Preparation of mitochondrial targeting liposome and application of mitochondrial targeting liposome in fat regulation
By developing mitochondrial targeting liposomes and using the phospholipid bilayer structure to combine with mitochondrial targeting peptides, the problem of existing drugs lacking targeting and side effects in the treatment of obesity is solved, and the efficient accumulation and specific targeting of drugs in the fat site is achieved, and the effective regulation of fat metabolism is effectively regulated.
Patent Information
- Application Number
- CN202510061692.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-25
AI Technical Summary
Existing drugs lack targeting in the treatment of obesity and related metabolic diseases, resulting in nonspecific effects and serious side effects.
A mitochondria-targeted liposome was developed, which uses the phospholipid bilayer structure to bind to the mitochondria-targeted peptide to form liposomes that specifically target mitochondria, and serve as a drug carrier for fat regulation.
It has achieved efficient accumulation and specific targeting of drugs in fat parts, effectively regulate fat metabolism, reduce side effects, and improve treatment effect.
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Figure CN119868274A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to liposome preparation and application thereof, and in particular to mitochondria-targeted liposome preparation and application thereof in fat regulation. 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 has become a global public health problem, which can cause a series of related metabolic diseases such as insulin resistance, hyperglycemia, hypertension, hyperlipidemia, etc., and has become an important risk factor for diabetes, cardiovascular disease and cancer. Studies have found that mitochondria play a vital role in the energy production of cells and also coordinate the stress response of cells. Excessive mitochondrial fission can impair their function, disrupt metabolism, and increase toxic byproducts associated with insulin resistance in certain tissue types.
[0004] Regulating the functions of mitochondria and cells can effectively solve the problem of obesity, but the high level of liver metabolism and lack of target specificity of drugs 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, including edema, local pain, bruising and numbness.
[0005] Therefore, it is of great research value to develop a liposome with good biocompatibility and the ability to specifically target mitochondria for efficient drug delivery. Summary of the invention
[0006] The present invention aims to provide a mitochondrial targeting liposome, which utilizes the phospholipid bilayer structure similar to that of biological cells, has the advantages of good water solubility and biocompatibility, and is combined with the mitochondrial targeting peptide through DSPE-PEG 2K After being connected with -COOH, it has the function of specifically targeting mitochondria; it can be used as a drug carrier, and 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. Therefore, the liposome can be used as a drug carrier for mitochondrial targeted therapy and is widely used in the treatment field.
[0007] To achieve the above object, the present invention provides the following technical solution: a liposome for mitochondrial targeting, the liposome comprising phospholipids, cholesterol, DSPE-PEG 2K-COOH and mitochondrial targeting peptide (SS-31), the phospholipid is preferably soybean lecithin, and the liposome is used as a drug carrier for fat regulation.
[0008] Specifically, a method for preparing mitochondrial-targeted liposomes comprises the following steps:
[0009] S1 combines phospholipids, cholesterol, DSPE-PEG 2K -COOH were dissolved in organic solvents and mixed, and a uniform lipid film was formed by rotary evaporation, and PBS was added for hydration at a temperature of 37°C for 30 to 120 minutes. After the hydration was completed, liposomes were formed by ultrasonication with an ice bath probe at a power of 80 to 150 W for 5 to 10 minutes;
[0010] S2. Add EDC to the liposomes formed in step S1 for activation for half an hour, then add NHS and mitochondrial targeting peptide SS-31, the sequence of which is DArg-Dmt-Lys-Phe-NH 2 After the reaction was successfully loaded, the free materials were removed by dialysis to obtain mitochondrial targeted liposomes, which were dialyzed overnight.
[0011] Specifically, in step S1, phospholipids, cholesterol and DSPE-PEG 2K The feed ratio of -COOH is 40:10:9.
[0012] Specifically, the organic solvent is chloroform, methanol, ethanol or dichloromethane, preferably chloroform.
[0013] Specifically, in step S2, the feed ratio of EDC, NHS and SS-31 is 2:2:5, the solvent of EDC, NHS and mitochondrial targeting peptide (SS-31) is PBS, and the magnetic stirring reaction time is not less than 12 hours.
[0014] Specifically, the use of mitochondria-targeted liposomes in targeting mitochondria.
[0015] Specifically, the application of mitochondria-targeted liposomes in fat regulation, especially in improving obesity and its related metabolic diseases.
[0016] Specifically, the mitochondria-targeted liposome achieves drug accumulation in fat parts by specifically targeting mitochondria, thereby effectively regulating fat metabolism.
[0017] The principle and beneficial effects of this technical solution:
[0018] The mitochondria-targeted liposome of the present invention has readily available materials, is non-toxic, environmentally friendly, biodegradable, and has excellent safety and biocompatibility.
