Temperature-sensitive liposome hydrogel targeting controlled release drug carrier and its preparation method and application

Abstract

The application discloses a temperature-sensitive liposome hydrogel for targeting controllable release of a drug carrier and a preparation method and application thereof, and belongs to the field of liposome hydrogels.The method comprises the following steps: (1) dispersing polysaccharide in water, then adding polyvinyl alcohol, stirring uniformly, and then freezing / thawing the obtained mixed solution for multiple times to obtain a physically cross-linked hydrogel, and then eluting and soaking the hydrogel to obtain the hydrogel; (2) uniformly oscillating a liposome emulsion and the hydrogel, and obtaining the liposome hydrogel through centrifugation and freeze-drying.The application solves the problems that the liposome as a drug carrier is influenced by external environment and temperature conditions, and the targeting characteristics for some diseases are not ideal, and the phenomena of drug leakage and poor effect are caused.The application also realizes the effects of controllable release of a drug and improvement of drug bioavailability, and has a higher drug release amount at a temperature of 42 DEG C.

Description

Technical Field

[0001] This invention belongs to the field of liposome hydrogels, and specifically relates to a temperature-sensitive liposome hydrogel for targeted and controllable release of drug carriers, its preparation method, and its application. Background Technology

[0002] Liposomes are spherical vesicles formed by a lipid bilayer. The membrane material is mainly natural lipid molecules such as lecithin and cholesterol, with a particle size between 1 nm and 10 μm. Liposomes possess excellent properties such as good biocompatibility, non-toxicity, sustained-release effect, and stimulus responsiveness, and are therefore widely used as drug carriers. However, when used as drug carriers, liposomes are easily affected by external environmental conditions such as pH and temperature, and have drawbacks such as unsatisfactory targeting characteristics for some diseases, leading to phenomena such as aggregation, drug leakage, and poor efficacy, which seriously limit their application.

[0003] Hydrogels are hydrophilic three-dimensional network structures capable of absorbing large amounts of water and retaining it within a matrix without dissolving. They possess advantages such as good biocompatibility, biodegradability, and soft tissue similarity, attracting widespread attention in the food industry, drug delivery systems, and tissue repair. Integrating natural polymer chains with liposomes through adsorption and hydrophobic interactions can enhance the stability of embedded liposomes, forming a dual barrier for drug release. The presence of hydrogels maintains the integrity and stability of liposomes, while liposomes can provide the sustained-release and responsive release effects of hydrogels, thus avoiding burst release effects, increasing local drug concentration, reducing side effects, and improving efficacy, thereby expanding the application range of liposomes. Patent application CN114191603 A discloses a method for preparing a composite liposome hydrogel medical dressing. This hydrogel medical dressing has multiple functions, including wound healing and scar repair, good mechanical properties, strong drug release capacity, natural antibacterial and anti-inflammatory properties, and biodegradability. However, the preparation process of this hydrogel is complex, requiring the addition of cross-linking agents and catalysts, and the resulting liposome hydrogel is only suitable for external wound healing. Patent application CN114767837 A discloses an oral insulin liposome hydrogel with pH-responsive characteristics, which can prevent the large-scale release of insulin under acidic conditions, promote intestinal permeation and absorption, and control blood sugar for a longer period. However, this invention only solves the technical problems of oral insulin being easily degraded, poorly absorbed, and having low bioavailability; its versatility is poor, and its application scope is limited. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a temperature-sensitive liposome hydrogel for targeted and controllable drug release, its preparation method, and its application. The liposome hydrogel prepared by the present invention can encapsulate various water-soluble drugs, achieving controlled drug release and improved drug bioavailability according to the application scenario, and exhibits a high drug release rate at 42°C.

[0005] The objective of this invention is achieved through the following scheme.

[0006] A method for preparing a temperature-sensitive liposome hydrogel with targeted and controllable drug release includes the following steps:

[0007] (1) Disperse polysaccharides in water, then add polyvinyl alcohol, stir evenly, and freeze / thaw the resulting mixed solution multiple times to obtain a physically cross-linked hydrogel. Then wash and soak to obtain a hydrogel.

[0008] (2) The liposome emulsion and the hydrogel were shaken evenly, and then centrifuged and freeze-dried to obtain the liposome hydrogel.

