Liposome for promoting transdermal delivery by double-layer loaded polysaccharide or protein as well as preparation method and application of liposome

Abstract

The invention discloses a double-layer loaded lipidosome capable of promoting transdermal delivery of polysaccharide or protein as well as a preparation method and application of the double-layer loaded lipidosome. According to the preparation method disclosed by the invention, cholesterol, soybean lecithin and the like are taken as raw materials, and the double-layer loaded polysaccharide and protein lipidosome is prepared by virtue of a high-speed homogenization and rotary evaporation process in a clockwise and anticlockwise alternate rotation manner and a micro-jet high-pressure method in sequence, and the lipidosome can promote the transdermal delivery of polysaccharide or protein; the utilization rate of polysaccharide and protein with the functions of treatment, repair and the like in skin is expected to be fully exerted, and the polysaccharide and protein can be used for preparing products for preventing or treating skin injury.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, relates to transdermal delivery technology, and specifically relates to a double-layer liposome loaded with polysaccharide or protein to promote transdermal delivery, and a preparation method and application thereof. Background Art

[0002] Transdermal drug delivery system refers to a new way of delivering drugs through the skin surface, so that the drug enters the human blood circulation at a constant rate through the capillaries to produce systemic or local therapeutic effects. As the largest organ of the human body, the skin has attracted widespread attention for transdermal drug delivery. Its advantages are significant, such as drug absorption can avoid the first-pass effect of the liver, control the speed of drug delivery, improve drug utilization, and non-invasive drug delivery. Polysaccharides and proteins, as indispensable components of the human body, play an important role. For example, hyaluronic acid can promote tissue regeneration and wound healing by participating in cell signal transduction; alginate can promote chronic wound healing through the production of cytokines by monocytes; collagen can delay skin aging by removing free radicals in the human body; immunoglobulins can regulate the function and activity of immune cells by binding to receptors on the surface of immune cells. However, the oral use of polysaccharides and proteins is limited by the pH of the gastrointestinal tract and the complex enzyme environment, resulting in low utilization. Therefore, transdermal devices based on polysaccharides and proteins have broad prospects for the treatment of related diseases.

[0003] At present, a variety of transdermal drug delivery carriers based on polysaccharides and proteins have been developed, such as hydrogels, films, microneedles, etc. For example, Wang et al. used Panax notoginseng polysaccharide to make a soluble microneedle, which can promote the transdermal delivery of drugs by activating skin dendritic cells. With the help of microneedles, it can penetrate the stratum corneum and target the ear skin, promoting the maturation and migration of immune cells (Pharmaceuticals, 2022, 15: 602); Anirudhan et al. made a chitosan / hyaluronic acid transdermal film device, which showed excellent control of the release of the drug lidocaine by modifying chitosan and covalently linking with hyaluronic acid, as well as good mechanical properties and biosafety (Carbohydrate Polymers, 2016, 152: 687-698); Donnely et al. prepared a hydrogel microneedle system through a silicone micromold, and successfully delivered proteins and peptides of various molecular weights transdermally using this system (Advanced Functional Materials, 2012, 22: 4879-4890). Many studies at home and abroad have proved that the delivery of polysaccharides and proteins can be achieved through transdermal systems that break through the skin barrier and bypass the digestive system. However, at this stage, there are still problems such as limited delivery quantity, delivery type, and delivery molecular weight. In addition, the skin, as the first barrier to pathogen invasion, has also become the main obstacle to the transdermal delivery of polysaccharides and proteins. Therefore, constructing a new type of material that can effectively penetrate the skin of polysaccharides and proteins has become an important scientific issue that needs to be solved urgently.

[0004] Liposomes are one or more closed vesicles formed by amphiphilic substances such as lecithin and cholesterol dispersed in the aqueous phase. They can encapsulate hydrophilic and lipophilic drugs. Their bilayer structure is the same as that of cell membranes and they are widely used in intravenous, skin and oral administration. The use of liposomes as carrier materials for transdermal administration has the following advantages: 1. They have a sustained-release effect, which can slowly release the encapsulated drugs, delay renal excretion and metabolism, and thus prolong the duration of drug use; 2. They have targeting and lymphatic orientation, which can avoid adverse reactions caused by systemic absorption of drugs; 3. Drugs encapsulated by liposomes are more stable; 4. They can reduce the toxicity of drugs, and liposomes themselves are also non-toxic, harmless, non-irritating, and safe to use. The synthesis method of liposomes largely determines their performance. In previous studies, our research group used high-speed homogenization, reverse evaporation and microfluidization high pressure to synthesize embedded liposomes, which significantly improved the transdermal utilization rate of high molecular weight hyaluronic acid in the treatment of acute and chronic photodamaged skin, providing a feasible way for the non-invasive application of large molecular active substances (Acta Biomaterialia, 2024).

[0005] However, for polysaccharides or proteins, existing methods cannot stably obtain liposomes that can truly achieve double-layer loading and promote transdermal delivery of polysaccharides or proteins. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a double-layer loaded liposome capable of promoting transdermal delivery of polysaccharides or proteins, and a preparation method and application thereof.

