A transfersome complex sugar microneedle patch and its preparation method and application

Abstract

The present invention discloses a transfersome complex sugar microneedle patch and a preparation method and application thereof, belonging to the field of microneedle technology, and the method comprises: adding a transfersome to a monosaccharide or disaccharide solution, fully mixing to obtain a transfersome-sugar solution, using the transfersome-sugar solution to fill the needle body part of a microneedle mold, drying to obtain the needle body, and then using a polymer solution to continue filling the remaining part of the microneedle mold, drying to obtain the transfersome complex sugar microneedle patch; wherein the transfersome includes cell exosomes, cell microvesicles, apoptotic bodies or liposomes, etc. The transfersome complex sugar microneedle patch prepared by the method of the present invention can maintain the structure and biological function of the transfersome for a long time, and has broad application prospects in the fields of drug delivery, medical treatment, health care or beauty.

Description

Technical Field

[0001] The present invention relates to the technical field of microneedles, and in particular to a transfersome complex sugar microneedle patch and a preparation method and application thereof. Background Art

[0002] Microneedle transdermal drug delivery is a new drug delivery technology that has emerged in recent years and has unique clinical application value. After puncturing the skin, the microneedle can release the payload into the epidermis and dermis of the skin without touching the blood vessels and nerves, achieving efficient delivery of the payload. Microneedle drug delivery has the advantages of being stable, rapid, painless, non-invasive and easy for patients to accept, and is expected to play an important role in chronic disease management, infectious disease prevention and control, beauty and skin care, and other fields.

[0003] With the continuous development and innovation of science and technology, the research on microneedle transdermal drug delivery technology is now mainly focused on the following two aspects: (1) By designing microneedles of different shapes, sizes or materials, precise control of drug release rate, targeting and biocompatibility can be achieved to meet the treatment needs of different diseases and patients; (2) combining microneedle drug delivery technology with other drug delivery systems, such as exosomes, liposomes, nanoparticles, etc., to form a multifunctional drug delivery platform to achieve combined drug therapy and personalized treatment.

[0004] A Chinese patent document with publication number CN117122552A discloses an exosome complex microneedle patch for hair regeneration and a preparation method thereof. The microneedle patch includes a microneedle body and a microneedle substrate. The material of the microneedle body is mainly composed of biodegradable polymer materials, exosomes, plant extracts, amino acids, vitamins and nucleic acids, wherein the biodegradable polymer material is methacryloyl carboxymethyl chitosan, hyaluronic acid, chitosan, etc.

[0005] The Chinese patent document with the publication number CN112336749A discloses a stem cell exosome microneedle patch for removing spots and wrinkles and its preparation method. The microneedle patch includes a microneedle and a waterproof backing layer for fixing the microneedle. The microneedle is solidified by a needle tip solution and a needle body solution; the needle tip solution includes stem cell exosomes, astaxanthin, trehalose, chitosan, bovine serum albumin, and phosphate buffer, and the needle body solution includes PVP K30, PEG400, squalane, and sterile water. Trehalose can form a protective film on the surface of the exosome to prevent the inactivation of proteins on the surface of the exosome, and chitosan has antiseptic properties; bovine serum albumin can provide a suitable protein concentration environment for the exosome, so that it can maintain biological activity for a long time; the phosphate buffer can adjust the osmotic pressure and ionic strength of the exosome preservation solution.

[0006] However, according to the records of the above invention, it is impossible to determine whether the corresponding microneedles can effectively maintain the structure of exosomes. The structural integrity of exosomes and other transfer bodies is very important for their delivery efficiency. Therefore, it is necessary to develop a method for preparing a composite microneedle patch that can maintain the biological structural integrity of transfer bodies including exosomes, vesicles and liposomes. Summary of the invention

[0007] The present invention provides a method for preparing a transfersome complex sugar microneedle patch. The prepared transfersome complex sugar microneedle patch can maintain the structure and biological function of the transfersome for a long time, and has broad application prospects in the fields of transfersome dry storage, drug delivery, medical treatment, health care or beauty.

