A method for preparing liposomes

Abstract

The application provides a preparation method of liposomes. The liposomes are prepared by using lecithin containing calcium citrate as a liposome film material and using a water-soluble solution containing a water-soluble active ingredient as a hydration solution. By mixing calcium citrate in the liposome film, the active ingredient in the liposomes can be quickly released, and the stability of the liposomes can be significantly improved. The raw materials used are non-toxic to human bodies, have high safety, and are suitable for preparing products such as medicines, oral care products and oral health care products.

Description

Technical Field

[0001] This invention belongs to the field of daily chemical products and biopharmaceutical manufacturing technology. More specifically, it relates to a method for preparing liposomes. Background Technology

[0002] In many application scenarios, people have an urgent need for the immediate effectiveness of medicines, oral care products, and oral health care products. For example: (1) For medicines, when patients are in emergency situations (such as angina emergency) or encounter acute pain (such as acute toothache), the active ingredients in the medicine need to be released quickly and take effect immediately; (2) For oral care products, after people eat strong-flavored food, there will be residual odor and a large number of bacteria in the mouth. If the active ingredients in oral care products such as mouthwash can be released quickly, they can freshen breath and kill bacteria in time, and achieve oral cleaning immediately; (3) For oral health care products, such as toothpaste and mouthwash, the active ingredients need to be released quickly in a short time when brushing teeth and rinsing mouth in order to effectively achieve oral health care.

[0003] Currently, the common methods to achieve immediate effects for these products are: (1) increasing the concentration of active ingredients, which allows for the release of more active ingredients during use and achieves immediate effects. However, excessively high concentrations of active ingredients may irritate oral mucosa and other tissues, causing discomfort or even damage. Furthermore, high concentrations of active ingredients are usually less stable and are prone to decomposition and deterioration during storage, affecting the quality and efficacy of the product. (2) adding penetration enhancers. Penetration enhancers can change the permeability of the mucosa, helping the active ingredients to penetrate the mucosa more quickly into the body or reach the site of action. However, the use of penetration enhancers may damage the normal physiological structure of the mucosa. Long-term use may lead to a decline in the mucosal barrier function, increasing the risk of infection. Moreover, some penetration enhancers themselves have certain toxicity and irritation, posing a potential threat to the health of users.

[0004] Therefore, there is an urgent need to find a way to quickly release active ingredients while ensuring safety and stability, which is crucial for the immediate effectiveness of pharmaceuticals, oral care products, and oral health products. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a method for preparing liposomes. By mixing calcium citrate into the liposome membrane, the active ingredients in the liposomes can be released rapidly, and the stability of the liposomes can be significantly improved. Moreover, all raw materials used are non-toxic to the human body and have high safety.

[0006] The primary objective of this invention is to provide a method for preparing liposomes.

[0007] A second objective of the present invention is to provide liposomes prepared by the above method.

[0008] A third objective of this invention is to provide a product.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides a method for preparing liposomes, specifically: using lecithin containing calcium citrate as the liposome film material and an aqueous solution containing water-soluble active ingredients as the hydration solution to obtain liposomes.

[0011] Preferably, the mass ratio of lecithin to calcium citrate is 10-45:1.5-12, more preferably 10-30:1.5-2.5 or 360:93-94, and most preferably 10:1.5-2.5 or 45:11.675.

[0012] Preferably, the water-soluble active ingredient is one or more of lysozyme, papain, and glucanase.

[0013] In a preferred embodiment, when the required volume of liposomes is less than 20 mL, the preparation method includes the following steps:

[0014] S1. Dissolve lecithin and calcium citrate in an organic solvent, dry, and obtain a liposome film:

[0015] S2. The liposome film and water-soluble active ingredients obtained in S1 are dissolved in xylitol aqueous solution, and then hydrated and sonicated in sequence to obtain liposomes.

[0016] Preferably, the organic solution in S1 is an aqueous solution of tert-butanol or an aqueous solution of ethanol.

