CN118767319B

CN118767319B - A transfersome complex sugar microneedle patch and its preparation method and application

Info

Publication number: CN118767319B
Application number: CN202410871702.0A
Authority: CN
Inventors: 常皓; 渠凤丽; 穆思佳
Original assignee: Hangzhou Institute Of Medicine Chinese Academy Of Sciences
Current assignee: Hangzhou Institute Of Medicine Chinese Academy Of Sciences
Priority date: 2024-07-01
Filing date: 2024-07-01
Publication date: 2025-04-22
Anticipated expiration: 2044-07-01
Also published as: CN118767319A

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

Transporter composite sugar microneedle patch and preparation method and application thereof

Technical Field

The invention relates to the technical field of microneedles, in particular to a transfersome composite sugar microneedle patch, a preparation method and application thereof.

Background

Microneedle transdermal drug delivery is a novel drug delivery technology which is emerging in recent years and has unique clinical application value. After the microneedle punctures the skin, the load can be released to the skin epidermis layer and the dermis layer without touching blood vessels and nerves, so that the high-efficiency delivery of the load is realized. The micro-needle administration has the advantages of stability, rapidness, painless, no wound, easy acceptance by patients and the like, and is expected to play an important role in the fields of chronic disease management, infectious disease prevention and control, beauty, skin care and the like.

Along with the continuous development and innovation of scientific technology, the research on the microneedle transdermal drug delivery technology is mainly focused on the following two aspects of (1) realizing accurate control on drug release rate, targeting and biocompatibility by designing microneedles with different shapes, sizes or materials so as to meet the treatment requirements of different diseases and patients, and (2) combining the microneedle drug delivery technology with other drug delivery systems, such as exosomes, liposomes, nanoparticles and the like, to form a multifunctional drug delivery platform so as to realize the combined treatment and personalized treatment of drugs.

The Chinese patent document with publication number CN117122552A discloses an exosome compound microneedle patch for hair regeneration and a preparation method thereof, wherein the microneedle patch comprises a microneedle body and a microneedle substrate, and the microneedle body is mainly made of biodegradable polymer materials, exosomes, plant extracts, amino acids, vitamins and nucleic acids, wherein the biodegradable polymer materials are methacryloyl carboxymethyl chitosan, hyaluronic acid, chitosan and the like.

The Chinese patent document with the publication number of CN112336749A discloses a micro-needle patch for removing spots and wrinkles of stem cell exosomes and a preparation method thereof, wherein the micro-needle patch comprises micro-needles and a waterproof backing layer for fixing the micro-needles, the micro-needles are obtained by solidifying needle tip solutions and needle body solutions, the needle tip solutions comprise stem cell exosomes, astaxanthin, trehalose, chitosan, bovine serum albumin and phosphate buffer solution, and the needle body solutions comprise PVP K30, PEG400, squalane and sterile water. Trehalose can be used for protecting the surface of an exosome from being deactivated, chitosan has antiseptic property, bovine serum albumin can provide an environment with proper protein concentration for the exosome, so that the exosome can keep bioactivity for a long time, and a phosphate buffer solution can be used for regulating the osmotic pressure and the ionic strength of the exosome preservation solution.

However, according to the above-described invention, it is not possible to determine whether or not the corresponding microneedle can effectively maintain the structure of the exosome. The structural integrity of the exosomes and other transmitters is important to their delivery efficiency. Therefore, there is a need to develop a method of preparing a composite microneedle patch that is capable of maintaining the biological structural integrity of the transfersomes including exosomes, vesicles and liposomes.

Disclosure of Invention

The invention provides a preparation method of a transfersome composite sugar microneedle patch, and the prepared transfersome composite sugar microneedle patch can keep the structure and biological function of a transfersome for a long time and has wide application prospects in the fields of transfersome dry storage, drug delivery, medical treatment, health care or cosmetology and the like.