[0007] The first aspect of the present invention is to provide a method for preparing a double-layer liposome loaded with polysaccharide or protein for transdermal delivery, comprising the following steps:

[0008] S1. Cholesterol and phospholipid are dissolved in an organic solvent at room temperature and stirred to obtain a mixed solution A;

[0009] S2. adding the polysaccharide or protein to the solvent until it is completely dissolved to obtain a solution, and then slowly adding the solution to the mixed solution A to mix evenly to obtain a mixed solution B, wherein the solvent is water;

[0010] S3. After mixing, the mixed solution B was evaporated by a rotary evaporator to remove the solvent, and the rotary evaporation process was alternately rotated clockwise and counterclockwise. When the mixed solution was rotary evaporated to a residual volume of 45%-55%, polysaccharide was added and rotary evaporation was continued to obtain a solid;

[0011] The clockwise speed is set to 90r / min-100r / min, the counterclockwise speed is set to 90r / min-100r / min, the rotation time in each direction is 9-11min, and the mass volume ratio of the added polysaccharide or protein to the remaining mixed solution is 0.8-1.2mg / mL;

[0012] S4. After adding pure water to the solid, ultrasonic hydration is performed, and the double-layer liposome suspension loaded with polysaccharide or protein is obtained by microfluidization high pressure process.

[0013] In some embodiments, in step S1, the phospholipid is soybean lecithin with a molecular weight of 700-1000.

[0014] In some embodiments, in step S1, the molecular weight of the cholesterol is 300-400.

[0015] In some embodiments, the mass ratio of phospholipid to cholesterol is 30:(1-2).

[0016] In some of the embodiments, the mass volume ratio of the soybean lecithin to the organic solvent A is 1-1.5 mg / mL.

[0017] In some embodiments, the organic solvent is at least one of anhydrous ethanol and chloroform. Preferably, the organic solvent is a mixture of anhydrous ethanol and chloroform. Furthermore, the ratio of anhydrous ethanol to chloroform is 1:1.

[0018] In some of the embodiments, the polysaccharide is galactoarabinan with a molecular weight of 10-15 kD, mannan with a molecular weight of 1000-1500 kD, hydroxyethyl chitosan with a molecular weight of 60-70 kD, or amylopectin with a molecular weight of 100-200 kD.

[0019] In some embodiments, the protein includes but is not limited to collagen with a molecular weight of 8000-10000 kD, or elastin with a molecular weight of 50-70 kD.

[0020] In some embodiments, the stirring in step S2 is stirring at room temperature for 10 to 20 minutes.

[0021] In some embodiments, the mass volume ratio of the polysaccharide to the solvent C is (10-20) mg:20 mL.

[0022] In some embodiments, the time of the slow addition in step S2 is 3.8-4.2 mL / min.

[0023] In some of the embodiments, in step S2, the mixing is carried out uniformly by a high shear dispersing emulsifier, the power of the high shear dispersing emulsifier is 200-300W, the rotation speed is 7500-8500rpm, and the time for each mixing is 4.5-5.5min.

[0024] In some embodiments, the temperature of the rotary evaporator is set at 60-65° C., the speed is set at 80-120 r / min, preferably 90-100 r / min, the rotary evaporation time is 30-50 min, and the vacuum degree of the evaporating flask is 70-100 kPa.

[0025] In some of the embodiments, the mass volume ratio of the solid E to pure water is 1 g: 8-12 mL, and the hydration time in the ultrasonic instrument is 5 to 10 min.

[0026] The second aspect of the present invention is to provide a double-layer liposome loaded with polysaccharide or protein for transdermal delivery obtained by the above preparation method.

[0027] The third aspect of the present invention is to provide the use of the double-layer liposome loaded with polysaccharide or protein for transdermal delivery in preparation.

[0028] In some embodiments, the product is a medicine or a skin care product.

[0029] The present invention uses rotary evaporation and, at the appropriate time during rotary evaporation, adds a suitable amount of polysaccharide or protein to obtain a liposome preparation method that truly achieves double-layer loading and can promote transdermal delivery of polysaccharides or proteins, as well as obtains corresponding liposomes, and is expected to give full play to the utilization rate of polysaccharides and proteins with therapeutic and repair functions in the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 Characterization of the double-layered polysaccharide- and protein-loaded liposomes prepared in Example 3 and Example 4; wherein A is a fluorescence morphology image of the double-layered polysaccharide-loaded liposome; B is a fluorescence morphology image of the double-layered protein-loaded liposome; C, D, E, and F are fluorescence morphology images of the liposomes prepared in Example 11 to Example 14, respectively.

[0032] Figure 2 The double-layered polysaccharide-loaded liposomes prepared in Example 3 were subjected to permeation verification using a Franz diffusion cell; A is the permeation experimental result of the double-layered polysaccharide-loaded liposomes; and B is the permeation experimental result of the fluorescently labeled polysaccharide.

[0033] Figure 3 The bilayer protein-loaded liposomes prepared in Example 4 were subjected to permeation verification using a Franz diffusion cell; A is the permeation experimental result of the bilayer protein-loaded liposomes; and B is the permeation experimental result of the fluorescently labeled protein.

[0034] Figure 4 The double-layered polysaccharide-loaded liposomes prepared in Example 3 were verified in vivo; A is the fluorescence intensity result of the back skin of mice; B is the penetration depth result of the liposomes in the back skin section of mice.

[0035] Figure 5 The double-layer protein-loaded liposomes prepared in Example 4 were verified in vivo; A is the fluorescence intensity result of the back of the mouse back skin; B is the penetration depth result of the liposomes in the mouse back skin section.

[0036] Figure 6 This is an in vitro cellular safety verification of the double-layered polysaccharide-loaded liposomes prepared in Example 1.

[0037] Figure 7 This is an in vitro cellular safety verification of the double-layered protein-loaded liposomes prepared in Example 2.