[0017] More preferably, in the aqueous solution of tert-butanol, the volume ratio of tert-butanol to water is 3 to 5:20, and most preferably 5:20.

[0018] More preferably, the volume concentration of ethanol in the aqueous ethanol solution is 45% to 55%, and most preferably 50%.

[0019] Preferably, the ratio of lecithin to organic solution in S1 is 10-30 mg:1 mL, and most preferably 10 mg:1 mL.

[0020] Preferably, the ratio of calcium citrate to organic solution in S1 is 1.5–2.5 mg: 1 mL.

[0021] Preferably, the drying in S1 is achieved by rotary evaporation, such as at 35-40 °C, and most preferably at 37 °C.

[0022] Preferably, the xylitol in S2 has a volume concentration of 1% to 20% in the xylitol aqueous solution, and most preferably 5%.

[0023] Preferably, the ratio of the water-soluble active ingredient in S2 to the xylitol aqueous solution is 0.8–1.2 mg: 8–12 mL, and most preferably 1 mg: 10 mL.

[0024] Preferably, the ratio of lecithin in S1 to xylitol aqueous solution in S2 is 10-30 mg:1 mL, and most preferably 10 mg:1 mL.

[0025] Preferably, the hydration in S2 is achieved by stirring.

[0026] More preferably, the stirring is carried out at 400–1300 rpm and 0–4 °C for 0.8–1.2 h, and most preferably at 1300 rpm and 0 °C for 1 h.

[0027] Preferably, the power of the ultrasound in S2 is 280-300 W, and most preferably 292.5 W.

[0028] Preferably, the ultrasound duration in S2 is 2.8–3.2 min. For example, the on-time is 2.8–3.2 min in the form of 1.5 s on / 1.5 s off.

[0029] In another preferred embodiment, when the required volume of liposomes is 10 mL or more, the preparation method includes the following steps:

[0030] S1. Dissolve lecithin and calcium citrate in an organic solvent;

[0031] S2. Dissolve the product obtained in S1 and the water-soluble active ingredient in water, then evaporate the organic solution, and finally perform shearing and homogenization to obtain liposomes.

[0032] Preferably, the organic solution in S1 is an aqueous solution of tert-butanol or an aqueous solution of ethanol.

[0033] More preferably, in the aqueous solution of tert-butanol, the volume ratio of tert-butanol to water is 3 to 5:20, and most preferably 1:4.

[0034] More preferably, the volume concentration of ethanol in the aqueous ethanol solution is 45% to 55%, and most preferably 50%.

[0035] Preferably, the ratio of lecithin to organic solution in S1 is 350–370 mg: 35–40 mL, and most preferably 360 mg: 37 mL.

[0036] Preferably, the ratio of calcium citrate to organic solution in S1 is 93-94 mg: 35-40 mL, and most preferably 93.4 mg: 37 mL.

[0037] Preferably, the ratio of lecithin in S1 to water in S2 is 350-370 mg: 80-120 mL, and most preferably 360 mg: 100 mL.

[0038] Preferably, the ratio of the water-soluble active ingredient to water in S2 is 4.5–5.5 mg: 80–120 mL, and most preferably 5 mg: 100 mL.

[0039] Preferably, the dissolution temperature in S2 is 45–55 °C, and most preferably 50 °C.

[0040] Optionally, the evaporation of the organic solution described in S2 can be achieved by conventional methods, such as heating or stirring.

[0041] Optionally, the shearing described in S2 is performed at 10,000 to 16,000 rpm for 5 to 15 minutes, and most preferably at 13,000 rpm for 10 minutes.

[0042] Preferably, the homogenization pressure in S2 is 950–1050 bar.

[0043] Preferably, the homogenization process in S2 is performed 5 to 20 times, more preferably 10 times.

[0044] The liposomes prepared by the above method have the advantages of rapid release of active ingredients and high stability, and are suitable for the preparation of pharmaceuticals, oral care products, oral health care products and other products. Therefore, the liposomes prepared by the above method and products containing the above liposomes should be within the protection scope of this invention.

