Nanometer supramolecular drug-loaded liposome and preparation method thereof

Abstract

The invention belongs to the field of medical biology, and relates to a medical preparation, in particular to a nano supramolecular drug-loaded liposome and a preparation method thereof. The nano supramolecular drug-loaded liposome is prepared from the following raw materials in percentage by weight: 1 to 20 percent of active ingredient, 5 to 15 percent of lecithin, 20 to 40 percent of eutectic solvent, 0.1 to 10 percent of grease, 0.1 to 0.5 percent of cholesterol, 0.1 to 5 percent of emulsifier, 0.1 to 1.5 percent of stabilizer and the balance of deionized water, and the active ingredient is water-soluble VC (prototype VC). According to the invention, the active ingredients and the deep eutectic solvent are subjected to ultrasonic treatment to form a ternary supramolecular VC structure, and then the ternary supramolecular VC structure is wrapped, so that the stability of VC molecules in a solution state is greatly improved by virtue of dual clamping of supramolecules and lipidosome, the problem of oxidation discoloration of the VC molecules in a long-term storage process is improved, and the effect of VC in the cosmetic industry is better played.

Description

Technical Field

[0001] The invention belongs to the field of medicine and biology, relates to medical preparations, and specifically relates to a nano supramolecular drug-loaded liposome and a preparation method thereof. Background Art

[0002] Natural deep eutectic solvents (NaDES) are defined as mixtures of two or more components, which can be either solid or liquid. Under certain compositions, they will show a high melting point depression phenomenon, that is, they are in a liquid state at room temperature. NaDES is a new type of solvent that green chemistry is actively looking for to replace traditional toxic and highly volatile organic solvents. It is mainly composed of hydrogen bond donors and hydrogen bond acceptors in a specific molar ratio. The charge delocalization mediated by intermolecular complexation is the main reason for the low melting point of NaDES. NaDES has many advantages, such as low volatility, high boiling point, high degradability, biocompatibility, compatibility with water, low or even non-toxicity, 100% atom economy, etc., so it is widely used in many fields such as biocatalysis, electrochemistry, material extraction and separation, and drug delivery.

[0003] Nano drug-loaded liposomes are closed multi-layer microcapsules formed by dispersing amphiphilic phospholipid molecules and cholesterol in water. The drug is mainly present in the inner phase and the middle phase of the microcapsule, thus forming a drug encapsulation structure. Liposomes can improve the solubility of the dissolved substances, enhance stability and improve bioavailability, and rely on the affinity of the lipid layer and the biological membrane to achieve the sustained release and targeted delivery effects of the active ingredients. The preparation methods of traditional liposomes mainly include reverse evaporation, thin film dispersion, freeze-thaw drying, spray drying, ethanol injection, ultrasonic dispersion, high-pressure homogenization and high-pressure microfluidization. These methods will inevitably involve organic solvents in the process of preparing liposomes, and it is difficult to remove them in the subsequent process, resulting in the problem of residual organic solvents, which limits their application in the fields of cosmetics and food.

[0004] Nano supramolecular drug-loaded liposomes mainly use green supramolecular solvents to disperse active ingredients, lecithin, and cholesterol, and change the existence form of active ingredients through physical modification of supramolecular and structural similarity between liposomes and cell membranes. With the dual support of supramolecular and liposomes, the encapsulation of active ingredients is greatly improved, and the degree of active ingredients entering the stratum corneum and epidermal lipids is also enhanced, achieving effective accumulation of active ingredients in local skin, thereby more efficiently exerting skin care effects.

[0005] Vitamin C (VC) is widely used in various whitening products. It can achieve the effect of whitening and lightening spots by inhibiting the activity of tyrosinase, reducing melanin in the skin and removing harmful free radicals. However, VC molecules are unstable and very sensitive to external environments such as light, heat, and oxygen. When VC is used in cosmetics, there are generally technical problems such as easy degradation and oxidation, poor skin permeability, poor storage stability, and strong irritation.

[0006] Patent CN110279590A discloses a vitamin C (VC) liposome and a preparation method and application thereof. The process adopts a reverse evaporation method to prepare VC liposomes. Although the preparation of nanoscale liposomes is achieved, toxic organic reagents such as chloroform are used in the preparation process. Although most of the organic solvents can be removed by subsequent vacuum distillation, some chloroform will still remain in the final liposomes, which greatly limits its application in the field of cosmetics. In addition, the nanocarriers prepared by the reverse evaporation method generally have poor stability and general dispersibility, and it is difficult to produce practical application value.

[0007] Patent CN101912388A discloses the preparation of medium-chain fatty acid-VC liposomes by reverse evaporation-high-pressure microjet. Although the preparation of nano-scale carriers can be achieved by relying on the cavitation and shear effects of high-pressure microjet, the final carrier has a VC loading of about 0.5%, and ethanol is used as the oil phase solvent in the preparation process, which still limits the application of the liposomes in certain scenarios.

[0008] Patent CN115400045A discloses a VC flexible liposome and its preparation method and application. The VC liposome prepared by this method has strong deformability and has certain advantages in promoting penetration. The addition of xanthan gum can indeed increase the suspension stability of the system. However, the final VC load of this method is less than 0.2%, and the PDI is large and the dispersibility is poor.

[0009] Patent CN116349868A discloses a VC-loaded emulsion and its application. This method uses two different types of emulsifiers to disperse one emulsion in another continuous phase emulsion. It is indeed a good choice in the short term, but the multiple emulsion itself is not a form that can exist stably. After being left for a long time, the internal oil-in-water structure is prone to phase transition, resulting in drug leakage.