[0038] Figure 8 It is a schematic diagram of the structure of the double-layer loaded polysaccharide / protein liposome of the present invention. DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0040] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0041] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0042] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] In the following examples, the preparation method of anhydrous ethanol follows the following operating steps: adding calcium hydride to anhydrous ethanol, stirring for 6 to 24 hours, and then distilling at atmospheric pressure to obtain anhydrous ethanol, wherein the amount of calcium hydride added is 1 to 2 grams per 500 mL of ethanol; the chloroform and anhydrous dimethyl sulfoxide are obtained by the same treatment.

[0045] The present invention aims at how to promote the effective penetration of proteins and polysaccharides of various molecular weights in the skin and improve their utilization rate. It intends to use cholesterol, soybean lecithin and the like with good biosafety as raw materials, and successively synthesize double-layer loaded polysaccharide and protein liposomes through high-speed homogenization, evaporation method of alternating clockwise and counterclockwise rotation during rotary evaporation and micro-jet high pressure, embed the polysaccharide and protein into the surface and cavity of the double-layer structure similar to the liposome membrane, so as to penetrate the epidermis through lipid fusion, skin appendages and the like, penetrate into the skin, realize the delivery of polysaccharides and proteins, and finally construct a double-layer loaded polysaccharide and protein liposome.

[0046] The transdermal delivery system of the present invention uses polysaccharides and proteins to participate in the construction of liposomes instead of simply encapsulating the polysaccharides and proteins, which is beneficial for the adaptive deformation of polysaccharides and proteins with liposomes and thus beneficial for transdermal delivery. The present invention overcomes the existing problems of limited transdermal delivery quantity, delivery types and delivery molecular weight of polysaccharides and proteins, and is expected to give full play to the utilization rate of polysaccharides and proteins with therapeutic and repair functions in the skin. The system can also be used as a transdermal drug delivery carrier, and can achieve the purpose of effective loading of various types of polysaccharides and proteins and efficient penetration of the skin. The selected components are all materials approved by the state for use as pharmaceutical excipients, and have significant biosafety.

[0047] In the following examples, the absolute ethanol (Macklin) used has a purity of AR, 95%; the soy lecithin (Shanghai Yuanye) used has a purity of ≥90%; the cholesterol (Macklin) used has a purity of AR, 99%; in the following examples, room temperature refers to 10-35°C.

[0048] The description will not be repeated below.

[0049] The preparation method of the present invention utilizes high-speed homogenization, reverse evaporation and microfluidization high pressure to obtain a double-layer liposome loaded with polysaccharide or protein for transdermal delivery, and its structural schematic diagram is shown as follows: Figure 8 As shown in the figure, the whole body is in the shape of a double-layered ring, and the polysaccharide and protein are embedded in the phospholipid layer outside the liposome and in the cavity of the double-layered phospholipid molecules, participating in the construction of the liposome.

[0050] The present invention is further described in detail below with reference to specific embodiments.

[0051] Example 1

[0052] The method for preparing the double-layer polysaccharide-loaded transdermal delivery liposome of this embodiment comprises the following steps:

[0053] (1) First, cholesterol (molecular weight 386) and phospholipid (soy lecithin with a molecular weight of 758) are dissolved in an organic solvent A at room temperature and stirred to obtain a mixed solution B, wherein the mass ratio of the phospholipid to the cholesterol is 30:1; and the mass volume ratio of the soybean lecithin to the organic solvent A is 1 mg / mL;

[0054] (2) adding polysaccharide to deionized water, stirring at room temperature for 15 min until completely dissolved to obtain polysaccharide solution D, then slowly adding polysaccharide solution D to mixed solution B, and mixing evenly with a high shear dispersing emulsifier to obtain a mixed solution; the high shear dispersing emulsifier has a power of 220 W, a rotation speed of 8000 rpm, and each time is 5 min;

[0055] (3) The mixed solvent obtained in step (2) is evaporated by a rotary evaporator to remove the mixed solvent, and the rotary evaporator rotates alternately clockwise and counterclockwise during the rotary evaporation. When the mixed solvent is evaporated to 1 / 2, the same polysaccharide as in step (2) is added, and the mass volume ratio of the added polysaccharide to the remaining mixed solvent is 1 mg / mL. The rotary evaporation is continued to obtain solid E. The temperature of the rotary evaporator is set to 60° C., the clockwise speed is set to 100 r / min, the counterclockwise speed is set to 100 r / min, the rotation time in each direction is 10 min, the rotary evaporation time is 45 min, and the vacuum degree of the evaporating flask is 70 kPa;

[0056] (4) Finally, pure water was added to the solid E for ultrasonic hydration, and the double-layer loaded polysaccharide liposome suspension was obtained by a microfluidizer and a microfluidizer high pressure process (the homogenization pressure was 1000 bar, the homogenization times were twice, and the voltage was 380 V, which was the same in the following embodiments).

[0057] In the above method, in step (2) and step (3), the polysaccharides are galactoarabinan with a molecular weight of 13.489 kD, mannan with a molecular weight of 1016 kD, hydroxyethyl deacetylated chitosan with a molecular weight of 69.513 kD, and amylopectin with a molecular weight of 150 kD; the organic solvent A is 15 ml of anhydrous ethanol and 15 ml of chloroform; the mass volume ratio of the polysaccharide to the pure water is 10 mg:20 mL; and the time for slow addition is 4 mL / min.

[0058] The mass volume ratio of the solid E to pure water is 1 g:10 mL, and the hydration time in the ultrasonic instrument is 10 min.