The technical scheme adopted is as follows:

A method for preparing a transfersome composite sugar microneedle patch, which comprises the following steps:

(1) Dissolving a saccharide substance in the solvent A to obtain a saccharide solution, wherein the saccharide substance is monosaccharide or disaccharide, and the concentration of the saccharide solution is 5-2000 mg/mL;

(2) Adding a carrier into a sugar solution, fully and uniformly mixing to obtain a carrier-sugar solution, filling a needle part of a microneedle mould by using the carrier-sugar solution, and drying, wherein the carrier is at least one of animal-derived extracellular body, animal-derived cell microvesicles, animal-derived apoptotic bodies, animal-derived outer membrane vesicles, animal-derived apoptotic vesicles and liposomes;

(3) And dissolving the polymer in the solvent B to obtain a polymer solution, continuously filling the rest part (back lining part) of the microneedle mould by using the polymer solution, and drying to obtain the transferor composite sugar microneedle patch, wherein the needle body and the back lining of the microneedle are not interfered with each other, and the transferor is only gathered at the needle body position.

The invention utilizes the combination of monosaccharide or disaccharide and the transfersome to prepare the microneedle patch, the monosaccharide or disaccharide has the function of stabilizing the protein and/or lipid bilayer of the transfersome, can form glassy protective cocoons on the surface of the transfersome to play a role of protection, can lead water molecules to be far away from the transfersome, reduce the hydration radius thereof and increase the compactness and stability thereof. The monosaccharide or disaccharide can be used as the needle material of the microneedle to help the carrier overcome the influence of the drying process on the protein and membrane structure of the carrier, and enhance the stability of the carrier.

The saccharide includes, but is not limited to, trehalose, sucrose, maltose, mannose, glucose, fructose, lactose, galactose, glucosamine, xylitol, nougat, israel, etc. Preferably, the saccharide is trehalose, and experiments prove that the trehalose has the most outstanding performance.

The solvent A is an aqueous phase solvent and is selected from deionized water or buffer solution.

Preferably, the concentration of the sugar solution is 10-1000 mg/mL, the adding amount of the transfersome in the sugar solution is 0.1-200 mg/mL, and the parameter conditions are favorable for maintaining the structure and the function of the micro-needle transfersome.

Further, the concentration of the sugar solution is 10 to 500mg/mL, and the amount of the mediator added to the sugar solution is 0.1 to 5mg/mL.

In the step (2), the carrier-sugar solution is uniformly filled in the needle part of the microneedle mould in a centrifugal mode or a vacuum mode, wherein the centrifugal condition is that the centrifugal speed is 100-20000 rpm, the centrifugal time is 10 s-4 h, the centrifugal temperature is 0-60 ℃, and the vacuum condition is that the vacuum degree is-0.1 to-1000 kPa, and the vacuum temperature is 0-60 ℃.

Such polymers include, but are not limited to, polyvinylpyrrolidone, gelatin, hyaluronic acid, carboxymethyl cellulose, silk fibroin, polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polystyrene, polyvinyl chloride, polyethylene, polyurethane, polycarbonate, polypropylene, polymethyl methacrylate, polyethersulfone, polyamide, acrylonitrile-butadiene-styrene copolymer, cellulose acetate, polyacrylic acid, polybutadiene, polyvinylfluoride, polyacrylate, polymethacrylate, polyacrylonitrile, or the like.

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

The concentration of the polymer solution is preferably 5-2000 mg/mL.

In the steps (2) and (3), the drying method comprises natural drying, centrifugal drying, vacuum drying or freeze drying.

The invention also provides the conveyer composite sugar microneedle patch prepared by the preparation method of the conveyer composite sugar microneedle patch. The complex carbohydrate microneedle patch can maintain the structure and biological function of the delivery body for a long time, and can be used for loading, delivering and long-term storage of the delivery body.

The invention also provides application of the transfersome composite sugar microneedle patch in the fields of drug delivery, medical treatment, health care or cosmetology.

The invention also provides a method for storing the transfersome, which is used for storing the transfersome by using the transfersome composite sugar microneedle patch. The transfersome is stored in the form of the transfersome composite sugar microneedle patch, and the transfersome is stored at the temperature of-80-60 ℃ so as to keep the structural integrity of the transfersome to be more than or equal to 30 days.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention improves the structural stability of the transfersome by utilizing monosaccharide or disaccharide, the effect is obviously better than that of common polysaccharide or hydrophilic polymer, and the prepared transfersome composite sugar microneedle patch can be applied to dry storage of the transfersome, and the stability of the transfersome is enhanced.