[0010] Patent CN111557912A discloses a method for preparing fatty acid-based VC liposomes, which uses sodium dodecyl sulfate to emulsify linoleic acid and other high-carbon oils to form a nanostructure that encapsulates VC. However, such oils are usually difficult to emulsify, and even after emulsification, the system formed is mostly milky white, which is not suitable for some transparent system application scenarios. Sodium dodecyl sulfate itself is also highly irritating, and the liposomes formed by self-emulsification are not uniform in particle size.

[0011] The invention of CN117481981A "A ginsenoside liposome encapsulating soluble collagen, its preparation method and application" discloses a ginsenoside liposome encapsulating soluble collagen, including 0.001-0.1 parts of soluble collagen and 0.2-2 parts of supramolecular rare ginsenoside crystals; the supramolecular rare ginsenoside crystals include diol rare ginsenosides, triol rare ginsenosides and supramolecular plant fermentation liquid; the mass ratio of the diol rare ginsenosides, triol rare ginsenosides and supramolecular plant fermentation liquid is (0.01-10): (0.01-10): (0.1-20); the preparation method of the supramolecular plant fermentation liquid comprises the following steps: inactivating the fermentation liquid obtained by fermentation and culture of lactic acid bacteria or the wet bacteria obtained by fermentation centrifugation, centrifuging to obtain supernatant, adding plant extract and supramolecular NaDES solvent to the obtained supernatant, heating and stirring until the solution is clear, cooling, and obtaining. The NaDES solvent comprises 0.1 to 10 parts of lactobionic acid, 0.1 to 10 parts of fructose, 0.1 to 10 parts of sorbitol and 0.5 to 50 parts of water. The preparation method of the supramolecular NaDES solvent is as follows: heating lactobionic acid, fructose, sorbitol and water at 46°C to 50°C for 2.5h to 3.5h, and drying at 58°C to 62°C for 71h to 73h according to the mass parts. Summary of the invention

[0012] The technical problem to be solved by the present invention is to provide a nano supramolecular drug-loaded liposome and a preparation method thereof.

[0013] In order to solve the above problems, the present invention provides a nano supramolecular drug-loaded liposome, the raw materials of which are composed of the following components by weight: Active ingredient: 1-20%, Lecithin: 5-15%, Deep eutectic solvent (NADES): 20-40%, Grease (liquid grease at room temperature): 0.1-10%, Cholesterol: 0.1-0.5%, Emulsifier: 0.1-5%, Stabilizer: 0.1-1.5%, The balance is deionized water; The active ingredient is water-soluble VC (prototype VC).

[0014] As a preference: Active ingredient: 3-17%, Lecithin: 5.5-15%, Deep eutectic solvent: 26-40%, Oil: 2-6.5%, Cholesterol: 0.125-0.35%, Emulsifier: 1.5-5%, Stabilizer: 0.55-0.9%, The balance was deionized water.

[0015] As an improvement of the nano-supramolecular drug-carrying liposome of the present invention, the lecithin is at least any one of the following: soybean lecithin, hydrogenated lecithin, egg yolk lecithin, lysolecithin, phosphatidylethanolamine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and dipalmitoylphosphatidylglycerol.

[0016] As a further improvement of the nano-supramolecular drug-carrying liposome of the present invention, the low eutectic solvent is at least any one of the following: Betaine glycerol supramolecular solvent, betaine propylene glycol supramolecular solvent, betaine butylene glycol supramolecular solvent, betaine isosorbide supramolecular solvent, betaine citric acid supramolecular solvent, choline chloride propylene glycol supramolecular solvent, choline chloride urea supramolecular solvent, choline chloride malic acid supramolecular solvent, choline chloride glucose supramolecular solvent, choline chloride glycerol supramolecular solvent, L-proline glycerol supramolecular solvent.

[0017] As a further improvement of the nano-supramolecular drug-carrying liposome of the present invention, the oil is at least any one of the following: caprylic capric triglyceride, isopropyl myristate, olive oil, camellia oil, jojoba oil, and meadowfoam seed oil.

[0018] As a further improvement of the nano supramolecular drug-loaded liposome of the present invention: The emulsifier is at least any one of the following: PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, polyglyceryl-3 cocoate, polyglyceryl-10 laurate, polyglyceryl-10 oleate, polyglyceryl-10 myristate, polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-80, sodium stearoyl glutamate; The stabilizer is at least any one of the following: EDTA-2Na, vitamin E acetate, vitamin E, sodium pyrosulfite, diethylhexyl syringylidenemalonate.

[0019] As a further improvement of the nano supramolecular drug-loaded liposome of the present invention, the raw materials of the nano supramolecular drug-loaded liposome are composed of the following components by weight: 5% water-soluble VC, 40% betaine glycerol supramolecular solvent, 0.25% cholesterol, 4% caprylic capric triglyceride, 10% soy lecithin, 0.5% Vitamin E acetate, 0.1% EDTA-2Na, 4% PEG-40 hydrogenated castor oil, 36.15% deionized water.