[0009] Preferably, the method for preparing the liposome emulsion in step (2) includes the following steps:

[0010] Nitrogen gas was used to purge the phospholipid mother liquor and cholesterol mother liquor at a constant speed until a uniform film was formed. The residual solvent was then removed by dialysis. The water-soluble drug solution was then added to the film. The resulting liposome emulsion was placed in 35-45°C for hydration to obtain the liposome emulsion.

[0011] The solvents in the phospholipid mother liquor and cholesterol mother liquor are a mixture of chloroform and methanol.

[0012] Preferably, the phospholipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), or dipalmitoylphosphatidylglycerol (DPPG), with DPPC being more preferred.

[0013] The volume ratio of phospholipids to cholesterol is 1:1 to 9:1.

[0014] The volume ratio of chloroform to methanol is 2:1 to 4:1; the concentration of the water-soluble drug solution is 0.01 to 0.05 M.

[0015] The water-soluble drug is 5(6)-carboxyfluorescein.

[0016] The hydration time is 12–24 hours.

[0017] Preferably, the polysaccharide in step (1) is one or more of xanthan gum, gellan gum and sodium alginate, with xanthan gum being more preferred.

[0018] Preferably, the mass ratio of the polysaccharide and polyvinyl alcohol in step (1) is 1:5 to 1:15.

[0019] Preferably, in step (1), the polysaccharide is dispersed in water, then polyvinyl alcohol is added, and the mixture is stirred at room temperature for 1-2 hours, then stirred at 4°C for 20-30 minutes. The resulting mixed solution is then frozen at -20°C for 18-24 hours, and then thawed at room temperature for 3-5 hours. This freezing / thawing cycle is repeated multiple times to obtain a physically cross-linked hydrogel. The hydrogel is then washed with water and soaked for 12-24 hours to obtain the hydrogel.

[0020] Preferably, the mass ratio of the liposome emulsion to the hydrogel in step (2) is 10:1 to 50:1.

[0021] Preferably, the oscillation conditions in step (2) are: 25°C, 150-200 rpm, 30-60 min.

[0022] The liposome hydrogel prepared by any of the above preparation methods.

[0023] The above-described applications of liposome hydrogels in drug carriers.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) This invention solves the problems of drug leakage and poor efficacy caused by the influence of external temperature conditions on liposomes as drug carriers and the unsatisfactory targeting characteristics for some diseases.

[0026] (2) The hydrogel prepared by the present invention has a porous network structure with strong mechanical strength, which can realize the stability of embedded liposomes and form a dual barrier for drug release.

[0027] (3) The present invention achieves the effects of sustained release, controlled release and improved drug bioavailability. Combining liposomes and hydrogels reduces drug leakage and increases the amount of drug released and accumulated at the target site. It has a high drug release at a temperature of 42°C. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the preparation process of the present invention.

[0029] Figure 2 The images show the cytotoxicity of the liposome hydrogels obtained in Examples 1-4 and Comparative Examples 1-2 of this invention at different concentrations.

[0030] Figure 3 The graph shows the effect of different temperatures on drug release from the liposome hydrogels obtained in Examples 1-4 and Comparative Examples 1-2 of this invention. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0032] Example 1

[0033] (1) Preparation of blank liposomes: DPPC stock solution and cholesterol stock solution of 18 mg / mL were prepared using a chloroform and methanol (2:1, v:v) mixed solvent. The DPPC stock solution and cholesterol stock solution of 1:1 volume ratio were purged with nitrogen gas at a constant speed until a uniform film was formed. The residual chloroform and methanol were removed by dialysis.

[0034] (2) Loading water-soluble drugs: Take 10 mL of 0.01 M 5(6)-carboxyfluorescein and add it to the film in step (1) to obtain liposome emulsion;

[0035] (3) The liposome emulsion obtained in step (2) was placed in a 40℃ constant temperature oven for 12 hours to hydrate, and stored at 4℃ for later use.

[0036] (4) Preparation of hydrogel: Weigh 1g xanthan gum and disperse it in 100mL of water, add 5g polyvinyl alcohol, stir at room temperature for 1h, continue stirring at 4℃ for 30min, then place the mixed solution at -20℃ to freeze, then thaw at room temperature, repeat the freeze / thaw cycle multiple times to obtain physically cross-linked hydrogel, then wash with distilled water and soak for 12h.

