Preparation and application of fat targeted cationic liposome

Abstract

The invention discloses a preparation method of a fat-targeted cationic liposome, which is characterized in that the liposome contains auxiliary phospholipid, cationic lipid and cholesterol, and the preparation method comprises the following steps: (1) preparing the auxiliary phospholipid, the cationic lipid and the cholesterol into the liposome through a film hydration method; and (2) adding deionized water into the lipidosome formed in the step (1) to dilute the lipidosome to a specific concentration, and removing the free material by a dialysis method to obtain the fat-targeted cationic lipidosome. The preparation method has the advantages that materials are easy to obtain, non-toxic, environmentally friendly and biodegradable, good safety and biocompatibility are achieved, the adipose tissue and cell specific targeting capacity is achieved, hydrophilic and hydrophobic drugs can be loaded through a hydrophobic shell layer or a hydrophilic core, and the universality of drug entrapment is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to the preparation and application of fat-targeted cationic liposomes. Background Art

[0002] Liposomes are bilayer structures spontaneously formed by lipid molecules or lipids in water. They have a structure similar to that of biological cells, so they have good biocompatibility and achieve targeted or efficient drug delivery. Ideal liposomes have good tissue compatibility, low toxicity, suitable drug encapsulation and release capabilities, and are widely used in many fields such as anti-tumor, antibacterial, anti-inflammatory drug carriers and vaccine carriers.

[0003] Obesity and overweight are causing a surge in global health challenges, with serious complications beyond obesity itself, including diabetes and cardiovascular disease, two major causes of death. Obesity is directly caused by the expansion of white adipose tissue (WAT) due to the formation and growth of adipocytes. During the development of obesity, the expansion of WAT is accompanied by an increased production of the extracellular matrix (ECM), which contains glycosaminoglycans, which are the most negatively charged biomacromolecules known.

[0004] Therefore, targeting white fat can effectively improve obesity, but the higher metabolism and off-target effects of drugs in the liver result in some clinical therapeutic drugs having nonspecific effects on adjacent tissues after being injected into local adipose tissue, which often leads to serious side effects. In addition, since the drugs cannot be retained in adipose tissue for a long time, they are quickly circulated to other tissues.

[0005] Therefore, it is of great research value to develop a liposome with good biocompatibility and the ability to specifically target adipose tissue and efficiently deliver drugs. Summary of the invention

[0006] The present invention provides a method for preparing fat-targeted cationic liposomes. The liposomes utilize a phospholipid bilayer structure similar to biological cells and have advantages such as good water solubility and biocompatibility. After the cationic lipid DOTAP is incorporated, the liposomes have the function of specifically targeting the extracellular matrix of adipose tissue.

[0007] The liposome can be used as a drug carrier. It has the characteristics of a hydrophobic shell and a hydrophilic core, and can be widely used for the encapsulation of various hydrophilic and hydrophobic drugs. Furthermore, the liposome can be used as a drug carrier for fat-targeted therapy and is widely used in the treatment field.

[0008] The technical solution provided by the present invention is: a cationic liposome for fat targeting: the liposome comprises auxiliary phospholipids, cholesterol, and cationic lipid (2,3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), and the preparation method comprises the following steps:

[0009] (1) preparing liposomes by thin film hydration method using auxiliary phospholipids, cationic lipids and cholesterol;

[0010] (2) adding deionized water to the liposomes formed in step (1) to dilute to a specific concentration, and removing free materials by dialysis to obtain fat-targeted cationic liposomes.

[0011] Furthermore, the organic solvent in step (1) is one of chloroform, methanol, ethanol and dichloromethane, preferably chloroform.

[0012] Furthermore, the auxiliary phospholipid in step (1) is DSPC.

[0013] Furthermore, the cationic lipid in step (2) is (2,3-dioleyloxypropyl)trimethylammonium chloride, namely DOTAP.

[0014] Furthermore, the feed ratio of DSPC to DOTAP in steps (1) and (2) is 3:5.

[0015] Furthermore, in step (1), the feed ratio of cationic lipid, cholesterol and auxiliary phospholipid is 5:2:3.

[0016] Furthermore, the thin film hydration method described in step (1) specifically comprises the following steps: dissolving auxiliary phospholipids, cationic lipids and cholesterol in chloroform respectively and mixing them, forming a uniform thin film by rotary evaporation, adding deionized water for hydration at a temperature of 40° C. for a hydration time of 30 to 120 min, and performing ice bath probe ultrasound after hydration at a power of 125 to 175 W for a time of 10 to 15 min.

[0017] Furthermore, the hydration time was 30 min, and the ultrasonic power of the ice bath probe was 150 W for 10 min.

[0018] Application of fat-targeted cationic liposomes as drug carriers.

[0019] Advantages of the present invention:

[0020] (1) The fat-targeted cationic liposomes of the present invention are readily available, non-toxic, environmentally friendly, biodegradable, and have good safety and biocompatibility.