(2) The method has simple process and easy operation, and experiments prove that the prepared transfersome composite sugar microneedle patch has the effect of storing the transfersome for a long time, and can keep the structural integrity of the transfersome to be more than or equal to 30 days.

Drawings

FIG. 1 is a flow chart of a method for preparing an exosome composite trehalose microneedle patch according to an embodiment.

Fig. 2 is a transmission electron microscope image of an exosome solution that cannot be stored for a long period of time at normal temperature or 4 ℃.

Fig. 3 is a transmission electron microscope image of the exosomes in the exosome-complex trehalose microneedle patch of example 1 stored for a long period of time at normal temperature or 4 ℃.

Fig. 4 is a graph comparing the particle size of exosomes in the exosome complex trehalose microneedle patch of example 1 with the exosome solution.

Fig. 5 is a graph comparing the marker proteins of exosomes in the exosome solution and exosome complex trehalose microneedle patch of example 1.

Fig. 6 is a transmission electron microscope image of the liposome in the liposome solution, dried liposome, and liposome composite sucrose microneedle patch of example 3.

Fig. 7 is a transmission electron microscope image of exosomes stored in the exosome complex trehalose microneedle patches of comparative examples 1-3 and example 1.

Detailed Description

The invention is further elucidated below in connection with the examples and the accompanying drawing. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The flow chart of the preparation method of the exosome composite trehalose microneedle patch in the embodiment is shown in figure 1, wherein exosome, apoptotic bodies and cell microvesicles are animal sources, in particular to exosome, apoptotic bodies and cell microvesicles obtained by in vitro culture of mouse melanoma B16F10 cells.

Example 1

Dissolving trehalose powder in a DPBS buffer solution, adding 50 mug of exosomes, wherein the adding amount of the exosomes in the trehalose solution is 1.25mg/mL, and fully and uniformly mixing to obtain an exosome-trehalose solution, wherein the concentration of the trehalose is 300mg/mL;

Dripping 40 mu L of exosome-trehalose solution into the needle part of the microneedle mould, centrifuging at 4 ℃ and 3000rpm for 3min to uniformly fill the exosome-trehalose solution into the needle part of the microneedle mould, and naturally drying overnight at room temperature in a drying oven;

And dissolving polyvinylpyrrolidone in absolute ethyl alcohol to obtain a polyvinylpyrrolidone solution with the concentration of 150mg/mL, continuously filling the rest part of the microneedle mould with the polyvinylpyrrolidone solution, naturally drying in a drying oven at room temperature for 2 days, and demoulding to obtain the exosome composite trehalose microneedle patch.

The exosomes are prepared according to the following extraction method, when cells are amplified to about 90% density in a culture dish, a complete culture medium containing serum is sucked and removed, DPBS is used for cleaning the cells for 2 times, a basic culture medium without serum is added, after culturing for 48 hours, cell supernatant is collected and subjected to the following steps of 300g centrifugation for 10min, supernatant is taken, 2000g centrifugation for 10min, supernatant is taken, 0.22 mu m polyethersulfone resin membrane is used for filtering once, 100kDa ultrafiltration tube is used for concentrating the supernatant to 5mL, each 3500g centrifugation for 10min, 10000g centrifugation for 1h, supernatant is taken, 100000g centrifugation for 2h, and precipitation is carried out, thus the exosomes are obtained.

Example 2

Dissolving trehalose powder in DPBS buffer solution, adding 50 μg exosome (extraction method of exosome is the same as that of example 1), adding exosome in trehalose solution in an amount of 1.25mg/mL, and mixing thoroughly to obtain exosome-trehalose solution with trehalose concentration of 40mg/mL;

Dripping 40 mu L of exosome-trehalose solution into the needle part of the microneedle mould, uniformly filling the exosome-trehalose solution into the needle part of the microneedle mould in a vacuum mode, wherein the vacuum condition is that the vacuum degree is 100kPa, the vacuum temperature is 25 ℃, and further drying in a vacuum overnight;

Dissolving hyaluronic acid in methanol to obtain hyaluronic acid solution with the concentration of 300mg/mL, continuously filling the rest part of the microneedle mould with the hyaluronic acid solution, naturally drying in a drying oven at room temperature for 2 days, and demolding to obtain the exosome composite trehalose microneedle patch.