[0008] The specific technical solutions adopted are as follows:

[0009] A method for preparing a transfersome complex sugar microneedle patch comprises the following steps:

[0010] (1) dissolving a sugar substance in solvent A to obtain a sugar solution, wherein the sugar substance is a monosaccharide or a disaccharide, and the concentration of the sugar solution is 5 to 2000 mg / mL;

[0011] (2) adding the transfersome to the sugar solution, mixing thoroughly to obtain a transfersome-sugar solution, using the transfersome-sugar solution to fill the needle body of the microneedle mold, and drying; the transfersome is at least one of an animal-derived cell exosome, an animal-derived cell microvesicle, an animal-derived apoptotic body, an animal-derived outer membrane vesicle, an animal-derived apoptotic vesicle, and a liposome;

[0012] (3) dissolving the polymer in solvent B to obtain a polymer solution, and using the polymer solution to continue filling the remaining part (backing part) of the microneedle mold, and drying to obtain the transfersome complex sugar microneedle patch; wherein the needle body and the backing of the microneedle do not interfere with each other, and the transfersome is only gathered at the needle body position.

[0013] The present invention uses monosaccharides or disaccharides to prepare microneedle patches in combination with transfersomes. Monosaccharides or disaccharides have the function of stabilizing the transfersome protein and / or lipid bilayer, and can form a glassy protective cocoon on the transfersome surface to play a protective role, and can keep water molecules away from the transfersome, reduce its hydration radius, and increase its density and stability. Monosaccharides or disaccharides as microneedle needle materials can help the transfersome overcome the effects of the drying process on its protein and membrane structure, and enhance the stability of the transfersome.

[0014] The sugar substances include but are not limited to trehalose, sucrose, maltose, mannose, glucose, fructose, lactose, galactose, glucosamine, xylitol, nodus, israeli sugar, etc. Preferably, the sugar substance is trehalose, and experiments have shown that trehalose has the most outstanding performance.

[0015] The solvent A is an aqueous solvent selected from deionized water or a buffer solution.

[0016] Preferably, the concentration of the sugar solution is 10-1000 mg / mL, and the amount of the transfersome added to the sugar solution is 0.1-200 mg / mL. The above parameters are conducive to the maintenance of the structure and function of the transfersome by microneedling.

[0017] Furthermore, the concentration of the sugar solution is 10 to 500 mg / mL, and the amount of the transfersome added to the sugar solution is 0.1 to 5 mg / mL.

[0018] In step (2), the transfersome-sugar solution is uniformly filled into the needle body of the microneedle mold by centrifugation or vacuum; the centrifugation conditions are: centrifugal speed 100 to 20000 rpm, centrifugal time 10 s to 4 h, centrifugal temperature 0 to 60°C; the vacuum conditions are: vacuum degree -0.1 to -1000 kPa, vacuum temperature 0 to 60°C.

[0019] The polymer includes, but is not limited to, polyvinyl pyrrolidone, gelatin, hyaluronic acid, carboxymethyl fiber, silk protein, polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polystyrene, polyvinyl chloride, polyethylene, polyurethane, polycarbonate, polypropylene, polymethyl methacrylate, polyether sulfone, polyamide, acrylonitrile-butadiene-styrene copolymer, cellulose acetate, polyacrylic acid, polybutadiene, polyvinyl fluoride, polyacrylate, polymethacrylate or polyacrylonitrile, etc.

[0020] The solvent B includes, but is not limited to, methanol, ethanol, acetone, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide, dichloromethane, toluene, benzene, petroleum ether, carbon disulfide, chloroform, 1,4-dioxane, dimethylaminoformamide, N,N-dimethylacetamide, hexane, and tetrachloromethane.

[0021] The concentration of the polymer solution is preferably 5 to 2000 mg / mL.

[0022] In steps (2) and (3), the drying method includes natural drying, centrifugal drying, vacuum drying or freeze drying.

[0023] The present invention also provides a transfersome conjugate microneedle patch prepared by the preparation method of the transfersome conjugate microneedle patch. The transfersome conjugate microneedle patch can maintain the structure and biological function of the transfersome for a long time, and the transfersome conjugate microneedle patch can be used for the loading, delivery and long-term storage of the transfersome.