[0059] Example 2

[0060] The preparation method of the double-layer protein-loaded liposome of this embodiment comprises the following steps:

[0061] (1) First, cholesterol and phospholipid are dissolved in an organic solvent A at room temperature and stirred to obtain a mixed solution B. The specific operation is as shown in Example 1;

[0062] (2) adding the protein to solvent C (pure water) until it is completely dissolved to obtain protein solution D, then slowly adding protein solution D to solution B, and mixing the two evenly using a high shear dispersing emulsifier to obtain a mixed solution; the high shear dispersing emulsifier has a power of 220 W, a rotation speed of 8000 rpm, and each time is 5 min;

[0063] (3) using a rotary evaporator to evaporate the mixed solution obtained in step (2) to remove the mixed solvent, rotating alternately clockwise and counterclockwise during the rotary evaporation process, and when the mixed solvent is evaporated to 1 / 2, adding the corresponding protein, and continuing the rotary evaporation, the mass volume ratio of the added protein to the remaining mixed solvent is 1 mg / mL, to obtain a solid, the temperature of the rotary evaporator is set to 60° C., the clockwise speed is set to 100 r / min, the counterclockwise speed is set to 100 r / min, the rotation time in each direction is 10 min, the rotary evaporation time is 45 min, and the vacuum degree of the evaporating flask is 70 kPa;

[0064] (4) Finally, pure water is added to the solid and ultrasonic hydration is performed, and finally the double-layered proteoliposome suspension is obtained by microfluidization high pressure process. The mass volume ratio of the solid to pure water is 1 g: 10 mL, and the hydration time in the ultrasonic instrument is 10 min.

[0065] In the above method, the proteins are collagen with a molecular weight of 9000kD and elastin with a molecular weight of 60kD; the organic solvent A is a mixture of anhydrous ethanol and chloroform, 15ml each; the mass volume ratio of the protein to the solvent C is 10mg:20mL; and the slow addition time is 4mL / min.

[0066] In order to verify the biological activity of the polysaccharide liposomes and proteoliposomes of Examples 1 and 2, we also prepared fluorescently labeled polysaccharide or proteoliposomes according to the same process. See Examples 3 and 4.

[0067] Example 3

[0068] The preparation method of the fluorescently labeled polysaccharide liposomes of this embodiment comprises the following steps:

[0069] (1) dissolving polysaccharide in pure water to obtain solution A;

[0070] At room temperature, a fluorescent marker is dissolved in an organic solvent B to obtain a solution C, wherein the organic solvent B is anhydrous dimethyl sulfoxide, the fluorescent marker is fluorescein isothiocyanate (FITC), and the mass volume ratio of FITC to anhydrous dimethyl sulfoxide is 1:1;

[0071] (2) Solution A and Solution C were fully stirred and dissolved to obtain Solution D, reacted at room temperature for 4 hours, dialyzed for 48 hours, and freeze-dried to obtain Solid E, i.e., the fluorescently labeled polysaccharide;

[0072] (3) Cholesterol (molecular weight 386) and phospholipid (soy lecithin with molecular weight 758) were dissolved in an organic solvent (a mixture of anhydrous ethanol and chloroform) at room temperature and stirred to obtain a mixed solution G, wherein the mass ratio of phospholipid to cholesterol was 30:1; the mass volume ratio of soybean lecithin to organic solvent (a mixture of anhydrous ethanol and chloroform) was 1 mg / mL;

[0073] The fluorescent labeled polysaccharide is added to solvent H (pure water) until it is completely dissolved to obtain solution I, and then solution G is slowly added to solution I and mixed evenly;

[0074] (4) The mixed solution obtained in step (3) is mixed uniformly with a high shear dispersing emulsifier, and the mixed solvent is evaporated away by a rotary evaporator, and the rotary evaporator rotates alternately clockwise and counterclockwise during the rotary evaporation process. When the mixed solution is rotary evaporated to a residual volume of 50%, the same polysaccharide as in step (2) is added to obtain solid J, and finally pure water is added to the solid J and ultrasonic hydration is performed, and finally the double-layer loaded polysaccharide liposome suspension is obtained by a microjet high-pressure process.

[0075] In step (2), the mass ratio of solution A to solution C is 50:1.

[0076] In step (3), the fluorescently labeled polysaccharides are: FITC-labeled galactosylarabinan, FITC-labeled mannan, FITC-labeled hydroxyethyl deacetylated chitosan, and FITC-labeled pullulan; the organic solvent F includes anhydrous ethanol, chloroform, and a mixture of the two; the mass volume ratio of the fluorescently labeled polysaccharide to the solvent H is 10 mg:20 mL; and the time for slow addition is 4 mL / min.

[0077] The parameters of various processes in this embodiment (including those not described) are the same as those in Embodiment 1.

[0078] The fluorescence appearance of the prepared double-layer polysaccharide-loaded liposomes is as follows Figure 1 As shown in A, the fluorescently labeled polysaccharide is embedded in the outer layer of liposome phospholipids and the cavity of the double-layer phospholipid molecules, forming a double-layer ring structure.

[0079] Example 4

[0080] The preparation method of the fluorescently labeled proteoliposomes of this embodiment comprises the following steps:

[0081] (1) respectively dissolving the proteins in pure water to obtain solution A, wherein the proteins are a mixture of collagen with a molecular weight of 9000 kD and elastin with a molecular weight of 60 kD;

[0082] At room temperature, a fluorescent marker (fluorescein isothiocyanate (FITC)) is dissolved in an organic solvent B, anhydrous dimethyl sulfoxide, at a mass volume ratio of 1:1 to obtain a solution C;

[0083] (2) Solution A and Solution C were fully stirred and dissolved to obtain Solution D, reacted at room temperature for 4 hours, dialyzed for 48 hours, and freeze-dried in a freeze dryer to obtain a solid E, that is, the fluorescent labeled protein;

[0084] (3) dissolving cholesterol (molecular weight 386) and phospholipid (soybean lecithin (molecular weight 758)) in organic solvent F at room temperature and stirring to obtain a mixed solution G after dissolution. The organic solvent F is a mixture of 15 ml of anhydrous ethanol and 15 ml of chloroform.