[0021] (2) The fat-targeted cationic liposomes of the present invention have the ability to specifically target fat tissue and cells, and can release drugs in fat tissue and cells, effectively increasing the accumulation of drugs in fat areas.

[0022] (3) The fat-targeted cationic liposomes of the present invention can load hydrophilic and hydrophobic drugs through the hydrophobic shell or the hydrophilic core, which greatly improves the universality of drug encapsulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison diagram of the hydrated particle sizes of the liposomes and fat-targeted liposomes in Example 1;

[0024] Figure 2 This is a comparison chart of the in vitro fat targeting ability of Lip-ATS;

[0025] Figure 3 The results of Lip-DOTAP's ability to target body fat at different times are shown in FIG. DETAILED DESCRIPTION

[0026] Example 1

[0027] This embodiment provides a method for preparing fat-targeted cationic liposomes, and method (1) is as follows:

[0028] Preparation of cationic liposomes for lipid targeting:

[0029] (1) preparing liposomes by thin film hydration method using auxiliary phospholipids, cationic lipids and cholesterol;

[0030] (2) adding deionized water to the liposomes formed in step (1) to dilute to a specific concentration, and removing free materials by dialysis to obtain fat-targeted cationic liposomes.

[0031] In the above liposome preparation process, the organic solvent described in method (1) is one of chloroform, methanol, ethanol or dichloromethane, preferably chloroform, and the phospholipids include cationic lipids, cholesterol, and auxiliary phospholipids in a feed ratio of 5:2:3.

[0032] In method (1), the hydration temperature is 40° C., the hydration time is 30 to 120 min, preferably 30 min, the ultrasonic power of the ice bath probe is 125 to 175 W, the time is 10 to 15 min, preferably 150 W, 10 min.

[0033] Specifically, 25 mg, 10 mg, and 15 mg of DOTAP, cholesterol, and DSPC were weighed and placed in a round-bottom flask, dissolved with 7 mL of chloroform, and then the organic solvent was removed by rotary evaporation in a water bath. After the solvent was completely evaporated, a uniform film was formed at the bottom of the round-bottom flask. After adding 5 mL of deionized water, the flask was placed in a 40° C. water bath for 30 minutes to fully hydrate the film to form a liposome solution. After being dialyzed by ice bath probe ultrasound (150 W, 10 minutes), the free dialysis bag specifications included 2000-3500 MW. The fat-targeted cationic liposomes were obtained by dialysis overnight.

[0034] Example 2

[0035] This example provides the preparation of common liposomes used as a control, method (2) is as follows:

[0036] (1) The auxiliary phospholipid, cholesterol, and DOTAP are dissolved in an organic solvent respectively and mixed, and a uniform lipid film is formed by rotary evaporation, and deionized water is added for hydration. After hydration, cationic liposomes (Lip-DOTAP) are formed by ice bath probe ultrasound.

[0037] (2) The auxiliary phospholipids and cholesterol are dissolved in organic solvents respectively and mixed, and a uniform lipid film is formed by rotary evaporation, and deionized water is added for hydration. After hydration, ordinary liposomes (Lip-DSPC) are formed by ice bath probe ultrasound.

[0038] The organic solvent described in method (2) is selected from chloroform, methanol, ethanol or dichloromethane, preferably chloroform. The phospholipids include auxiliary phospholipids and cholesterol in a feed ratio of 4:1.

[0039] Furthermore, in method (2), the hydration temperature is 37° C., the hydration time is 30 to 120 min, preferably 30 min, the ultrasonic power of the ice bath probe is 100 to 150 W, and the time is 10 to 15 min, preferably 100 W for 10 min.

[0040] Specifically, DSPC and cholesterol, 40 mg and 10 mg, were weighed and placed in a round-bottom flask, dissolved with 5 mL of chloroform, and then the organic solvent was removed by rotary evaporation in a water bath, and a uniform film was formed at the bottom of the round-bottom flask after the solvent was completely evaporated, and 5 mL of deionized water was added and placed in a 37° C. water bath for 30 min, and the film was fully hydrated to fall off to form a liposome solution, and then dialyzed to remove free liposomes by ice bath probe ultrasound (100 W, 10 min), and the specifications of the dialysis bag for removing free liposomes included 2000-3500 MW, and dialyzed overnight to obtain fat-targeted cationic liposomes.

[0041] Liposome size

[0042] The hydrated particle sizes of the liposomes (Lip-DOTAP) prepared by method (1) in Example 1 and the fat-targeted liposomes (Lip-DSPC) prepared by method (2) in Example 2 are similar. The Zeta potential of the cationic liposomes is +33.9 mV, while the Zeta potential of the ordinary liposomes used for comparison is -2.3 mV. It can be seen from PDI that both can be evenly distributed in deionized water. Figure 1 .

