A high drug loading tetrandrine liposome, a preparation method and application thereof

Abstract

The application discloses a high-drug-loading tetrandrine liposome as well as a preparation method and application thereof. The liposome comprises the following components in parts by weight: tetrandrine 1.5-2.5 parts, a pH regulator 0.6-1 part, lecithin 3-5 parts, cholesterol 0.1-0.3 parts, a cosolvent 3-6 parts, an antioxidant 0.5-1 part, oil 6-10 parts, water 25-30 parts and a polyhydric alcohol 35-55 parts. The drug loading of the tetrandrine liposome prepared by the application can be as high as 1.5-2.5 %, which is significantly higher than that of the tetrandrine liposome prepared by the prior art. The tetrandrine liposome has the following test results: a clear appearance, good stability, a raw solution particle size of 100-400 nm, a raw solution PDI (polydispersity index) of 0.5 or less and an encapsulation rate of 80 % or more, and meets the requirements for cosmetic use. Moreover, the tetrandrine liposome is proved to be completely applicable to cosmetics through tests on irritation, hyaluronidase inhibition effect, transdermal absorption effect and inflammatory factor secretion inhibition.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a high drug-loading tetrandrine liposome, its preparation method and application, and particularly to a high drug-loading liposome containing tetrandrine, its preparation method, and the application of the tetrandrine-containing nanoliposome in cosmetics. Background Technology

[0002] Fangji is the dried root of *Stephania tetrandra* S. Moore, a plant in the Menispermaceae family. It possesses properties that dispel wind and relieve pain, promote diuresis and reduce swelling, and is used for rheumatic pain, edema, beriberi, dysuria, eczema, and sores. The main active ingredients of *Stephania tetrandra* are alkaloids, with tetrandrine and fangchinorline being the most abundant. In the cosmetics field, tetrandrine primarily functions as an anti-allergic, anti-inflammatory, analgesic, anti-swelling, and anti-redness / itch-relieving agent, mainly used for the repair of various sensitive skin types.

[0003] Tetrandrine is poorly soluble in water and has low bioavailability, which greatly limits its application in cosmetics. Currently, the content of tetrandrine in various commercially available cosmetic ingredients is low. When added to cosmetics as an active ingredient, the tetrandrine content is approximately 10-100 ppm, which affects its efficacy.

[0004] Liposomes are spherical, closed vesicles with an internal aqueous phase, formed by phospholipids dispersed in water. Due to their phospholipid bilayer structure, similar to that of cell membranes, liposomes have become an important carrier for improving the solubility and bioavailability of tetrandrine. For example, Zhao Xiaodan [Zhao Xiaodan. Preparation of tetrandrine nanoliposome carriers [J]. China Health Care and Nutrition, 2012, (11): 4686-4687] prepared tetrandrine solid lipid nanoparticles (drug loading of 0.45%, encapsulation rate of 70%-90%) using emulsification-ultrasonic dispersion method; Hu Chunmei [Hu Chunmei, Zhu Li, Zhao Junyi, et al. Preparation and stability study of tetrandrine liposomes [J]. China Pharmacy, 2008, 19(13): 995-997] prepared tetrandrine liposomes using ammonium sulfate gradient method (using chloroform as a second solvent in the process, drug loading of 0.16%, encapsulation rate of 80.4%-81.6%); Chen Hongxuan [Chen Hongxuan, Wang Yanli. Determination of drug content and encapsulation rate of tetrandrine liposomes [J]. Journal of Henan University: Medical Edition, 200 7,26(4):38-40】Preparation of tetrandrine liposomes by thin film dispersion-ultrasound method (the process uses anhydrous ethanol as organic solvent, and the encapsulation rate is 69.2%-70.5%); Hu Jieru [Hu Jieru, Fu Junxia, ​​Zhu Yayan, et al. Experimental study on the prevention of diabetic cardiomyopathy by activation of Nrf2 / HO-1 signaling channel by novel tetrandrine nanoliposomes [J]. Chinese Pharmacist, 2021, 24(4):664-669]Preparation of tetrandrine nanoliposomes by thin film dispersion combined with ultrasonic hydration technology (the process uses anhydrous ethanol as organic solvent, the drug loading is 0.1%, and the encapsulation rate is (87.23±2.21)%); Fan Chao [Fan Chao, Li Xinru, Zhou Yanxia, ​​et al. Preparation, characterization and anti-arthritis efficacy of tetrandrine nanoliposomes [J]. Journal of Chinese Pharmaceutical Sciences, 2012, 21(4):311-320.】The pH gradient method was used to prepare tetrandrine liposomes (drug loading of 0.15%, encapsulation rate of (52.87±3.81)%); Li Su [Li Su. Study on tetrandrine lipid nanoparticles [D]. Shenyang Pharmaceutical University, 2011] prepared tetrandrine solid lipid nanoparticles (drug loading of 0.54%, encapsulation rate of (89.01±1.61)%) and tetrandrine nanostructure lipid carriers (drug loading of 0.54%, encapsulation rate of (86.48±1.13)%) by emulsification and ultrasonic dispersion method. At present, there are three problems with tetrandrine liposomes:

[0005] (1) The preparation process uses toxic solvents such as chloroform, which is not suitable for use in the cosmetics field.

[0006] (2) The current liposome preparation methods are more suitable for laboratory use than for industrial-scale preparation. The reasons are as follows: a. Thin-film dispersion involves dissolving the drug in an organic solvent, removing the solvent under reduced pressure, allowing the lipids to form a thin film on the container wall, and then adding a buffer solution containing water-soluble drugs. After thorough shaking, liposomes are obtained. However, this method requires a large amount of organic solvent, is time-consuming, has a low encapsulation rate, and results in a large particle size. Furthermore, industrially prepared liposomes are subject to significant batch-to-batch variations due to the influence of factors such as negative pressure, temperature, and solvent volume. b. Ultrasonic dispersion: Ultrasonic treatment is one of the earliest mechanical methods used to treat amphiphilic lipids. One method involves inserting the probe of an ultrasonic instrument into the liposome dispersion; another involves placing the sample in a test tube or beaker and then placing it in a water bath ultrasonic instrument. This method is difficult to implement in industrial production, and the ultrasonic intensity received at different locations within the equipment container varies greatly. Therefore, the preparation of liposomes results in large differences in particle size and poor process repeatability; c. Chemical gradient method: This method utilizes the characteristic that amphiphilic drugs are lipophilic in the non-ionic state and can cross the lipid bilayer, but are hydrophilic in the ionized form and cannot cross the lipid bilayer to achieve drug encapsulation. It mainly includes pH gradient method, ammonium sulfate gradient method and calcium acetate gradient method; This method can solve the problem of industrial production to a certain extent; However, it requires the introduction of organic reagents and ionic reagents, which can easily lead to low encapsulation rate and high ionic strength of the product, especially affecting the preparation of cosmetic creams, lotions and other dosage forms.

[0007] (3) The drug loading of tetrandrine in the current preparation process is low, all less than 0.6%.

[0008] Therefore, it is necessary to conduct research to address the problems existing in the prior art, in order to provide a high drug-loading liposome containing tetrandrine and its preparation method, and to apply the tetrandrine-containing liposome to cosmetics, so as to broaden the application field of tetrandrine liposomes. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the purpose of this invention is to provide a high-drug-loading tetrandrine liposome, its preparation method, and its applications.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention provides a high-drug-loading tetrandrine liposome, comprising the following components in parts by weight:

[0012]

[0013] Preferably, the pH adjuster is selected from at least one of lactic acid, citric acid, tartaric acid, and succinic acid.

[0014] More preferably, the pH adjuster is lactic acid.

[0015] Preferably, the mass ratio of the tetrandrine to the pH adjuster is 1:0.4.

[0016] Preferably, the lecithin is soybean lecithin; the soybean lecithin is soybean phospholipid CS-95.

[0017] Preferably, the co-solvent is selected from at least one of PEG-40 hydrogenated castor oil, PPG-13-decyltetradecyl alcohol polyether-24, and polyoxyethylene sorbitan monooleate.