[0085] The fluorescently labeled protein is added to the solvent H until it is completely dissolved to obtain a solution I, and then the solution G is slowly added to the solution I and mixed evenly to obtain a mixed solution; wherein the mass volume ratio of the fluorescently labeled protein to the solvent H is 10 mg:20 mL;

[0086] (4) The mixed solution obtained in step (3) is mixed uniformly with a high shear dispersing emulsifier, and the mixed solvent is evaporated away by a rotary evaporator, and the rotary evaporator rotates alternately clockwise and counterclockwise during the rotary evaporation process. When the mixed solution is rotary evaporated to a residual volume of 50%, the same protein as in step (2) is added to obtain solid J, and pure water is added to the solid J to perform ultrasonic hydration, and finally the double-layer protein-loaded liposome suspension is obtained by a microfluidization high-pressure process.

[0087] In the above step (2), the mass ratio of solution A to solution C is 50:1

[0088] The fluorescently labeled protein includes: FITC-labeled collagen and FITC-labeled elastin; the organic solvent F includes anhydrous ethanol, chloroform and a mixture of the two; the stirring time at room temperature is 15 minutes; the mass volume ratio of the fluorescently labeled protein to the solvent H is 10 mg:20 mL.

[0089] Other parameters not specifically noted are the same as those in Example 2.

[0090] The fluorescence appearance of the prepared double-layer protein-loaded liposomes is shown in Figure 1 As shown in B, the fluorescently labeled protein is embedded in the cavity of the outer layer of phospholipids and the double-layer phospholipid molecules to form a double-layer loading structure.

[0091] Example 5

[0092] The Franz diffusion cell was used to verify the permeation of the double-layer galactose arabinan liposomes, double-layer mannan liposomes, double-layer hydroxyethyl chitosan liposomes, double-layer pullulan liposomes, FITC-labeled galactose arabinan, FITC-labeled mannan, FITC-labeled hydroxyethyl chitosan, and FITC-labeled pullulan obtained in Example 3. 12 mL of PBS buffer solution (pH = 7.4) was injected into the receiving chamber and stirred continuously at a speed of 200 rpm / min. The diffusion cell temperature was always maintained at 37°C by circulating heating in a water bath. A mouse skin of appropriate size was placed between the receiving chamber and the supply chamber, and the supply chamber was sealed. 2 mL of liposome suspension and FITC-labeled polysaccharide solution were respectively added dropwise to the upper supply chamber of the Franz diffusion cell skin. After 5 hours, the mouse skin was taken out of the Franz diffusion cell, washed with PBS (pH = 7.4) to remove residual materials and dried, and embedded and fixed with an embedding agent (OTC compound). The frozen skin tissue was then cut vertically into skin slices with a thickness of approximately 10 μm at -20°C using a cryostat. The fluorescence distribution in the skin was observed using an AxioCamMRc inverted fluorescence microscope. Figure 2 As shown, the polysaccharides in the double-layer loaded polysaccharide liposome structure can penetrate through the epidermis and enter the dermis, while the pure polysaccharide solution without the double-layer loading technology can only stay on the surface of the skin tissue.

[0093] Example 6

[0094] The Franz diffusion cell was used to verify the penetration of the bilayer collagen-loaded liposomes, bilayer elastin-loaded liposomes, FITC-labeled collagen, and FITC-labeled elastin obtained in Example 4. 12 mL of PBS buffer solution (pH = 7.4) was injected into the receiving chamber and stirred continuously at a speed of 200 rpm / min. The temperature of the diffusion cell was always maintained at 37°C by circulating heating in a water bath. A mouse skin of appropriate size was placed between the receiving chamber and the supply chamber, and the supply chamber was ensured to be sealed. 2 mL of liposome suspension and fluorescently labeled protein solution were respectively added dropwise to the upper supply chamber of the Franz diffusion cell skin. After 5 hours, the mouse skin was removed from the Franz diffusion cell, washed with PBS (pH = 7.4) to remove residual materials and dried, and embedded and fixed with an embedding agent (OTC compound). Afterwards, the frozen skin tissue was vertically cut into skin slices with a thickness of about 10 μm at -20°C using a freezing microtome. The fluorescence distribution in the skin was observed using an inverted fluorescence microscope. The results are as follows Figure 3 As shown, the protein can penetrate through the epidermis and enter the dermis, while the pure protein solution without the double-layer loading technology of the present invention can only stay on the surface of the skin tissue.

[0095] Example 7

[0096] The bilayer polysaccharide-loaded liposomes obtained in Example 3 were subjected to in vivo verification, and the steps were as follows: anesthetize the mouse, remove the hair on the back of the mouse using a depilatory cream, apply 200 μL of the liposome suspension evenly on the back of the mouse, and remove a 1 cm 2 For the same area of ​​skin tissue, the residual liquid was removed with physiological saline and dried with filter paper. The fluorescence intensity on the back of the skin was observed using a small animal live imaging device, and the fluorescence distribution in the skin was observed using an inverted fluorescence microscope. Figure 4 As shown, the back of the mouse back skin exhibits a strong fluorescence intensity, and under a microscope it can be observed that the liposomes penetrate the epidermis and enter the dermis, indicating that the liposomes can deliver polysaccharides to the dermis of the skin in vivo.

