Liposome-exosome hybrid vesicle as well as preparation method and application thereof

Abstract

The invention provides a liposome-exosome hybrid vesicle as well as a preparation method and application thereof, and relates to the field of biomedicine. The preparation method comprises the following steps: mixing an organic phase containing lipidosome and a water phase containing anionic polysaccharide, emulsifying to form a water-in-oil emulsion, then removing an unemulsified organic phase in the water-in-oil emulsion, and then sequentially hydrating and homogenizing to obtain the anionic polysaccharide lipidosome, the exosome and the anionic polysaccharide lipidosome are fused, then cationic polysaccharide is added, and after incubation, membrane extrusion is performed to obtain the lipidosome-exosome hybrid vesicle. According to the preparation method and the liposome-exosome hybrid vesicle prepared by the preparation method, the defects in exosome application are relieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a liposome-exosome hybrid vesicle and a preparation method and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Exosomes (Exo) are extracellular vesicles secreted by almost all types of cells. They have a diameter of about 100nm. They are derived from the body and have the characteristics of low immunogenicity and high biocompatibility. More importantly, Exo has potential targeting ability, which helps to improve the targeting ability of drugs. There have been many studies in the fields of drug delivery, tissue repair and regeneration. Studies have shown that Exo can participate in various processes of skin tissue repair and regeneration, so it has great application value in the fields of anti-inflammatory relief, wrinkle removal, anti-aging, burns and scalds, difficult-to-heal ulcers and postoperative trauma repair. However, due to the barrier function of the skin, Exo cannot penetrate into the skin through the skin very well. In particular, the amount of Exo obtained is small and relatively precious, and the barrier function of the skin will cause unnecessary waste. At present, most animal models use local injection of exosomes to treat melasma, which has problems such as high pain, unfavorable diffusion of exosomes, and long recovery period.

[0004] Exo has poor stability due to its own biological origin. Hybridization of artificially synthesized stable liposomes and Exo can improve the stability of exosomes on the one hand, retain the biological characteristics of exosomes on the other hand, and achieve the scale-up production of hybrid Exo based on Exo and liposomes. At present, the commonly used methods for hybridization of liposomes and Exo mainly include freeze-thaw method, natural incubation method, polyethylene glycol method, ultrasound method and thin film dispersion method. However, it has been found in practice that these methods are difficult to control accurately, easily destroy the integrity of the Exo membrane, have low hybridization efficiency and affect the biological activity of Exo.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing a liposome-exosome hybrid vesicle and a liposome-exosome hybrid vesicle to alleviate the defects in the application of exosomes.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] In a first aspect, a method for preparing a liposome-exosome hybrid vesicle is provided, the preparation method comprising:

[0009] An organic phase containing liposomes and an aqueous phase containing anionic polysaccharides are mixed and emulsified to form an oil-in-water emulsion, and then the unemulsified organic phase in the oil-in-water emulsion is removed, followed by hydration and homogenization to obtain anionic polysaccharide liposomes;

[0010] The exosomes and the anionic polysaccharide liposomes are fused, and then cationic polysaccharides are added, and after incubation, the liposome-exosome hybrid vesicles are obtained by membrane extrusion.

[0011] In a second aspect, a liposome-exosome hybrid vesicle is provided, wherein the liposome-exosome hybrid vesicle contains liposomes, exosomes, anionic polysaccharides and cationic polysaccharides; the liposomes hybridize exosomes and anionic polysaccharides, and the anionic polysaccharides are embedded in the liposome membrane, and the cationic polysaccharides are located on the surface of the liposomes.

[0012] In a third aspect, a method for preparing the liposome-exosome hybrid vesicles of the first aspect, or use of the liposome-exosome hybrid vesicles of the second aspect in preparing products for skin problems is also provided.

[0013] In a fourth aspect, a product for skin problems is also provided, the product comprising the liposome-exosome hybrid vesicles of the first aspect and optional auxiliary materials.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The method for preparing liposome-exosome hybrid vesicles provided by the present invention has the advantages of high fusion rate between exosomes and liposomes, high stability of exosomes, and also maintains the integrity of the exosome membrane. At the same time, the size of the prepared liposome-exosome hybrid vesicles can be controlled at 200-400nm, and the particle size is uniform.