[0045] Preferably, the product is one of the following: pharmaceuticals, oral care products, or oral health products.

[0046] More preferably, the drug is one or more of the following: oral liquid, tincture, tincture, aerosol, powder, injection, sterile powder for injection, and suppository. The types of the above preparations can be understood according to the relevant definitions in *Pharmaceutics* (6th Edition, People's Medical Publishing House, Cui Fude), and the preparation of the above preparations can be carried out according to the relevant preparation methods in *Pharmaceutics* (6th Edition, People's Medical Publishing House, Cui Fude).

[0047] Furthermore, the medicine also contains excipients.

[0048] Furthermore, the excipients are solid lubricants (such as stearic acid and / or magnesium stearate, used to reduce friction and ensure successful drug manufacturing), vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and / or cocoa butter, used as solvents or carriers to help drug components dissolve or disperse better), polyols (such as propylene glycol, glycerin, sorbitol, mannitol, and / or polyethylene glycol, used to improve drug stability, prevent drug dehydration or deterioration during storage, and also to improve drug taste and solubility), and alginic acid (to maintain the physical stability of the drug and prevent...). Drug components include: (1) prevents drug particle sedimentation or aggregation; (2) emulsifiers (such as Tween and / or polyoxyethylene castor oil, used to reduce interfacial tension, promote emulsification of the dispersed phase, and maintain emulsion stability); (3) wetting agents (such as sodium lauryl sulfate, used to help drug components disperse and dissolve better); (4) colorants (used to improve the appearance and color of drugs, making them easier to identify and distinguish); (5) flavoring agents (used to improve the taste of drugs and increase patient medication compliance); and (6) tableting agents (facilitating the compression of loose granular substances into solid tablets, making them convenient for patients to take and carry, and also improving drug stability and bioavailability). The following are listed: stabilizers (to increase the physical stability of the drug and prevent it from deteriorating or degrading during storage or transportation), antioxidants (to prevent the drug from deteriorating due to oxidation during storage or use and extend its shelf life), isotonic salt solutions (to adjust the osmotic pressure of the drug to meet the physiological requirements of the human body, thereby reducing irritation and adverse reactions), phosphate buffer solutions (to adjust the pH value of the drug to meet the physiological requirements of the human body, thereby reducing irritation and adverse reactions), sugars (such as lactose, glucose and / or sucrose, to help the drug components disperse and dissolve better, while providing necessary energy support), starches (such as corn starch and / or potato starch, to help tablets disintegrate rapidly after administration and release the drug components, thereby improving the bioavailability of the drug), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and / or methyl cellulose, to improve the compressibility and formability of the drug, and also to control the release rate of the drug), binders (such as gelatin, to help the drug components bind together better), and lubricants (such as talc, to reduce friction in the drug). The type of excipient can be selected based on the need to improve drug stability, activity, and / or bioavailability.

[0049] More preferably, the oral care product is one or more of the following: oral care liquid, oral lozenges, oral sprays, oral patches, oral powders, oral gels, oral sustained-release agents, mouthwash, toothpaste, and mouthwash effervescent tablets.

[0050] More preferably, the oral care product is one or more of the following: oral care solution, oral lozenges, oral sprays, oral patches, oral powders, oral gels, oral sustained-release agents, mouthwash, toothpaste, and mouthwash effervescent tablets.

[0051] Furthermore, the oral care products and oral health products also contain excipients.