[0020] The present invention also provides a method for preparing the nano supramolecular drug-loaded liposome, comprising the following steps: Step 1: Add the active ingredient to a low eutectic solvent, and ultrasonicate or stir at 45-75°C until the active ingredient is dissolved to obtain solution A; Step 2: Add cholesterol to the oil and ultrasonicate at 45-75°C until the cholesterol is dissolved to obtain solution B; Step 3: at 45-75°C and under stirring conditions, add solution B obtained in step 2 dropwise to solution A obtained in step 1 (addition time is about 1-2 minutes), then add lecithin and stabilizer, keep warm and stir until lecithin and stabilizer are dispersed, to obtain dispersion 1; Step 4: Add the emulsifier into deionized water, stir at 45-75°C until the emulsifier is dispersed, and obtain dispersion 2; Step 5: Slowly add the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing conditions (addition time is about 5-8 minutes), the shearing speed is 2000-5000rpm, the shearing temperature is 45-75°C, and the shearing time is 5-30min to obtain a supramolecular liposome primary emulsion; Step 6: subjecting the supramolecular liposome primary emulsion obtained in step 5 to high pressure homogenization or high pressure microfluidization to obtain nano supramolecular drug-loaded liposomes.

[0021] As an improvement to the preparation method of the nano-supramolecular drug-loaded liposomes of the present invention: the pressure of the high-pressure homogenization in step 6 is 500-800 bar, the number of high-pressure homogenization is 3-8 times, and the temperature of the high-pressure homogenization is 15-45°C.

[0022] As a further improvement of the method for preparing the nano-supramolecular drug-loaded liposomes of the present invention: the stirring speeds of step 3 and step 4 are both 500-1500 rpm.

[0023] The present invention uses green supramolecular solvents (deep eutectic solvents) and active substances to form a multi-supramolecular structure to achieve supramolecular physical modification, and then uses liposomes to achieve dual-phase encapsulation. The formation of the multi-supramolecular structure can improve the encapsulation rate of the original liposomes, and also has a gain effect on the stability and drug loading of the liposomes. The present invention uses the dual penetration-enhancing effects of supramolecular and liposomes to achieve efficient transdermal effects of active substances.

[0024] The nano supramolecular drug-carrying liposome of the present invention is "nano" as can be seen from its particle size.

[0025] It should be emphasized that the low eutectic solvent of the present invention is significantly different from the supramolecular NaDES solvent in CN117481981A as follows: 1. The low eutectic solvent of the present invention is a typical low eutectic solvent formed by hydrogen bond acceptor quaternary ammonium salt and zwitterion and hydrogen bond donor, while the supramolecular NaDES solvent in patent CN117481981A is formed by adding sugar to sugar, and the preparation methods of the two are also different. Patent CN117481981A is obviously prepared by simple heating and mixing, and water needs to be added to dissolve the two sugars first, and then dried. Since the water in NaDES exists in the form of bonded water, it is difficult to evaporate and remove. Therefore, it can be inferred that the supramolecular NaDES solvent in CN117481981A contains water.

[0026] 2. Improve the penetration ability of active substances. The role of supramolecular NaDES solvent is mainly to form hydrogen bonds with ginsenosides to stabilize the encapsulation structure, while the present invention mainly uses supramolecular solvents and active substances to form a multi-supramolecular structure to achieve physical modification of VC, so that VC can be dispersed in the middle oil phase of liposomes and can also be dispersed in the water phase, thereby improving the encapsulation rate.

[0027] Compared with the prior art, the present invention has the following technical effects: 1. The present invention makes VC compatible with the original low eutectic solvent by means of physical modification of the supramolecular solvent (low eutectic solvent), forming a multi-component supramolecular structure containing VC. The formation of the multi-component supramolecular structure can stabilize the VC molecules on the one hand, and secondly allow the VC molecules to occupy the inner phase and the middle phase space of the liposome at the same time, thereby improving the encapsulation rate and loading capacity of the liposome. For the VC molecules encapsulated by traditional liposomes, VC only exists in the inner phase of the liposome, so the encapsulation rate is generally below 50%. The VC after the supramolecular modification of the present invention can more reasonably utilize the cavity of the liposome, the encapsulation rate can be increased to more than 80%, and the loading capacity can reach up to about 15%.

[0028] 2. The present invention ultrasonically treats the active ingredient and the low eutectic solvent to form a ternary supramolecular VC structure and then encapsulates it. With the dual support of supramolecular and liposome, the stability of VC molecules in solution can be greatly improved, and the oxidative discoloration problem during long-term storage can be improved, so as to better play the role of VC in the cosmetics industry.

[0029] Description: Liposome is an encapsulation structure formed by the present invention with the help of lecithin, a material. VC forms a multi-supramolecular structure through a supramolecular solvent (low eutectic solvent), which is the supramolecular blessing. Then lecithin, a membrane material, forms a liposome structure at the interface of the supramolecular and water to encapsulate VC, which is the liposome blessing, so it is called double blessing.

[0030] 3. The supramolecular solvent can improve the penetration-promoting ability of the drug. The formation of the multi-supramolecular structure containing VC can accelerate the transdermal absorption of VC molecules in the liquid preparation, so that the final nano supramolecular drug-loaded liposome has a better drug delivery effect than the traditional liposome. That is, after the supramolecular solvent in the present invention forms a multi-supramolecular structure with VC, the transdermal ability can be further increased.

[0031] 4. The nano-supramolecular structured liposomes prepared with the aid of supramolecular solvents have better stability. They can weaken the system collapse caused by Oswald ripening during long-term storage of traditional liposomes, especially the stability in response to high temperatures.