[0018] More preferably, the cosolvent is PEG-40 hydrogenated castor oil.

[0019] Preferably, when the content of tetrandrine is 2-2.5%, the mass ratio of lecithin to cosolvent is 1:1.

[0020] Preferably, the antioxidant is selected from at least one of tocopherol, tocopheryl acetate, ascorbate stearate, ascorbate tetraisopalmitate, tetrahexyldecyl ascorbate, and di-tert-butyl-p-cresol.

[0021] Preferably, the oil is selected from at least one of caprylic / capric triglyceride, glyceryl monostearate, glyceryl behenate, and glyceryl palmitate stearate.

[0022] Preferably, the polyol is selected from at least one of glycerol, propylene glycol, butylene glycol, dipropylene glycol, and PEG400.

[0023] Preferably, the high-drug-loading tetrandrine liposomes also contain a preservative; the preservative is present in 1 to 3.5 parts by weight.

[0024] Preferably, the preservative is selected from at least one of 1,2-pentanediol, 1,2-hexanediol, p-hydroxyacetophenone, and phenoxyethanol.

[0025] Secondly, this invention provides a method for preparing tetrandrine liposomes with high drug loading capacity, comprising the following steps:

[0026] A1. Lecithin, solubilizer, antioxidant, preservative, oil, cholesterol, and some polyol are stirred in a water bath at 70-75℃ until completely dissolved to obtain the oil phase;

[0027] A2. After sterilizing the water, add glycerin and stir well; cool to below 50℃ to obtain the aqueous phase;

[0028] A3. Add tetrandrine and pH adjuster to the oil phase, stir until completely dissolved, and then start cooling to ≤50℃;

[0029] A4. Slowly add the mixture obtained after step A3 into the aqueous phase, stir and mix evenly to form a transparent liquid;

[0030] A5. The transparent liquid is homogenized under high pressure to obtain tetrandrine liposomes.

[0031] Secondly, the present invention provides an application of the aforementioned high drug-loading tetrandrine liposomes in the preparation of cosmetics.

[0032] Fourthly, the present invention provides the use of the aforementioned high-drug-loading tetrandrine liposomes in the preparation of soothing compositions.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The drug loading capacity of the tetrandrine liposomes prepared by the present invention can be as high as 1.5-2.5%, which is significantly higher than that of tetrandrine liposomes prepared by existing methods.

[0035] 2. The tetrandrine liposomes prepared by this invention also have the following test results: clear appearance, good stability, particle size of the original solution in the range of 100-400 nm, PDI of the original solution ≤0.5 (more preferably PDI ≤0.3), and encapsulation rate ≥80% (more preferably encapsulation rate ≥90%), which meet the requirements for use in cosmetics.

[0036] 3. After undergoing irritation tests, hyaluronidase inhibition tests, transdermal absorption tests, and tests on the inhibition of RAW264.7 cell inflammatory factor secretion, the tetrandrine liposomes prepared by this invention have been proven to be fully applicable in cosmetics, thus broadening the application field of tetrandrine liposomes. Attached Figure Description

[0037] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0038] Figure 1 The transdermal absorption effect of the tetrandrine liposomes prepared in Example 1 is shown; where "*" indicates that compared with the tetrandrine extract solution (2.0%), *P<0.05;

[0039] Figure 2 The results show the effect of the tetrandrine liposomes prepared in Example 1 on the activity of RAW264.7 cells; where "###" indicates that compared with the Control group, P<0.001; "*" indicates that compared with the LPS control group, P<0.05; "**" indicates that compared with the LPS control group, P<0.01; and "***" indicates that compared with the LPS control group, P<0.001. Detailed Implementation

[0040] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0041] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0042] It should be understood that, except in any operational instance or otherwise indicated, all figures representing the amounts of ingredients used, for example, in the specification and claims, should be understood to be modified in all cases by the term "about". Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximations varying with the desired performance to be obtained according to the invention. It is not at all an attempt to limit the application of the doctrine of equivalents to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0043] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​listed in the specific examples are reported as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in their respective test measurements.

