Fat-targeting liposome, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of pharmaceutical preparations, and discloses a fat-targeting liposome, and a preparation method therefor and a use thereof. The liposome is composed of phospholipid, cholesterol, DSPE-PEG2k-MAL, and ATS peptide. The preparation method comprises: S1, preparing Lip-MAL by means of a thin-film hydration method; and S2, adding ATS to the Lip-MAL, and removing a free material by means of dialysis to obtain Lip-ATS. Provided is a use of the Lip-ATS in a drug carrier and in fat targeting. In the present invention, a phospholipid bilayer similar to a biological cell is linked to a fat-targeting peptide by means of maleimide, so that the phospholipid bilayer has the function of specifically targeting an adipocyte resistin receptor; the phospholipid bilayer is used as a drug carrier; and due to having the characteristics of a hydrophobic shell and a hydrophilic core, the phospholipid bilayer can be widely applied to the encapsulation of various hydrophilic and hydrophobic drugs, and can be used as a drug carrier for fat-targeted therapy.

Description

Fat targeting liposome and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a fat targeting liposome and preparation method and application thereof. BACKGROUND

[0002] Liposomes are bilayer structures formed spontaneously by lipid molecules or lipids in water, which have a similar structure to biological cells and thus have good biocompatibility, enabling effective targeted or efficient delivery of drugs. Ideal liposomes have good tissue compatibility, low toxicity, appropriate drug encapsulation and release capacity, and are widely used in drug carriers and vaccine carriers for anti-tumor, antibacterial, anti-inflammatory and other drugs.

[0003] Obesity has become one of the most concerned public health problems worldwide, which can cause insulin resistance, hyperglycemia, hypertension, hyperlipidemia and a series of related metabolic diseases, and has become an important risk factor for diabetes, cardiovascular disease and cancer. Related studies have found that adipose tissue is a metabolically active organ that plays a key role in regulating whole-body energy homeostasis, including food intake, glucose handling, insulin sensitivity, thermogenesis, and immune response regulation. Therefore, regulating the function of adipose tissue and cells can effectively improve obesity problems, but high-level liver metabolism of drugs and lack of target specificity often lead to serious side effects (including edema, local pain, bruising and numbness) in the clinical treatment of some drugs injected into local adipose tissue, resulting in treatment failure and even threatening the safety of patients' lives in severe cases.

[0004] Therefore, how to obtain a liposome with good biocompatibility and specific targeting of adipocytes for efficient drug delivery is one of the problems that needs to be solved by those skilled in the art.

[0005] SUMMARY

[0006] The present application aims to provide a fat targeting liposome and preparation method and application thereof, which utilizes a phospholipid bilayer structure similar to biological cells, has good water solubility and biocompatibility, and has the function of specifically targeting adipocyte somatostatin receptors after being connected with a fat targeting peptide through a maleimide. It can be used as a drug carrier, which has a hydrophobic shell and a hydrophilic core, and can be widely used for the encapsulation of various hydrophilic and hydrophobic drugs, and can be used as a fat targeting therapeutic drug carrier. The problems in the background art are solved.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] A fat-targeting liposome, which is composed of phospholipid, cholesterol, distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide and a fat-targeting peptide.

[0009] Further, the phospholipid is soybean lecithin; and the fat-targeting peptide sequence comprises CKGGRAKDC.

[0010] Further, the mass ratio of the phospholipid, cholesterol, distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide is 4:1:1.5-3; and the mass ratio of the distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide to the fat-targeting peptide is 1:1.

[0011] The preparation method of the fat-targeting liposome comprises the following steps:

[0012] S1. A certain amount of phospholipid, cholesterol, distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide is used to prepare a liposome by a thin film hydration method;

[0013] S2. The fat-targeting peptide is added to the liposome prepared in step S1, and after successful loading, free materials are removed by dialysis to obtain the required fat-targeting liposome.

[0014] Further, in S1, the organic solvent used in the thin film hydration method for preparing the liposome is one of trichloromethane, methanol, ethanol or dichloromethane.

[0015] Further, the organic solvent used in the thin film hydration method for preparing the liposome is trichloromethane.

[0016] Further, in S1, the specific method for preparing the liposome is as follows: the phospholipid, cholesterol and distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide are dissolved in trichloromethane respectively and mixed, and a uniform film is formed by rotary evaporation; then PBS is added for hydration, the hydration temperature is 37°C, and the hydration time is 30-120 min; after hydration, ice bath probe ultrasonic is performed, the ultrasonic power is 80-150 W, and the ultrasonic time is 5-10 min.