[0032] 5. The present invention utilizes green supramolecular solvents to replace traditional organic solvents to dissolve lecithin, which has safer ingredients, can effectively avoid allergenic problems, and is more suitable for the production and use of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0034] Figure 1 This is a diagram showing the appearance changes of the nano supramolecular drug-loaded liposomes prepared by the present invention during storage; Figure 2 This is a graph showing the particle size stability of the nano supramolecular drug-loaded liposomes prepared by the present invention; Figure 3 This is a dispersion stability diagram of the nano supramolecular drug-loaded liposome prepared by the present invention; Figure 4 This is a diagram showing the stability of the encapsulation efficiency of the nano-supramolecular drug-loaded liposomes prepared by the present invention; Figure 5 This is the infrared spectrum of the ternary supramolecular containing VC prepared by the present invention; Figure 6 This is a comparison chart of the cumulative permeability test results of Example 1, Comparative Example 1 and Comparative Example 2; Figure 7 This is a graph showing the cumulative retention test results of Example 1, Comparative Example 1 and Comparative Example 2. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto: The low eutectic solvents involved in the present invention are shown in Table 1 below.

[0036] Table 1

[0037] Taking betaine glycerol supramolecular solvent as an example, its preparation method is: betaine and glycerol are mixed in a molar ratio of 1:2, and then heated in an oil bath at 80-100°C and continuously stirred until a transparent liquid is formed (stirring time is about 3 hours) to prepare the betaine glycerol supramolecular solvent.

[0038] The remaining supramolecular solvents were prepared in a similar manner according to the formulations described in Table 1.

[0039] In the following cases, stirring speeds that are not clearly limited are all normal stirring speeds.