[0024] The present invention also provides the application of the transfersome complex sugar microneedle patch in the fields of drug delivery, medical treatment, health care or beauty.

[0025] The present invention also provides a transfersome storage method, using the transfersome conjugate microneedle patch to store transfersomes. The transfersomes are stored in the form of the transfersome conjugate microneedle patch and stored at a temperature of -80 to 60°C, and the transfersome structural integrity can be maintained for ≥30 days.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention utilizes monosaccharides or disaccharides to improve the structural stability of transfersomes, and the effect is significantly better than that of common polysaccharides or hydrophilic polymers. The prepared transfersome complex sugar microneedle patch can be used for dry storage of transfersomes to enhance the stability of transfersomes.

[0028] (2) The method of the present invention is simple in process and easy to operate. Experimental verification shows that the prepared transfersome complex sugar microneedle patch has the effect of long-term storage of transfersomes and can maintain the structural integrity of transfersomes for ≥30 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Flow chart of the preparation method of the exosome composite trehalose microneedle patch in the embodiment.

[0030] Figure 2 This is a transmission electron microscopy image showing that exosome solutions cannot be stored for a long time at room temperature or 4°C.

[0031] Figure 3 This is a transmission electron microscopy image of the exosomes in the exosome composite trehalose microneedle patch of Example 1 stored for a long time at room temperature or 4°C.

[0032] Figure 4 This is a comparison picture of the particle size of the exosomes in the exosome solution and the exosome-complex trehalose microneedle patch in Example 1.

[0033] Figure 5 This is a comparison picture of the marker proteins of exosomes in the exosome solution and the exosome-complex trehalose microneedle patch of Example 1.

[0034] Figure 6 These are transmission electron microscopy images of the liposome solution, the dried liposomes, and the liposome-complexed sucrose microneedle patch of Example 3.

[0035] Figure 7 These are transmission electron microscopy images of exosomes stored in the exosome composite trehalose microneedle patches of Comparative Examples 1-3 and Example 1. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the embodiments and drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0037] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0038] The flow chart of the preparation method of the exosome composite trehalose microneedle patch in the embodiment is as follows Figure 1 As shown; exosomes, apoptotic bodies, and cell microvesicles are of animal origin, specifically exosomes, apoptotic bodies, and cell microvesicles obtained by culturing mouse melanoma B16F10 cells in vitro.

[0039] Example 1

[0040] Dissolve trehalose powder in DPBS buffer, add 50 μg of exosomes, the amount of exosomes added to the trehalose solution is 1.25 mg / mL, mix thoroughly to obtain an exosome-trehalose solution, wherein the trehalose concentration is 300 mg / mL;

[0041] 40 μL of exosome-trehalose solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 3000 rpm for 3 min at 4°C to evenly fill the needle body of the microneedle mold with the exosome-trehalose solution, and then dried naturally in a drying oven at room temperature overnight;

[0042] Polyvinyl pyrrolidone was dissolved in anhydrous ethanol to obtain a polyvinyl pyrrolidone solution with a concentration of 150 mg / mL. The polyvinyl pyrrolidone solution was used to continue filling the remaining part of the microneedle mold. The mold was naturally dried at room temperature in a drying oven for 2 days. After demolding, an exosome-complexed trehalose microneedle patch was obtained.

[0043] Exosomes were prepared according to the following extraction method: when the cells expanded to a density of about 90% in the culture dish, the complete medium containing serum was discarded, the cells were washed twice with DPBS, and a serum-free basal medium was added. After culturing for 48 hours, the cell supernatant was collected and the following steps were performed: centrifugation at 300g for 10 minutes, and the supernatant was taken; centrifugation at 2000g for 10 minutes, and the supernatant was taken; 0.22μm polyethersulfone resin membrane was filtered once; the supernatant was concentrated to 5mL using a 100kDa ultrafiltration tube, and centrifuged at 3500g for 10 minutes each time; centrifugation at 10000g for 1 hour, and the supernatant was taken; centrifugation at 100000g for 2 hours, the supernatant was removed, and the precipitate was the exosomes.