[0044] In the following examples, all raw materials used were commercially available. The specific lecithin and preservatives used are described below:

[0045] Lecithin 1 is soybean lecithin CS-95, purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.

[0046] Lecithin 2 is soybean lecithin purchased from Senrui Company;

[0047] Preservative 1 is a mixture of 1,2-pentanediol and 1,2-hexanediol, with a mixing ratio of 3:1;

[0048] Preservative 2 is a mixture of p-hydroxyacetophenone and 1,2-hexanediol in a 1:1 ratio.

[0049] Example 1

[0050] This embodiment provides a liposome with high drug loading capacity of tetrandrine, the composition and weight of each raw material are shown in Table 1.

[0051] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0052] 1) Dissolve lecithin 1, PPG-13-decyltetradecyl alcohol polyether-24, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0053] 2) Sterilize purified water by heating at 80-90℃ for 15 minutes, then add glycerol and stir well; cool to below 50℃ to obtain the aqueous phase;

[0054] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0055] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0056] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0057] Example 2

[0058] This embodiment provides a liposome with high drug loading capacity of tetrandrine, the composition and weight of each raw material are shown in Table 1.

[0059] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0060] 1) Dissolve lecithin 1, PPG-13-decyltetradecyl alcohol polyether-24, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0061] 2) Sterilize purified water by heating at 80-90℃ for 15 minutes, then add glycerol and stir well; cool to below 50℃ to obtain the aqueous phase;

[0062] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0063] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0064] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0065] Example 3

[0066] This embodiment provides a liposome with high drug loading capacity of tetrandrine, the composition and weight of each raw material are shown in Table 1.

[0067] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0068] 1) Dissolve lecithin 1, PPG-13-decyltetradecyl alcohol polyether-24, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0069] 2) Sterilize purified water by heating at 80-90℃ for 15 minutes, then add glycerol and stir well; cool to below 50℃ to obtain the aqueous phase;

[0070] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0071] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0072] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0073] Example 4

[0074] This embodiment provides a high drug loading liposome of tetrandrine, the raw material components and their weight parts are shown in Table 1.

[0075] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0076] 1) Dissolve lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0077] 2) Sterilize purified water by heating at 80-90℃ for 15 minutes, then add glycerol and stir well; cool to below 50℃ to obtain the aqueous phase;

[0078] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0079] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0080] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0081] Example 5

[0082] This embodiment provides a high drug loading liposome of tetrandrine, the raw material components and their weight parts are shown in Table 1.

[0083] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0084] 1) Dissolve lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0085] 2) Sterilize purified water by heating at 80-90℃ for 15 minutes, then add glycerol and stir well; cool to below 50℃ to obtain the aqueous phase;

[0086] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0087] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0088] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0089] Example 6

[0090] This embodiment provides a high drug loading liposome of tetrandrine, the raw material components and their weight parts are shown in Table 1.

[0091] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0092] 1) Dissolve lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0093] 2) Sterilize purified water by heating at 80-90℃ for 15 minutes, then add glycerol and stir well; cool to below 50℃ to obtain the aqueous phase;

[0094] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0095] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0096] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0097] Example 7

[0098] This embodiment provides a high drug loading liposome of tetrandrine, the raw material components and their weight parts are shown in Table 1.

[0099] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0100] 1) Dissolve lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0101] 2) Sterilize purified water by heating at 80-90℃ for 15 minutes, then add glycerol and stir well; cool to below 50℃ to obtain the aqueous phase;

[0102] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0103] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0104] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0105] Table 1

[0106]

[0107]

[0108] Comparative Example 1

[0109] This comparative example provides a liposome with a high drug loading capacity of tetrandrine, the components and weight proportions of which are shown in Table 2. The only difference from Example 4 is that lecithin 1 is replaced with lecithin 2.

[0110] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0111] 1) Dissolve 2 lecithin, PEG-40 hydrogenated castor oil, tocopheryl acetate, 1 preservative, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0112] 2) Sterilize purified water by heating it to 80-90℃ for 15 minutes, then add glycerol and stir well; cool it down to below 50℃ to obtain the aqueous phase;

[0113] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0114] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0115] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0116] Comparative Example 2

[0117] This comparative example provides a liposome with a high drug loading capacity of tetrandrine, the components and weight proportions of which are shown in Table 2. The only difference from Example 4 is that lactic acid is not added.