[0017] Further, the specific method for preparing the fat-targeting liposome is as follows: the fat-targeting peptide is completely dissolved in PBS, then the liposome prepared in step S1 is added, and magnetic stirring is performed for more than 12 h, and dialysis is performed overnight to obtain the required fat-targeting liposome.

[0018] The fat-targeting liposome is used as a drug carrier.

[0019] The fat-targeting liposome is used in fat targeting.

[0020] The technical scheme has the following beneficial effects:

[0021] 1. The fat-targeting liposome provided by the application has the advantages of easy availability of component raw materials, non-toxicity, environmental protection, biodegradability, good safety and biocompatibility;

[0022] 2. The fat-targeting peptide liposome provided by the application has the advantages of fat tissue and cell-specific targeting capability, drug release in fat tissue and cells, and effective improvement of drug accumulation in fat parts.

[0023] 3. The fat-targeting liposome provided by the application has the advantages of application in a drug carrier, loading of hydrophobic and hydrophilic drugs through a hydrophobic shell or a hydrophilic core, great improvement of the universality of drug loading, and wide application in the treatment field. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a hydration particle size diagram of the liposome prepared in step (1) in the embodiment 1 of the application;

[0025] Fig. 2 is a hydration particle size diagram of the fat-targeting liposome prepared in step (2) in the experimental example 1 of the application;

[0026] Fig. 3 is a fluorescence intensity column chart of &FITC@Lip-ATS, FITC, FITC@Lip, FITC@Lip-ATS in the experimental example 2 of the application;

[0027] Fig. 4 is a fluorescence chart of Control, DiD@Lip-ATS 10mg , DiD@Lip-ATS 5mg , DiD@Lip-ATS 2.5mg in the experimental example 3 of the application. DETAILED DESCRIPTION

[0028] The application will be further described in detail in combination with the drawings and embodiments:

[0029] Example 1: Preparation of fat-targeting liposome

[0030] (1) 40 mg of soybean lecithin, 10 mg of cholesterol and 15 mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide (DSPE-PEG 2k -MAL) are weighed respectively and placed in a round-bottom flask, dissolved in 5 mL of chloroform, and then the organic solvent is removed by rotary evaporation in a water bath. After the solvent is completely volatilized, a uniform film is formed at the bottom of the round-bottom flask. After 4 mL of PBS solution is added, the film is placed in a 37℃ water bath for 30 min, and then the film is completely hydrated to form a liposome solution. Finally, the MAL-modified liposome (Lip-MAL) is prepared by ice bath probe ultrasonication (ultrasonic power is 100 W, and ultrasonic time is 10 min).

[0031] (2) After 15 mg of fat targeting peptide (ATS) was completely dissolved in PBS, it was added dropwise to the liposomes prepared in step (1) above, and incubated at room temperature for 12 h. The reaction completed liposomes were placed in a 2000 MW dialysis bag and dialyzed for 12 h to obtain fat targeting liposomes (Lip-ATS).

[0032] Experimental Example 1: Determination of liposome size

[0033] The liposome (Lip-MAL) prepared in step (1) and the fat targeting liposome (Lip-ATS) prepared in step (2) in Example 1 were subjected to water hydration particle size analysis, and the results are shown in FIGS. 1 and 2.

[0034] Experimental Example 2: Determination of in vitro fat targeting ability of Lip-ATS

[0035] The fat targeting liposome (Lip-ATS) was subjected to flow cytometry experiment to investigate its targeting ability to mature adipocytes (3T3-L1).

[0036] In Example 1, 2 mg of fluorescent agent fluorescein isothiocyanate (FITC) was added to a round bottom flask, and was loaded into the hydrophobic shell of the liposome. Subsequently, step (2) was performed to finally prepare the FITC-loaded fat targeting liposome (FITC@Lip-ATS).

[0037] The FITC-loaded liposome (FITC@Lip) was prepared by the same method as in Example 1.

[0038] After preadipocytes were inoculated in a 6-well plate, they were induced to become mature adipocytes. Equal amounts of FITC, FITC@Lip, and FITC@Lip-ATS were added to the mature adipocytes, and equal amounts of FITC@Lip-ATS were added to the preadipocytes. All were incubated at 37°C, 5% CO2, and saturated humidity for 4 h in the dark. After free drugs were washed with PBS, they were resuspended and loaded onto the machine. The results are shown in FIG. 3.

