Preparation method of liposome / cellulose hydrogel loaded with large amount of water-soluble drospirenone
By incorporating drospirenone liposomes with cellulose hydrogel, the problems of poor water solubility and low bioavailability of drospirenone are solved, the stability and sustained release performance of high drug loading are achieved, and the application of drugs is expanded scope.
Patent Information
- Application Number
- CN202310801027.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-03
- Publication Date
- 2025-01-07
AI Technical Summary
The poor water solubility and low bioavailability of drospirenone lead to instability of high drug-loaded liposomes, limiting its application range.
Cellulose hydrogel is used to incorporate drospirenone liposomes, and a stable liposome hydrogel drug-loading system is formed through the combination of soy lecithin and cholesterol, and the lipids are absorbed and fixed by using the three-dimensional network structure of cellulose. body, improve the solubility and stability of drospirenone.
The stability and sustained release performance of high drug loading are achieved, the sudden release effect is avoided, the local concentration and bioavailability of the drug are improved, the side effects are reduced, and the scope of application is expanded.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure HDA0004316374880000011
Abstract
Description
Technical Field
[0002] The invention belongs to the field of drospirenone solubilization and sustained release applications, and particularly relates to a method for preparing a liposome / cellulose hydrogel carrying a large amount of water-soluble drospirenone. Background Art
[0003] Liposomes are closed vesicles with a bilayer-encapsulated aqueous structure. Because their structure is similar to that of biological membranes, they are also called artificial biological membranes. They are usually composed of amphiphilic molecules such as soybean lecithin and cholesterol, and their particle sizes range from 10nm to 2μm. Liposomes have excellent properties such as good biocompatibility, non-toxicity, sustained and controlled release effects, and stimulus responsiveness, and are widely used for drug incorporation and sustained release. However, drug-loaded liposomes are greatly affected by temperature and pH during the preparation process. The liposome microencapsulation technology was used to significantly solubilize drospirenone. Studies have found that liposomes with high drug loading levels settle in a short period of time, and their physical and chemical properties are not stable. This seriously limits the application of liposome drug delivery systems.
[0004] Hydrogel is a hydrophilic three-dimensional network structure, which has the ability to absorb a large amount of water and keep it in the matrix without being dissolved. It has the advantages of good biocompatibility, degradability and soft tissue similarity, and has attracted wide attention in the fields of food industry, drug carrier, tissue repair, etc. The presence of hydrogel can maintain the integrity of liposomes and improve the stability of liposomes. Liposomes can also provide the effect of hydrogel on drugs, thereby avoiding the occurrence of burst effect, increasing local drug concentration, reducing side effects, and improving drug efficacy, thereby expanding the application range of liposomes. The drospirenone liposome / cellulose hydrogel first uses soybean lecithin to encapsulate it to increase the water solubility of drospirenone, and then prepares hydrogel with cellulose as the main chain to absorb, disperse and fix the drospirenone liposome inclusion compound, thereby preparing a drug-carrying system with a large drug loading capacity, sustained release performance and the ability to release soluble drospirenone. Summary of the invention
[0005] In order to overcome the problems of poor water solubility, low bioavailability and instability of high-drug-loaded liposomes of drospirenone, the present invention aims to solve the above problems and provide a stable high-drug-loaded sustained-release liposome hydrogel drug delivery system and a preparation method thereof.
[0006] The object of the present invention is achieved through the following solutions.
[0007] A method for preparing a stable, high-drug-loaded, sustained-release liposome hydrogel comprises the following steps:
[0008] (1) Dissolve a certain amount of cellulose in a certain amount of distilled water, and add a certain amount of sodium hydroxide solution of a certain concentration after the solution is completely dissolved. Then add epichlorohydrin and seal the container for stirring. After stirring for a certain period of time, let it stand for cross-linking. Then wash, soak, and freeze-dry to obtain a hydrogel;
[0009] (2) placing the weighed dry hydrogel in the centrifuged liposome emulsion, and waiting for the supernatant to be completely absorbed to obtain the drospirenone liposome / cellulose hydrogel.
[0010] Preferably, the method for preparing the liposome emulsion in step (2) comprises the following steps:
[0011] Weigh phospholipids, drospirenone drugs, and cholesterol according to a certain mass ratio, add a certain volume of anhydrous ethanol solution, fully dissolve and mix in a 30°C water bath to form an oil phase, then measure a certain volume of PBS in a magnetic stirrer to form a water phase, inject the prepared oil phase into the water phase evenly and quickly with a syringe, stir for a certain time in a water bath at a certain temperature to completely evaporate the ethanol, and after ultrasonication in an ice bath on an ultrasonicator for a certain time, obtain a liposome emulsion.