[0118] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0119] 1) Dissolve 2 lecithin, PEG-40 hydrogenated castor oil, tocopheryl acetate, 1 preservative, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0120] 2) Sterilize purified water by heating it to 80-90℃ for 15 minutes, then add glycerol and stir well; cool it down to below 50℃ to obtain the aqueous phase;

[0121] 3) Add tetrandrine to the oil phase and stir until completely dissolved for ≤10 min;

[0122] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0123] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0124] Comparative Example 3

[0125] This comparative example provides a liposome with a high drug loading capacity of tetrandrine, the components and weight proportions of which are shown in Table 2. The only difference from Example 4 is the weight proportion of lactic acid added.

[0126] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0127] 1) Dissolve 2 lecithin, PEG-40 hydrogenated castor oil, tocopheryl acetate, 1 preservative, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0128] 2) Sterilize purified water by heating it to 80-90℃ for 15 minutes, then add glycerol and stir well; cool it down to below 50℃ to obtain the aqueous phase;

[0129] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0130] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0131] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0132] Comparative Example 4

[0133] This comparative example provides a liposome with a high drug loading capacity of tetrandrine, the components and weight proportions of which are shown in Table 2. The only difference from Example 4 is the weight proportion of lactic acid added.

[0134] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0135] 1) Dissolve 2 lecithin, PEG-40 hydrogenated castor oil, tocopheryl acetate, 1 preservative, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0136] 2) Sterilize purified water by heating it to 80-90℃ for 15 minutes, then add glycerol and stir well; cool it down to below 50℃ to obtain the aqueous phase;

[0137] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0138] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0139] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0140] Comparative Example 5

[0141] This comparative example provides a liposome with a high drug loading capacity of tetrandrine, the components and weight proportions of which are shown in Table 2. The only difference from Example 5 is the weight proportion of PEG-40 hydrogenated castor oil added.

[0142] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0143] 1) Dissolve 2 lecithin, PEG-40 hydrogenated castor oil, tocopheryl acetate, 1 preservative, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0144] 2) Sterilize purified water by heating it to 80-90℃ for 15 minutes, then add glycerol and stir well; cool it down to below 50℃ to obtain the aqueous phase;

[0145] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0146] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0147] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0148] Comparative Example 6

[0149] This comparative example provides a liposome with a high drug loading capacity of tetrandrine, the components and weight proportions of which are shown in Table 2. The only difference from Example 5 is the weight proportion of PEG-40 hydrogenated castor oil added.

[0150] The preparation steps of the high drug-loading powder tetrandrine liposomes are as follows:

[0151] 1) Dissolve 2 lecithin, PEG-40 hydrogenated castor oil, tocopheryl acetate, 1 preservative, caprylic / capric triglyceride, cholesterol, and propylene glycol in a water bath at 70-75°C until completely dissolved to obtain the oil phase;

[0152] 2) Sterilize purified water by heating it to 80-90℃ for 15 minutes, then add glycerol and stir well; cool it down to below 50℃ to obtain the aqueous phase;

[0153] 3) Add tetrandrine and lactic acid together to the oil phase, stir until completely dissolved, and stir for ≤10 min;

[0154] 4) Add the aqueous phase to the mixture after step 3, stir for about 10 minutes, and mix to form a transparent liquid;

[0155] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, at 500 bar once, and at 800 bar three times, to obtain tetrandrine liposomes with high drug loading.

[0156] Table 2

[0157]

[0158] Performance testing:

[0159] The tetrandrine liposomes prepared in Examples 1-7 and Comparative Examples 1-6 were tested for appearance, pH value, particle size, PDI, zeta potential, stability, and encapsulation efficiency. The pH value was measured using a Leici pH meter, and the particle size and zeta potential were measured using an NS-90Z nanoparticle size and potential analyzer.