[0040] Example 1: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 5.00 g of VC (levorotatory VC) and add it to 40.00 g of betaine glycerol supramolecular solvent, and ultrasonicate at 70° C. until VC is completely dissolved to obtain a transparent solution A (a ternary supramolecular structure); Step 2, add 0.25 g of cholesterol to 4.00 g of caprylic capric triglyceride, heat and stir at 70°C until the cholesterol is dissolved to obtain solution B (transparent homogeneous phase); Step 3, under stirring conditions of 1500 rpm and 70° C., solution B is added dropwise (the dropping time is about 1-2 minutes) to solution A. After the dropping is completed, 10.00 g of soybean lecithin, 0.50 g of vitamin E acetate, and 0.10 g of EDTA-2Na are added, and stirring is continued at 70° C. and 1500 rpm until the soybean lecithin, vitamin E acetate, and EDTA-2Na are dispersed and mixed to obtain dispersion 1; Step 4, weigh 4.00 g of PEG-40 hydrogenated castor oil and add it to 36.15 g of deionized water at 70° C., keep the temperature at 70° C. and stir at 1500 rpm until the PEG-40 hydrogenated castor oil is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing (dropwise for about 5-8 minutes), the shearing speed is 4500 rpm, the shearing temperature is 70°C, and the shearing is continued for 15 minutes after the addition is completed (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 750 bar, the homogenization temperature is 25° C., and the number of cycles (number of homogenizations) is 7 times, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0041] Example 2: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 10.00 g of VC and add it to 40.00 g of betaine glycerol supramolecular solvent, and ultrasonicate at 75° C. until VC is completely dissolved to obtain a transparent solution A; Step 2, adding 0.35 g of cholesterol to 6.45 g of caprylic capric triglyceride, and ultrasonicating at 75° C. until the cholesterol is dissolved to obtain solution B; Step 3, under stirring conditions of 1500 rpm and 75° C., solution B was added dropwise to solution A. After the addition was completed, 15.00 g of soybean lecithin, 0.50 g of vitamin E acetate, and 0.10 g of EDTA-2Na were added, and stirring was continued at 75° C. and 1500 rpm until the soybean lecithin, vitamin E acetate, and EDTA-2Na were dispersed and mixed to obtain dispersion 1; Step 4, weigh 5.00 g of PEG-40 hydrogenated castor oil and add it to 22.60 g of deionized water at 75° C., keep the temperature at 75° C. and stir at 1500 rpm until the PEG-40 hydrogenated castor oil is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing, the shearing speed is 2000 rpm, the shearing temperature is 75°C, and after the addition is completed, the shearing is continued for 25 minutes (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 750 bar, the homogenization temperature is 35° C., and the number of cycles is 7, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0042] Example 3: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 4.00 g of VC and add it to 30.00 g of proline glycerol supramolecular solvent, and ultrasonicate at 65° C. until VC is completely dissolved to obtain a transparent solution A; Step 2, adding 0.20 g of cholesterol to 3.20 g of caprylic capric triglyceride, and ultrasonicating at 65° C. until the cholesterol is dissolved to obtain solution B; Step 3, under stirring conditions of 1200 rpm and 65° C., solution B was added dropwise to solution A. After the addition was completed, 8.00 g of soybean lecithin, 0.50 g of vitamin E acetate, and 0.05 g of EDTA-2Na were added, and stirring was continued at 65° C. and 1200 rpm until the soybean lecithin, vitamin E acetate, and EDTA-2Na were dispersed and mixed to obtain dispersion 1; Step 4, weigh 3.00 g of polysorbate-80 and add it to 51.05 g of deionized water at 65° C., keep the temperature at 65° C. and stir at 1200 rpm until the polysorbate-80 is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing, the shearing speed is 4200 rpm, the shearing temperature is 65°C, and the shearing is continued for 20 minutes after the addition is completed (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 700 bar, the homogenization temperature is 30° C., and the number of cycles is 6, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0043] Example 4: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 3.00 g of VC and add it to 30.00 g of choline chloride glycerol supramolecular solvent, and ultrasonicate at 55° C. until VC is completely dissolved to obtain a transparent solution A; Step 2, adding 0.15 g of cholesterol to 2.40 g of caprylic capric triglyceride, and ultrasonicating at 65° C. until the cholesterol is dissolved to obtain a transparent homogeneous phase B (solution B); Step 3, under the stirring condition of 1000rpm and 55°C, solution B was added dropwise to solution A. After the addition was completed, 7.00g of soybean lecithin, 0.75g of vitamin E, and 0.15g of EDTA-2Na were added, and stirring was continued at 55°C and 1000rpm until the soybean lecithin, vitamin E, and EDTA-2Na were dispersed and mixed to obtain dispersion 1; Step 4, weigh 2.50 g of polyglycerol-10 myristate and add it to 54.05 g of deionized water at 55° C., keep the temperature at 55° C. and stir at 1000 rpm until the polyglycerol-10 myristate is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing, the shearing speed is 3000 rpm, the shearing temperature is 65°C, and the shearing is continued for 20 minutes after the addition is completed (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 800 bar, the homogenization temperature is 15° C., and the number of cycles is 5, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0044] Example 5: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 10.00 g of VC and add it to 30.00 g of betaine 1,3-propylene glycol supramolecular solvent, and ultrasonicate at 75° C. until VC is completely dissolved to obtain a transparent solution A; Step 2, adding 0.25 g of cholesterol to 4.00 g of caprylic capric triglyceride, and ultrasonicating at 75° C. until the cholesterol is dissolved to obtain a transparent homogeneous phase B (solution B); Step 3, under the stirring condition of 1500rpm and 75°C, solution B was added dropwise to solution A. After the addition was completed, 12.00g of soybean lecithin, 0.50g of vitamin E, and 0.10g of EDTA-2Na were added, and stirring was continued at 75°C and 1500rpm until the soybean lecithin, vitamin E, and EDTA-2Na were dispersed and mixed to obtain dispersion 1; Step 4, weigh 8.0 g of PEG-40 hydrogenated castor oil, add it to 35.15 g of deionized water at 75° C., keep the temperature at 75° C. and stir at 1000 rpm until the PEG-40 hydrogenated castor oil is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing, the shearing speed is 4500 rpm, the shearing temperature is 75°C, and the shearing is continued for 25 minutes after the addition is completed (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 750 bar, the homogenization temperature is 25° C., and the number of cycles is 7, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0045] Example 6: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 1.50 g of VC and add it to a mixed supramolecular solvent consisting of 20.00 g of betaine glycerol and 6.00 g of betaine lactic acid, and ultrasonicate at 50° C. until VC is completely dissolved to obtain a transparent solution A; Step 2, adding 0.125 g of cholesterol to 2.00 g of caprylic capric triglyceride, and ultrasonicating at 50° C. until the cholesterol is dissolved to obtain a transparent homogeneous phase B (solution B); Step 3, adding solution B dropwise to solution A at 800 rpm stirring and 50° C., after the addition is complete, adding 5.50 g of soybean lecithin, 0.50 g of vitamin E, and 0.10 g of EDTA-2Na, and continuing to stir at 50° C. and 800 rpm until the soybean lecithin, vitamin E, and EDTA-2Na are dispersed and mixed to obtain dispersion 1; Step 4, weigh 2.0 g of polyglycerol-10 oleate, add it to 62.275 g of deionized water at 50° C., keep the temperature at 50° C. and stir at 800 rpm until the polyglycerol-10 oleate is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing, the shearing speed is 3000 rpm, the shearing temperature is 50°C, and the shearing is continued for 15 minutes after the addition is completed (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 750 bar, the homogenization temperature is 20° C., and the number of cycles is 6, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0046] Example 7: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 1.50 g of VC and add it to 40.00 g of betaine isosorbide supramolecular solvent, and ultrasonicate at 70° C. until VC is completely dissolved to obtain a transparent solution A; Step 2, adding 0.25 g of cholesterol to 4.00 g of caprylic capric triglyceride, and ultrasonicating at 70° C. until the cholesterol is dissolved to obtain a transparent homogeneous phase B (solution B); Step 3, adding solution B dropwise to solution A at 1400 rpm and 70° C., after the addition is complete, adding 10.0 g of soybean lecithin, 0.50 g of vitamin E acetate, and 0.10 g of EDTA-2Na, and continuing to stir at 70° C. and 1400 rpm until the soybean lecithin, vitamin E acetate, and EDTA-2Na are dispersed and mixed to obtain dispersion 1; Step 4, weigh 1.5 g of polyglycerol-10 laurate, add it to 42.15 g of deionized water at 70° C., keep the temperature at 70° C. and stir at 1400 rpm until the polyglycerol-10 laurate is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing, the shearing speed is 2500 rpm, the shearing temperature is 70°C, and the shearing is continued for 25 minutes after the addition is completed (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 700 bar, the homogenization temperature is 30° C., and the number of cycles is 7, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0047] Example 8: A method for preparing nano supramolecular drug-loaded liposomes, comprising the following steps: Step 1, weigh 17.00 g of VC and add it to 40.00 g of betaine glycerol supramolecular solvent, and ultrasonicate at 75° C. until VC is completely dissolved to obtain a transparent solution A; Step 2, add 0.35 g of cholesterol to 4.00 g of caprylic capric triglyceride, and sonicate at 75° C. until the cholesterol is dissolved to obtain a transparent homogeneous phase B (solution B); Step 3, adding solution B dropwise to solution A at 1500 rpm stirring and 75° C., after the addition is complete, adding 13.00 g of soybean lecithin, 0.50 g of vitamin E acetate, and 0.10 g of EDTA-2Na, and continuing to stir at 75° C. and 1500 rpm until the soybean lecithin, vitamin E acetate, and EDTA-2Na are dispersed and mixed to obtain dispersion 1; Step 4, weigh 5.00 g of polysorbate-80 and add it to 20.05 g of deionized water at 75° C., keep the temperature at 75° C. and stir at 1500 rpm until the polysorbate-80 is dispersed and mixed, to obtain dispersion 2; Step 5, slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing, the shearing speed is 5000 rpm, the shearing temperature is 75°C, and the shearing is continued for 15 minutes after the addition is completed (the shearing parameters are the same as above), to obtain a supramolecular liposome primary emulsion; Step 6: The supramolecular liposome primary emulsion obtained in step 5 is subjected to high-pressure homogenization, the homogenization pressure is 750 bar, the homogenization temperature is 40° C., and the number of cycles is 7, to obtain nano supramolecular drug-loaded liposomes containing VC.

