Double-layer liposome loaded with polysaccharide or protein to promote transdermal delivery and its preparation method and application

Abstract

The present invention discloses a bilayer-loaded liposome capable of promoting transdermal delivery of polysaccharides or proteins, as well as its preparation method and application. The bilayer-loaded polysaccharide and protein liposomes are prepared using cholesterol, soy lecithin, and other raw materials through high-speed homogenization, evaporation using alternating clockwise and counterclockwise rotation during rotary evaporation, and microfluidization with high pressure. These liposomes are capable of promoting transdermal delivery of polysaccharides or proteins, and are expected to fully utilize the therapeutic and repairing polysaccharides and proteins in the skin. They can be used to prepare products for preventing or treating skin damage.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to transdermal delivery technology, in particular 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 systems are novel approaches for administering drugs through the skin, allowing them to enter the human bloodstream at a constant rate through capillaries, producing systemic or local therapeutic effects. The skin, as the body's largest organ, has garnered widespread attention for transdermal drug delivery, offering significant advantages such as avoiding the first-pass effect in the liver, controlling drug delivery rate, improving drug utilization, and providing non-invasive delivery. Polysaccharides and proteins, as essential components of the human body, play important roles. For example, hyaluronic acid promotes tissue regeneration and wound healing by participating in cell signaling; alginate promotes chronic wound healing by inducing cytokine production in monocytes; collagen scavenges free radicals and slows skin aging; and immunoglobulins regulate immune cell function and activity by binding to surface receptors on immune cells. However, oral administration of polysaccharides and proteins is limited by the gastrointestinal pH and complex enzymatic environment, resulting in low utilization rates. Therefore, transdermal devices based on polysaccharides and proteins hold great promise for treating related diseases.

[0003] Currently, a variety of transdermal drug delivery vehicles based on polysaccharides and proteins have been developed, such as hydrogels, films, and microneedles. For example, Wang et al. used Panax notoginseng polysaccharide to create a soluble microneedle that can activate skin dendritic cells to promote transdermal drug delivery. With the help of the microneedle, 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. created a chitosan / hyaluronic acid transdermal film device. By modifying chitosan and covalently linking it with hyaluronic acid, it demonstrated excellent control over the release of the drug lidocaine, as well as good mechanical properties and biosafety (Carbohydrate Polymers, 2016, 152:687-698). Donnely et al. prepared a hydrogel microneedle system using silicone micromolds and successfully used this system to transdermally deliver proteins and peptides of various molecular weights (Advanced Functional Materials, 2012, 22:4879-4890). Many studies at home and abroad have shown that polysaccharides and proteins can be delivered through transdermal systems by breaking through the skin barrier and bypassing 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 a major obstacle to the transdermal delivery of polysaccharides and proteins. Therefore, constructing a new class of materials that can effectively penetrate the skin for polysaccharides and proteins has become an important scientific issue that needs to be solved urgently.

[0004] Liposomes are one or more closed vesicles composed of amphiphilic substances such as lecithin and cholesterol dispersed in an aqueous phase. They can encapsulate both hydrophilic and lipophilic drugs. Their bilayer structure is similar to that of cell membranes and is widely used for intravenous, transdermal, and oral administration. Using liposomes as a carrier material for transdermal drug delivery offers the following advantages: 1. They have a sustained-release effect, slowly releasing encapsulated drugs, delaying renal excretion and metabolism, thereby extending the duration of drug use; 2. They are targeted and lymphatic-directed, preventing adverse reactions caused by systemic drug absorption; 3. Liposome-encapsulated drugs are more stable; 4. They can reduce drug toxicity. Liposomes themselves are 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-molecule 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 that can promote the transdermal delivery of polysaccharides or proteins, as well as 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. The polysaccharide or protein is added to a solvent until completely dissolved to obtain a solution, and then the solution is slowly added to the mixed solution A and mixed to obtain a mixed solution B, wherein the solvent is water;

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

[0011] The clockwise speed is set to 90 r / min-100 r / min, the counterclockwise speed is set to 90 r / min-100 r / min, the rotation time in each direction is 9-11 min, and the mass volume ratio of the added polysaccharide or protein to the remaining mixed solution is 0.8-1.2 mg / 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 the phospholipid to cholesterol is 30:(1-2).