Example 3

Dissolving sucrose powder in water, adding 200 mug of liposome, wherein the adding amount of the liposome in the sucrose solution is 5mg/mL, and fully and uniformly mixing to obtain liposome-sucrose solution, wherein the sucrose concentration is 300mg/mL;

Dripping 40 mu L of liposome-sucrose solution into the needle part of the microneedle mould, centrifuging at 25 ℃ and 3000rpm for 10min to uniformly fill the liposome-sucrose solution into the needle part of the microneedle mould, and naturally drying overnight at room temperature in a drying oven;

and dissolving polylactic acid in acetone to obtain a polylactic acid solution with the concentration of 200mg/mL, continuously filling the rest part of the microneedle mould by using the polylactic acid solution, naturally drying in a drying oven at room temperature for 1 day, and demolding to obtain the liposome composite sucrose microneedle patch.

The liposome is prepared by dissolving 3.8mg of POPC palmitoyl oleoyl phosphatidylcholine, 3.5mg of DOTAP (2, 3-dioleoxypropyl) trimethyl ammonium chloride and 1.62mg of cholesterol in 1mL of chloroform, uniformly mixing, and performing rotary evaporation to form a lipid film. Add 1mL DPBS to resuspend and shake on a 37 ℃ shaker for 1h. And then the lipid mixture is fully extruded by using a 100nm film to form a uniform liposome solution.

Example 4

Dissolving glucose powder in DPBS buffer solution, adding 100 mug of cell microvesicles, wherein the adding amount of the cell microvesicles in the glucose solution is 2.5mg/mL, and fully and uniformly mixing to obtain cell microvesicle-glucose solution, wherein the glucose concentration is 1000mg/mL;

dripping 40 mu L of cell microvesicle-glucose solution into the needle part of a microneedle mould, centrifuging at 4 ℃ and 3500rpm for 30min to uniformly fill the cell microvesicle-glucose solution into the needle part of the microneedle mould, and naturally drying overnight at room temperature in a drying oven;

and dissolving carboxymethyl cellulose in methanol to obtain carboxymethyl cellulose solution with the concentration of 150mg/mL, continuously filling the rest part of the microneedle mould by using the carboxymethyl cellulose solution, naturally drying in a drying oven at room temperature for 1 day, and demoulding to obtain the cell microvesicle composite glucose microneedle patch.

The cell microvesicles are prepared according to the following extraction method, when cells are amplified to about 90% density in a culture dish, the complete culture medium containing serum is sucked and removed, the cells are washed 2 times by DPBS, the basic culture medium without serum is added, after culturing for 48 hours, the cell supernatant is collected and subjected to the following steps of 300g centrifugation for 10min, supernatant is taken, 2000g centrifugation for 10min, supernatant is taken, 16500g centrifugation for 30min, and the supernatant is removed, thus obtaining the cell microvesicles.

Example 5

Dissolving maltose powder in DPBS buffer solution, adding 20 mug exosomes, wherein the adding amount of the exosomes in the maltose solution is 0.5mg/mL, and fully and uniformly mixing to obtain exosome-maltose solution, wherein the maltose concentration is 40mg/mL;

dripping 40 mu L of exosome-maltose solution into the needle part of the microneedle mould, uniformly filling the exosome-trehalose solution into the needle part of the microneedle mould in a vacuum mode, wherein the vacuum condition is that the vacuum degree is 50kPa, the vacuum temperature is 25 ℃, and drying is carried out in vacuum overnight;

dissolving silk fibroin in absolute ethyl alcohol to obtain silk fibroin solution with the concentration of 500mg/mL, continuously filling the rest part of the microneedle mould with the silk fibroin solution, naturally drying in a drying box at room temperature for 5 hours, and demoulding to obtain the exosome composite maltose microneedle patch.

Example 6

Dissolving fructose powder in DPBS buffer solution, adding 10 mug of apoptotic bodies, wherein the addition amount of the apoptotic bodies in the fructose solution is 0.25mg/mL, and fully and uniformly mixing to obtain apoptotic body-fructose solution, wherein the fructose concentration is 150mg/mL;

Dripping 40 mu L of apoptotic body-fructose solution into the needle part of the microneedle mould, centrifuging at 4 ℃ and 2000rpm for 5min to uniformly fill the apoptotic body-fructose solution into the needle part of the microneedle mould, standing at room temperature for 24h, and naturally drying;

And dissolving polyvinylpyrrolidone in absolute ethyl alcohol to obtain a polyvinylpyrrolidone solution with the concentration of 400mg/mL, continuously filling the rest part of the microneedle mould by using the polyvinylpyrrolidone solution, standing at room temperature for 3 days, naturally drying, and demoulding to obtain the apoptotic body composite fructose microneedle patch.