[0012] Preferably, the phospholipid is soybean lecithin.
[0013] Preferably, the mass ratio of the phospholipid to cholesterol is 1:0 to 6:1.
[0014] Preferably, the drug-to-lipid ratio is 0.25:1 to 5:1.
[0015] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:5 to 1:10;
[0016] Preferably, the hydration time is 0.5 to 1.5 hours, and the hydration temperature is 30 to 80°C.
[0017] Preferably, the ultrasonic time is 5 to 15 minutes.
[0018] Preferably, the polysaccharide in step (1) is one or more of hydroxyethyl cellulose, carboxymethyl cellulose and sodium alginate, and hydroxyethyl cellulose is more preferably used.
[0019] Preferably, in step (1), the volume of epichlorohydrin is 1.5 to 5.5 mL, the concentration of the sodium hydroxide solution is 0.2% to 1%, and the volume of distilled water is 2.5 to 10 mL.
[0020] Preferably, in step (1), cellulose is dispersed in distilled water, sodium hydroxide solution is added, and stirred at room temperature for 0.5 to 1.5 hours. The resulting mixed solution is then allowed to stand at room temperature for 8 to 24 hours, and then washed with water and soaked for 12 to 24 hours to obtain a hydrogel.
[0021] Preferably, the mass ratio of the liposome emulsion to the hydrogel in step (2) is 20:1 to 60:1.
[0022] The liposome hydrogel is prepared by any of the preparation methods described above.
[0023] Application of the above-mentioned liposome hydrogel in drug carrier.
[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0025] (1) The problem of poor water solubility of drospirenone was solved by using liposome solubilization method.
[0026] (2) The problem of poor stability of homodrospirenone / liposome inclusion complex was solved by using cellulose hydrogel immobilization method. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is a preparation flow chart of the present invention.
[0028] Figure 2 To optimize the preparation process of liposome emulsion.
[0029] Figure 3 The release of drospirenone in the liposome hydrogel drug delivery system. DETAILED DESCRIPTION
[0030] The present invention will be further described below in conjunction with the embodiments.
[0031] Example 1
[0032] (1) Preparation of drospirenone liposomes: Drospirenone liposome inclusion complexes were prepared by ethanol injection method. Soy lecithin, drospirenone drug, and cholesterol were dissolved in 10 mL of anhydrous ethanol solution at a ratio of 2:2:1 and fully dissolved and mixed in a 30°C water bath to form an oil phase. The solution was then slowly injected into phosphate buffered saline (PBS, pH 7.4) with a syringe and stirred continuously, and the remaining ethanol was evaporated at 50°C. After ultrasonication on an ice bath for 5 minutes, the mixture was stored at 4°C in the dark.
[0033] (2) Preparation of hydrogel: Weigh 3 g of hydroxyethyl cellulose and disperse it in 30 mL of sodium hydroxide solution. Add 1 mL of epichlorohydrin and stir at room temperature for 1 h. Allow to stand for crosslinking for 8 h. Then, wash with distilled water, soak for 12 h, and finally freeze-dry to obtain hydroxyethyl cellulose hydrogel.
[0034] (3) Preparation of liposome hydrogel: 10 mL of the liposome emulsion prepared in step (1) was added to 1 g of the hydrogel prepared in step (2), and the supernatant was completely absorbed to obtain drospirenone liposome cellulose hydrogel.
[0035] Example 2
[0036] (1) Preparation of drospirenone liposomes: The drospirenone liposome inclusion complex was prepared by ethanol injection method. Soy lecithin, drospirenone drug, and cholesterol were dissolved in 10 mL of anhydrous ethanol solution at a ratio of 4:2:1 and fully dissolved and mixed in a 30°C water bath to form an oil phase. The solution was then slowly injected into phosphate buffered saline (PBS, pH 7.4) with a syringe and stirred continuously, and the remaining ethanol was evaporated at 50°C. After ultrasonication on an ice bath for 5 minutes, the mixture was stored at 4°C in the dark.
[0037] (2) Preparation of hydrogel: Weigh 3 g of hydroxyethyl cellulose and disperse it in 30 mL of sodium hydroxide solution. Add 2 mL of epichlorohydrin and stir at room temperature for 1 h. Allow to stand for crosslinking for 8 h. Then, wash with distilled water, soak for 12 h, and finally freeze-dry to obtain hydroxyethyl cellulose hydrogel.
