Preparation and application of multivesicular liposome based on carbon dot labeled protein

Abstract

The invention provides a preparation method of a multi-vesicular liposome based on carbon dot labeled protein, and aims to improve the biological stability and the treatment effect of a medicine. The preparation process comprises the steps of dissolving the mixed lipid, mixing and emulsifying the carbon quantum dots and the protein, preparing multiple emulsion and the like. The multivesicular liposome has a fluorescent label, is convenient for monitoring in-vivo drug distribution and release, and is beneficial to optimizing a drug administration scheme. The specific implementation mode comprises stability verification, encapsulation rate calculation, cytotoxicity and uptake testing and morphological observation.

Description

Technical Field

[0001] The present invention relates to the technical field of carbon quantum dots and multivesicular liposome applications, and in particular provides the preparation and application of multivesicular liposomes based on carbon dot-labeled proteins, which can detect fluorescent signals, facilitate in vivo monitoring, and more accurately track the distribution and release of drugs in the body, providing important value for optimizing drug administration regimens. Background Art

[0002] In recent years, protein / peptide drugs have special curative effects on the prevention and treatment of diseases. They have the advantages of small dosage and few side effects, and have become biomacromolecules with broad prospects. However, the chemical structure that maintains its activity, such as amino acids and their order, terminal groups, peptide chains and disulfide bond positions, is often affected by certain factors. In addition, its two-dimensional structure and three-dimensional structure will also affect its activity. Moreover, protein / peptide drugs are prone to physical and chemical degradation, causing protein coagulation, precipitation or denaturation and loss of activity, and the efficacy of the drug is reduced. Therefore, protein / peptide drugs often have the disadvantages of poor biological stability, short half-life, high in vivo clearance rate, and susceptibility to enzyme damage. These problems greatly affect the delivery and in vivo distribution of protein drugs, and also produce other adverse reactions. Therefore, carriers that can load protein / peptide drugs play an important role in the prevention and treatment of current diseases.

[0003] Multivesicular liposomes have been a hot topic in recent years. They have a carrier function and can carry drugs to the disease site to exert a therapeutic effect. They play an important role in the prevention and treatment of diseases. As a new type of drug delivery carrier, multivesicular liposomes not only retain the biocompatibility, biodegradability and sustained release effect of traditional liposomes, but also show significant advantages in drug loading, drug leakage control, drug release rate and burst reduction, so the bioavailability of drugs can be significantly improved. Moreover, multivesicular liposomes also have good biocompatibility, which means that the integrity of the protein is maintained. However, since multivesicular liposomes of protein / peptide drugs lack detectable signals, their distribution and delivery in cells lack detection methods, so it is necessary to add suitable fluorescent dyes to track them, so as to achieve precise delivery and release of drugs, thereby improving the therapeutic effect and reducing drug side effects; moreover, by introducing fluorescently labeled multivesicular liposomes into cells, researchers can observe their movement and interaction in cells. This helps to better understand biological processes in cells, such as cell phagocytosis, endocytosis and cell signaling. Summary of the invention

[0004] The purpose of the present invention is to solve the problem that multivesicular liposomes lack detectable signals and are difficult to achieve ideal precise delivery and release of drugs, and to provide a method for preparing multivesicular liposomes based on carbon dot labeled proteins. The present invention provides a method for preparing multivesicular liposomes based on carbon dot labeled proteins, which can accurately deliver drugs to lesions, reduce the distribution of drugs in normal tissues, thereby reducing side effects and improving therapeutic effects. In addition, fluorescent labels are also helpful for biological imaging and diagnosis. The distribution and accumulation of drugs in the body are observed by fluorescent imaging technology. This non-invasive imaging method can be used for early diagnosis of diseases and monitoring of therapeutic effects.

[0005] The technical solution of the present invention is as follows:

[0006] 1. Preparation and application of multivesicular liposomes based on carbon dot-labeled proteins, characterized in that the carbon quantum dot-labeled multivesicular liposomes prepared by us have a particle size of 100 μm, and carbon quantum dots can label lipid or protein drugs according to the principle of like dissolves like, and the preparation process includes the following steps:

[0007] (1) Soybean lecithin, cholesterol, dipalmitoylphosphatidylglycerol and triolein were weighed in proportion and dissolved in dichloromethane.

[0008] (2) 50 μL of laboratory carbon quantum dots were mixed with 50 μL BSA by vortex mixing for 1 min, dissolved in sucrose buffer solution, and the mixed solution was added to the mixed lipids of (1) above and emulsified to obtain W / O colostrum.

[0009] (3) 10 mL of a mixed solution containing glucose and 40 mmol / L lysine aqueous solution was added to the colostrum and emulsified to obtain a W / O / W double emulsion. Finally, carbon quantum dot-labeled multivesicular liposomes (CDs-BSA-MVLs) were obtained.

[0010] The ratio in step (1) is 19:35:5:5-21:35:5:5; wherein the amount of soybean lecithin used is 0.0761-0.0841 g.

[0011] The carbon quantum dots in step (2) include water-soluble quantum dots / oil-soluble carbon quantum dots and amphiphilic carbon quantum dots. The emulsification conditions are 3000 rpm / min and mechanical stirring for 12 min.

[0012] The emulsification conditions in step (3) are 3000 rpm / min, mechanical stirring for 2 min, and the concentration of glucose is 3.0%-3.5% (m / v).

