CN112251222B - Lipid drop induction and imaging reagent and preparation method and application thereof - Google Patents

Abstract

The invention discloses a lipid drop induction and imaging reagent, a preparation method and application thereof. The liposome has good water dispersibility, can effectively induce cells to generate a large amount of lipid droplets under the condition of not influencing the activity of the cells, and has greatly improved lipid droplet generation efficiency compared with the prior induction strategy. More importantly, the reagent can also carry out fluorescent labeling on intracellular lipid drops, thereby realizing the imaging tracing of the lipid drops.

Description

Lipid drop induction and imaging reagent and preparation method and application thereof

Technical Field

The invention relates to biotechnology, in particular to a lipid drop induction and imaging reagent and a preparation method and application thereof.

Background

Lipid Droplets (LDs) are the main storage sites for neutral lipids in cells, can store triglyceride, sterol ester, retinyl ester and the like, and are multifunctional organelles with a monolayer structure and surface-coated proteins. Recent studies have found that lipid droplets, in addition to being responsible for fat storage, are also essential for many cellular functions, such as lipid metabolism, membrane biosynthesis, cell signaling, inflammation, and the like. In addition, there is increasing evidence that cancer development is associated with abnormalities in lipid droplets. These important biological functions of lipid droplets urgently require researchers to develop reagents that are capable of efficiently regulating and monitoring cellular lipid droplets. However, current lipid droplet inducing agents, such as cyclosporine, are now classified as a class of carcinogens; the clinically used oxidized low-density lipoprotein is limited by short shelf life (generally, the storage time is about 6 weeks at 2-8 ℃); as far as representative oleic acid of the long-chain fatty acids which are most commonly used at present is concerned, it is poorly water-soluble and is unable to label lipid droplets. Therefore, it is necessary to develop an agent that can effectively induce lipid droplet production in cells and perform specific fluorescence imaging thereof.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a lipid drop high-efficiency inducing and fluorescence labeling reagent, which can effectively overcome the defects that the existing lipid drop inducing reagent has poor water solubility, cannot carry out specific tracing on lipid drops, has poor safety and the like.

The invention also provides a preparation method and application of the lipid drop inducing and imaging reagent.

The technical scheme is as follows: in order to achieve the above purpose, the lipid drop inducing and imaging agent of the present invention is a liposome composed of negatively charged unsaturated glycerophospholipid and phospholipid with tail labeled fluorescence.

Wherein the negatively charged unsaturated glycerophospholipid is a negatively charged unsaturated glycerophospholipid whose head negatively charged group is Phosphatidylserine (PS), Phosphatidylglycerol (PG), Phosphatidylinositol (PI), Phosphatidic Acid (PA) or diphosphatidylglycerol (CA).

Wherein the negatively charged unsaturated glycerophospholipid is a single-chain or multi-chain negatively charged unsaturated glycerophospholipid having an alkane chain length of 14-18 carbon atoms and containing at least one unsaturated cis-double bond.

Wherein the negatively charged unsaturated glycerophospholipid is a single phospholipid or a mixed phospholipid in any proportion in the phospholipids.

Wherein the head group of the tail-labeled fluorescent phospholipid is Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylglycerol (PG), Phosphatidylinositol (PI), Phosphatidic Acid (PA) or other phospholipid molecule head groups.

Wherein, the fluorophore contained in the phospholipid with the tail labeled fluorescence is 7-nitrobenz-2-oxa-1, 3-diazolyl (NBD), Pyrene (Pyrene), dipyrromethene boron Difluoride (DPMB), Diphenylhexatriene (DPH) or other fluorescent molecules capable of being grafted on the phospholipid tail chain.

Wherein the mass ratio of the tail part labeled fluorescent phospholipid to the electronegative unsaturated glycerophospholipid is 1:20-1: 500.

The preparation method of the lipid drop inducing and imaging reagent comprises the following steps:

uniformly mixing phospholipid with tail part marked fluorescence and negatively charged unsaturated glycerophospholipid in chloroform or methanol, drying the mixture in vacuum after drying a solvent, adding phosphate buffer solution, oscillating and hydrating, and ultrasonically preparing liposome at room temperature by using a probe, namely the lipid drop inducing and imaging reagent.

The invention relates to an application of a lipid drop induction and imaging reagent in lipid drop induction.

The application of the lipid drop inducing and imaging reagent in lipid drop specific fluorescence imaging is provided.

The invention provides a bi-component liposome reagent, which is a lipid drop high-efficiency induction and fluorescence labeling imaging reagent. The structure of the lipid drop inducing and imaging liposome is shown in figure 1. The tail-labeled fluorescent phospholipid utilized by the invention has the characteristics similar to the common phospholipid in structure, can form a multi-component liposome with the common phospholipid (non-fluorescent label) and realize the positioning and fluorescence imaging of a specific area along with the common phospholipid, and is combined with the negatively-charged unsaturated glycerophospholipid. Because the electronegative unsaturated glycerophospholipid in the reagent can induce cells to generate a large number of lipid droplets and stably stay in the lipid droplets, the fluorescent phospholipid forming the liposome with the electronegative unsaturated glycerophospholipid is also positioned at the lipid droplets, and finally, the efficient induction of the lipid droplets and the specific fluorescent labeling of the lipid droplets can be simultaneously realized.