[0038] (3) Preparation of liposome hydrogel: 15 mL of the liposome emulsion prepared in step (1) was added to 1 g of the hydrogel prepared in step (2), and the supernatant was completely absorbed to obtain drospirenone liposome cellulose hydrogel.
[0039] Example 3
[0040] (1) Preparation of drospirenone liposomes: The drospirenone liposome inclusion complex was prepared by the ethanol injection method. Soy lecithin, drospirenone drug, and cholesterol were dissolved in 10 mL of anhydrous ethanol solution at a ratio of 6:2:1 and fully dissolved and mixed in a 30°C water bath to form an oil phase. The solution was then slowly injected into phosphate buffered saline (PBS, pH 7.4) with a syringe and stirred continuously, and the remaining ethanol was evaporated at 50°C. After ultrasonication on an ice bath for 5 minutes, the mixture was stored at 4°C in the dark.
[0041] (2) Preparation of hydrogel: Weigh 3 g of hydroxyethyl cellulose and disperse it in 30 mL of sodium hydroxide solution. Add 3 mL of epichlorohydrin, stir at room temperature for 1 h, stand for crosslinking for 8 h, then wash with distilled water, soak for 12 h, and finally freeze-dry to obtain hydroxyethyl cellulose hydrogel.
[0042] (3) Preparation of liposome hydrogel: 20 mL of the liposome emulsion prepared in step (1) was added to 1 g of the hydrogel prepared in step (2), and the supernatant was completely absorbed to obtain drospirenone liposome cellulose hydrogel.
[0043] Example 4
[0044] (1) Preparation of drospirenone liposomes: Drospirenone liposome inclusion complexes were prepared by ethanol injection method. Soy lecithin, drospirenone drug, and cholesterol were dissolved in 10 mL of anhydrous ethanol solution at a ratio of 8:2:1 and fully dissolved and mixed in a 30°C water bath to form an oil phase. The solution was then slowly injected into phosphate buffered saline (PBS, pH 7.4) with a syringe and stirred continuously, and the remaining ethanol was evaporated at 50°C. After ultrasonication on an ice bath for 5 minutes, the mixture was stored at 4°C in the dark.
[0045] (2) Preparation of hydrogel: Weigh 3 g of hydroxyethyl cellulose and disperse it in 30 mL of sodium hydroxide solution. Add 4 mL of epichlorohydrin, stir at room temperature for 1 h, let stand for crosslinking for 8 h, then wash with distilled water, soak for 12 h, and finally freeze-dry to obtain hydroxyethyl cellulose hydrogel.
[0046] (3) Preparation of liposome hydrogel: 25 mL of the liposome emulsion prepared in step (1) was added to 1 g of the hydrogel prepared in step (2), and the supernatant was completely absorbed to obtain drospirenone liposome cellulose hydrogel.
[0047] 1. Determination of drug encapsulation efficiency
[0048] The photometric measurement of drospirenone liposomes was performed by ultraviolet spectrophotometry to calculate the encapsulation efficiency. The specific operation steps are as follows:
[0049] (1) Determination of the maximum absorption wavelength: Accurately pipette 5 mL of the drospirenone reference stock solution into a 25 mL volumetric flask, dilute to the mark with methanol, shake well, use methanol as a blank, scan the wavelength in the range of 200-400 nm, and measure its maximum absorption wavelength. Then, measure the absorbance of the blank liposomes and the drospirenone liposomes after demulsification at the maximum absorption wavelength (263 nm) to study the effect of the blank liposomes and unencapsulated drospirenone on the detection of encapsulated drospirenone.
[0050] (2) Preparation of drospirenone calibration curve: prepare gradient solutions with concentrations of 2, 4, 8, 12, and 16 mg / L, measure their absorbances respectively, with the ordinate being the absorbance and the abscissa being the drospirenone concentration, draw a standard curve and perform linear fitting.
[0051] (3) Determination of encapsulation efficiency: The drospirenone liposome emulsions with different drug-lipid ratios were centrifuged at 7000-10000 r / min for 8-10 min, and the supernatant (liposome inclusion complex) was taken and fixed to volume in a 25 mL volumetric flask for photometric measurement. The encapsulation efficiency of drospirenone in the liposomes was calculated based on the drospirenone calibration curve. The calculation formula for the encapsulation efficiency of drospirenone in the liposomes is:
[0052]
[0053] The encapsulation efficiency of the liposomes obtained in Examples 1-4 of the present invention is as follows: Figure 2 As shown by Figure 2 It can be seen that as the ratio of soybean lecithin, drospirenone drug and cholesterol changes, the encapsulation rate also changes, and can reach up to 84%. This shows that the liposome prepared by the present invention can greatly improve the solubility of drospirenone.