[0044] Example 2

[0045] Dissolve trehalose powder in DPBS buffer, add 50 μg of exosomes (the method for extracting exosomes is the same as in Example 1), the amount of exosomes added to the trehalose solution is 1.25 mg / mL, and mix thoroughly to obtain an exosome-trehalose solution, wherein the trehalose concentration is 40 mg / mL;

[0046] 40 μL of exosome-trehalose solution was added dropwise to the needle body of the microneedle mold, and the exosome-trehalose solution was evenly filled in the needle body of the microneedle mold by vacuum. The vacuum conditions were: vacuum degree 100 kPa, vacuum temperature 25 °C, and further vacuum dried overnight;

[0047] Hyaluronic acid was dissolved in methanol to obtain a hyaluronic acid solution with a concentration of 300 mg / mL. The remaining part of the microneedle mold was continued to be filled with the hyaluronic acid solution, and the mold was naturally dried at room temperature in a drying oven for 2 days. After demolding, an exosome-complexed trehalose microneedle patch was obtained.

[0048] Example 3

[0049] Dissolve sucrose powder in water, add 200 μg liposomes, the amount of liposomes added to the sucrose solution is 5 mg / mL, mix thoroughly to obtain a liposome-sucrose solution, wherein the sucrose concentration is 300 mg / mL;

[0050] 40 μL of liposome-sucrose solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 25°C, 3000 rpm for 10 min to evenly fill the needle body of the microneedle mold with the liposome-sucrose solution, and then dried naturally in a drying oven at room temperature overnight;

[0051] Polylactic acid was dissolved in acetone to obtain a polylactic acid solution with a concentration of 200 mg / mL. The remaining part of the microneedle mold was continuously filled with the polylactic acid solution. The mold was naturally dried at room temperature in a drying oven for 1 day. After demolding, a liposome-complexed sucrose microneedle patch was obtained.

[0052] Among them, liposomes were prepared according to the following method: 3.8 mg POPC palmitoyloleoylphosphatidylcholine, 3.5 mg DOTAP (2,3-dioleyloxypropyl) trimethylammonium chloride, and 1.62 mg cholesterol were dissolved in 1 mL chloroform, mixed and rotary evaporated to form a lipid film. 1 mL DPBS was added to resuspend and placed on a shaker at 37 ° C for 1 hour. Then, the lipid mixture was fully squeezed using a 100 nm film to form a uniform liposome solution.

[0053] Example 4

[0054] Dissolve glucose powder in DPBS buffer, add 100 μg of cell microvesicles, the amount of cell microvesicles added to the glucose solution is 2.5 mg / mL, mix thoroughly to obtain a cell microvesicle-glucose solution, wherein the glucose concentration is 1000 mg / mL;

[0055] 40 μL of cell microvesicle-glucose solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 3500 rpm for 30 min at 4°C to evenly fill the needle body of the microneedle mold with the cell microvesicle-glucose solution, and then dried naturally in a drying oven at room temperature overnight;

[0056] Carboxymethyl cellulose was dissolved in methanol to obtain a carboxymethyl cellulose solution with a concentration of 150 mg / mL. The remaining part of the microneedle mold was filled with the carboxymethyl cellulose solution and naturally dried in a drying oven at room temperature for 1 day. After demolding, a cell microvesicle composite glucose microneedle patch was obtained.

[0057] The cell microvesicles were prepared according to the following extraction method: when the cells were expanded to a density of about 90% in the culture dish, the complete medium containing serum was discarded, the cells were washed twice with DPBS, and a serum-free basal medium was added. After culturing for 48 hours, the cell supernatant was collected and the following steps were performed: centrifugation at 300g for 10 minutes, and the supernatant was taken; centrifugation at 2000g for 10 minutes, and the supernatant was taken; centrifugation at 16500g for 30 minutes, the supernatant was removed, and the precipitate was the cell microvesicles.