[0160] The specific method for stability testing is as follows: Place the sample of the example in a constant temperature chamber at 4℃ and a light chamber at 45℃ and leave it for 3 months. Observe whether the properties of the sample change. If there is no change under the above conditions, the stability is recorded as "stable"; if there is a change, record the state of the change.

[0161] The specific test method for encapsulation rate is as follows:

[0162] (1) Method for determining total content

[0163] Accurately measure 1.0 mL of tetrandrine liposomes and place them in a 20 mL volumetric flask. Add an appropriate amount of methanol and sonicate for 5 min to completely destroy the liposomes. Then dilute to the mark with methanol, shake well, and filter through a 0.45 μm microporous membrane. Use the filtrate as the test solution. Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph. Record the chromatogram and measure the peak area of ​​tetrandrine. Calculate the total content based on the peak area using the external standard method.

[0164] (2) Encapsulation efficiency determination method

[0165] Accurately measure 0.5 g of tetrandrine liposomes and place them in a centrifuge ultrafiltration tube. Centrifuge at 4°C and 7000 rpm. -1Centrifuge for 30 min. Transfer the filtrate to a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, and mix well. Filter through a 0.45 μm microporous membrane. Inject 20 μL into the liquid chromatograph, record the chromatogram, and measure the peak area of ​​tetrandrine. Calculate the free drug content in the supernatant using the external standard method based on the peak area.

[0166] The encapsulation efficiency of tetrandrine liposomes was calculated using the formula: (Total content - Free content in supernatant) / Total content × 100%.

[0167] Furthermore, the drug loading refers to the percentage of tetrandrine added when preparing tetrandrine liposomes. For example, in Example 1, if 2 parts by weight of tetrandrine were used to prepare 100 parts by weight of tetrandrine liposomes, then the drug loading in Example 1 would be 2%.

[0168] The test results are shown in Table 3.

[0169] Table 3

[0170]

[0171]

[0172] Cosmetic application testing:

[0173] 1) The irritation test of the tetrandrine liposomes prepared in Examples 1-3 was conducted using the chicken embryo chorioallantoic membrane (CAM) vascular test. The specific test method was as follows: Chicken embryos aged 10-11 days were selected. The air cell location was marked using an egg candler. The upper shell portion of the air cell was peeled off with tweezers to expose the white shell membrane. A suitable amount of physiological saline was added to moisten the shell membrane. The shell membrane was carefully removed with tweezers, ensuring no damage to the vascular membrane. A Teflon ring was placed in the area of ​​dense blood vessels. 40 μl of the test sample was placed into the ring, and the samples were incubated in a constant temperature and humidity incubator for 30 min. The extent of vascular damage within the Teflon ring was evaluated and recorded. A score was calculated based on the vascular response of the chicken embryo chorioallantoic membrane (CAM). 4-10 valid chicken embryos were tested for each sample. The irritation score of each sample was recorded, and the weighted average was calculated using the following formula. This average value is the NC value (normal usage concentration irritation value).

[0174] The test results are shown in Table 4 below.

[0175] Table 4

[0176] Liposomes concentration Stimulus rating in conclusion Example 1 10％ NC=0 No vascular injury stimulation Example 2 10％ NC=0 No vascular injury stimulation Example 3 10％ NC=0 No vascular injury stimulation

[0177] As shown in Table 4, the tetrandrine liposomes prepared in Examples 1-3 with drug loadings of 2%, 2.5%, and 1.5% respectively, when diluted to a concentration of 10% with PBS solution, showed no vascular irritation in the chicken embryo chorioallantoic membrane vascular experiment.

[0178] 2) The hyaluronidase inhibition effect of the tetrandrine liposomes prepared in Examples 1-3 was tested. The specific test method was as follows:

[0179] a) Before the experiment, prepare hyaluronidase 500U / mL, sodium hyaluronate 0.5mg / mL, pH 5.6 sodium acetate buffer, acetylacetone solution, P-DAB colorimetric reagent, calcium chloride, and sodium hydroxide solution in sequence according to the laboratory hyaluronidase test method.