[0016] In the preparation method provided by the present invention, anionic polysaccharides are self-assembled with phospholipids before hybridization to form anionic polysaccharide liposomes. Anionic polysaccharide liposomes have good active ingredient encapsulation ability and ability to promote transdermal absorption, enhance affinity for cells and prolong circulation time, and achieve efficient penetration and long-term retention of exosomes in the skin. Cationic polysaccharides are added to the vesicle system after hybridization to play a fixing role on the outer layer of the vesicle. The amphoteric electrolyte properties of cationic polysaccharides are utilized to physically cross-link them with anionic polysaccharides, and through hydrogen bonds and electrostatic interactions, the stability of the hybrid vesicles is further improved, and the cryoprotective performance is improved. During the production and storage process, the use of charged poly / oligosaccharides can delay the denaturation of exosomes to various environmental stresses, so that the liposome-exosome hybrid vesicles will not lose their biological activity and have a longer shelf life.

[0017] In summary, the preparation method provided by the present invention enables the obtained liposome-exosome hybrid vesicles to have significant transdermal absorption performance, and also has the advantages of high stability, controllable particle size, easier freeze-drying and storage, and significantly promoting transdermal absorption effect. The liposome-exosome hybrid vesicles show a positive promoting effect in improving oxidative stress, inhibiting apoptosis and regulating inflammatory response, and have significant effects in improving skin damage, repairing skin barrier function, and promoting protein expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Potential diagrams of Exo, hL, and hcL-Exo;

[0020] Figure 2 The particle size diagrams of Exo, hL, and hcL-Exo;

[0021] Figure 3 The morphology and structure of Exo, hL, and hcL-Exo, where a is the TEM image of Exo, hL, and hcL-Exo, scale bar = 200 nm, and b is the schematic diagram of confocal fluorescence microscopy analysis of Exo, hL, and hcL-Exo, scale bar = 5 μm;

[0022] Figure 4 Western blot analysis of CD81 and TSG101 in Exo and hcL-Exo;

[0023] Figure 5 To quantify the fluorescence intensity of Exo and hcL-Exo penetrating the skin at different time intervals of 1h, 3h, 5h, and 7h;

[0024] Figure 6 The permeation distribution of Exo and hcL-Exo in the skin after 7 h of in vitro permeation in Franz diffusion cells, scale bar = 100 μm;

[0025] Figure 7 This is the quantitative analysis result of epidermal thickness;

[0026] Figure 8 This is the quantitative analysis result of K1 expression;

[0027] Fig. 9 is the content of TNF-α in the skin of mice in each group;

[0028] Fig.10 is the expression level of SOD activity in the skin of mice in each group;

[0029] Fig.11 The freeze-drying effect diagram of liposome-exosome without sodium hyaluronate and carboxymethyl chitosan (left) and liposome-exosome with sodium hyaluronate and carboxymethyl chitosan (right);

[0030] Fig.12 The stability graphs are of the liposome-exosomes without sodium hyaluronate and carboxymethyl chitosan after freeze-drying and re-dissolving (top) and the liposome-exosomes with sodium hyaluronate and carboxymethyl chitosan after freeze-drying and re-dissolving (bottom). DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] It should be noted that:

[0033] In the present invention, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution.

[0034] In the present invention, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0035] In the present invention, unless otherwise specified, percentage (%) or part refers to the weight percentage or weight part relative to the composition.

[0036] In the present invention, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution.

[0037] In the present invention, unless otherwise specified, the numerical range "a-b" represents an abbreviation of any real number combination between a and b, where a and b are real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" have been listed in this document, and "6-22" is just an abbreviation of these numerical combinations.

[0038] The “range” disclosed in the present invention is in the form of a lower limit and an upper limit, which can be one or more lower limits, and one or more upper limits, respectively.

[0039] In the present invention, unless otherwise specified, each reaction or operation step may be performed in sequence or not.