[0097] Example 8

[0098] The bilayer protein-loaded liposomes obtained in Example 4 were subjected to in vivo verification, and the steps were as follows: anesthetize the mouse, remove the hair on the back of the mouse using a depilatory cream, apply 200 μL of the liposome suspension evenly on the back of the mouse, and remove 1 cm 2 For the same area of ​​skin tissue, the residual liquid was removed with physiological saline and dried with filter paper. The fluorescence intensity on the back of the skin was observed using a small animal live imaging device, and the fluorescence distribution in the skin was observed using an AxioCamMRc inverted fluorescence microscope. Figure 5 As shown, the back of the mouse back skin exhibits a strong fluorescence intensity, and under a microscope it can be observed that the liposomes penetrate the epidermis and enter the dermis, indicating that the liposomes can deliver proteins to the dermis of the skin in vivo.

[0099] Example 9

[0100] The bilayer loaded polysaccharide liposomes prepared in Example 1 were sterilized by ultraviolet and then prepared into solutions with a concentration gradient of 25, 50, 100, and 200 μg / ml using culture medium. The solutions were co-incubated with rat fibroblasts with a confluence of 70% for 24 hours and then the cytotoxicity of the materials was determined using the CCK-8 method. The results are as follows: Figure 6 As shown in the figure, when the sample concentration reaches 200μg / ml, the cell survival rate still remains above 95%, and there is a certain phenomenon of promoting cell growth. Compared with the cells in the pure polysaccharide and pure liposome groups, the survival rate of the double-layer loaded polysaccharide liposomes is close to that of the cells in the pure polysaccharide and pure liposome groups, and there is no significant difference. The embedded polysaccharide liposomes have good biocompatibility.

[0101] Example 10

[0102] The bilayer proteoliposomes prepared in Example 2 were sterilized by ultraviolet and prepared into solutions with a concentration gradient of 25, 50, 100, and 200 μg / ml using culture medium. The solutions were incubated with rat fibroblasts with a confluence of 70% for 24 hours and then the cytotoxicity of the materials was determined using the CCK-8 method. The results are as follows: Figure 7 As shown, when the sample concentration reaches 200μg / ml, the cell survival rate is still maintained above 95%, and there is a certain phenomenon of promoting cell growth. Compared with the cells in the pure protein and pure liposome groups, the survival rate of the double-layer protein-loaded liposomes is close to that of the cells in the pure protein and pure liposome groups, and there is no significant difference. The double-layer protein-loaded liposomes do not change the good biocompatibility of the protein.

[0103] Embodiment 11

[0104] The preparation method of the liposomes encapsulating fluorescently labeled proteosomes of this embodiment comprises the following steps:

[0105] (1) dissolving a protein in pure water to obtain solution A, wherein the protein is collagen with a molecular weight of 9000 kD; dissolving a fluorescent marker (fluorescein isothiocyanate (FITC)) in an organic solvent B, anhydrous dimethyl sulfoxide, at a mass-volume ratio of 1:1 at room temperature to obtain solution C;

[0106] (2) Solution A and Solution C were fully stirred and dissolved to obtain Solution D, reacted at room temperature for 4 hours, dialyzed for 48 hours, and freeze-dried in a freeze dryer to obtain a solid E, that is, the fluorescent labeled protein;

[0107] (3) dissolving cholesterol (molecular weight 386) and phospholipid (soybean lecithin (molecular weight 758)) in an organic solvent F at room temperature and stirring to obtain a mixed solution G after dissolution is completed. The organic solvent F is anhydrous ethanol and chloroform in a volume ratio of 1:1;

[0108] The fluorescently labeled protein is added to the solvent H until it is completely dissolved to obtain a solution I, and then the solution G is slowly added to the solution I and mixed evenly to obtain a mixed solution; wherein the mass volume ratio of the fluorescently labeled protein to the solvent H is 10 mg:20 mL;

[0109] (4) The mixed solution obtained in step (3) is mixed uniformly using a high shear dispersing emulsifier, and the mixed solvent is evaporated and removed by a rotary evaporator to obtain a solid J. Pure water is added to the solid J and ultrasonic hydration is performed to obtain a reconstituted solution, and collagen is added to the obtained solution (the mass volume ratio of the reconstituted solution to the added protein is 1 mg / ml). Finally, the liposome suspension encapsulating the fluorescently labeled protein is obtained by a microfluidization high pressure process.

[0110] Various process parameters are the same as those in Example 4.

[0111] The main difference between this embodiment and embodiment 4 is that in step (4), collagen is not added during the rotary evaporation process, but is added after re-dissolving. Figure 1 As shown in Figure C, this liposome is different from the liposome prepared in Example 2. It simply encapsulates the fluorescently labeled collagen inside the liposome, and does not involve the protein in the construction of the liposome.