[0019] The mitochondrial targeting peptide liposomes of the present invention have mitochondrial targeting capability, can release drugs at mitochondria, and effectively increase the accumulation of drugs at fat sites; and can load hydrophilic and hydrophobic drugs through a hydrophobic shell or a hydrophilic core, greatly improving the universality of drug encapsulation. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a diagram of the hydrated particle size of the liposomes and fat-targeted liposomes in Example 1 of the present invention;
[0021] Figure 2 This is a graph showing the experimental results of the targeting ability of liposomes to mitochondria of mature adipocytes (3T3-L1) in Example 1 of the present invention;
[0022] Figure 3 This is a diagram showing the experimental results of restoring mitochondrial homeostasis of preadipocytes and mature adipocytes using mitochondrial-targeted liposomes containing mitochondrial regulators in Example 1 of the present invention. DETAILED DESCRIPTION
[0023] The present invention is further described in detail below with reference to the accompanying drawings and embodiments:
[0024] Embodiment 1:
[0025] This embodiment provides a mitochondrial targeting liposome, and the preparation method is as follows:
[0026] Weigh soybean lecithin, cholesterol, DSPE-PEG 2K -COOH 40mg, 10mg, and 9mg were placed in a round-bottom flask, dissolved with 5mL 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 4mL of PBS solution, it was placed in a 37°C water bath for 30min to fully hydrate the film to form a liposome solution. Finally, the liposomes were prepared by ice bath probe ultrasound (100W, 10min).
[0027] S2: After adding 2 mg EDC to the liposomes formed in step S1 for activation for half an hour, 2 mg NHS and 5 mg mitochondrial targeting peptide SS-31 were added, and the mixture was incubated at room temperature with stirring for 12 hours. The liposomes after the reaction were placed in a 2000 MW dialysis bag and dialyzed for 12 hours to obtain mitochondrial targeting liposomes.
[0028] Liposome size: The hydrated particle size of the liposomes (PEG-Lip) prepared in step S1 and the fat-targeted liposomes (PEP-Lip) prepared in step S2, see Figure 1 .
[0029] In vitro mitochondrial targeting ability of Lip-SS-31: The targeting ability of mitochondrial targeted liposomes (PEP-Lip) to mitochondria of mature adipocytes (3T3-L1) was investigated by laser confocal microscopy.
[0030] In step S1, 2 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 S2 was followed to finally prepare mitochondrial targeting liposomes (FITC@PEP-Lip) encapsulating FITC. The same liposome preparation method as in Example 1 was used to prepare liposomes encapsulating only FITC (FITC@PEG-Lip).
[0031] After preadipocytes were inoculated into 6-well plates, adipocytes were induced to obtain mature adipocytes. Equal amounts of FITC@PEG-Lip and FITC@PEP-Lip were added to the mature adipocytes, respectively, and incubated for 4 h at 37°C, 5% CO2 and saturated humidity in the dark. The cell culture medium was removed, and then JC-1 working solution sufficient to cover all cells was added. The culture medium was incubated at 37°C, 5% CO2 and saturated humidity for 4 h. 2 Incubate in a culture box for 15-30 min, wash with PBS three times, and observe under a confocal microscope.
[0032] Figure 2 The results showed that liposomes loaded with mitochondrial targeting peptide SS-31 (FITC@PEP-Lip) had higher mitochondrial targeting ability.
[0033] Application of PEP-Lip in restoring mitochondrial homeostasis in adipocytes: Fluorescence co-localization analysis of preadipocytes and mature adipocytes after administration was performed using laser confocal microscopy to explore the application of mitochondrial targeted liposomes in restoring mitochondrial homeostasis in adipocytes.
[0034] 4 mg of mitochondrial regulator Empagliflozin was dissolved in 4 ml of PBS, and after forming a thin film in step S1 in Example 1, the Empagliflozin solution was added to form liposomes (Em@PEG-Lip) encapsulating Empagliflozin. Subsequently, the drug-loaded liposomes (Em@PEP-Lip) connected with mitochondrial targeting peptide SS-31 were finally prepared in the same manner as step S2.
[0035] Preadipocytes were inoculated into 6-well plates and induced to adipogenesis to obtain mature adipocytes. Equal amounts of Empagliflozin and Em@PEP-Lip were added to preadipocytes and mature adipocytes, respectively, and the plates were placed in a dark environment at 37°C and 5% CO. 2Incubate for 12 h under saturated humidity. Remove the cell culture medium and add enough JC-1 working solution to cover all cells. Incubate at 37°C, 5% CO 2 Incubate in a culture box for 15-30 min, wash with PBS three times, and observe under a confocal microscope.