[0040] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0041] In a first aspect, a method for preparing a liposome-exosome hybrid vesicle is provided, the preparation method comprising:

[0042] An organic phase containing liposomes and an aqueous phase containing anionic polysaccharides are mixed and emulsified to form an oil-in-water emulsion, and then the unemulsified organic phase in the oil-in-water emulsion is removed, followed by hydration and homogenization to obtain anionic polysaccharide liposomes;

[0043] The exosomes and the anionic polysaccharide liposomes are fused, and then cationic polysaccharides are added, and after incubation, the liposome-exosome hybrid vesicles are obtained by membrane extrusion.

[0044] In an optional embodiment, the liposomes are soybean lecithin and cholesterol, and the feed ratio of soybean lecithin to cholesterol is (20-40):1, preferably 30:1.

[0045] In an alternative embodiment, the anionic polysaccharide comprises sodium hyaluronate.

[0046] In an optional embodiment, the molecular weight of the sodium hyaluronate is 1 to 1.5M.

[0047] In an alternative embodiment, the cationic polysaccharide comprises carboxymethyl chitosan.

[0048] In an optional embodiment, the mass ratio of anionic polysaccharide to cationic polysaccharide is 1:(1-5), preferably 1:2.

[0049] In an optional embodiment, the organic phase is a mixed system of ethanol and chloroform.

[0050] In an optional embodiment, the volume ratio of ethanol to chloroform is (1-2):(1-2), preferably 1:1.

[0051] In an optional embodiment, the aqueous phase is a buffer, preferably a phosphate buffer, more preferably a phosphate buffer with a pH of 7.4.

[0052] In an optional embodiment, the concentration of the anionic polysaccharide in the aqueous phase is 0.1-0.5% w / v, preferably 0.1% w / v.

[0053] In an optional embodiment, the emulsification includes mixing the organic phase containing the liposomes and the aqueous phase containing the anionic polysaccharide under stirring, and then stirring and dissolving them using a high shear dispersing emulsifier to form an oil-in-water emulsion.

[0054] In an alternative embodiment, rotary evaporation is used to remove the unemulsified organic phase of the water-in-oil emulsion.

[0055] In an alternative embodiment, the hydration comprises hydration using a phosphate buffer.

[0056] In an optional embodiment, the homogenization is performed by microfluidization.

[0057] In an optional embodiment, the preparation method includes using vortexing and ultrasonic treatment to promote the fusion of exosomes and the anionic polysaccharide liposomes. When exosomes and anionic polysaccharide liposomes exist in a unified system, they will fuse, and vortexing and ultrasound will make the two fuse more fully, and the number of successful fusions will be greater. The fusion rate of exosomes and liposomes and the stability of exosomes are further improved by combining ultrasound, incubation and membrane extrusion.

[0058] In an optional embodiment, the parameters of the ultrasonic treatment include 20-50% maximum amplitude, 2-8 seconds pulse on / off, and treatment for 2-5 minutes.

[0059] In an optional embodiment, the parameters of the ultrasonic treatment include 30% maximum amplitude, 5-second pulse on / off, and treatment for 3 minutes.

[0060] In an optional embodiment, the incubation includes incubating at 30-40° C. for 0.5-2 h after adding the cationic polysaccharide.

[0061] In an optional embodiment, the incubation includes incubating at 37° C. for 1 hour after adding the cationic polysaccharide.

[0062] In an optional embodiment, the membrane extrusion includes sequentially passing through a 300-500 nm membrane and a 100-200 nm membrane, and the liposome-exosome hybrid vesicles are obtained after extrusion 20 to 100 times.

[0063] In an optional embodiment, the membrane extrusion includes passing through a 400 nm and a 200 nm membrane in sequence, and the liposome-exosome hybrid vesicles are obtained after extrusion 50 times.

[0064] In an alternative embodiment, the film comprises a polycarbonate film.

[0065] In an optional embodiment, the preparation method further comprises freeze-drying the liposome-exosome hybrid vesicles obtained after membrane extrusion.