[0052] Furthermore, the excipients include solvents (such as glycerin, propylene glycol, and / or ethanol, used to dissolve raw materials for ease of use and storage), flavorings (such as peppermint flavoring, used to enhance the aroma and taste of oral care and oral health products, improving the user's sensory experience), preservatives (such as potassium sorbate, sodium benzoate, methylparaben, and / or propylparaben, used to prevent the growth of microorganisms in oral care and oral health products, extending their shelf life), antibacterial agents (such as cetylpyridinium chloride, chlorhexidine gluconate, and / or benzalkonium chloride, used to inhibit or kill harmful microorganisms in the oral cavity, helping to maintain oral health), and pH adjusters (such as disodium hydrogen phosphate, sodium dihydrogen phosphate, and / or sodium citrate, used to adjust the pH of oral care and oral health products to meet their requirements). The excipients used in oral care and hygiene products include one or more of the following: excipients to meet the physiological needs of the oral environment and reduce irritation to oral tissues; thickeners (such as poloxamer 407, used to increase the viscosity of oral care and hygiene products, improve the user experience, and also to improve the stability of oral care and hygiene products, preventing separation or sedimentation); sweeteners (such as sucralose, sodium saccharin, sorbitol, xylitol, and / or maltitol, used to increase the sweetness of oral care and hygiene products, enhancing the user's sensory experience); cooling agents (such as menthol and its derivatives, used to increase the cooling sensation of oral care and hygiene products, enhancing the user's sensory experience); and surfactants (such as sodium lauryl sulfate and / or sodium lauroyl sarcosinate, used to reduce the surface tension of oral care and hygiene products, enhancing cleaning effectiveness). The type of excipient can be selected based on the need to improve the stability, safety, effectiveness, or user experience of oral care and hygiene products.

[0053] The present invention has the following beneficial effects:

[0054] This invention, by incorporating calcium citrate into liposome membranes, not only rapidly releases the active ingredients in the liposomes but also significantly improves their stability. Furthermore, all raw materials used are non-toxic to the human body and possess high safety, making it suitable for the preparation of pharmaceuticals, oral care products, oral health products, and other related products. Attached Figure Description

[0055] Figure 1The graph shows the effects of tert-butanol-water ratio, lecithin concentration, and stirring speed on liposome uniformity.

[0056] Figure 2 The graph shows the changes in drug content of liposomes containing different concentrations of calcium citrate (0.05%, 0.1%, 0.25%, 0.5%, 1%) on days 7, 15, and 30 of storage.

[0057] Figure 3 The figure shows the drug release of liposomes containing different concentrations of calcium citrate (0.05%, 0.1%, 0.25%, 0.5%, 1%) at 1, 5, 15, 30, 60, and 120 h after storage.

[0058] Figure 4 for Figure 3 Enlarged view of drug release from liposomes at 1, 5 and 15 h.

[0059] Figure 5 The graph shows the changes in drug content of liposomes containing different concentrations of calcium citrate (0.1%, 0.125%, 0.15%, 0.2%, 0.25%) on days 7, 15, and 30 of storage.

[0060] Figure 6 This is a photograph of the liposomes in Example 3.

[0061] Figure 7 This is a graph showing the detection results of the average particle size of liposomes in Example 3.

[0062] Figure 8 Images of liposomes from 5 batches.

[0063] Figure 9 This is a graph showing the detection results of the average particle size of liposomes in Example 4.

[0064] Figure 10 This is a graph showing the drug content detection results of liposomes in Example 4. Detailed Implementation

[0065] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0066] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0067] D H The detection methods for (average particle size) and PDI (polydispersity index) are as follows: Dilute the liposomes by 5 times with ultrapure water and then place them in the particle size measurement sample cell of the Malvern nanoparticle size analyzer for detection.

[0068] Method for detecting drug content: Take 1 mL of liposomes, add 1.5 mL of DMSO to break the emulsion, dilute 5-20 times with PBS buffer (pH=7.5) and filter. Take the filtrate and analyze it according to the BCA protein content assay kit (Pierce). TM The instructions for the BCA Protein Assay Kits are for testing the content of lysozyme.

[0069] Method for detecting drug release: Liposomes were placed in dialysis bags, and 200 mL of PBS buffer (pH=7.5) was used as the release medium. The release temperature was set to 37±0.5 ℃. During the release process, 10 mL of sample was taken to detect the drug content in the release medium, and the liquid was replenished immediately after sampling. The method for detecting the drug content in the release medium was as follows: after lyophilizing the sample, 0.1 mL of PBS buffer (pH=7.5) was added, and the drug content was determined according to the BCA protein content assay kit (Pierce). TM The instructions for the BCA Protein Assay Kit are used to test for lysozyme content.