[0016] In some 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 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 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 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 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 minutes.

[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 an appropriate 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 corresponding liposomes. This 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0031] Figure 1 Characterization of the double-layered polysaccharide- and protein-loaded liposomes prepared in Examples 3 and 4; A is a fluorescence morphology image of the double-layered polysaccharide-loaded liposomes; B is a fluorescence morphology image of the double-layered protein-loaded liposomes; C, D, E, and F are fluorescence morphologies of the liposomes prepared in Examples 11 to 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; B is the permeation experimental result of the fluorescently labeled polysaccharide.

[0033] Figure 3 The permeation test of the bilayer protein-loaded liposomes prepared in Example 4 was performed using a Franz diffusion cell; A is the permeation test result of the bilayer protein-loaded liposomes; B is the permeation test result of the fluorescent-labeled protein.

[0034] Figure 4 The double-layered polysaccharide-loaded liposomes prepared in Example 3 were subjected to in vivo verification; 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.

[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 the in vitro cell safety verification of the double-layered polysaccharide-loaded liposomes prepared in Example 1.

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

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

[0039] To facilitate 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 present disclosure more thorough and comprehensive.

[0040] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

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

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

[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 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, soy lecithin and the like with good biosafety as raw materials, and sequentially 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. The polysaccharide and protein are embedded in the surface and cavity of a double-layer structure similar to the liposome membrane, thereby penetrating through the epidermis through lipid fusion, skin appendages and other pathways, penetrating into the skin, and finally constructing 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 polysaccharides and proteins, which is beneficial for the adaptive deformation of polysaccharides and proteins with liposomes, thereby facilitating transdermal delivery. The present invention overcomes the current difficulties in the limited amount, type and molecular weight of transdermal delivery 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 to 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 in pharmaceutical excipients, and have significant biosafety.

[0047] In the following examples, the purity of the anhydrous ethanol (Macklin) used is AR, 95%; the purity of the soy lecithin (Shanghai Yuanye) used is ≥90%; the purity of the cholesterol (Macklin) used is 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, the structural diagram of which is shown in FIG. Figure 8 As shown in the figure, the whole body is in the shape of a double-layered ring, with polysaccharides and proteins 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 liposomes of this embodiment comprises the following steps:

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

[0054] (2) Add the polysaccharide to deionized water and stir at room temperature for 15 minutes until the polysaccharide solution D is completely dissolved. Then, slowly add the polysaccharide solution D to the mixed solution B and mix evenly using a high-shear dispersing emulsifier to obtain a mixed solution; the high-shear dispersing emulsifier has a power of 220W and a speed of 8000 rpm, and each time is 5 minutes;

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

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

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

[0061] (1) First, cholesterol and phospholipid are dissolved in 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 completely dissolved to obtain protein solution D, then slowly adding protein solution D to solution B, and mixing the two using a high shear dispersing emulsifier to obtain a mixed solution; the high shear dispersing emulsifier has a power of 220W, a speed of 8000rpm, and each time is 5 minutes;

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

[0064] (4) Finally, pure water is added to the solid and ultrasonic hydration is performed, and finally the bilayer 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 minutes.

[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, each 15ml; the mass volume ratio of the protein to the solvent C is 10mg:20mL; and the slow addition time is 4mL / min.

[0066] To verify the biological activities 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) Dissolve the 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 thoroughly stirred and dissolved to obtain Solution D, reacted at room temperature for 4 h, dialyzed for 48 h, and freeze-dried to obtain Solid E, i.e., the fluorescently labeled polysaccharide;

[0072] (3) Cholesterol (molecular weight 386) and phospholipid (soy lecithin with a molecular weight of 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. The mass ratio of the phospholipid to the cholesterol was 30:1; the mass volume ratio of the soybean lecithin to the organic solvent (a mixture of anhydrous ethanol and chloroform) was 1 mg / mL.

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

[0074] (4) The mixed solution obtained in step (3) is mixed uniformly with the mixed solution using a high shear dispersing emulsifier, and the mixed solvent is evaporated away using 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 the 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 Example 1.