[0066] In a specific embodiment, the method for preparing the liposome-exosome hybrid vesicles comprises:

[0067] (a) Soybean lecithin and cholesterol in a mass ratio of 1:1 were added to anhydrous ethanol and chloroform in a volume ratio of 1:1 in a ratio of 30:1, and heated in a water bath with stirring for 2 h;

[0068] (b) weighing sodium hyaluronate powder with a molecular weight of 1 to 1.5 M and adding it to a phosphate buffer solution (pH = 7.4), stirring for 4 hours to obtain a sodium hyaluronate solution, wherein the sodium hyaluronate concentration is 0.1%, w / v;

[0069] (c) mixing the products of step (a) and (b) uniformly under magnetic stirring, stirring and dissolving them in a high shear dispersing emulsifier to form a water-in-oil emulsion, and finally rotary evaporating, hydrating with a phosphate buffer solution, and microfluidizing to obtain hyaluronic acid liposomes;

[0070] (d) fusing the exosomes with the hyaluronic acid liposomes obtained in step (c) at an equal mass (mg) to volume (mL) ratio and vortexing and sonicating, wherein the vortexing and sonicating parameters are 30% maximum amplitude, 5-second pulse on / off, and 3 min;

[0071] (e) adding carboxymethyl chitosan to the product of step (d), with the mass ratio of sodium hyaluronate to carboxymethyl chitosan being 1:2, mixing well and placing the mixture in a shaker at 37° C. for incubation for 1 h, then passing through 400 nm and 200 nm polycarbonate membranes in turn, manually pushing the mixture forward and backward 50 times to obtain the liposome-exosome hybrid vesicles, and then freeze-drying the mixture.

[0072] In a second aspect, a liposome-exosome hybrid vesicle is also provided, which contains liposomes, exosomes, anionic polysaccharides and cationic polysaccharides; the liposomes hybridize exosomes and anionic polysaccharides, and the anionic polysaccharides are embedded in the liposome membrane, and the cationic polysaccharides are located on the surface of the liposomes.

[0073] In an optional embodiment, the liposome comprises soybean lecithin and cholesterol; the anionic polysaccharide comprises sodium hyaluronate; the cationic polysaccharide comprises carboxymethyl chitosan;

[0074] In an optional embodiment, the molecular weight of the sodium hyaluronate is 1 to 1.5M.

[0075] In an optional embodiment, the particle size of the liposome-exosome hybrid vesicle is 200-400 nM.

[0076] In an optional embodiment, the liposome-exosome hybrid vesicles of the second aspect are prepared by the preparation method of the first aspect.

[0077] In a third aspect, a method for preparing the liposome-exosome hybrid vesicles of the first aspect, or use of the liposome-exosome hybrid vesicles of the second aspect in preparing products for skin problems is also provided.

[0078] In a fourth aspect, a product for skin problems is also provided, the product comprising the liposome-exosome hybrid vesicles of the first aspect and optional auxiliary materials.

[0079] The uses of the products for skin problems in the third and fourth aspects include, but are not limited to, one or more of anti-inflammatory, soothing, wrinkle removal, anti-aging and wound repair, wherein the wound repair includes, but is not limited to, burns, scalds, difficult-to-heal ulcers and postoperative wound repair.

[0080] The optional excipients may be, but are not limited to, any known and acceptable excipients in the field of cosmetics, including, but not limited to, one or more of lyoprotectants, solvents, solubilizers, cosolvents, colorants, adhesives, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, antibacterial agents, fragrances, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, protective agents and moisturizers.

[0081] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0082] Example 1 Preparation of hcL-Exo

[0083] Soybean lecithin and cholesterol were added to anhydrous ethanol and chloroform in a ratio of 30:1 (W / W), heated in a water bath and stirred for 2 hours, and sodium hyaluronate powder (0.1%, W / V) with a molecular weight of 1-1.5M was weighed and added to a phosphate buffer solution (pH=7.4), and stirred for 4 hours to obtain a sodium hyaluronate solution. The two were slowly mixed evenly under magnetic stirring, stirred and dissolved by a high shear dispersing emulsifier to form a stable W / O (water-in-oil) emulsion, and finally rotary evaporated, hydrated with a phosphate buffer solution, and microfluidized to obtain hL (sodium hyaluronate liposomes). Exo and hL were fused in the same ratio and treated with vortex and ultrasound (30% maximum amplitude, 5-second pulse on / off, 3 min), and then carboxymethyl chitosan (HA: carboxymethyl chitosan = 1:2, W / W) was added and mixed evenly. The mixture was placed in a shaker at 37°C and incubated for 1 h. After that, it was passed through 400 nm and 200 nm polycarbonate membranes in sequence, and it was manually pushed forward and backward 50 times to obtain hcL-Exo, which was then freeze-dried.