[0037] (5) Preparation of liposome hydrogel: Weigh 10g of the liposome emulsion prepared in step (3) and add it to 1g of the hydrogel prepared in step (4). Shake at 25℃, 150rpm for 30min for 1h. After centrifugation and freeze drying, obtain liposome hydrogel.

[0038] Example 2

[0039] (1) Preparation of blank liposomes: DPPC stock solution and cholesterol stock solution of 18 mg / mL were prepared using a mixture of chloroform and methanol (2:1, v:v). The DPPC stock solution and cholesterol stock solution of 3:1 volume ratio were purged with nitrogen gas at a constant speed until a uniform film was formed. The residual chloroform and methanol were removed by dialysis.

[0040] (2) Loading water-soluble drugs: Take 15 mL of 0.02 M 5(6)-carboxyfluorescein and add it to the film in step (1) to obtain liposome emulsion;

[0041] (3) The liposome emulsion obtained in step (2) was placed in a 40℃ constant temperature oven for 24 hours to hydrate, and stored at 4℃ for later use.

[0042] (4) Preparation of hydrogel: Weigh 1g xanthan gum and disperse it in 100mL of water, add 10g polyvinyl alcohol, stir at room temperature for 1h, continue stirring at 4℃ for 30min, then place the mixed solution at -20℃ to freeze, then thaw at room temperature, repeat the freeze / thaw cycle multiple times to obtain physically cross-linked hydrogel, then wash with distilled water and soak for 12h.

[0043] (5) Preparation of liposome hydrogel: Weigh 20g of the liposome emulsion prepared in step (3) and add it to 1g of the hydrogel prepared in step (4). Shake at 25℃, 170rpm for 40min for 1h. After centrifugation and freeze drying, obtain liposome hydrogel.

[0044] Example 3

[0045] (1) Preparation of blank liposomes: DPPC stock solution and cholesterol stock solution of 18 mg / mL were prepared using a mixture of chloroform and methanol (2:1, v:v). The DPPC stock solution and cholesterol stock solution of 9:1 volume ratio were purged with nitrogen gas at a constant speed until a uniform film was formed. The residual chloroform and methanol were removed by dialysis.

[0046] (2) Loading water-soluble drugs: Take 20 mL of 0.04 M 5(6)-carboxyfluorescein and add it to the film in step (1) to obtain liposome emulsion;

[0047] (3) The liposome emulsion obtained in step (2) was placed in a 40℃ constant temperature oven for 12 hours to hydrate, and stored at 4℃ for later use.

[0048] (4) Preparation of hydrogel: Weigh 1g xanthan gum and disperse it in 100mL of water, add 15g polyvinyl alcohol, stir at room temperature for 1h, continue stirring at 4℃ for 30min, then place the mixed solution at -20℃ to freeze, then thaw at room temperature, repeat the freeze / thaw cycle multiple times to obtain physically cross-linked hydrogel, then wash with distilled water and soak for 12h.

[0049] (5) Preparation of liposome hydrogel: Weigh 40g of the liposome emulsion prepared in step (3) and add it to 1g of the hydrogel prepared in step (4). Shake at 25℃, 180rpm for 50min for 1h. After centrifugation and freeze drying, obtain liposome hydrogel.

[0050] Example 4

[0051] (1) Preparation of blank liposomes: DPPC stock solution and cholesterol stock solution of 18 mg / mL were prepared using a mixture of chloroform and methanol (2:1, v:v). The DPPC stock solution and cholesterol stock solution of 3:1 volume ratio were purged with nitrogen gas at a constant speed until a uniform film was formed. The residual chloroform and methanol were removed by dialysis.

[0052] (2) Loading water-soluble drugs: Take 15 mL of 0.05 M 5(6)-carboxyfluorescein and add it to the film in step (1) to obtain liposome emulsion;

[0053] (3) The liposome emulsion obtained in step (2) was placed in a 40℃ constant temperature oven for 24 hours to hydrate, and stored at 4℃ for later use.