[0078] The fluorescence appearance of the prepared double-layer loaded polysaccharide liposomes is as follows Figure 1 As shown in A, the fluorescently labeled polysaccharide is embedded in the outer layer of liposomes 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) 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)) was dissolved in an organic solvent B, anhydrous dimethyl sulfoxide, at a mass-to-volume ratio of 1:1 to obtain solution C.

[0083] (2) Solution A and Solution C were thoroughly stirred and dissolved to obtain Solution D, reacted at room temperature for 4 h, dialyzed for 48 h, and freeze-dried in a freeze dryer to obtain Solid E, i.e., the fluorescent labeled protein;

[0084] (3) Cholesterol (molecular weight 386) and phospholipid (soy lecithin (molecular weight 758)) were dissolved in organic solvent F at room temperature and stirred to dissolve, thereby obtaining a mixed solution G. The organic solvent F was a mixture of 15 ml of anhydrous ethanol and 15 ml of chloroform.

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

[0086] (4) The mixed solution obtained in step (3) is mixed uniformly with the mixed solution using a high shear dispersing emulsifier, and the mixed solvent is evaporated away using 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. Finally, pure water is added to the solid J and ultrasonic hydration is performed. Finally, the double-layer protein-loaded liposome suspension is obtained by a microjet high-pressure process.

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

[0088] The fluorescent labeled proteins include: FITC labeled collagen, 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 fluorescent 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, as shown in Figure 1 As shown in B, the fluorescent marker protein is embedded in the outer layer of phospholipids and the cavity of the double-layer phospholipid molecules to form a double-layer loading structure.

[0091] Example 5

[0092] A Franz diffusion cell was used to verify the permeation of the bilayer galactoarabinan-loaded liposomes, bilayer mannan-loaded liposomes, bilayer hydroxyethyl chitosan-loaded liposomes, bilayer pullulan-loaded liposomes, FITC-labeled galactoarabinan, 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 200 rpm / min. The diffusion cell temperature was maintained at 37°C using a circulating water bath. A mouse skin of appropriate size was placed between the receiving chamber and the donor chamber, ensuring that the donor chamber was sealed. 2 mL of each liposome suspension and FITC-labeled polysaccharide solution were added dropwise to the donor chamber on the skin of the Franz diffusion cell. After 5 hours, the mouse skin was removed from the Franz diffusion cell, washed with PBS (pH = 7.4) to remove residual material, 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 using a freezing microtome at -20°C. The fluorescence distribution in the skin was observed using an AxioCam MRc inverted fluorescence microscope. Figure 2 As shown, the polysaccharide in the double-layer loaded polysaccharide liposome structure can penetrate 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 double-layer collagen-loaded liposomes, double-layer 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 each 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). The frozen skin tissue was then cut vertically 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 shown in FIG. Figure 3 As shown, the protein can penetrate 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 double-layer polysaccharide-loaded liposomes obtained in Example 3 were subjected to in vivo verification. 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 The same area of ​​skin tissue 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 into the dermis, indicating that the liposomes can deliver polysaccharides to the dermis of the skin in vivo.

[0097] Example 8

[0098] The double-layer protein-loaded liposomes obtained in Example 4 were subjected to in vivo verification. 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 The same area of ​​skin tissue 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 double-layer 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 incubated with rat fibroblasts with a confluence of 70% for 24 hours and the cytotoxicity of the materials was determined using the CCK-8 method. The results are as follows: Figure 6 As shown, at a sample concentration of 200 μg / ml, cell viability remained above 95%, with some cell growth promotion observed. Compared to cells in the pure polysaccharide and pure liposome groups, the survival rates of bilayer-loaded polysaccharide liposomes were similar, with no significant difference, demonstrating the excellent biocompatibility of embedded polysaccharide liposomes.