[0112] Example 12

[0113] The preparation method of the fluorescently labeled polysaccharide liposomes of this embodiment comprises the following steps:

[0114] (2) dissolving a polysaccharide in pure water to obtain solution A, wherein the polysaccharide is mannan with a molecular weight of 1016 kD; dissolving a fluorescent marker (fluorescein isothiocyanate (FITC)) in an organic solvent B, anhydrous dimethyl sulfoxide, at a mass volume ratio of 1:1 at room temperature to obtain solution C;

[0115] (2) Solution A and Solution C were fully stirred and dissolved to obtain Solution D, reacted at room temperature for 4 hours, dialyzed for 48 hours, and freeze-dried in a freeze dryer to obtain a solid E, that is, the fluorescently labeled polysaccharide;

[0116] (3) dissolving cholesterol (molecular weight 386) and phospholipid (soybean lecithin (molecular weight 758)) in an organic solvent F at room temperature and stirring to obtain a mixed solution G after dissolution is completed. The organic solvent F is anhydrous ethanol and chloroform in a volume ratio of 1:1;

[0117] The fluorescently labeled polysaccharide is added to the solvent H until it is completely dissolved to obtain a solution I, and then the solution G is slowly added to the solution I and mixed evenly to obtain a mixed solution; wherein the mass volume ratio of the fluorescently labeled polysaccharide to the solvent H is 10 mg:20 mL;

[0118] (4) The mixed solution obtained in step (3) is mixed uniformly using a high shear dispersing emulsifier, and the mixed solvent is evaporated and removed using a rotary evaporator to obtain a solid J. Pure water is added to the solid J to perform ultrasonic hydration, and finally the suspension of the liposomes encapsulating the fluorescently labeled polysaccharide is obtained by a microfluidic high-pressure process. The temperature of the rotary evaporator is set to 60° C., the clockwise speed is set to 100 r / min, the rotary evaporation time is 45 min, and the vacuum degree of the evaporating flask is 70 kPa.

[0119] Various process parameters are the same as those in Example 3.

[0120] The main difference between this embodiment and embodiment 3 is that in step (4), no polysaccharide is added during the rotary evaporation process, and the rotary evaporation process is performed in a single direction. Figure 1 As shown in D, the liposome does not form a double-layer ring structure, and the fluorescently labeled polysaccharide is encapsulated in the liposome, but is not embedded in the outer layer of the liposome or the inner cavity to participate in the formation of the liposome.

[0121] Example 13

[0122] The preparation method of the liposomes encapsulating fluorescently labeled proteosomes of this embodiment comprises the following steps:

[0123] (3) dissolving a protein in pure water to obtain solution A, wherein the protein is collagen with a molecular weight of 9000 kD; dissolving a fluorescent marker (fluorescein isothiocyanate (FITC)) in an organic solvent B anhydrous dimethyl sulfoxide at a mass volume ratio of 1:1 at room temperature to obtain solution C;

[0124] (2) Solution A and Solution C were fully stirred and dissolved to obtain Solution D, reacted at room temperature for 4 hours, dialyzed for 48 hours, and freeze-dried in a freeze dryer to obtain a solid E, that is, the fluorescent labeled protein;

[0125] (3) dissolving cholesterol (molecular weight 386) and phospholipid (soybean lecithin (molecular weight 758)) in an organic solvent F at room temperature and stirring to obtain a mixed solution G after dissolution is completed. The organic solvent F is anhydrous ethanol and chloroform in a volume ratio of 1:1;

[0126] The fluorescently labeled protein is added to the solvent H until it is completely dissolved to obtain a solution I, and then the solution G is slowly added to the solution I and mixed evenly to obtain a mixed solution; wherein the mass volume ratio of the fluorescently labeled protein to the solvent H is 10 mg:20 mL;

[0127] (4) The mixed solution obtained in step (3) is mixed evenly using a high shear dispersing emulsifier, and the mixed solvent is evaporated and removed by a rotary evaporator. The mixed solvent is rotated alternately clockwise and counterclockwise during the rotary evaporation. When the mixed solvent is evaporated to 1 / 2, collagen is added, and the rotary evaporation is continued to obtain solid J. Pure water is added to the solid J and ultrasonic hydration is performed. Finally, the suspension of liposomes encapsulating fluorescent labeled protein is obtained by microfluidization high pressure process. The mass volume ratio of the solid J to pure water is 1g:10mL, and the hydration time in the ultrasonic instrument is 10min. The temperature of the rotary evaporator is set to 60°C, the clockwise speed is set to 30r / min, the counterclockwise speed is set to 30r / min, the rotation time in each direction is 10min, the rotary evaporation time is 45min, the vacuum degree of the evaporating bottle is 70kPa, and the mass volume ratio of the added protein to the remaining mixed solvent is 1mg / mL.

[0128] The parameters of the above preparation method are basically the same as those of Example 2 and Example 4, except that the rotation speed in step 4 is different. The fluorescence morphology of the prepared liposomes encapsulating fluorescently labeled proteoliposomes is as follows: Figure 1 As shown in E, the liposome encapsulates the fluorescently labeled protein inside the cavity of the phospholipid molecules and does not achieve double-layer loading.

[0129] Embodiment 14

[0130] The preparation method of the fluorescently labeled polysaccharide liposomes of this embodiment comprises the following steps:

[0131] (1) dissolving a polysaccharide in pure water to obtain solution A, wherein the polysaccharide is mannan with a molecular weight of 1016 kD; dissolving a fluorescent marker (fluorescein isothiocyanate (FITC)) in an organic solvent B anhydrous dimethyl sulfoxide at a mass volume ratio of 1:1 at room temperature to obtain solution C;

[0132] (2) Solution A and Solution C were fully stirred and dissolved to obtain Solution D, reacted at room temperature for 4 hours, dialyzed for 48 hours, and freeze-dried in a freeze dryer to obtain solid E, i.e., the fluorescent labeled polysaccharide; the mass ratio of Solution A to Solution C was 50:1;

[0133] (3) dissolving cholesterol (molecular weight 386) and phospholipid (soybean lecithin (molecular weight 758)) in an organic solvent F (anhydrous ethanol and chloroform in a volume ratio of 1:1) at room temperature, stirring and dissolving to obtain a mixed solution G, wherein the organic solvent F is anhydrous ethanol and chloroform in a volume ratio of 1:1;