[0084] Effect Example 1 Characterization of hcL-Exo:

[0085] Fluorescence labeling was used to label the liposomes and exosomes in hcL-Exo. 50 μL of PKH-26 dye working solution was added to 200 μg Exo equivalent solution, and 5 μL of PKH-67 dye working solution was added to 100 μL hL. After mixing, the mixture was incubated at room temperature for 10 min. After centrifugation at 4 °C, the supernatant was removed to remove excess dye and the precipitate was resuspended in phosphate buffer solution (pH = 7.4) as required to obtain fluorescently labeled Exo dye complex and hL dye complex. The experiment was kept away from light. The same operation steps were followed to obtain fluorescently labeled hcL-Exo.

[0086] The zeta potential and particle size changes of hL, Exo and hcL-Exo were measured using a laser nanoparticle size analyzer (Malvern). Figure 1 and Figure 2 ).

[0087] The morphological changes of hL, Exo and hcL-Exo were observed using a high-power transmission electron microscope (JEOL TEM-1210) with an operating voltage of 120KV (the samples were stained with 1% phosphotungstic acid before being dropped onto the copper grid). The results are shown in Figure 3 The structures of Exo labeled with PKH-26 and hL labeled with PKH-67 were characterized by laser confocal microscopy (LSM 880). Figure 3 As shown in b.

[0088] The expression of CD81, TSG101 and β-actin in Exo and hcL-Exo was verified by Western Blotting experiment, and β-actin was used as the internal reference of protein expression. After incubation, the chemiluminescence imaging system was used to detect ( Figure 4 ).

[0089] Effect Example 2 Kinetic Analysis in Skin Tissue

[0090] In vitro simulated skin penetration for quantitative analysis: using a penetration area of ​​1.767 cm 2Franz diffusion cell was used to conduct in vitro simulated skin penetration experiment on Exo and hcL-Exo labeled with specific dye PKH-26. The receiving chamber was filled with phosphate buffer solution (pH=7.4) and stirred continuously at a speed of 200rpm / min. The temperature in the diffusion cell was kept constant at 37°C by circulating water bath. Mouse skin of appropriate size was sandwiched between the donor chamber and the receiving chamber, and the dermis of the mouse skin was contacted with the receptor solution of phosphate buffer solution (pH 7.4). 2mL of freshly prepared PKH-26-labeled Exo and hcL-Exo sample solution (Exo concentration was 0.1%) was given to the stratum corneum facing the donor chamber, and then 1mL of receptor solution was taken out through the sampling port at different time intervals (1h, 3h, 5h, 7h), and then replaced with an equal volume of phosphate buffer solution (pH=7.4). The fluorescence intensity at 551nm was measured by a multifunctional microplate reader (Synergy H1) to analyze the content of Exo in the receptor solution ( Figure 5 ).

[0091] Skin penetration depth and distribution: Analysis After 7 h of in vitro penetration using the Franz diffusion cell in the above experiment, the excess sample solution on the skin surface was removed, the skin was removed from the Franz diffusion cell, and the skin surface was washed with saline to ensure that no sample remained. The skin tissue was dried with filter paper and cut into pieces with an area of ​​approximately 1 cm 2 Thin slices were obtained from the skin tissue, embedded with a cryosection embedding medium (OTC compound) and fixed with liquid nitrogen. The frozen skin tissue was then cut vertically into skin slices with a thickness of about 10 μm at -20°C using a cryostat. The skin tissue slices were adhered to a glass slide and an advanced inverted fluorescence microscope (AxioCamMRc) was used to observe the fluorescence distribution in the skin (using a filter with an emission wavelength of 567 nm and an excitation wavelength of 551 nm). The results are shown in Figure 2. Figure 6 shown.