[0101] Example 10

[0102] The double-layered proteoliposomes prepared in Example 2 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 incubated with rat fibroblasts with a confluence of 70% for 24 hours and the cytotoxicity of the materials was determined using the CCK-8 method. The results are shown in Figure 2. Figure 7 As shown, at a sample concentration of 200 μg / ml, cell viability remained above 95%, with some cell growth promotion observed. Compared to cells in the pure protein and pure liposome groups, the survival rates of bilayer protein-loaded liposomes were similar, with no significant difference. Bilayer protein-loaded liposomes do not alter the protein's excellent biocompatibility.

[0103] Example 11

[0104] The preparation method of the fluorescently labeled proteoliposomes of this embodiment includes 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-to-volume ratio of 1:1 at room temperature to obtain solution C;

[0106] (2) Solution A and Solution C were thoroughly stirred and dissolved to obtain Solution D, reacted at room temperature for 4 h, dialyzed for 48 h, and freeze-dried in a freeze dryer to obtain Solid E, i.e., the fluorescent labeled protein;

[0107] (3) Cholesterol (molecular weight 386) and phospholipid (soy lecithin (molecular weight 758)) were dissolved in an organic solvent F at room temperature and stirred to dissolve, thereby obtaining a mixed solution G. The organic solvent F was anhydrous ethanol and chloroform in a volume ratio of 1:1.

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

[0109] (4) The mixed solution obtained in step (3) is mixed uniformly with the mixed solution using a high shear dispersing emulsifier, and the mixed solvent is evaporated away using a rotary evaporator to obtain solid J. Pure water is added to the solid J and ultrasonic hydration is performed to obtain a reconstituted solution. Collagen is then added to the obtained solution (the mass volume ratio of the reconstituted solution to the added protein is 1 mg / ml), and finally the liposome suspension containing 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 the resolubilization. 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-to-volume ratio of 1:1 at room temperature to obtain solution C;

[0115] (2) Solution A and Solution C were thoroughly stirred and dissolved to obtain Solution D, reacted at room temperature for 4 h, dialyzed for 48 h, and freeze-dried in a freeze dryer to obtain Solid E, i.e., the fluorescently labeled polysaccharide;

[0116] (3) Cholesterol (molecular weight 386) and phospholipid (soy lecithin (molecular weight 758)) were dissolved in an organic solvent F at room temperature and stirred to dissolve, thereby obtaining a mixed solution G. The organic solvent F was anhydrous ethanol and chloroform in a volume ratio of 1:1.

[0117] The fluorescently labeled polysaccharide is added to solvent H until completely dissolved to obtain solution I, and then solution G is slowly added to 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 away using a rotary evaporator to obtain a solid J. Pure water is added to the solid J and ultrasonic hydration is performed. Finally, the suspension of the liposomes encapsulating the fluorescently labeled polysaccharide is obtained by a microfluidization high-pressure process. The rotary evaporator is set at a temperature of 60° C., a clockwise speed of 100 r / min, a rotary evaporation time of 45 min, and a vacuum degree of 70 kPa in the evaporating flask.

[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 carried out in a single direction. Figure 1 As shown in D, the liposome does not form a double-layer ring structure. The fluorescently labeled polysaccharide is encapsulated inside the liposome and does not embed in the outer layer and the inner cavity of the liposome to participate in the formation of the liposome.

[0121] Example 13

[0122] The preparation method of the fluorescently labeled proteoliposomes of this embodiment includes the following steps:

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

[0124] (2) Solution A and Solution C were thoroughly stirred and dissolved to obtain Solution D, reacted at room temperature for 4 h, dialyzed for 48 h, and freeze-dried in a freeze dryer to obtain Solid E, i.e., the fluorescent labeled protein;

[0125] (3) Cholesterol (molecular weight 386) and phospholipid (soy lecithin (molecular weight 758)) were dissolved in an organic solvent F at room temperature and stirred to dissolve, thereby obtaining a mixed solution G. The organic solvent F was anhydrous ethanol and chloroform in a volume ratio of 1:1.