[0039] As shown in FIG. 3, the liposome loaded with fat targeting peptide (Lip-ATS) has higher targeting ability to mature adipocytes, but weaker targeting ability to preadipocytes. The reason is that preadipocytes lack plasminogen receptors compared to mature adipocytes.

[0040] Experimental Example 3: Determination of in vivo fat targeting ability of Lip-ATS

[0041] The in vivo fat targeting ability and distribution of the fat targeting liposome were investigated by analyzing the fluorescence of the ex vivo tissue by in vivo imaging technology.

[0042] In step (1) of Example 1, 5 mg of fluorescent agent DiD was added to a round bottom flask, and was loaded into the hydrophobic shell of the liposome, and then in step (2) by changing the loading amount of ATS, three kinds of DiD-loaded fat-targeting liposomes (DiD@Lip-ATS 10mg , DiD@Lip-ATS 5mg , DiD@Lip-ATS 2.5mg ) were finally prepared.

[0043] The liposomes loaded with only DiD (DiD@Lip) were prepared by the method of Example 1 as the Control group.

[0044] C57 BL / 6J mice were used to obtain an obese mouse model by feeding 60% high-fat feed, and the same amount of DiD@Lip, DiD@Lip-ATS 10mg , DiD@Lip-ATS 5mg , DiD@Lip-ATS 2.5mg was injected into the tail vein respectively. At 8 h, 12 h, and 24 h after injection, the epididymal fat, perirenal fat, mesenteric fat, inguinal fat, axillary brown fat, scapular brown fat, heart, liver, spleen, lung, and kidney of the mice were collected and photographed, and the fluorescence intensity was observed, as shown in Figure 4.

[0045] As can be seen from Figure 4, the liposomes loaded with fat-targeting peptides have higher fluorescence intensity in fat tissues at 8 h, 12 h, and 24 h compared with those without loading, that is, the fat-targeting liposomes have better fat targeting ability and can accumulate more in fat tissues.

[0046] The above is only an embodiment of the present application, and the specific technical solutions or characteristics of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the application. The protection scope of the present application should be subject to the content of its claims, and the specific embodiments described in the specification can be used to explain the content of the claims.

Claims

1. A fat targeting liposome, characterized by, The liposome is composed of phospholipid, cholesterol, distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide and fat targeting peptide.

2. The fat targeting liposome according to claim 1, wherein, The phospholipid is soybean lecithin; and the fat targeting peptide sequence comprises CKGGRAKDC.

3. The fat targeting liposome according to claim 1, wherein, The mass ratio of the phospholipid, cholesterol, distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide is 4:1:1.5-3; and the mass ratio of the distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide to the fat targeting peptide is 1:

1.

4. The method of claim 1 to 3, wherein the method is characterized in that, The method comprises the following steps: S1, preparing liposome by thin film hydration method by weighing the phospholipid, cholesterol, distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide; S2, adding fat targeting peptide to the liposome prepared in step S1, removing free materials by dialysis method after successful loading, and obtaining the required fat targeting liposome.

5. The method of claim 4, wherein the liposome is a fat targeting liposome. In S1, the organic solvent for preparing the liposome by thin film hydration method is one of trichloromethane, methanol, ethanol or dichloromethane.

6. The method of claim 5, wherein the liposome is a fat targeting liposome. The organic solvent for preparing the liposome by thin film hydration method is trichloromethane.

7. The method of claim 6, wherein the liposome is a fat targeting liposome. In S1, the specific method for preparing the liposome is: first, dissolving the phospholipid, cholesterol, distearoylphosphatidyl ethanolamine-polyethylene glycol-maleimide in trichloromethane respectively and mixing, forming a uniform film by rotary evaporation; then adding PBS for hydration, the hydration temperature is 37℃, and the hydration time is 30-120min; after hydration, ice bath probe ultrasonic is performed, the ultrasonic power is 80-150W, and the ultrasonic time is 5-10min.

8. The method of claim 7, wherein the liposome is a fat targeting liposome. The specific method for preparing the fat targeting liposome is: first, completely dissolving the fat targeting peptide in PBS, then adding the liposome prepared in step S1, and magnetically stirring for more than 12h, dialyzing overnight, and obtaining the required fat targeting liposome.

9. The fat targeting liposome according to any one of claims 1-3 for use as a drug carrier.

10. The fat targeting liposome according to any one of claims 1-3 for use in fat targeting.

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