[0092] In summary, Franz diffusion cell was used to simulate in vitro permeation to determine the permeation efficiency of Exo and hcL-Exo in the skin, such as Figure 5 As shown in the figure, the intensity of fluorescently labeled Exo and hcL-Exo was measured after 1h, 3h, 5h, and 7h, respectively. Compared with Exo, hcL-Exo showed good drug penetration at 1h, and the cumulative penetration of hcL-Exo after acting on the skin within 7h was significantly higher than that of Exo. The skin was then washed, embedded, and made into frozen sections for observation under a fluorescence microscope. The results are shown in the figure. Figure 6As shown in the figure, it can be clearly seen through the longitudinal section of the mouse skin after 7 hours that compared with the Exo group, the hcL-Exo group has a stronger fluorescence intensity, higher drug permeability and higher resident content. Therefore, this study verified that the hL hybrid fusion Exo greatly improved the transdermal efficiency of Exo by using the Franze diffusion cell and the skin penetration depth and distribution.

[0093] Effect Example 3: Therapeutic application of hcL-Exo in an animal model of acute light damage

[0094] Establishment of laser dot matrix acute skin injury: The modeling method simulates the laser dot matrix of human skin. After anesthesia, the mice were depilated with a depilatory cream. 24 hours after depilation, a carbon dioxide laser dot matrix instrument (Keying Laser) was used to evenly scan and irradiate the depilated area to induce an acute skin photoinjury model in mice. The laser dot matrix energy was 2.5mJ / g, and the dot matrix range was 10×15mm. This was repeated once. The appearance of red spots or a small amount of exudate on the back skin of the mice was considered to be a successful modeling.

[0095] Mouse skin repair and histological evaluation: According to the different drugs used for laser-damaged skin wounds in mice, the mice after successful modeling were randomly divided into five groups, with 5 mice in each group, namely the healthy group, model group (PBS), Exo group, hL group and hcL-Exo group. The healthy group was treated blankly, and the model group was given phosphate buffer solution on the first day after modeling. The Exo group, hL group, and hcL-Exo group began to be treated for 7 days. After the experiment, the repaired skin tissues on the backs of mice in each group were collected and fixed in 4% paraformaldehyde solution, and then the tissue samples were dehydrated, paraffin-embedded and cut into 4μm thickness. The epidermal thickness and protein expression intensity of the skin of each group were measured by Image J software, and each sample was calculated as the average value of three random sites in the sample.

[0096] Mouse skin tissue ELISA: Double antibody sandwich ELISA is used to detect TNF-α and SOD antibodies pre-coated on high affinity ELISA plates. Standards, samples to be tested and biotinylated detection antibodies are added to the wells of the ELISA plates. After incubation, TNF-α and SOD in the samples bind to the solid phase antibodies and detection antibodies. After washing to remove unbound substances, horseradish peroxidase-labeled streptavidin is added. After washing, the color substrate TMB is added and the color is developed in the dark. The depth of the color reaction is proportional to the concentration in the sample. The stop solution is added to stop the reaction, and the absorbance values ​​are measured at specific wavelengths.

[0097] To determine the regulation of skin inflammation by hcL-Exo and the deep repair of mouse skin tissue, the treated skin samples were analyzed in detail. Figure 7As shown in the figure, epidermal thickness was used as a quantitative parameter to evaluate the degree of skin photodamage. Compared with the healthy group, the epidermal thickness of the mice in the model group increased significantly, reaching more than 6 times that of the healthy group. The skin layers of the healthy group were evenly distributed, and the junctions between the epidermis were wavy. However, the connection between the epidermis and the dermis in the model group was disturbed, and there was inflammatory infiltration in the dermis. At the same time, compared with other treatment groups, the improvement of epidermal thickness in the hcL-Exo group was the most significant. Figure 8 As shown in the figure, in the model group, keratin K1 expression decreased significantly, while the keratin K1 content in the hcL-Exo group was closer to normal levels compared with the other treatment groups. Therefore, topical application of hcL-Exo can significantly regulate protein expression and skin healing. In summary, the successful construction of an acute light-damaged animal model verified that hcL-Exo promotes local repair of skin light damage and wound healing of traumatized skin by improving the skin microenvironment.