[0048] Comparative Example 1, Preparation of VC aqueous solution: Prepare a VC aqueous solution with a mass concentration of 5%.

[0049] Comparative Example 2: Preparation of traditional VC liposomes: Preparation of oil phase: 10 g soybean lecithin, 40 g glycerol, 0.25 g cholesterol, 4.0 g caprylic capric triglyceride, 0.5 g vitamin E acetate, and 0.10 g EDTA-2Na were stirred in a water bath at 1400 rpm and 70°C until well mixed and set aside.

[0050] Preparation of aqueous phase: 5.0 g VC, 4.00 g PEG-40 hydrogenated castor oil, and 36.15 g deionized water were stirred in a water bath at 1400 rpm and 70° C. until mixed uniformly and set aside.

[0051] Shear mixing: Slowly add the oil phase into the water phase under shear conditions, the shear speed is 4000 rpm, and the shear time is 10 min to obtain VC liposome colostrum.

[0052] High-pressure homogenization: The VC liposome colostrum was subjected to high-pressure homogenization, with a homogenization pressure of 750 bar, a homogenization temperature of 25°C, and 7 cycles to obtain traditional VC liposomes.

[0053] Experiment 1: Appearance stability test: The nano-supramolecular drug-loaded liposomes prepared in Example 1 were placed at room temperature for 2 months and the appearance changes were recorded. The experimental results are as follows: Figure 1 As shown, since lecithin itself presents a certain yellow color, the nano supramolecular drug-loaded liposomes will present a light yellow to yellow state at the beginning of preparation. During the storage for 2 months, it can be found that the appearance of the nano supramolecular drug-loaded liposomes has no obvious deepening, stratification, or agglomeration, and still maintains a good light-transmitting state; the carrier has not changed to the brown or brownish yellow direction, proving that most of the VC has not undergone oxidative degradation. The formation of the ternary supramolecular structure makes VC a part of the low eutectic solvent, which, on the one hand, realizes the physical modification of VC, and secondly increases the stability of VC molecules. Therefore, the nano supramolecular drug-loaded liposomes prepared by the method of the present invention have good appearance stability.

[0054] Note: In Examples 2 to 8, the appearance did not show obvious deepening and stratification, and still maintained a good light transmittance state, which indicates that the nano-supramolecular drug-loaded liposomes prepared by the present invention have good stability.

[0055] Experiment 2: The particle size, dispersibility and encapsulation efficiency of the nano-supramolecular drug-loaded liposomes prepared in Examples 1 to 8 were tested: Take 1.00g of nano supramolecular drug-loaded liposomes, add 9.00g of deionized water, and disperse evenly. Then, the particle size and dispersibility are measured by a British Malvern laser particle size scattering instrument as shown in Table 2 below; the encapsulation efficiency is measured by ultrafiltration centrifugation as shown in Table 2 below.

[0056] Note: For ultrafiltration centrifugation method, please refer to the following references: Yang S, Liu W, Liu C, et al. Characterization and bioavailability ofvitamin C nanoliposomes prepared by film evaporation-dynamic high pressure microfluidization[J]. Journal of dispersion science and technology, 2012, 33(11): 1608-1614. Encapsulation efficiency = (W 总 -W 游离 ) / W 总 ; Load = (W 总 -W 游离 ) / (WW 游离 ); W总 represents the quality of VC joining, W 游离 represents the mass of unencapsulated VC, and W represents the total mass of supramolecular drug-loaded liposomes.

[0057] Table 2. Particle size, dispersibility and encapsulation efficiency of nano supramolecular drug-loaded liposomes prepared by the present invention

[0058] As shown in Table 2, the supramolecular nano drug-loaded liposomes prepared in Examples 1 to 8 have a relatively small particle size, most of which are below 50 nm, and are relatively evenly dispersed, with an encapsulation rate generally above 80%, which is higher than the encapsulation rate of about 50% reported in the literature. This is mainly because VC and the low eutectic solvent form a ternary supramolecular structure under ultrasound or heating conditions, which allows VC molecules to simultaneously occupy the internal water phase layer of the liposome and the oil phase layer between the double-layer membrane, so that the supramolecular nano drug-loaded liposomes can achieve efficient encapsulation of VC.

[0059] Experiment 3, stability test: The nano-supramolecular drug-loaded liposomes prepared in Example 1 were sealed in a transparent glass bottle and placed at 25° C. and 37° C. for 2 months to track the particle size stability, dispersion stability and encapsulation efficiency stability. The experimental results are shown in FIG. Figures 2~4 As shown, the particle size, dispersibility and encapsulation efficiency of the samples maintained good stability for up to 2 months, whether stored at room temperature or at high temperature. This is due to the huge and complex hydrogen bond network of the supramolecular solvent, which can weaken the system collapse caused by Ostwald ripening during long-term storage of traditional liposomes, especially reduce the acceleration effect of high temperature on Ostwald rate, so the supramolecular nano drug-loaded liposomes prepared by the present invention have good thermodynamic stability.