[0180] b) The experiment was conducted on 96-well plates, divided into sample group (A), sample zeroing group (B), normal group (C), and normal zeroing group (D). At the beginning, reagents were added to each group (Group A: 25 μL sample + 25 μL hyaluronidase; Group B: 25 μL sample + 25 μL buffer; Group C: 25 μL distilled water + 25 μL hyaluronidase; Group D: 25 μL hyaluronidase + 25 μL buffer), and then incubated at 37°C for 20 min.

[0181] c) After adding 5 μL of calcium chloride, continue to place at 37°C for 20 min.

[0182] d) Add 25 μL of sodium hyaluronate to the wells of groups A and C, and add 25 μL of buffer solution to the wells of groups B and D. Incubate at 37°C for 40 min.

[0183] e) Add 25 μL of distilled water, 5 μL of sodium hydroxide solution, and 25 μL of acetylacetone solution sequentially. Incubate in a boiling water bath for 15 min, an ice bath for 10 min, and then at room temperature for 10 min.

[0184] f) Add 50 μL of P-DAB solution, shake thoroughly, add 100 μL of anhydrous ethanol, and let stand at room temperature for 30 min. Measure the absorbance at 570 nm.

[0185] The results of the hyaluronidase inhibition rate (%) at different concentrations are shown in Table 5 below.

[0186] Table 5

[0187] Liposomes 2.5% concentration 5% concentration 10% concentration Example 1 24.22 71.09 72.66 Example 2 26.56 50.78 78.13 Example 3 16.25 31.40 83.72

[0188] As can be seen from the results in Table 5, the tetrandrine liposomes prepared in Examples 1-3 with drug loading of 2%, 2.5%, and 1.5% respectively can significantly inhibit hyaluronidase activity at different concentrations (diluted to different concentrations with PBS solution) and have skin-soothing effects.

[0189] 3) The transdermal efficacy of the tetrandrine liposomes prepared in Example 1 (with a tetrandrine loading of 2%) was tested. The specific test method was as follows:

[0190] a) The biomimetic skin used in the experiment was Milpore Strat-MTM Membrane, with a diameter of 25 mm and an area of ​​4.9 cm². 2 b) Fix the Strat-MTM Membrane between the receiving and supply chambers. Add 1000 μL each of *Stephania tetrandra* liposome (2.0%) solution and *Stephania tetrandra* extract solution (2.0%) to the supply chamber. Use 0.1 M PBS buffer as the receiving solution and stir at 300 rpm at 32°C for diffusion. Collect 1 mL of the receiving solution at 0 h, 2 h, 4 h, 6 h, 8 h, and 24 h. At 24 h, cut the Strat-MTM Membrane into small pieces, place them in a centrifuge tube, add 1 mL of PBS, soak for 30 min, centrifuge, and collect the supernatant.

[0191] c) Use HPLC analysis to calculate the cumulative drug permeation at different times.

[0192] A tetrandrine solution (obtained by dissolving tetrandrine in 0.8% lactic acid and 97.2% purified water) was used as a control. Transdermal absorption rate results are as follows: Figure 1 As shown, by Figure 1 It is evident that the transdermal effect of the tetrandrine liposomes prepared in this invention is superior to that of the tetrandrine solution.

[0193] 4) The tetrandrine liposomes prepared in Example 1 (with a tetrandrine loading of 2%) were used to test the secretion of the inflammatory factor TNF-α in RAW264.7 cells induced by LPS (lipopolysaccharide). The specific test method was as follows:

[0194] a) Take cells in good exponential growth phase, digest them, and seed them in 12-well plates for 24 hours.

[0195] b) Aspirate the complete culture medium from the wells, add PBS for starvation treatment for 3 h, add complete culture medium to the blank control group, add LPS and complete culture medium to the model group, and add LPS, complete culture medium and diluted sample to the sample group, and incubate for 48 h.

[0196] c) Absorb the cell supernatant culture medium, centrifuge to obtain the supernatant, and store it in a -80℃ freezer.