[0070] Example 1: Effects of tert-butanol-water ratio, lecithin concentration, and stirring speed on liposome uniformity.

[0071] I. Preparation of Liposomes

[0072] S1. Weigh a specific mass of lecithin and 15 mg of calcium citrate into a round-bottom flask, add 10 mL of a specific concentration of tert-butanol aqueous solution, shake the round-bottom flask to dissolve the lecithin and calcium citrate, and evaporate to dryness using a rotary evaporator at 37 °C until a light yellow film is visible at the bottom of the round-bottom flask, thus obtaining the liposome film.

[0073] S2. Weigh 1 mg of lysozyme and dissolve it in 10 mL of 5% ( v / v The liposomes were dissolved in a xylitol aqueous solution and then added to a round-bottom flask in S1 to dissolve the light yellow film. After hydration in a 0 °C ice bath magnetic stirrer at a specific speed for 1 h, the liposomes were ultrasonically treated with an ultrasonic instrument at 292.5 W, 1.5 s on / 1.5 s off (so that the on time reaches 3 min) to obtain the liposomes.

[0074] II. Effects of tert-butanol-water ratio, lecithin concentration, and stirring speed on liposome uniformity

[0075] The effects of tert-butanol-water ratio (volume ratio of tert-butanol to water in the tert-butanol aqueous solution), lecithin concentration (lecithin mass ÷ volume of tert-butanol aqueous solution × 100%), and stirring speed (S2 stirring speed for hydration) on the average particle size and PDI of liposomes were investigated as single variables. Specifically, when the tert-butanol-water ratio (3:20, 1:5, 1:4) was used as a single variable, the lecithin concentration was controlled at 1% and the stirring speed at 1300 rpm; when the lecithin concentration (1%, 2%, 3%) was used as a single variable, the tert-butanol-water ratio was controlled at 1:4 and the stirring speed at 1300 rpm; and when the stirring speed (400, 900, 1300 rpm) was used as a single variable, the tert-butanol-water ratio was controlled at 1:4 and the lecithin concentration at 1%.

[0076] The results are as follows Figure 1 As shown, the average particle size and PDI of liposomes gradually decrease with the increase of tert-butanol-water ratio, the decrease of lecithin concentration, and the increase of stirring speed. When the tert-butanol-water ratio is 1:4, the lecithin concentration is 1%, and the stirring speed is 1300 rpm, the PDI of liposomes is already small enough. Therefore, when the tert-butanol-water ratio is ≥1:4, the lecithin concentration is ≤1%, and the stirring speed is ≥1300 rpm, the particle size of liposomes is uniform and stable, and they have good uniformity.

[0077] For the purpose of saving energy, the following examples use tert-butanol-water ratio of 1:4, lecithin concentration of 1%, and stirring speed of 1300 rpm to prepare liposomes.

[0078] Example 2: Effect of calcium citrate concentration on liposome stability and drug release

[0079] I. Preparation of Liposomes

[0080] S1. Weigh 100 mg of lecithin and a specific mass of calcium citrate into a round-bottom flask, add 10 mL of tert-butanol aqueous solution (tert-butanol-water ratio is 1:4), shake the round-bottom flask to dissolve the lecithin and calcium citrate, and evaporate to dryness using a rotary evaporator at 37 ℃ until a light yellow film is visible at the bottom of the round-bottom flask, thus obtaining the liposome film.

[0081] S2. Weigh 1 mg of lysozyme and dissolve it in 10 mL of 5% ( v / v The liposomes were dissolved in a xylitol aqueous solution and then added to a round-bottom flask in S1. The solution was stirred and hydrated in a 0 °C ice bath magnetic stirrer at 1300 rpm for 1 h. The solution was then sonicated at 292.5 W with 1.5 s on and 1.5 s off conditions (so that the on time reaches 3 min) to obtain liposomes.