[0054] (4) Preparation of hydrogel: Weigh 1g xanthan gum and disperse it in 100mL of water, add 15g polyvinyl alcohol, stir at room temperature for 1h, continue stirring at 4℃ for 30min, then place the mixed solution at -20℃ to freeze, then thaw at room temperature, repeat the freeze / thaw cycle multiple times to obtain physically cross-linked hydrogel, then wash with distilled water and soak for 12h.

[0055] (5) Preparation of liposome hydrogel: Weigh 50g of the liposome emulsion prepared in step (3) and add it to 1g of the hydrogel prepared in step (4). Shake at 25℃, 200rpm for 60min for 1h. After centrifugation and freeze drying, obtain liposome hydrogel.

[0056] Comparative Example 1

[0057] (1) Preparation of blank liposomes: DPPC stock solution and cholesterol stock solution of 18 mg / mL were prepared using a mixture of chloroform and methanol (2:1, v:v). The DPPC stock solution and cholesterol stock solution of 3:1 volume ratio were purged with nitrogen gas at a constant speed until a uniform film was formed. The residual chloroform and methanol were removed by dialysis.

[0058] (2) Loading water-soluble drugs: Take 15 mL of 0.05 M 5(6)-carboxyfluorescein and add it to the film in step (1) to obtain liposome emulsion;

[0059] (3) The liposome emulsion obtained in step (2) was placed in a 40°C constant temperature incubator for 24 hours to hydrate, and then stored at 4°C for later use.

[0060] Comparative Example 2

[0061] (1) Preparation of blank liposomes: DPPC stock solution and cholesterol stock solution of 18 mg / mL were prepared using a mixture of chloroform and methanol (2:1, v:v). The DPPC stock solution and cholesterol stock solution of 3:1 volume ratio were purged with nitrogen gas at a constant speed until a uniform film was formed. The residual chloroform and methanol were removed by dialysis.

[0062] (2) Loading water-soluble drugs: Take 15 mL of 0.05 M 5(6)-carboxyfluorescein and add it to the film in step (1) to obtain liposome emulsion;

[0063] (3) The liposome emulsion obtained in step (2) was placed in a 40℃ constant temperature oven for 24 hours to hydrate, and stored at 4℃ for later use.

[0064] (4) Preparation of hydrogel: Weigh 1g xanthan gum and disperse it in 100mL of water. Stir at room temperature for 1h, continue stirring at 4℃ for 30min, then place the xanthan gum dispersion at -20℃ and freeze it at room temperature. Repeat the freezing / thawing cycle multiple times to obtain hydrogel, then wash with distilled water and soak for 12h.

[0065] (5) Preparation of liposome hydrogel: Weigh 50g of the liposome emulsion prepared in step (3) and add it to 1g of the hydrogel prepared in step (4). Shake at 25℃, 200rpm for 60min for 1h. After centrifugation and freeze drying, obtain liposome hydrogel.

[0066] 1. Cytotoxicity assay

[0067] The cytotoxic effect of liposome hydrogels was determined using methylene blue staining. HepG2 cells with a confluence density of 90% and in good condition were digested, centrifuged, stained with trypan blue, and counted using a hemocytometer under an inverted microscope. They were then seeded into 96-well plates (4 × 10⁶ cells / well). 4 Cells / well. After 24 h of incubation, the culture medium was discarded, and cells were washed with PBS. Then, 100 μL of liposome hydrogels (diluted with complete culture medium) at different concentration gradients (0, 120, 240, 360, and 480 mg / L) were added to each well. 100 μL of complete culture medium was added to the blank control group, and the cells were incubated at 37 °C. After incubation, the culture medium was discarded, and cells were washed with PBS. Immediately afterwards, 50 μL of methylene blue staining solution was added to each well, and the cells were incubated at 37 °C for another 1 h. After the reaction, the plates were rinsed with water, dried, and tapped on absorbent paper until no significant moisture remained. 100 μL of EB buffer was added, and the plates were shaken for 15 min to dissolve the methylene blue staining solution bound to the cells. The absorbance of each well was measured at 570 nm. Cell viability was calculated using the OD values ​​for each group. The calculation method is as follows:

[0068]

[0069] If the sample inhibits cell activity by ≥10%, it is considered that the sample will be toxic to cells at concentrations exceeding this level, and the toxicity range of the sample concentration will be determined.