[0058] Example 5

[0059] Dissolve maltose powder in DPBS buffer, add 20 μg of exosomes, the amount of exosomes added to the maltose solution is 0.5 mg / mL, mix thoroughly to obtain an exosome-maltose solution, in which the maltose concentration is 40 mg / mL;

[0060] 40 μL of exosome-maltose solution was added dropwise to the needle body of the microneedle mold, and the exosome-trehalose solution was evenly filled in the needle body of the microneedle mold by vacuum. The vacuum conditions were: vacuum degree 50 kPa, vacuum temperature 25 °C, and vacuum drying overnight;

[0061] The silk fibroin was dissolved in anhydrous ethanol to obtain a silk fibroin solution with a concentration of 500 mg / mL. The silk fibroin solution was used to continue filling the remaining part of the microneedle mold, and the mold was naturally dried at room temperature in a drying oven for 5 h. After demolding, the exosome-complex maltose microneedle patch was obtained.

[0062] Example 6

[0063] Dissolve fructose powder in DPBS buffer, add 10 μg of apoptotic bodies, the amount of apoptotic bodies added to the fructose solution is 0.25 mg / mL, mix thoroughly to obtain an apoptotic body-fructose solution, wherein the fructose concentration is 150 mg / mL;

[0064] 40 μL of apoptotic body-fructose solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 2000 rpm for 5 min at 4°C to evenly fill the needle body of the microneedle mold with the apoptotic body-fructose solution, and then allowed to stand at room temperature for 24 h to dry naturally;

[0065] Polyvinyl pyrrolidone was dissolved in anhydrous ethanol to obtain a polyvinyl pyrrolidone solution with a concentration of 400 mg / mL. The polyvinyl pyrrolidone solution was used to continue filling the remaining part of the microneedle mold. The mold was left to stand at room temperature for 3 days to dry naturally. After demolding, an apoptotic body complex fructose microneedle patch was obtained.

[0066] Method for extracting apoptotic bodies: When the cells expand to a density of about 90% in the culture dish, the complete medium containing serum is discarded, the cells are washed twice with DPBS, and the basal medium without serum is added. After culturing for 48 hours, the cell supernatant is collected and the following steps are performed: centrifugation at 300g for 10 minutes, and the supernatant is taken; centrifugation at 2000g for 10 minutes, the supernatant is removed, and the precipitate is the apoptotic body.

[0067] Example 7

[0068] Dissolve lactose powder in water, add 200 μg liposomes (the preparation method of liposomes is the same as in Example 3), the amount of liposomes added to the lactose solution is 5 mg / mL, and mix thoroughly to obtain a liposome-lactose solution, wherein the lactose concentration is 300 mg / mL;

[0069] 40 μL of liposome-lactose solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 25°C, 3000 rpm for 10 min to evenly fill the needle body of the microneedle mold with the liposome-lactose solution, and then allowed to stand at room temperature for 24 h to dry naturally;

[0070] Polyethersulfone was dissolved in ethyl acetate to obtain a polyethersulfone solution with a concentration of 200 mg / mL. The polyethersulfone solution was used to continue filling the remaining part of the microneedle mold. The mold was naturally dried at room temperature in a drying oven for 2 days. After demolding, a liposome-complexed lactose microneedle patch was obtained.

[0071] Comparative Example 1

[0072] Dissolve hyaluronic acid powder in DPBS buffer, add 50 μg of exosomes, the amount of exosomes added to the hyaluronic acid solution is 1.25 mg / mL, mix thoroughly to obtain an exosome-hyaluronic acid solution, wherein the hyaluronic acid concentration is 300 mg / mL;

[0073] 40 μL of exosome-hyaluronic acid solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 3000 rpm for 3 min at 4°C to evenly fill the needle body of the microneedle mold with the exosome-hyaluronic acid solution, and then dried naturally in a drying oven at room temperature overnight;

[0074] Polyvinyl pyrrolidone was dissolved in anhydrous ethanol to obtain a polyvinyl pyrrolidone solution with a concentration of 150 mg / mL. The polyvinyl pyrrolidone solution was used to continue filling the remaining part of the microneedle mold. The mold was naturally dried at room temperature in a drying oven for 2 days. After demolding, an exosome-composite hyaluronic acid microneedle patch was obtained.