[0082] II. Effects of calcium citrate concentration on liposome stability and drug release

[0083] (1) Using calcium citrate concentration (calcium citrate mass ÷ volume of tert-butanol aqueous solution × 100%) as a single variable, the changes in drug content of liposomes stored at 30±1 ℃ for 7, 15, and 30 days were studied when the concentration was 0.05%, 0.1%, 0.25%, 0.5%, and 1%, respectively. The results are as follows: Figure 2 As shown, when the calcium citrate concentration is above 0.1%, the drug content in the liposomes remains basically stable within 30 days, exhibiting high stability.

[0084] (2) Using calcium citrate concentration as the single variable, the drug release of liposomes stored at 30±1 ℃ for 1, 5, 15, 30, 60, and 120 h was studied when the concentrations were 0.05%, 0.1%, 0.25%, 0.5%, and 1%, respectively. The results are as follows: Figures 3-4 As shown, where, Figure 3 This is a graph showing drug release from liposomes at 1, 5, 15, 30, 60, and 120 hours. Figure 4 for Figure 3 Enlarged images of drug release from liposomes at 1, 5, and 15 h. It can be seen that the amount of drug released decreases with increasing calcium citrate concentration in the liposomes. When the calcium citrate concentration exceeds 0.25%, the drug release from the liposomes within 120 h is less than 45.43%, indicating poor release behavior.

[0085] (3) Using calcium citrate concentration as a single variable, the changes in drug content of liposomes stored at 30±1 ℃ for 7, 15, and 30 days were studied when the concentrations were 0.1%, 0.125%, 0.15%, 0.2%, and 0.25%, respectively. The results are as follows: Figure 5 As shown, when the calcium citrate concentration is above 0.15%, the drug content in the liposomes can be maintained at over 90% within 30 days, exhibiting higher stability.

[0086] In summary, when the calcium citrate concentration is 0.15% to 0.25%, the liposomes of the present invention have the advantages of rapid release of active ingredients and high stability.

[0087] Example 3: Stability of liposomes

[0088] I. Preparation of Liposomes

[0089] S1. Weigh 100 mg of lecithin and 25 mg of calcium citrate into a round-bottom flask, add 10 mL of tert-butanol aqueous solution (tert-butanol-water ratio is 1:4), shake the round-bottom flask to dissolve the lecithin and calcium citrate, and evaporate to dryness using a rotary evaporator at 37 ℃ until a light yellow film is visible at the bottom of the round-bottom flask, thus obtaining the liposome film.

[0090] S2. Weigh 1 mg of lysozyme and dissolve it in 10 mL of 5% ( v / v The xylitol aqueous solution was added to a round-bottom flask (S1) to dissolve a pale yellow film. The mixture was then hydrated by stirring at 1300 rpm in a 0 °C ice bath with a magnetic stirrer for 1 h. Following this, the mixture was sonicated at 292.5 W with a 1.5 s on / 1.5 s off cycle (ensuring the on time reaches 3 min) to obtain liposomes. (The liposome image at this point is shown in the image.) Figure 6 (As shown).

[0091] II. Liposome Stability Test

[0092] Liposomes were placed in environments of 4, 20, and 30 °C, and their D content was measured on days 5 and 10. H (Average particle size).

[0093] The results are as follows Figure 7 As shown, the average particle size of liposomes remained within the range of 140–170 nm over 10 days, without significant increase. p >0.05), indicating that the liposomes of the present invention have high stability.

[0094] Example 4 Reproducibility of the liposome preparation method

[0095] I. Preparation of Liposomes

[0096] Five batches of liposomes were prepared according to the following method:

[0097] S1. Weigh 100 mg of lecithin and 15 mg of calcium citrate into a round-bottom flask, add 10 mL of tert-butanol aqueous solution (tert-butanol-water ratio is 1:4), shake the round-bottom flask to dissolve the lecithin and calcium citrate, and evaporate to dryness using a rotary evaporator at 37 ℃ until a light yellow film is visible at the bottom of the round-bottom flask, thus obtaining the liposome film.