In addition, as the phospholipid contains hydrophilic groups, the inner cavity and the outer part of the liposome formed by the phospholipid are both in aqueous solution environment, and the water dispersibility is good; the fluorescent phospholipid with different colors can be actually replaced, so that fluorescent imaging of lipid drops with different colors is realized.

Has the advantages that: compared with the prior art, the reagent has low toxicity to cells, and the efficiency of inducing lipid droplets is greatly improved. In addition, the invention also realizes the specific fluorescent imaging tracing of intracellular lipid drops.

In particular, the agents of the invention have the following advantages:

(1) compared with the traditional lipid drop inducing reagents such as oleic acid and the like, the liposome disclosed by the invention is good in water dispersibility, and can effectively induce cells to generate a large amount of lipid drops under the condition of not influencing the activity of the cells, so that the application of the liposome to the field of cell biology is facilitated;

(2) the main component glycerophospholipid of the lipid drop inducing reagent is a component contained in cells, so the lipid drop inducing reagent has good cell compatibility;

(3) the fluorescent phospholipid can replace fluorescent phospholipid with different colors to emit light according to requirements, so that fluorescent imaging of lipid drops with different colors is realized;

(4) the lipid drop induction and imaging reagent has the advantages of wide raw material source, high bioavailability, simple preparation process and potential for large-scale production and application.

Drawings

FIG. 1 is a block diagram of a lipid droplet inducing and imaging liposome of the present invention;

FIG. 2 is a molecular structural formula of unsaturated glycerophospholipid and fluorescent phospholipid used in example 1 of the present invention;

FIG. 3 is a diagram of the structure of lipid droplet-inducing and imaging liposomes obtained in example 1 of the present invention;

FIG. 4 is a graph showing the toxicity evaluation of dioleoylphosphatidylserine/7-nitrobenzo-2-oxa-1, 3-diazolyl-labeled phosphatidylserine (DOPS/NBD-PS) liposomes against A549 lung cancer cells;

FIG. 5 shows the observation of lipid droplet induction of A549 lung cancer cells by commercial lipid droplet inducer oleic acid;

FIG. 6 is the lipid droplet induction microscopic observation result of DOPS/NBD-PS liposome on A549 lung cancer cells;

FIG. 7 shows the results of lipid drop fluorescence imaging of DOPS/NBD-PS liposomes on A549 lung cancer cells.

Detailed Description

The invention is further illustrated by the following figures and examples.

Materials, reagents and the like used in examples are commercially available unless otherwise specified. Negatively charged unsaturated glycerophospholipids and tail-labeled fluorescent phospholipids referred to in the examples were purchased from Avanti corporation, usa. The phosphate buffers in the examples were all phosphate buffers for cells, pH 7.4, and ionic strength 150 mM.

Example 1

(1) 10mg of dioleoyl phosphatidylserine (DOPS, structural formula shown in figure 2) was weighed and dissolved in 0.5mL of chloroform, and the solvent was dried with nitrogen gas in a vacuum oven at room temperature for 4 hours. Adding 1mL of phosphate buffer solution, oscillating, hydrating and dispersing, and preparing a liposome named DOPS liposome by using probe ultrasound (163W for 4 minutes);

(2) 0.2mL of 1mg/mL fluorescent phospholipid (named as NBD-PS, structural formula shown in figure 2) with NBD mark at tail and phosphatidylserine group at head (solvent is chloroform) is mixed with DOPS (10mg) dissolved in 0.5mL of chloroform, and the mixture is dried in a vacuum drying oven for 4 hours at room temperature after nitrogen blow-drying. Adding 1mL phosphate buffer solution, hydrating and dispersing, and performing ultrasonic probe (163W, 4 min) to obtain liposome, named DOPS/NBD-PS liposome (structure shown in figure 3).

Example 2

Construction of lipid drop induction and imaging liposomes of different saturation:

the preparation method was the same as in example 1, except that DOPS used in (2) of example 1 was replaced with Palmitoyl Oleoyl Phosphatidylserine (POPS) of equal mass.

Example 3

Lipid drop induction and imaging liposome construction of different head groups:

the preparation method was the same as in example 1, except that DOPS used in (2) of example 1 was replaced with equal mass of Dioleoylphosphatidylglycerol (DOPG), Dioleoylphosphatidylglycerol (DOPI), dioleoylphosphatidic acid (DOPA) or Dioleoylphosphatidylglycerol (DOCA).