[0075] Comparative Example 2

[0076] Gelatin powder was dissolved in DPBS buffer, 50 μg of exosomes were added, and the amount of exosomes added to the gelatin solution was 1.25 mg / mL. After thorough mixing, an exosome-gelatin solution was obtained, in which the gelatin concentration was 300 mg / mL;

[0077] 40 μL of exosome-gelatin solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 37°C and 3000 rpm for 3 min to evenly fill the needle body of the microneedle mold with the exosome-gelatin solution, and then dried naturally in a drying oven at 37°C overnight;

[0078] Hyaluronic acid was dissolved in water to obtain a hyaluronic acid solution with a concentration of 200 mg / mL. The remaining part of the microneedle mold was continued to be filled with the hyaluronic acid solution. The mold was naturally dried at room temperature in a drying oven for 2 days. After demolding, an exosome-composite gelatin microneedle patch was obtained.

[0079] Comparative Example 3

[0080] Dissolve polyvinyl pyrrolidone powder in DPBS buffer, add 50 μg of exosomes, the amount of exosomes added to the polyvinyl pyrrolidone solution is 1.25 mg / mL, mix thoroughly to obtain an exosome-polyvinyl pyrrolidone solution, wherein the concentration of polyvinyl pyrrolidone is 300 mg / mL;

[0081] 40 μL of exosome-polyvinyl pyrrolidone solution was added dropwise to the needle body of the microneedle mold, and centrifuged at 37°C, 3000 rpm for 3 min to evenly fill the needle body of the microneedle mold with the exosome-polyvinyl pyrrolidone solution, and then dried naturally in a drying oven at room temperature overnight;

[0082] Polyvinyl pyrrolidone was dissolved in anhydrous ethanol to obtain a polyvinyl pyrrolidone solution with a concentration of 150 mg / mL. The polyvinyl pyrrolidone solution was used to continue filling the remaining part of the microneedle mold. The mold was naturally dried at room temperature in a drying oven for 2 days. After demolding, an exosome-composite polyvinyl pyrrolidone microneedle patch was obtained.

[0083] Sample analysis

[0084] The morphology of the exosome solution stored at room temperature or 4°C and the exosomes in the exosome composite trehalose microneedle patch of Example 1 were compared by transmission electron microscopy, and the storage effect within 1 month was observed. Figure 2As shown in the figure, the exosome solution showed outer membrane fragmentation and shrinkage after only 3 days at room temperature or 4°C, and the typical exosome morphology could not be observed after 7 days. However, the exosomes in the microneedle patch maintained a complete membrane structure for 31 days under storage conditions at room temperature or 4°C, indicating that the microneedle patch can improve the stability of exosomes and maintain the integrity of the structure ( Figure 3 ).

[0085] The size of the exosomes released from the exosome composite trehalose microneedle patch of Example 1 was characterized by a nanoparticle tracking analyzer and compared with fresh exosomes. The results showed that the median particle size of fresh exosomes was 126.5nm, and that of the exosomes in the microneedle patch was 121.5nm. The hydrated particle sizes of the two were distributed between 40-200nm, with no significant difference. The slightly smaller size of the exosomes in the microneedle patch may be due to the insufficient re-dissolution process after drying ( Figure 4 ).

[0086] The marker proteins (CD63, Alix, CD81 and TSG101) of the exosomes released from the exosome composite trehalose microneedle patch of Example 1 were characterized by Western blotting and compared with fresh exosomes. The results showed that both fresh exosomes and microneedle patches contained representative protein markers of exosomes, and the bands were basically the same ( Figure 5 ).

[0087] Compare the morphology of freshly prepared liposomes, dried liposomes and liposomes in sucrose liposome-complex microneedle patch. Figure 6 As shown, the liposome membrane structure in the microneedle patch is intact, which is basically consistent with that of the freshly prepared one, while the liposomes under drying stimulation cannot maintain their three-dimensional structure, indicating that trehalose has a protective effect on liposomes.