[0060] Experiment 4: Infrared proof of supramolecular VC structure: First, a betaine glycerol supramolecular solvent (denoted as Bet-Gly low eutectic solvent, Bet is the abbreviation of betaine, and Gly represents glycerol) is used, and then a certain amount of VC is added to the Bet-Gly low eutectic solvent and continued to heat and stir until a clear, transparent, colorless solution is formed, which is a ternary supramolecular solvent containing VC (denoted as Bet-Gly-VC). Specifically, 5.0 g of VC is added to 40 g of Bet-Glycerol low eutectic solvent, wherein Bet:Glycerol=1:2 (molar ratio).

[0061] Fourier transform infrared spectroscopy was performed on betaine, glycerol, Bet-Gly low eutectic solvent, Bet-Gly-VC ternary supramolecular solvent and pure VC. The experimental results are as follows Figure 5As shown, the purple line at the bottom represents the infrared absorption spectrum of betaine, the red line at the top represents the infrared spectrum of glycerol, the green line is the infrared absorption peak of the sweet Bet-Gly low eutectic solvent, the yellow line represents the infrared absorption peak of the ternary supramolecular structure containing VC, and the top blue line represents the infrared absorption peak of free VC. Because the VC molecule is in a solid state, several hydroxyl peaks are not significantly affected by hydrogen bonds and appear to be separated to a certain extent. From the infrared spectrum of the supramolecular system formed by betaine-glycerol, due to the formation of a complex hydrogen bond network, the hydroxyl stretching vibration of the hydrogen bond donor undergoes an obvious red shift, from the 3374cm -1 Red shifted to 3347 cm in Bet-Gly -1 With the addition of VC, the stretching vibration of the hydroxyl group further red-shifts, forming a more complex ternary supramolecular structure containing VC. The formation of hydrogen bonds will lead to the average density of the electron cloud, thereby reducing the force constant between the hydrogen and oxygen bonds, resulting in a decrease in the energy absorbed by the vibrational-rotational spectrum, thus causing a red-shift phenomenon. The formation of the ternary supramolecular structure can, on the one hand, achieve a solubilization effect on the internal dissolved substances, and secondly, the formation of a complex hydrogen bond network can incorporate VC into the entire supramolecular system, which increases the stability of VC to a certain extent.

[0062] Experiment 5: In vitro transdermal absorption experiment: In this experiment, the abdominal skin of SD rats was used as the material for in vitro transdermal absorption. After the rats were anesthetized and killed, the abdominal skin was depilated with a depilatory cream. The abdominal skin was then removed, subcutaneous fat was removed, and the skin was placed in physiological saline for later use.

[0063] A vertical Franz diffusion device was used, with an effective volume of 6.4 mL and an effective penetration area of ​​2.2 m 2, cut the abdominal skin of SD rats of appropriate size and fix it between the supply tank and the receiving tank, and the receiving tank is filled with PBS buffer with pH=7.4. The stratum corneum faces the supply tank, and the dermis layer faces the receiving tank to ensure that the dermis layer and the liquid surface of the receiving liquid are tightly connected, and no obvious bubbles appear. Take 5.0g of each of the nano-supramolecular drug-loaded liposomes obtained in Example 1, Comparative Example 1, and Comparative Example 2, add them to a 100mL volumetric flask, and dilute to 100mL with deionized water to obtain different sample solutions with a VC concentration of 2.5mg / mL. Take 1.0mL of different sample solutions respectively and place them on the supply tank, and the receiving tank is continuously stirred at 37.0±0.5℃ and 300rmp. 1.0 mL of the receiving solution was taken at 1, 2, 3, 4, 5, 6, 7, 10, 24, and 48 h after the start of the experiment. 1.0 mL of pH 7.4 PBS buffer was added at the same time. The cumulative permeation of the samples was analyzed according to the method in Journal of dispersion science and technology, 2012, 33(11):1608-1614.

[0064] After the experiment was carried out for 48 hours, the mouse skin was removed and repeatedly rinsed with deionized water. The lipids completely removed the sample solution on the skin surface. The skin at the transdermal site was cut into pieces, and 3 mL of 75% ethanol aqueous solution was added. After ultrasonication for 30 minutes, the mixture was centrifuged at 8000 r / min for 5 minutes, and the supernatant was collected. 2 mL of 75% ethanol aqueous solution was added to the precipitate, and the above operation was repeated. The supernatants were combined twice. The retention amount of the sample in the mouse skin was analyzed by referring to the method in Journal of dispersion science and technology, 2012, 33(11): 1608-1614.

[0065] The experimental results are as follows Figure 6 and Figure 7 As shown: as time goes by, the cumulative permeation amount of Example 1, Comparative Example 1, and Comparative Example 2 all increase significantly, but the cumulative permeation amount of Example 1 is significantly higher than that of Comparative Example 1 and Comparative Example 2. Therefore, it is shown that the transdermal absorption effect of VC in nano supramolecular drug-loaded liposomes is better than that of traditional liposomes and free VC samples (VC aqueous solution). By analyzing the cumulative retention amount of VC in SD rat skin, it is found that the retention amount of VC in Example 1 in rat skin is 2.40 times and 1.38 times that of Comparative Example 1 and Comparative Example 2, respectively, indicating that the delivery effect of supramolecular nano drug-loaded liposomes on VC is better than that of traditional liposomes and unencapsulated VC samples.