[0134] The fluorescently labeled polysaccharide is added to the solvent H until it is completely dissolved to obtain a solution I, and then the solution G is slowly added to the solution I and mixed evenly to obtain a mixed solution; wherein the mass volume ratio of the fluorescently labeled polysaccharide to the solvent H is 10 mg:20 mL;

[0135] (4) The mixed solution obtained in step (3) is mixed evenly using a high shear dispersing emulsifier, and the mixed solvent is evaporated and removed by a rotary evaporator, and the mixed solvent is evaporated and removed by a rotary evaporator, and the rotary evaporation process rotates alternately clockwise and counterclockwise. When the mixed solvent is evaporated to 1 / 2, mannan is added, and the rotary evaporation is continued to obtain solid J. Pure water is added to the solid J and ultrasonic hydration is performed, and finally the encapsulated fluorescent labeled polysaccharide liposome suspension is obtained by a micro-jet high pressure process. The temperature of the rotary evaporator is set at 60°C, the clockwise speed is set at 100r / min, the counterclockwise speed is set at 100r / min, the rotation time in each direction is 10min, the rotary evaporation time is 45min, and the vacuum degree of the evaporating bottle is 70kPa.

[0136] The difference from Example 3 is that the mass volume ratio of the polysaccharide added in step (4) to the remaining mixed solvent is 0.2 mg / mL. The other parameters are the same as those of Examples 1 and 3.

[0137] The fluorescence appearance of the prepared liposomes encapsulating fluorescently labeled polysaccharides is shown in Figure 1 As shown in F, the liposome only shows fluorescence inside the cavity, indicating that the fluorescently labeled polysaccharide is simply encapsulated in the liposome and does not participate in the construction of the liposome.

[0138] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a double-layer liposome loaded with polysaccharide or protein for transdermal delivery, characterized in that: The following steps are involved: S1. Cholesterol and phospholipid are dissolved in an organic solvent at room temperature and stirred to obtain a mixed solution A; S2. adding the polysaccharide or protein to the solvent until it is completely dissolved to obtain a solution, and then slowly adding the solution to the mixed solution A and mixing evenly to obtain a mixed solution B, wherein the solvent is water; S3. After mixing, the mixed solution B is evaporated by a rotary evaporator to remove the solvent, and the rotary evaporation process is alternately rotated clockwise and counterclockwise. When the mixed solution is rotary evaporated to a residual volume of 45%-55%, the same polysaccharide or protein as in step S2 is added, and the rotary evaporation is continued to obtain a solid; The clockwise speed is set to 90r / min-100r / min, the counterclockwise speed is set to 90r / min-100r / min, the rotation time in each direction is 9-11min, and the mass volume ratio of the added polysaccharide or protein to the remaining mixed solution is 0.8-1.2mg / mL; S4. After adding pure water to the obtained solid, ultrasonic hydration is performed, and the double-layer liposome suspension loaded with polysaccharide or protein is obtained by microfluidization high pressure process.

2. The method for preparing liposomes according to claim 1, characterized in that: The polysaccharide is galactoarabinan with a molecular weight of 10 to 15 kD, mannan with a molecular weight of 1000 to 1500 kD, hydroxyethyl chitosan with a molecular weight of 60 to 70 kD, or amylopectin with a molecular weight of 100 to 200 kD; Or the protein is collagen with a molecular weight of 8000-10000 kD, or elastin with a molecular weight of 50-70 kD.

3. The method for preparing liposomes according to claim 1, characterized in that: The mass ratio of the phospholipid to cholesterol is 30:(1-2); and or The mass volume ratio of the phospholipid to the organic solvent is 1-1.5 mg / mL; and or the organic solvent A is at least one of anhydrous ethanol and chloroform.

4. The method for preparing liposomes according to claim 1, characterized in that: The mass volume ratio of the polysaccharide or protein to the solvent is (10-20) mg:20 mL.

5. The method for preparing liposomes according to any one of claims 1 to 4, characterized in that: In step S2, the mixture is uniformly mixed by a high shear dispersing emulsifier, wherein the power of the high shear dispersing emulsifier is 200-300 W, the rotation speed is 7500-8500 rpm, and the mixing time is 4.5-5.5 min each time.

6. The method for preparing liposomes according to any one of claims 1 to 4, characterized in that: The temperature of the rotary evaporator is set at 60-65° C., the rotation speed is set at 80-120 r / min, preferably 90-100 r / min, the rotary evaporation time is 30-50 min, and the vacuum degree of the evaporating flask is 70-100 kPa.

7. The method for preparing liposomes according to any one of claims 1 to 4, characterized in that: In step S4, the mass volume ratio of the solid to pure water is 1 g: 8-12 mL, and the hydration time is 5 to 10 min.

8. The method for preparing liposomes according to any one of claims 1 to 4, characterized in that: The stirring in step S2 is stirring at room temperature for 10 to 20 minutes; and or The time of the slow addition in step S2 is 3.8-4.2 mL / min; In step S1, the phospholipid is soybean lecithin with a molecular weight of 700-1000; and / or The molecular weight of the cholesterol is 300-400.

9. A double-layer polysaccharide-loaded transdermal liposome or a double-layer protein-loaded transdermal liposome obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the double-layered polysaccharide-loaded transdermal liposome or the double-layered protein-loaded transdermal liposome of claim 9 in the preparation of a product for preventing or treating skin damage.

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