[0088] The transmission electron microscopy images of the exosomes stored in the exosome composite trehalose microneedle patch of Comparative Examples 1-3 and Example 1 are as follows Figure 7 As shown in the figure, the exosomes dried under the protection of hyaluronic acid, gelatin, and polyvinyl pyrrolidone retain a certain morphology, but show different degrees of membrane damage and shrinkage. However, the exosomes dried under the protection of trehalose are consistent with the morphology of fresh exosomes, with a typical cup-shaped structure ( Figure 7 ).

[0089] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a transfersome complex sugar microneedle patch, characterized in that: The following steps are involved: (1) dissolving a sugar substance in solvent A to obtain a sugar solution, wherein the sugar substance is a monosaccharide or a disaccharide, and the concentration of the sugar solution is 5 to 2000 mg / mL; and the amount of the transfersome added to the sugar solution is 0.1 to 200 mg / mL; (2) adding the transfersome to the sugar solution, mixing thoroughly to obtain a transfersome-sugar solution, filling the needle body of the microneedle mold with the transfersome-sugar solution, and drying naturally; the transfersome is at least one of an exosome, a cell microvesicle, and an apoptotic body; (3) dissolving the polymer in solvent B to obtain a polymer solution, using the polymer solution to continue filling the remaining part of the microneedle mold, and drying to obtain the transfersome complex sugar microneedle patch; Storing the transfersome in the form of the transfersome conjugate microneedle patch at a temperature of -80 to 25° C. can maintain the transfersome structural integrity for ≥30 days.

2. The method for preparing the transfersome conjugate microneedle patch according to claim 1, characterized in that: The sugar substances include trehalose, sucrose, maltose, mannose, glucose, fructose, lactose, galactose, glucosamine, xylitol, nodules or israeli sugar.

3. The method for preparing the transfersome conjugate microneedle patch according to claim 1, characterized in that: The solvent A is an aqueous solvent selected from deionized water or a buffer solution.

4. The method for preparing the transfersome conjugate microneedle patch according to claim 1, characterized in that: The concentration of sugar solution is 10~500 mg / mL.

5. The method for preparing the transfersome conjugate microneedle patch according to claim 1, characterized in that: In step (2), the transfersome-sugar solution is uniformly filled into the needle body of the microneedle mold by centrifugation or vacuum. The centrifugation conditions are: centrifugal speed 100~20000 rpm, centrifugal time 10 s~4 h, centrifugal temperature 0~60 °C; the vacuum conditions are: vacuum degree -0.1~-1000 kPa, vacuum temperature 0~60 °C.

6. The method for preparing the transfersome conjugate microneedle patch according to claim 1, characterized in that: The polymer includes at least one of polyvinyl pyrrolidone, gelatin, hyaluronic acid, carboxymethyl cellulose, silk protein, polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polystyrene, polyvinyl chloride, polyethylene, polyurethane, polycarbonate, polypropylene, polymethyl methacrylate, polyether sulfone, polyamide, acrylonitrile-butadiene-styrene copolymer, cellulose acetate, polyacrylic acid, polybutadiene, polyvinyl fluoride, polyacrylate, polymethacrylate or polyacrylonitrile; the concentration of the polymer solution is 5-2000 mg / mL; solvent B includes at least one of methanol, ethanol, acetone, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide, dichloromethane, toluene, benzene, petroleum ether, carbon disulfide, chloroform, 1,4-dioxane, dimethylaminoformamide, N,N-dimethylacetamide, hexane and tetrachloromethane.

7. The method for preparing the transfersome conjugate microneedle patch according to claim 1, characterized in that: In step (3), the drying method includes natural drying, centrifugal drying, vacuum drying or freeze drying.

8. A transfersome conjugate microneedle patch prepared according to the method for preparing a transfersome conjugate microneedle patch according to any one of claims 1 to 7.

9. A transfersome storage method, characterized in that: The transfersome conjugate microneedle patch of claim 8 is used to store transfersomes.

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