The apoptotic body extracting process includes sucking complete culture medium containing serum to eliminate when the cell is amplified to 90% density in a culture dish, washing the cell with DPBS for 2 times, adding basic culture medium without serum, culturing for 48 hr, collecting supernatant, centrifuging for 10min 300g, collecting supernatant, centrifuging for 10min 2000g, eliminating supernatant and depositing to obtain apoptotic body.

Example 7

Dissolving lactose powder in water, adding 200 μg of liposome (the preparation method of the liposome is the same as that of example 3), wherein the adding amount of the liposome in lactose solution is 5mg/mL, and fully mixing to obtain liposome-lactose solution, wherein the lactose concentration is 300mg/mL;

dripping 40 mu L of liposome-lactose solution into the needle part of the microneedle mould, centrifuging at 25 ℃ and 3000rpm for 10min to uniformly fill the liposome-lactose solution into the needle part of the microneedle mould, standing at room temperature for 24h, and naturally drying;

and dissolving polyethersulfone in ethyl acetate to obtain a polyethersulfone solution with the concentration of 200mg/mL, continuously filling the rest part of the microneedle mould with the polyethersulfone solution, naturally drying in a drying oven at room temperature for 2 days, and demoulding to obtain the liposome composite lactose microneedle patch.

Comparative example 1

Dissolving hyaluronic acid powder in DPBS buffer solution, adding 50 mug exosomes, wherein the adding amount of the exosomes in the hyaluronic acid solution is 1.25mg/mL, and fully and uniformly mixing to obtain exosome-hyaluronic acid solution, wherein the concentration of the hyaluronic acid is 300mg/mL;

Dripping 40 mu L of exosome-hyaluronic acid solution into the needle part of the microneedle mould, centrifuging at 4 ℃ and 3000rpm for 3min to uniformly fill the exosome-hyaluronic acid solution into the needle part of the microneedle mould, and naturally drying overnight at room temperature in a drying oven;

And dissolving polyvinylpyrrolidone in absolute ethyl alcohol to obtain a polyvinylpyrrolidone solution with the concentration of 150mg/mL, continuously filling the rest part of the microneedle mould with the polyvinylpyrrolidone solution, naturally drying in a drying oven at room temperature for 2 days, and demoulding to obtain the exosome composite hyaluronic acid microneedle patch.

Comparative example 2

Dissolving gelatin powder in DPBS buffer solution, adding 50 mug exosomes, wherein the adding amount of the exosomes in the gelatin solution is 1.25mg/mL, and fully and uniformly mixing to obtain exosome-gelatin solution, wherein the gelatin concentration is 300mg/mL;

Dripping 40 mu L of exosome-gelatin solution into the needle part of the microneedle mould, centrifuging at 37 ℃ for 3min at 3000rpm to uniformly fill the exosome-gelatin solution into the needle part of the microneedle mould, and naturally drying overnight at 37 ℃ in a drying oven;

and dissolving hyaluronic acid in water to obtain hyaluronic acid solution with the concentration of 200mg/mL, continuously filling the rest part of the microneedle mould by using the hyaluronic acid solution, naturally drying in a drying oven at room temperature for 2 days, and demolding to obtain the exosome composite gelatin microneedle patch.

Comparative example 3

Dissolving polyvinylpyrrolidone powder in DPBS buffer solution, adding 50 mug exosomes, wherein the adding amount of the exosomes in the polyvinylpyrrolidone solution is 1.25mg/mL, and fully and uniformly mixing to obtain exosome-polyvinylpyrrolidone solution, wherein the concentration of polyvinylpyrrolidone is 300mg/mL;

Dripping 40 mu L of exosome-polyvinylpyrrolidone solution into the needle part of the microneedle mould, centrifuging at 37 ℃ and 3000rpm for 3min to uniformly fill the exosome-polyvinylpyrrolidone solution into the needle part of the microneedle mould, and naturally drying overnight at room temperature in a drying oven;

And dissolving polyvinylpyrrolidone in absolute ethyl alcohol to obtain a polyvinylpyrrolidone solution with the concentration of 150mg/mL, continuously filling the rest part of the microneedle mould with the polyvinylpyrrolidone solution, naturally drying in a drying oven at room temperature for 2 days, and demolding to obtain the exosome composite polyvinylpyrrolidone microneedle patch.