[0054] 2. Drug release study
[0055] The in vitro release of drospirenone from drospirenone liposomes / cellulose hydrogels was studied by diffusion technique in dialysis bags using PBS buffer (0.1 mmol L -1 The release medium was 0.75 g of lyophilized hydrogel, after absorbing the aqueous solution of drospirenone liposome inclusion complex, was placed in a dialysis bag, clamped at the end with a clamp, and immersed in 230.0 mL of PBS buffer. The release was carried out at 37°C. 3.0 mL of PBS release solution was taken every hour, and 3.0 mL of new PBS buffer solution was added at the same time. The amount of drospirenone released was determined by UV spectrophotometry.
[0056]
[0057] The drug release process of the liposome hydrogels with different drug loadings obtained in Examples 1-4 of the present invention is as follows: Figure 3 As shown, Figure 3 As shown in the figure, the release trend of the drug in the liposome hydrogel system with different drug loadings is basically the same, and the sustained release rate of the drug gradually becomes flat from fast to slow. Calculation by the kinetic equation shows that the sustained release system can achieve a long-term release of more than half a year.
[0058] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A method for preparing a liposome / cellulose hydrogel carrying a large amount of water-soluble drospirenone, characterized in that: The following steps are involved: (1) Dissolve a certain amount of cellulose in a certain amount of distilled water, and add a certain amount of sodium hydroxide solution of a certain concentration after the solution is completely dissolved. Then add epichlorohydrin and seal the container for stirring. After stirring for a certain period of time, let it stand for cross-linking. Then wash, soak, and freeze-dry to obtain a hydrogel; (2) placing the weighed dry hydrogel in the centrifuged liposome emulsion, and waiting for the supernatant to be completely absorbed to obtain the drospirenone liposome / cellulose hydrogel.
2. The preparation method according to claim 1, characterized in that: Preparation method of liposome emulsion described in step (2) The following steps are involved: Weigh phospholipids, drospirenone drugs, and cholesterol according to a certain mass ratio, add a certain volume of anhydrous ethanol solution, fully dissolve and mix in a 30-40°C water bath to form an oil phase, then measure a certain volume of PBS in a magnetic stirrer to form an aqueous phase, inject the prepared oil phase into the aqueous phase evenly and quickly with a syringe, stir for a certain time in a water bath at a certain temperature to completely evaporate the ethanol, ultrasonicate in an ice bath on an ultrasonicator for a certain time, and then store at 4°C away from light.
3. The preparation method according to claim 2, characterized in that: The phospholipid is soybean lecithin; the mass ratio of the phospholipid to cholesterol is 1:0-6:1; the drug-lipid ratio is 0.25:1-5:1; the volume ratio of the organic phase to the aqueous phase is 1:5-1:10; the hydration temperature is 30-80°C; the hydration time is 0.5-1.5h, and the ultrasonic time is 5-15min.
4. The preparation method according to any one of claims 1 to 3, characterized in that: The cellulose in step (1) is one or more of hydroxyethyl cellulose, carboxymethyl cellulose and sodium alginate.
5. The preparation method according to any one of claims 1 to 3, characterized in that: The volume of epichlorohydrin in step (1) is 1.5-5.5 mL, the concentration of the sodium hydroxide solution is 0.2%-1%, and the volume of distilled water is 2.5-10 mL.
6. The preparation method according to any one of claims 1 to 3, characterized in that: In step (1), cellulose is dispersed in distilled water, and then sodium hydroxide solution is added, stirred at room temperature for 0.5 to 1.5 hours, and then the resulting mixed solution is allowed to stand at room temperature for 8 to 24 hours, and then washed with water and soaked for 12 to 24 hours to obtain a hydrogel.
7. The preparation method according to any one of claims 1 to 3, characterized in that: The liposome emulsion of step (2) and The mass ratio of the hydrogel is 20:1 to 60:
1.
8. The liposome hydrogel prepared by the preparation method according to any one of claims 1 to 7.
9. Use of the liposome hydrogel according to claim 8 in a drug carrier.