[0197] d) Set up separate wells for standards and test samples. Add 100 μL of standard or test sample to each well, gently mix, cover with a plate patch, and incubate at 37°C for 2 hours. Each sample should have 3-4 replicates. Discard the liquid, spin dry, and wash the plate 5 times. Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate patch, and incubate at 37°C for 1 hour. Discard the liquid in the wells, spin dry, and wash the plate 5 times, soaking for 1 minute each time (200 μL / well). Spin dry. Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate patch, and incubate at 37°C for 1 hour. Discard the liquid in the wells, spin dry, and wash the plate 5 times, soaking for 1 minute each time (200 μL / well). Spin dry. Add 100 μL of substrate solution to each well sequentially, and develop color at 37°C in the dark for 15-30 minutes. Add 100 μL of stop solution to each well sequentially to terminate the reaction. Within 15 minutes after the reaction was terminated, the OD value of each well was measured sequentially at 450 nm using an ELISA reader.

[0198] LPS was used as the control group, and TNF-α secretion was also tested. The results are as follows: Figure 2 As shown, by Figure 2 It is evident that the tetrandrine liposomes (2.0%) prepared in this invention significantly reduced TNF-α content at all tested concentrations, suggesting that the tetrandrine liposomes (2.0%) have an inhibitory effect on TNF-α secretion.

[0199] In summary, tetrandrine liposomes (2.0%) have a soothing effect.

[0200] As can be seen from the above cosmetic application test results, the high drug loading liposomes prepared by this invention can be used in cosmetics.

[0201] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A liposome with high drug loading capacity of tetrandrine, characterized in that, The components include the following parts by weight: Tetrandrine 1.5–2.5 parts, pH adjuster 0.6 to 1 part, 3-5 parts lecithin Cholesterol 0.1–0.3 parts, 3-6 parts of cosolvent Antioxidant 0.5 to 1 part, 6-10 parts oil, 25-30 parts water 35-55 parts of polyols; The lecithin is soybean lecithin CS-95; The mass ratio of tetrandrine to pH adjuster is 1:0.4; The pH adjuster is lactic acid; the cosolvent is selected from at least one of PEG-40 hydrogenated castor oil, PPG-13-decyltetradecyl alcohol polyether-24, and polyoxyethylene sorbitan monooleate. When the content of tetrandrine is 2-2.5%, the mass ratio of lecithin to cosolvent is 1:1; The polyol is a combination of glycerol and other polyols, wherein the other polyols are selected from at least one of propylene glycol, butylene glycol, dipropylene glycol, and PEG400; The high-drug-loading tetrandrine liposomes also contain preservatives; The method for preparing the high-drug-loading tetrandrine liposomes includes the following steps: A1. Lecithin, solubilizer, antioxidant, preservative, oil, cholesterol, and some polyol are stirred in a water bath at 70-75℃ until completely dissolved to obtain the oil phase; A2. After sterilizing the water, add glycerin and stir well; cool to below 50℃ to obtain the aqueous phase; A3. Add tetrandrine and pH adjuster to the oil phase, stir until completely dissolved, and then start cooling to ≤50℃; A4. Slowly add the mixture obtained after step A3 into the aqueous phase, stir and mix evenly to form a transparent liquid; A5. The transparent liquid is homogenized under high pressure to obtain tetrandrine liposomes.

2. The high drug-loading tetrandrine liposome according to claim 1, characterized in that, The antioxidant is selected from at least one of tocopherol, tocopheryl acetate, ascorbate stearate, ascorbate tetraisopalmitate, tetrahexyldecyl ascorbate, and di-tert-butyl-p-cresol. The oil is selected from at least one of caprylic / capric triglyceride, glyceryl monostearate, glyceryl behenate, and glyceryl palmitate stearate.

3. The high drug-loading tetrandrine liposome according to claim 1, characterized in that, The preservative is present in parts by weight of 1 to 3.5 parts. The preservative is selected from at least one of 1,2-pentanediol, 1,2-hexanediol, p-hydroxyacetophenone, and phenoxyethanol.

4. The use of a high-drug-loading tetrandrine liposome according to any one of claims 1-3 in the preparation of cosmetics.

5. The use of a high-drug-loading tetrandrine liposome according to any one of claims 1-3 in the preparation of a soothing composition.

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