[0126] The fluorescent labeled protein is added to solvent H until completely dissolved to obtain solution I, and then solution G is slowly added to solution I and mixed evenly to obtain a mixed solution; wherein the mass volume ratio of the fluorescent 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 using a rotary evaporator. The rotary evaporation process rotates alternately clockwise and counterclockwise. 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 liposome suspension containing fluorescently labeled proteins 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 at 60°C, the clockwise speed is set at 30r / min, the counterclockwise speed is set at 30r / min, the rotation time in each direction is 10min, the rotary evaporation time is 45min, the vacuum degree of the evaporating flask 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 is different in step 4. The fluorescence appearance of the prepared fluorescent-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] Example 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 anhydrous dimethyl sulfoxide (DMSO) in an organic solvent B at a mass-to-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 h, dialyzed for 48 h, and freeze-dried in a freeze dryer to obtain Solid E, i.e., the fluorescently labeled polysaccharide; the mass ratio of Solution A to Solution C was 50:1;

[0133] (3) Cholesterol (molecular weight 386) and phospholipid (soy lecithin (molecular weight 758)) were dissolved in an organic solvent F (anhydrous ethanol and chloroform in a volume ratio of 1:1) at room temperature and stirred to obtain a mixed solution G after dissolution. The organic solvent F was anhydrous ethanol and chloroform in a volume ratio of 1:1.

[0134] The fluorescently labeled polysaccharide is added to solvent H until completely dissolved to obtain solution I, and then solution G is slowly added to 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 uniformly using a high shear dispersing emulsifier, and the mixed solvent is evaporated and removed using a rotary evaporator. The mixed solvent is evaporated and removed using a rotary evaporator, rotating alternately clockwise and counterclockwise during the rotary evaporation process. When the mixed solvent is evaporated to 1 / 2 of the remaining 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. Finally, the encapsulated fluorescent labeled polysaccharide liposome suspension is obtained by microfluidization high pressure process. The temperature of the rotary evaporator is set at 60°C, the clockwise speed is set at 100 r / min, the counterclockwise speed is set at 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.

[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. Other parameters are the same as those of Examples 1 and 3.

[0137] The fluorescence appearance of the prepared fluorescently labeled polysaccharide liposomes is shown in Figure 2. Figure 1 As shown in Figure 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-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended 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. The polysaccharide or protein is added to a solvent until completely dissolved to obtain a solution, and then the solution is slowly added to the mixed solution A and mixed to obtain a mixed solution B, wherein the solvent is water; S3. After mixing, the mixed solution B was evaporated to remove the solvent by rotary evaporation, and the rotary evaporation process was rotated alternately clockwise and counterclockwise. When the mixed solution was evaporated to a volume of 45%-55%, the same polysaccharide or protein as in step S2 was added, and the rotary evaporation was continued to obtain a solid; The clockwise speed is set to 90 r / min-100 r / min, the counterclockwise speed is set to 90 r / min-100 r / min, the rotation time in each direction is 9-11 min, and the mass volume ratio of the added polysaccharide or protein to the remaining mixed solution is 0.8-1.2 mg / mL; S4. After adding pure water to the obtained solid, ultrasonic hydration was performed, and the bilayer liposome suspension loaded with polysaccharide or protein was obtained by microfluidization high pressure process; 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; Or the protein is collagen with a molecular weight of 8000-10000 kD, or elastin with a molecular weight of 50-70 kD.

2. The method for preparing liposomes according to claim 1, wherein 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 to 1.5 mg / mL; and / or The organic solvent is at least one of anhydrous ethanol and chloroform.

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

4. The method for preparing liposomes according to any one of claims 1 to 3, characterized in that: In step S2, the mixture is uniformly mixed using 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.

5. The method for preparing liposomes according to any one of claims 1 to 3, characterized in that: The temperature of the rotary evaporator is set at 60-65°C, the speed is set at 80-120 r / min, the rotary evaporation time is 30-50 min, and the vacuum degree of the evaporating flask is 70-100 kPa.

6. The method for preparing liposomes according to claim 5, wherein The set speed is 90-100 r / min.

7. The method for preparing liposomes according to any one of claims 1 to 3, 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 5-10 min.

8. The method for preparing liposomes according to any one of claims 1 to 3, characterized in that: The stirring in step S1 is stirring at room temperature for 10 to 20 min; 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 double-layered protein-loaded transdermal liposome according to claim 9 in the preparation of a product for preventing or treating skin damage.

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