[0013] The beneficial effects of the present invention are as follows: the multivesicular liposomes of carbon dot-labeled proteins can detect fluorescent signals, which is convenient for monitoring in vivo, and can more accurately track the distribution and release of drugs in vivo, providing important value for optimizing the dosing regimen. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The morphology of multivesicular liposomes under a 40x microscope

[0015] Figure 2 TEM image of multivesicular liposomes DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings. It should not be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above invention.

[0017] The instruments and equipment used in the following examples are as follows:

[0018] Electronic balance (ME104E), multi-point magnetic stirrer (CJB-S-10D), centrifuge (Centrifuge JIDI-16R),

[0019] Fluorescence inverted microscope (Nikon Ti-S), Malvern particle size analyzer (ZEN3600), ultrasonic cleaning machine (SB-5200DT, UV-visible spectrophotometer (UV2600), pH meter (PHS-3C), three-purpose UV analyzer (ZF-7).

[0020] The specific historical aspects of the present invention are further described in detail as follows:

[0021] Example 1

[0022] Preparation of multivesicular liposomes labeled with carbon quantum dots: 0.0801 g of soybean lecithin, 0.0580 g of cholesterol, 0.0158 g of dipalmitoyl phosphatidylglycerol and 0.0166 g of triolein were weighed in a ratio of 4:7:1:1 and dissolved in 5 mL of dichloromethane. 50 μL of amphiphilic carbon quantum dots (CPC-h CDs) available in the laboratory were selected, and 50 μL of each was vortex mixed with 50 μL of BSA for 1 min, dissolved in 4% (m / v) sucrose buffer solution, and the mixed solution was added to the above-mentioned mixed lipids. The emulsification conditions were 3000 rpm / min and mechanical stirring was performed for 12 min to obtain W / O colostrum. 10 mL of a mixed solution containing 3.2% (m / v) glucose and 40 mmol / L lysine aqueous solution was added to the colostrum. The emulsification conditions were 3000 rpm / min and mechanical stirring was performed for 2 min to obtain a W / O / W double emulsion. Amphiphilic carbon quantum dots-labeled BSA multivesicular liposomes (CPC-hCDs-BSA-MVLs) were prepared.

[0023] Example 2

[0024] Stability verification: The prepared multivesicular liposomes were sealed and placed at room temperature for seven consecutive days. 100 μL of the sample was taken every day and diluted 1-fold before making slices. The morphological changes were observed under a fluorescent inverted microscope.

[0025] Example 3

[0026] Calculation of encapsulation efficiency: Weigh 0.1898 g of soybean lecithin, 0.1365 g of cholesterol, 0.0453 g of dipalmitoylphosphatidylglycerol and 0.0296 g of triolein, set the protein lipid ratio to 0.02, 0.03, 0.04, 0.05 and 0.06, respectively, and prepare multivesicular liposomes using different protein lipid ratios. Then centrifuge at 600 × g, draw the supernatant, measure the absorbance using a UV-visible spectrophotometer, bring it into the standard curve (the standard curve is obtained by measuring the absorbance of 6 concentration gradients of BSA solution), and obtain the concentration of free drug. The encapsulation efficiency is further calculated by the formula encapsulation efficiency = (total drug content - free drug content) / total drug content × 100%.

[0027] Example 4

[0028] Cytotoxicity test: Use 96-well plates for cell plating, set up 1 blank control group, and the remaining 8 groups were dosed with 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL, 500 mg / mL, and 1000 mg / mL for one day. After the drug administration, MTT solution was added and cultured for 4 hours, and finally the OD value was measured using an enzyme reader.

[0030] Example 5

[0031] Cell uptake: The BSA multivesicular liposomes labeled with A-CDs were diluted at a ratio of 1:20. A blank control group was set up, and the remaining 5 groups were dosed and cultured at time points of 1h, 2h, 4h, 6h, and 8h. After the administration, the excitation and emission in the fluorescence spectrum were photographed using a confocal microscope to observe the cellular uptake of the multivesicular liposomes.

Claims

1. Preparation and application of multivesicular liposomes based on carbon dot labeled proteins, characterized in that The following steps are involved: (1) Soybean lecithin, cholesterol, dipalmitoylphosphatidylglycerol and triolein were weighed in proportion and dissolved in dichloromethane. (2) 50 μL of laboratory carbon quantum dots and 50 μL of BSA were vortex mixed for 1 min and dissolved in sucrose buffer solution. The mixed solution was added to the mixed lipids of (1) above and emulsified to obtain W / O colostrum. (3) 10 mL of a mixed solution containing glucose and 40 mmol / L lysine aqueous solution was added to the colostrum and emulsified to obtain a W / O / W double emulsion. Finally, carbon quantum dot-labeled multivesicular liposomes (CDs-BSA-MVLs) were obtained.

2. According to claim 1, it is characterized in that: The ratio in step (1) is 19:35:5:5-21:35:5:5; wherein the amount of soybean lecithin used is 0.0761-0.0841 g.

3. According to claim 1, it is characterized in that: The carbon quantum dots in step (2) include water-soluble quantum dots / oil-soluble carbon quantum dots and amphiphilic carbon quantum dots. The emulsification conditions are 3000 rpm / min and mechanical stirring for 12 min.

4. According to claim 1, it is characterized in that: The emulsification conditions in step (3) are 3000-10000 rpm / min, mechanical stirring for 2-10 min, and the concentration of glucose is 3.0%-3.5% (m / v).

5. The multivesicular liposomes containing carbon dot-labeled proteins prepared according to claim 1 can detect fluorescent signals, which is convenient for monitoring in vivo, and can more accurately track the distribution and release of drugs in vivo, providing important value for optimizing the dosing regimen.

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