[0098] In response to skin photodamage, keratinocytes in the skin produce a variety of pro-inflammatory factors, such as tumor necrosis factor (TNF-α), which triggers an inflammatory response and induces inflammatory cells to infiltrate the injured skin tissue. In this experiment, the ELISA test of mouse skin tissue was used to determine the content of TNF-α in the mouse skin after treatment with each group of materials. Fig. 9 As shown in the figure, the levels of inflammatory factors in the hcL-Exo group mice were closest to those in the healthy group mice, indicating that the skin inflammation of mice treated with hcL-Exo for seven days was effectively suppressed and reached a level comparable to that of the healthy group. Superoxide dismutase (SOD) is an antioxidant enzyme that can scavenge superoxide free radicals. In previous cell experiments, hcL-Exo has shown outstanding antioxidant capacity. In order to further explore the regulatory role of hcL-Exo in skin tissue, the SOD index was determined by skin tissue enzyme-linked immunosorbent assay, as shown in Figure 2. Fig.10 As shown, compared with the model group, the SOD content detected in the hcL-Exo group was significantly increased, and the SOD content in the hcL-Exo group of mice was close to that in the healthy group, indicating that hcL-Exo reduced the effect of acute light damage on the content of antioxidant enzymes in mice, promoted the production of SOD, and successfully regulated the oxidative stress in the skin of acute light-damaged mice, thereby alleviating or even reversing the damage caused by oxidative stress.

[0099] Comparative Example 1

[0100] HA and carboxymethyl chitosan were not added during the preparation process, and the rest was the same as in Example 1. The specific steps were as follows: soybean lecithin and cholesterol were added to anhydrous ethanol and chloroform in a ratio of 30:1 (W / W), heated in a water bath and stirred for 2 h, and the two were slowly mixed and uniformly mixed under the action of magnetic stirring, stirred and dissolved by a high shear dispersing emulsifier to form a stable W / O emulsion, and finally rotary evaporated, hydrated with a phosphate buffer solution, and microfluidized to obtain blank liposomes. Exo and blank liposomes were fused in the same proportion and vortexed and ultrasonicated (30% maximum amplitude, 5-second pulse on / off, 3 min), placed in a 37°C shaker for incubation for 1 h, and then passed through 400 nm and 200 nm polycarbonate membranes in turn, manually pushed forward and backward 50 times, and then freeze-dried.

[0101] like Fig.11 As shown in the figure, if HA and carboxymethyl chitosan are not added, hcL-Exo tends to clump into larger particles after freeze-drying; Fig.12 It can be seen that the transmittance of the liposome-exosome spectrum without the addition of HA and carboxymethyl chitosan is reduced, indicating that the particle concentration is increased; and the spectral morphology of hcL-Exo with the addition of HA and carboxymethyl chitosan is almost unchanged, indicating its high stability.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing liposome-exosome hybrid vesicles, characterized in that: include: An organic phase containing liposomes and an aqueous phase containing anionic polysaccharides are mixed and emulsified to form an oil-in-water emulsion, and then the unemulsified organic phase in the oil-in-water emulsion is removed, followed by hydration and homogenization to obtain anionic polysaccharide liposomes; The exosomes and the anionic polysaccharide liposomes are fused, and then cationic polysaccharides are added, and after incubation, the liposome-exosome hybrid vesicles are obtained by membrane extrusion.

2. The liposome-exosome hybrid vesicle according to claim 1, characterized in that The liposomes are soybean lecithin and cholesterol, and the feed ratio of soybean lecithin to cholesterol is (20-50):1; Optionally, the anionic polysaccharide comprises sodium hyaluronate; Optionally, the molecular weight of the sodium hyaluronate is 1 to 1.5M; Optionally, the cationic polysaccharide comprises carboxymethyl chitosan; Optionally, the mass ratio of anionic polysaccharide to cationic polysaccharide is 1:(1-5).

3. The liposome-exosome hybrid vesicle according to claim 1, characterized in that The organic phase is a mixed system of ethanol and chloroform; Optionally, the volume ratio of ethanol to chloroform is (1-2):(1-2); Optionally, the aqueous phase is a buffer, preferably a phosphate buffer; Optionally, the concentration of the anionic polysaccharide in the aqueous phase is 0.1-0.5% w / v; Optionally, the emulsification comprises mixing the organic phase containing the liposomes and the aqueous phase containing the anionic polysaccharide under stirring, and then stirring and dissolving them using a high shear dispersing emulsifier to form a water-in-oil emulsion; Optionally, the unemulsified organic phase of the water-in-oil emulsion is removed using rotary evaporation; Optionally, the hydrating comprises hydrating with a phosphate buffer; Optionally, the homogenization is performed by microfluidization.