[0066] In summary, it can be seen that the present invention achieves a highly efficient delivery effect of active substances; and the delivery effect of nano-supramolecular drug-loaded liposomes is better than that of traditional liposomes.

[0067] Comparative Example 3-1: The amount of the "betaine glycerol supramolecular solvent" in Example 1 was changed from 40 g to 15 g, and the amount of deionized water was increased accordingly, that is, the total amount of the raw materials was ensured to be still 100 g, and the rest was the same as Example 1.

[0068] Comparative Example 3-2: The amount of the "betaine glycerol supramolecular solvent" in Example 1 was changed from 40 g to 50 g, and the amount of deionized water was reduced accordingly, that is, the total amount of the raw materials was ensured to be still 100 g, and the rest was the same as Example 1.

[0069] Comparative Example 4: Steps 1 to 5 of Example 1 are omitted, all the raw materials described in Example 1 are simply mixed, and then the high-pressure homogenization of Step 6 of Example 1 is directly performed.

[0070] The above-mentioned comparative examples 3-1, 3-2 and 4 were tested according to the above-mentioned experiments 2 and 5, and the obtained results were compared with those of Example 1 as shown in Table 3 below.

[0071] Table 3. Comparison of particle size, dispersibility and encapsulation efficiency

[0072] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Nano supramolecular drug-loaded liposomes, characterized in that The raw materials of nano supramolecular drug-loaded liposomes are composed of the following components by weight: Active ingredient: 1-20%, Lecithin: 5-15%, Deep eutectic solvent: 20-40%, Grease: 0.1-10%, Cholesterol: 0.1-0.5%, Emulsifier: 0.1-5%, Stabilizer: 0.1-1.5%, The balance is deionized water; The active ingredient is water-soluble VC.

2. The nano supramolecular drug-loaded liposome according to claim 1, characterized in that The lecithin is at least any one of the following: soybean lecithin, hydrogenated lecithin, egg yolk lecithin, lysolecithin, phosphatidylethanolamine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and dipalmitoylphosphatidylglycerol.

3. The nano-supramolecular drug-loaded liposome according to claim 2, characterized in that: The deep eutectic solvent is a supramolecular solvent; The supramolecular solvent is at least any one of the following: Betaine glycerol supramolecular solvent, betaine propylene glycol supramolecular solvent, betaine butylene glycol supramolecular solvent, betaine isosorbide supramolecular solvent, betaine citric acid supramolecular solvent, choline chloride propylene glycol supramolecular solvent, choline chloride urea supramolecular solvent, choline chloride malic acid supramolecular solvent, choline chloride glucose supramolecular solvent, choline chloride glycerol supramolecular solvent, proline glycerol supramolecular solvent.

4. The nano supramolecular drug-loaded liposome according to claim 3, characterized in that The oil is at least any one of the following: caprylic capric triglyceride, isopropyl myristate, olive oil, camellia oil, jojoba oil, and meadowfoam seed oil.

5. The nano-supramolecular drug-loaded liposome according to claim 4, characterized in that: The emulsifier is at least any one of the following: PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, polyglyceryl-3 cocoate, polyglyceryl-10 laurate, polyglyceryl-10 oleate, polyglyceryl-10 myristate, polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-80, sodium stearoyl glutamate; The stabilizer is at least any one of the following: EDTA-2Na, vitamin E acetate, vitamin E, sodium pyrosulfite, diethylhexyl syringylidenemalonate.

6. The nano supramolecular drug-loaded liposome according to claim 1, characterized in that The raw materials of nano supramolecular drug-loaded liposomes are composed of the following components by weight: 5% water-soluble VC, 40% betaine glycerol supramolecular solvent, 0.25% cholesterol, 4% caprylic capric triglyceride, 10% soy lecithin, 0.5% Vitamin E acetate, 0.1% EDTA-2Na, 4% PEG-40 hydrogenated castor oil, 36.15% deionized water.

7. The method for preparing the nano supramolecular drug-loaded liposome according to any one of claims 1 to 6, characterized in that The following steps are involved: Step 1: Add the active ingredient to a low eutectic solvent, and ultrasonicate or stir at 45-75°C until the active ingredient is dissolved to obtain solution A; Step 2: Add cholesterol to the oil and ultrasonicate at 45-75°C until the cholesterol is dissolved to obtain solution B; Step 3: under the condition of 45-75° C. and stirring, add the solution B obtained in step 2 dropwise to the solution A obtained in step 1, and then add lecithin and stabilizer, and stir while keeping warm until the lecithin and stabilizer are dispersed to obtain dispersion 1; Step 4: Add the emulsifier into deionized water, stir at 45-75°C until the emulsifier is dispersed, and obtain dispersion 2; Step 5: slowly adding the dispersion 1 obtained in step 3 to the dispersion 2 obtained in step 4 under shearing conditions, the shearing speed is 2000-5000 rpm, the shearing temperature is 45-75° C., and the shearing is continued for 5-30 minutes after the addition is completed to obtain a supramolecular liposome primary emulsion; Step 6: subjecting the supramolecular liposome primary emulsion obtained in step 5 to high pressure homogenization or high pressure microfluidization to obtain nano supramolecular drug-loaded liposomes.

8. The method for preparing the nano supramolecular drug-loaded liposome according to claim 7, characterized in that: The pressure of the high-pressure homogenization in step 6 is 500-800 bar, the number of high-pressure homogenization is 3-8 times, and the temperature of the high-pressure homogenization is 15-45°C.

9. The method for preparing nano supramolecular drug-loaded liposomes according to claim 7 or 8, characterized in that: The stirring speeds in step 3 and step 4 are both 500-1500 rpm.

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