[0043] In vitro fat targeting ability of Lip-ATS

[0044] Flow cytometry experiments were performed to investigate the targeting ability of fat-targeted liposomes (Lip-DOTAP) to mature adipocytes (3T3-L1).

[0045] In step (1) of Example 1, 1 mg of fluorescent agent FITC was added to a round-bottom flask and encapsulated into the hydrophobic shell of the liposomes. Subsequently, the same step (2) was performed to finally prepare the FITC-encapsulated fat-targeted cationic liposomes (FITC@Lip-DOTAP). The same liposome preparation method as in Example 1 was used to prepare ordinary liposomes (FITC@Lip-DSPC) encapsulating only FITC.

[0046] Preadipocytes were inoculated into 6-well plates and induced to adipogenesis to obtain mature adipocytes. Equal amounts of FITC, FITC@Lip-DOTAP, and FITC@Lip-DSPC were added to the mature adipocytes, respectively, and incubated for 4 hours at 37°C, 5% CO2, and saturated humidity in the dark. The free drugs were washed with PBS and resuspended in the microplate. Figure 2 .

[0047] Figure 2 The results showed that liposomes loaded with cationic lipids (FITC@Lip-DOTAP) had a higher ability to target mature adipocytes, but a weaker ability to target pre-adipocytes. The reason is that compared with mature adipocytes, the extracellular matrix of pre-adipocytes did not expand significantly and carried less negative charge.

[0048] In vivo fat targeting ability of Lip-DOTAP

[0049] Fluorescence analysis of ex vivo tissues was performed using in vivo imaging technology to investigate the fat-targeting ability and distribution of fat-targeted cationic liposomes in vivo.

[0050] In step (1) of Example 1, 4 mg of fluorescent agent DiD was added to a round-bottom flask and encapsulated into the hydrophobic shell of the liposome. Subsequently, the same method as step (2) was used to prepare two types of liposomes (DiD@DOT-Lip and DiD@DS-Lip) encapsulating DiD by adding or not adding cationic lipids.

[0051] C57 BL / 6J mice were fed a 60% high-fat diet to obtain an obese mouse model. DiD@Lip-DOTAP and DiD@DSPC were injected with equal amounts of DiD via intraperitoneal injection. Epididymal fat, perirenal fat, mesenteric fat, inguinal fat, heart, liver, spleen, lung, and kidney were collected and photographed at 8h, 12h, and 24h after injection to observe their fluorescence intensity. The results are shown in Figure 3 .

[0052] Figure 3 The results showed that the liposomes doped with cationic lipids had higher fluorescence intensity in adipose tissue at 4h, 8h, 12h and 24h compared with the undoped liposomes, that is, the fat-targeted liposomes had better fat-targeting ability and could accumulate more in adipose tissue.

[0053] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for preparing fat-targeted cationic liposomes, characterized in that: The liposome comprises auxiliary phospholipids, cationic lipids and cholesterol, and the preparation method comprises the following steps: (1) preparing liposomes by thin film hydration method using auxiliary phospholipids, cationic lipids and cholesterol; (2) adding deionized water to the liposomes formed in step (1) to dilute to a specific concentration, and removing free materials by dialysis to obtain fat-targeted cationic liposomes.

2. The preparation of cationic liposomes for fat targeting according to claim 1, characterized in that: The organic solvent described in step (1) is one of chloroform, methanol, ethanol and dichloromethane.

3. The preparation of cationic liposomes for fat targeting according to claim 1, characterized in that: The organic solvent described in step (1) is chloroform.

4. The preparation of cationic liposomes for fat targeting according to claim 1, characterized in that: The auxiliary phospholipid in step (1) is DSPC.

5. The preparation of fat-targeted cationic liposomes according to claim 1, characterized in that: The cationic lipid in step (2) is DOTAP.

6. The preparation of cationic liposomes for fat targeting according to claim 1, characterized in that: The feed ratio of DSPC to DOTAP in steps (1) and (2) is 3:

5.

7. The preparation of fat-targeted cationic liposomes according to claim 1, characterized in that: In step (1), the feed ratio of cationic lipid, cholesterol and auxiliary phospholipid is 5:2:

3.

8. The preparation of cationic liposomes for fat targeting according to claim 1, characterized in that: The specific steps of the thin film hydration method described in step (1) are as follows: the auxiliary phospholipid, cationic lipid and cholesterol are dissolved in chloroform respectively and mixed, a uniform thin film is formed by rotary evaporation, deionized water is added for hydration at a temperature of 40° C. for a hydration time of 30 to 120 min, and after the hydration is completed, ice bath probe ultrasound is performed at a power of 125 to 175 W for a time of 10 to 15 min.

9. The preparation of cationic liposomes for fat targeting according to claim 7, characterized in that: The hydration time was 30 min, the ultrasonic power of the ice bath probe was 150 W, and the time was 10 min.

10. Use of the fat-targeted cationic liposomes prepared according to the preparation method of claims 1 to 9 as a drug carrier.

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