4. The liposome-exosome hybrid vesicle according to claim 1, characterized in that The method comprises using vortexing and ultrasonic treatment to promote the fusion of exosomes and the anionic polysaccharide liposomes; Optionally, the parameters of the ultrasonic treatment include 20-50% maximum amplitude, 2-8 seconds pulse on / off, and treatment for 2-5 minutes; Optionally, the incubation includes incubating at 30 to 40° C. for 0.5 to 2 h after adding the cationic polysaccharide; Optionally, the membrane extrusion includes sequentially passing through a 300-500 nm membrane and a 100-200 nm membrane, and extruding 20-100 times to obtain the liposome-exosome hybrid vesicles; Optionally, the membrane comprises a polycarbonate membrane; Optionally, the preparation method further comprises freeze-drying the liposome-exosome hybrid vesicles obtained after membrane extrusion.

5. The liposome-exosome hybrid vesicle according to any one of claims 1 to 4, characterized in that The preparation method comprises: (a) Soybean lecithin and cholesterol in a mass ratio of 1:1 were added to anhydrous ethanol and chloroform in a volume ratio of 1:1 in a ratio of 30:1, and heated in a water bath with stirring for 2 h; (b) weighing sodium hyaluronate powder with a molecular weight of 1 to 1.5 M and adding it to a phosphate buffer solution with a pH of 7.4, stirring for 4 hours to obtain a sodium hyaluronate solution, wherein the sodium hyaluronate concentration is 0.1% w / v; (c) mixing the products of step (a) and (b) uniformly under magnetic stirring, stirring and dissolving them in a high shear dispersing emulsifier to form a water-in-oil emulsion, and finally rotary evaporating, hydrating with a phosphate buffer solution, and microfluidizing to obtain hyaluronic acid liposomes; (d) fusing the exosomes with the hyaluronic acid liposomes obtained in step (c) at an equal mass-volume ratio and vortexing and sonicating, wherein the sonication parameters are 30% maximum amplitude, 5-second pulse on / off, and 3 min; (e) adding carboxymethyl chitosan to the product of step (d), with the mass ratio of sodium hyaluronate to carboxymethyl chitosan being 1:2, mixing well and placing the mixture in a shaker at 37° C. for incubation for 1 h, then passing through 400 nm and 200 nm polycarbonate membranes in turn, manually pushing the mixture forward and backward 50 times to obtain the liposome-exosome hybrid vesicles, and then freeze-drying the mixture.

6. A liposome-exosome hybrid vesicle, characterized in that: The liposome-exosome hybrid vesicle contains liposomes, exosomes, anionic polysaccharides and cationic polysaccharides; the liposomes hybridize exosomes and anionic polysaccharides, and the anionic polysaccharides are embedded in the liposome membrane, and the cationic polysaccharides are located on the surface of the liposomes.

7. The liposome-exosome hybrid vesicle according to claim 1, characterized in that The liposomes include soybean lecithin and cholesterol; the anionic polysaccharide includes sodium hyaluronate; the cationic polysaccharide includes carboxymethyl chitosan; Optionally, the molecular weight of the sodium hyaluronate is 1 to 1.5M; Optionally, the particle size of the liposome-exosome hybrid vesicle is 200-400 nM.

8. The liposome-exosome hybrid vesicle according to claim 6 or 7, characterized in that: The compound is prepared by the preparation method described in any one of claims 1 to 5.

9. A method for preparing the liposome-exosome hybrid vesicle according to any one of claims 1 to 5, or use of the liposome-exosome hybrid vesicle according to any one of claims 6 to 8 in preparing a product for skin problems.

10. A product for skin problems, characterized in that Contains the liposome-exosome hybrid vesicle according to any one of claims 6 to 8 and optional auxiliary materials.

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