[0098] S2. Weigh 1 mg of lysozyme and dissolve it in 10 mL of 5% ( v / v The liposomes were dissolved in a xylitol aqueous solution and then added to a round-bottom flask in S1. The solution was stirred and hydrated in a 0 °C ice bath magnetic stirrer at 1300 rpm for 1 h. The solution was then sonicated at 292.5 W with 1.5 s on and 1.5 s off conditions (so that the on time reaches 3 min) to obtain liposomes.

[0099] II. Liposome Stability Test

[0100] Five batches of liposomes were photographed. Figure 8After that, the liposomes were stored at 30±1 ℃ for 90 days, and the average particle size and drug content of the liposomes were detected at 0, 30, 60 and 90 days.

[0101] The results are as follows Figures 9-10 As shown, where, Figure 9 This is a graph showing the detection results of the average particle size of liposomes. Figure 10 The image shows the drug content detection results for the liposomes. It can be seen that the average particle size of the liposomes remained within the range of 150–170 nm over 90 days, and the drug content remained stable at over 85%, indicating that the liposomes of this invention have high stability and the preparation method has good reproducibility.

[0102] Example 5 Drug encapsulation efficiency of liposomes

[0103] I. Preparation of Liposomes

[0104] S1. Weigh 360 mg of lecithin and 93.4 mg of calcium citrate and dissolve them in 37 mL of 50% ( v / v In ethanol;

[0105] S2. Weigh 5 mg of lysozyme and dissolve it in 100 mL of water at 50 °C. Then add it to the product obtained in S1 and stir for 30 min to evaporate the ethanol. Then shear it for 10 min at 13000 rpm using a high-speed emulsifier. Finally, homogenize it 10 times at 1000 bar using a nanoscale ultra-high pressure homogenizer (EmulsFlex-C3, purchased from Guangzhou Aolong Biotechnology Co., Ltd.) to obtain the liposomes.

[0106] II. Drug Encapsulation Efficiency Detection of Liposomes

[0107] After lyophilizing the liposomes, 100 mg was dissolved in 2 mL of PBS buffer (pH=7.5), and 0.5 mL of this solution was added to an ultrafiltration centrifuge tube. After ultrafiltration centrifugation at 10000 rpm for 30 min, the drug content in the lower supernatant was measured. The drug encapsulation efficiency of the liposomes was calculated using the formula: "Drug encapsulation efficiency (%) = (Drug addition amount - Drug content in lower supernatant) / Drug addition amount × 100%". Three parallel samples were set up, and the average value of the results was taken.

[0108] The results showed that the drug encapsulation efficiency of the liposomes was 89.26% ± 0.24%, indicating that the liposome preparation method of the present invention can achieve a high drug encapsulation efficiency.

[0109] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing liposomes, characterized in that, Includes the following steps: S1. Lecithin and calcium citrate are dissolved in an aqueous solution of tert-butanol and dried to obtain a liposome film: the mass ratio of lecithin to calcium citrate is 10:1.5-2.5; the volume ratio of tert-butanol to water in the aqueous solution of tert-butanol is 5:20; the volume ratio of lecithin to the aqueous solution of tert-butanol is 10 mg:1 mL. S2. The liposome film and water-soluble active ingredients obtained in S1 are dissolved in xylitol aqueous solution, and then hydrated and sonicated in sequence to obtain liposomes; The water-soluble active ingredient is one or more of lysozyme, papain, and glucanase.

2. The preparation method according to claim 1, characterized in that, The power of the ultrasound described in S2 is 280–300 W.

3. Liposomes prepared by the method according to any one of claims 1 to 2.

4. A product characterized in that, Contains the liposomes as described in claim 3.

5. The product according to claim 4, characterized in that, The product is one of the following: pharmaceuticals or oral care products.

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