) 56.28 37.50% Example 5 (concentration 0.25wt%) 79.52 94.28% Example 6 (concentration 0.25wt%) 80.63 96.99% Example 7 (concentration 0.25wt%) 76.18 86.12% PDRN control group (concentration 1 wt%) 51.96 26.95% 3.2. Test on the effect of HSF cell COL-III content (1) Experimental materials: HSF cells (modeling induction: UVA intensity 5J / cm)2 ); (2) Experimental method: ELISA method; (3) Test samples: Examples 2, 4-7, Spirulina-derived PDRN, TGF-β1; (4) Grouping: Blank control group (no induction and no drug administration), negative control group (induction only and no drug administration), positive control group (induction + 100 ng / mL TGF-β1 co-incubation), Example 2 group (induction + 0.25% Example 2 co-incubation), Example 4, 5, 6, 7 groups (induction + 0.25% Example 4, 5, 6, 7 co-incubation), PDRN control (induction + 1% Spirulina-derived PDRN co-incubation).

[0057] (5) Results: See Table 4 for details.

[0058] Table 4 Results of the Influence of COL-III Content Group <![CDATA[COL-III / pg·mL -1 ]]> Improvement rate (compared to negative control) Blank control group 53.19 / negative control group 27.82 / 100 ng / mL TGF-β1 54.43 95.65% Example 2 (concentration 0.25wt%) 47.25 69.84% Example 4 (concentration 0.25wt%) 34.01 22.25% Example 5 (concentration 0.25wt%) 38.97 40.08% Example 6 (concentration 0.25wt%) 39.15 40.73% Example 7 (concentration 0.25wt%) 36.78 32.21% PDRN control group (concentration 1 wt%) 31.26 12.37% 3.3, Raw264.7 Cellular Inflammatory Factor NO Effect Test (1) Experimental materials: HSF cells (modeling induction: 200 ng / mL LPS); (2) Experimental method: ELISA method; (3) Test samples: Examples 2, 4-7, Spirulina-derived PDRN, and dexamethasone; (4) Grouping: Blank control group (no induction and no drug administration), negative control group (induction only and no drug administration), positive control group (induction + 10 μg / mL dexamethasone co-incubation), Example 2 group (induction + 0.25% Example 2 co-incubation), Example 4, 5, 6, 7 groups (induction + 0.25% Example 4, 5, 6, 7 co-incubation), PDRN control (induction + 1% Spirulina-derived PDRN co-incubation).

[0059] (5) Results: See Table 5 for details.

[0060] Table 5 Results of NO test for inflammatory factors Group <![CDATA[NO / μmol·L -1 ]]> Inhibition rate (compared to negative control) Blank control group 2.34 / negative control group 16.52 / 10 μg / mL dexamethasone 6.11 63.01% Example 2 (concentration 0.25wt%) 10.48 36.56% Example 4 (concentration 0.25wt%) 13.50 18.28% Example 5 (concentration 0.25wt%) 12.61 23.67% Example 6 (concentration 0.25wt%) 12.08 26.88% Example 7 (concentration 0.25wt%) 12.97 21.49% PDRN control group (concentration 1 wt%) 14.29 13.50% 3.4 Results Analysis As shown in Tables 3, 4, and 5, in the SLS-induced HaCat cell damage experiment, the cell damage improvement rate of the 0.25wt% Example 2 group reached 129.03%, far exceeding that of the 1wt% high-concentration free PDRN group (26.95%). In the UVA-induced HSF cell photoaging experiment, the improvement rate of type III collagen synthesis in the Example 2 group reached 69.84%, more than 5 times that of the free PDRN group. In the LPS-induced Raw264.7 cell inflammation experiment, the inhibition rate of the pro-inflammatory factor NO in the Example 2 group reached 36.56%, 2.7 times that of the free PDRN group. The groups lacking the Spirulina vesicle core carrier and the Substances Bacillus subtilis lipopeptide sodium / avocado oil excipients showed a significant decline in all efficacy indicators.

[0061] Example 2 is the optimal formulation of the present invention, which can efficiently encapsulate and deliver PDRN transdermally. It has excellent barrier repair, collagen promotion and anti-aging, anti-inflammatory and soothing effects, and can significantly improve skin damage, photoaging, sensitivity and redness, etc., and achieve the core skin care effects of anti-aging, firming and repair.

[0062] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for preparing a cell membrane vesicle self-assembly loaded with spirulina PDRN, characterized in that, The preparation method comprises the following steps: Step 1, the spirulina is dispersed in a buffer solution at a concentration of 5-20 w / v%, and the mixture is uniformly mixed to obtain an algal body dispersion liquid, and the dispersion liquid is placed in an environment at 4-10 DEG C for homogenization crushing, and the mixed solution after homogenization is filtered through 1 mu m and 0.22 mu m filter membranes in sequence, and the filtrate is collected to obtain a spirulina vesicle suspension; Step 2, the spirulina vesicle suspension, spirulina-derived PDRN solution, hydrogenated soybean phosphatidylcholine, bacillus subtilis lipopeptide sodium, avocado oil, glycerol and solvent are mixed to obtain a drug-loaded vesicle premix; Step 3, the drug-loaded vesicle premix is first subjected to pre-homogenization treatment, the rotation speed is controlled at 8000-15000 rpm, and the treatment time is 2-5 min, and then fine homogenization is carried out at 4-15 DEG C, the homogenization pressure is controlled at 689.5-2068.5 bar, and the cycle treatment is 3-8 times; Step 4, the homogenized vesicle emulsion is sequentially filtered through 0.45 mu m and 0.22 mu m microporous filter membranes to obtain the spirulina PDRN-loaded cell membrane vesicle self-assembly.

2. The production method according to claim 1, characterized by, In step 1, the spirulina is selected from at least one of spirulina wet algal body and resuspended spirulina dry powder.

3. The preparation method according to claim 1, characterized in that, In step 1, the buffer solution is selected from at least one of Tris-HCl buffer solution, PBS buffer solution and sterile water, and the pH value of the buffer solution is 7.0-8.

5.

4. The method of claim 1, wherein, In the step 1, the homogenization pressure is controlled at 800-1500 bar, and the cycle treatment is 3-10 times.

5. The preparation method according to claim 1, characterized in that, In step 2, the solvent is at least one of sterile water for injection, PBS buffer solution and Tris-HCl buffer solution.

6. The method of claim 1, wherein, The amount of spirulina vesicles is 38-88% of the total lipid, the amount of hydrogenated soybean phosphatidylcholine is 10%-40 w / w% of the total lipid, the amount of bacillus subtilis lipopeptide sodium is 1-7 w / w% of the total lipid, the amount of avocado oil is 1-15 w / w% of the total lipid, and the concentration of glycerol is 0.5-80 v / v%.

7. The preparation method according to claim 1, characterized in that, In step 2, the mass ratio of spirulina-derived PDRN to total lipid is 1:20 to 1:

100.

8. A cell membrane vesicle self-assembly loaded with spirulina PDRN, characterized in that, The cell membrane vesicle self-assembly is prepared by the preparation method of any one of claims 1-7.

9. The use of the cell membrane vesicle self-assembly of claim 8 in the preparation of skin care products with anti-aging, firming, anti-wrinkle and whitening effects.