[0070] The cytotoxicity assay results of the liposome hydrogels obtained in Examples 1-4 and Comparative Examples 1-2 at different concentrations are as follows: Figure 2 As shown. By Figure 2 It can be seen that, within the experimental concentration range of 0–480 mg / L, the cell viability of the liposome hydrogels in Examples 1–4 and Comparative Examples 1–2 all exceeded 96%, indicating that the liposome hydrogels prepared in this invention are non-toxic.

[0071] 2. Determination of drug release

[0072] 150 mg of liposome hydrogel was added to 150 mL of PBS buffer solution and shaken in shakers at 25 °C, 42 °C and 60 °C for 3 h. The concentration of 5(6)-carboxyfluorescein released was measured using a fluorescence spectrophotometer, and the amount of 5(6)-carboxyfluorescein released by the liposome hydrogel at different temperatures was calculated.

[0073]

[0074] In the formula: Q e C represents the amount of drug released (mg / g). e V represents the drug concentration (mg / L) before release. e C represents the volume of the drug before release. O V represents the concentration of the drug after release (mg / L). O M represents the volume of the drug after adsorption, and M represents the mass (g) of the added liposome hydrogel.

[0075] The effects of different temperatures on drug release from the liposome hydrogels obtained in Examples 1-4 and Comparative Examples 1-2 of this invention are as follows: Figure 3 As shown. By Figure 3 It can be seen that the drug release amount of the liposome hydrogel at different temperatures is: 42℃>60℃>25℃, indicating that the liposome hydrogel prepared in this invention has a high drug release amount at a temperature of 42℃, and belongs to a temperature-sensitive liposome hydrogel.

[0076] 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 a temperature-sensitive liposome hydrogel with targeted and controllable drug release, characterized in that, Includes the following steps: (1) Disperse polysaccharides in water, then add polyvinyl alcohol, stir evenly, and freeze / thaw the resulting mixed solution multiple times to obtain a physically cross-linked hydrogel. Then wash and soak to obtain a hydrogel. (2) The liposome emulsion and the hydrogel were shaken evenly, and then centrifuged and freeze-dried to obtain the liposome hydrogel; The polysaccharide mentioned in step (1) is one or more of xanthan gum, gellan gum, and sodium alginate; The method for preparing the liposome emulsion in step (2) includes the following steps: The phospholipid mother liquor and cholesterol mother liquor were purged with nitrogen gas at a constant speed until a uniform film was formed. The residual solvent was then removed by dialysis. The water-soluble drug solution was then added to the film. The resulting liposome emulsion was placed in 35-45°C for hydration to obtain a liposome emulsion. The solvents in the phospholipid mother liquor and cholesterol mother liquor were a mixture of chloroform and methanol. The mass ratio of polysaccharide to polyvinyl alcohol in step (1) is 1:5 to 1:15; The mass ratio of the liposome emulsion to the hydrogel in step (2) is 10:1 to 50:

1.

2. The preparation method according to claim 1, characterized in that, The phospholipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, or dipalmitoylphosphatidylglycerol; the volume ratio of the phospholipid to cholesterol is 1:1 to 9:1; the volume ratio of chloroform to methanol is 2:1 to 4:1; the concentration of the water-soluble drug solution is 0.01 to 0.05 M; the water-soluble drug is 5(6)-carboxyfluorescein; and the hydration time is 12 to 24 h.

3. The preparation method according to any one of claims 1-2, characterized in that, In step (1), polysaccharides are dispersed in water, polyvinyl alcohol is added, and the mixture is stirred at room temperature for 1-2 h, then stirred at 4 ℃ for 20-30 min. The resulting mixed solution is then frozen at -20 ℃ for 18-24 h, and then thawed at room temperature for 3-5 h. This freezing / thawing cycle is repeated multiple times to obtain a physically cross-linked hydrogel. The hydrogel is then washed with water and soaked for 12-24 h to obtain the hydrogel.

4. The preparation method according to any one of claims 1-2, characterized in that, The oscillation conditions described in step (2) are: 25℃, 150~200 rpm, 30~60 min.

5. A liposome hydrogel prepared by the preparation method according to any one of claims 1-4.

6. The use of the liposome hydrogel according to claim 5 in the preparation of drug carriers.

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