[0036] Figure 3 The results showed that the liposomes loaded with mitochondrial targeting peptide SS-31 had a higher red fluorescence intensity than those without liposomes, that is, the mitochondrial targeting liposomes could be released more at the mitochondria, and the encapsulated mitochondrial regulators acted more effectively on the mitochondria and restored the homeostasis of the mitochondria in adipocytes more effectively.
[0037] In summary, the phospholipid, cholesterol, DSPE-PEG 2K -COOH and mitochondrial targeting peptide (SS-31) liposomes. These liposomes have a suitable hydrated particle size and are suitable as drug carriers. The targeting ability of mitochondrial targeted liposomes (PEP-Lip) to mitochondria of mature adipocytes (3T3-L1) was verified by laser confocal microscopy co-localization experiments. Liposomes loaded with fat targeting peptides (PEG-Lip) have higher mitochondrial targeting ability. The application of mitochondrial targeted liposomes (PEP-Lip) in restoring mitochondrial homeostasis of adipocytes was investigated by immunofluorescence experiments. Compared with liposomes without mitochondrial targeting peptide SS-31, liposomes loaded with mitochondrial targeting peptide SS-31 have higher red fluorescence intensity, that is, mitochondrial targeted liposomes can be released more at mitochondria, and the encapsulated mitochondrial regulators act more effectively on mitochondria and more effectively restore the homeostasis of adipocyte mitochondria.
[0038] Mitochondrial-targeted liposomes achieve drug accumulation in fat areas by specifically targeting mitochondria, thereby effectively regulating fat metabolism. This has important clinical significance for improving obesity and related metabolic diseases. Mitochondrial-targeted liposome materials are easily available, non-toxic, environmentally friendly, biodegradable, and have good safety and biocompatibility, which provides a basis for clinical application. Mitochondrial-targeted liposomes can be loaded with hydrophilic and hydrophobic drugs through the hydrophobic shell or hydrophilic core, greatly improving the universality of drug encapsulation.
[0039] In summary, the present invention not only provides an efficient and safe method for preparing mitochondrial targeted liposomes, but also verifies its targeting ability in vitro and in vivo through experiments, providing a new strategy for the treatment of obesity and related metabolic diseases. These results show that the present invention has important scientific significance and application prospects.
[0040] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions or characteristics in the solution is not described in detail here. For those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A liposome for mitochondrial targeting, characterized in that: The liposomes contain phospholipids, cholesterol, DSPE-PEG 2K -COOH and mitochondrial targeting peptide (SS-31), the phospholipid is preferably soybean lecithin, and the liposome is used as a drug carrier for fat regulation.
2. A method for preparing mitochondrial-targeted liposomes according to claim 1, characterized in that: The following steps are involved: S1, phospholipids, cholesterol, DSPE-PEG 2K -COOH were dissolved in organic solvents and mixed, and a uniform lipid film was formed by rotary evaporation, and PBS was added for hydration at a temperature of 37°C for 30 to 120 minutes. After the hydration was completed, liposomes were formed by ultrasonication with an ice bath probe at a power of 80 to 150 W for 5 to 10 minutes; S2 adds EDC to the liposomes formed in step S1 for activation for half an hour, then adds NHS and mitochondrial targeting peptide SS-31, wherein the sequence of the mitochondrial targeting peptide SS-31 is DArg-Dmt-Lys-Phe-NH2, and after the reaction is successfully loaded by magnetic stirring, removes free materials by dialysis to obtain mitochondrial targeting liposomes, and dialyzes overnight.
3. The method for preparing mitochondrial-targeted liposomes according to claim 2, characterized in that: In step S1, phospholipids, cholesterol and DSPE-PEG 2K The feed ratio of -COOH is 40:10:
9.
4. The method for preparing mitochondrial-targeted liposomes according to claim 2, characterized in that: The organic solvent is chloroform, methanol, ethanol or dichloromethane, preferably chloroform.
5. The method for preparing mitochondrial-targeted liposomes according to claim 2, characterized in that: In step S2, the feed ratio of EDC, NHS and SS-31 is 2:2:5, the solvent of EDC, NHS and mitochondrial targeting peptide (SS-31) is PBS, and the magnetic stirring reaction time is not less than 12 hours.
6. Use of the mitochondrial targeting liposome prepared by the preparation method according to any one of claims 1 to 4 in targeting mitochondria.
7. Use of the mitochondrial targeted liposome according to any one of claims 1 to 4 in fat regulation, especially in improving obesity and related metabolic diseases.
8. The use according to claim 6, characterized in that: The mitochondria-targeted liposome achieves the accumulation of drugs in fat parts by specifically targeting mitochondria, thereby effectively regulating fat metabolism.