Sample analysis

The morphology of the exosomes stored in the exosome composite trehalose microneedle patch of example 1 and the exosome solution stored at normal temperature or 4 ℃ were compared by transmission electron microscopy, and the storage effect was observed within 1 month. As shown in fig. 2, the exosome solution was subject to outer membrane fragmentation and shrinkage at normal temperature or 4 ℃ for only 3 days, and typical exosome forms could not be observed after 7 days. The exosomes in the microneedle patch always maintain a complete membrane structure within 31 days under normal temperature or 4 ℃ storage conditions, which indicates that the microneedle patch can promote the stability of the exosomes and maintain the structural integrity (fig. 3).

The size of the exosomes released in the exosome-complex trehalose microneedle patches of example 1 was characterized by a nanoparticle tracking analyzer and compared to fresh exosomes. The result shows that the median value of the particle size of the fresh exosomes is 126.5nm, the exosomes in the microneedle patch is 121.5nm, and the hydrated particle sizes of the fresh exosomes and the exosomes are distributed between 40 nm and 200nm without obvious difference. The exosomes in the microneedle patches were slightly smaller in size, probably due to insufficient reconstitution process after drying (fig. 4).

The marker proteins (CD 63, alix, CD81 and TSG 101) of the exosomes released in the exosome complex trehalose microneedle patches of example 1 were characterized by western blotting and compared to fresh exosomes. The results showed that both fresh exosomes and microneedle patches contained representative protein markers of exosomes and the bands were substantially identical (fig. 5).

And comparing the appearance of the newly prepared liposome, the dried liposome and the liposome in the liposome composite sucrose microneedle patch. As shown in fig. 6, the liposome membrane in the microneedle patch has a complete structure, which is basically consistent with that of the newly prepared liposome, and the liposome under dry stimulation cannot maintain a three-dimensional structure, which indicates that trehalose has a protective effect on the liposome.

The transmission electron microscopy pictures of the exosomes stored in the exosome composite trehalose microneedle patches of comparative examples 1-3 and example 1 are shown in fig. 7, and the exosomes dried under the protection of hyaluronic acid, gelatin, polyvinylpyrrolidone materials retain a certain morphology, but show different degrees of membrane damage and shrinkage, but the exosomes dried under the protection of trehalose are consistent with the fresh exosomes morphology, with a typical cup-shaped structure (fig. 7).

While the foregoing embodiments have been described in detail in connection with the embodiments of the invention, it should be understood that the foregoing embodiments are merely illustrative of the invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like made within the principles of the invention are intended to be included within the scope of the invention.

Claims

1. A method for preparing a transfersome conjugate microneedle patch, comprising the following steps:

(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, and 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 a transfersome complex sugar microneedle patch according to claim 1, wherein the sugar substance comprises trehalose, sucrose, maltose, mannose, glucose, fructose, lactose, galactose, glucosamine, xylitol, nodules or israeliose.

3. The method for preparing the transfersome complex sugar 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 a transfersome complex sugar microneedle patch according to claim 1, wherein the concentration of the sugar solution is 10-500 mg/mL.

5. The method for preparing a transfersome complex sugar microneedle patch according to claim 1, characterized in that in step (2), the transfersome-sugar solution is uniformly filled into the needle body part 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 a transfersome complex sugar microneedle patch according to claim 1, characterized in that the polymer comprises at least one of polyvinyl pyrrolidone, gelatin, hyaluronic acid, carboxymethyl cellulose, silk fibroin, 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; and the solvent B comprises 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 a transfersome conjugate microneedle patch according to claim 1, characterized in that in step (3), the drying method comprises 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 method for storing transfersomes, characterized in that the transfersomes are stored using the transfersome conjugate microneedle patch of claim 8.