Example 4

Construction of lipid drop induction and imaging liposomes of different fluorescent labels:

the preparation method was the same as in example 1, except that NBD-PS used in (2) of example 1 was replaced with equal mass of Pyrene-PS (Tail Pyrene-labeled PS), DPMB-PS (Tail DPMB-labeled PS), or DPH-PS (Tail DPH-labeled PS).

Example 5

The construction of lipid drop induction and imaging liposome with different proportions:

the preparation method was the same as in example 1, except that the mass ratio of the fluorescent phospholipid to the negatively charged unsaturated glycerophospholipid used in (2) of example 1 was changed from 1:50 (i.e., 0.2mg:10mg) to 1:20 (i.e., 0.5mg:10mg) or 1:500 (i.e., 0.02mg:10 mg).

Example 6

The cytotoxicity of the reagent prepared in example 1 was evaluated by the following procedure:

a549 lung cancer cell in 96-holeAfter 24 hours on-plate culture (5% CO)₂37 ℃ C.), the original DMEM medium in each well is replaced by 100 μ L of DMEM complete medium containing DOPS liposomes or DOPS/NBD-PS liposomes (DOPS concentration in each well is 0, 10, 20, 50, 80, 100, 120, 150 μ g/mL respectively; the corresponding NBD-PS concentrations were 0, 0.2, 0.4, 1.0, 1.6, 2.0, 2.4, 3.0. mu.g/mL, respectively, in an incubator (5% CO)₂Incubation was continued for 24 hours at 37 ℃. The cell viability was then measured by the MTT assay and the results are shown in FIG. 4.

As can be seen from FIG. 4, the toxicity of the agent of the present invention to A549 cells is very low, which proves that the agent of the present invention has good cell compatibility and good safety.

Example 7

The agent prepared in example 1 was evaluated for its cell lipid droplet-inducing effect by the following procedure:

to understand the lipid drop profile of the cells, A549 lung carcinoma cells were first cultured on confocal plates for 24 hours (5% CO)₂37 ℃), and then 100 μ L of DMEM complete medium containing oleic acid was replaced with the original DMEM medium per well (oleic acid: 120 μ M, i.e. the concentration of the substance following 100 μ g/mL DOPS) for a further 24 hours, stained with the commercial lipid drop dye BODIPY 493/503(sigma) (10 minutes, 2nM) and observed using a confocal microscope (excitation wavelength 488nM, reception band 495 to 555nM), the results are shown in fig. 5. As can be seen from FIG. 5, the bubble-like structure in the bright field co-localized well with the stained region of the commercial lipid droplet dye BODIPY 493/503, demonstrating that the bubble-like structure observed in the bright field is a lipid droplet in A549 cells, indicating that no fluorescent co-staining with the commercial lipid droplet reagent is required in the later stage, the location of the lipid droplet can be indicated in the bright field, and reference was made for imaging the lipid droplet of this reagent in example 8 (the commercial lipid droplet dye overlaps or interferes with the fluorescence of this reagent).

To evaluate the effect of the agent of the present invention on the induction of lipid droplets in cells, A549 lung cancer cells were first cultured on a confocal culture plate for 24 hours, then the original DMEM medium in each well was replaced with 100. mu.L of DMEM complete medium containing DOPS liposomes (DOPS: 100. mu.g/mL), and then returned to the incubator (5% CO)₂Incubation was continued for 24 hours at 37 ℃. Followed byThereafter, the cells were stained with the commercial lipid drop dye BODIPY 493/503 (10 min, 2nM) and observed using a confocal microscope, the results of which are shown in FIG. 6. As can be seen from FIG. 6, a large number of bubble-like structures were present in both the bright and fluorescent fields, demonstrating the large production of intracellular lipid droplets. Comparing fig. 5 and 6, it can be seen that DOPS liposome can induce cells to produce a large amount of lipid droplets significantly, and the efficiency of induction is higher than that of oleic acid at the same amount concentration as the substance.

Example 8

The reagents prepared in example 1 were evaluated for their lipid droplet fluorescence imaging effect by the following procedure:

a549 Lung cancer cells were cultured on confocal plates for 24 hours (5% CO)₂37 ℃ C.), the original DMEM medium was replaced with 100. mu.L of DMEM complete medium containing DOPS/NBD-PS liposomes (DOPS: 100 mu g/mL; corresponding NBD-PS: 2. mu.g/mL), and returned to the incubator (5% CO)₂Incubation was continued for 24 hours at 37 ℃. Then, observation was carried out by a confocal microscope (excitation wavelength: 488nm, and reception band: 495 to 580nm), and the results are shown in FIG. 7.

As can be seen from FIGS. 5 and 7, the agent of the present invention enables the production of a large number of lipid droplets in cells, induces a higher efficiency than that of oleic acid at the same amount as the substance, and also specifically fluorescently labels lipid droplets produced by the cells.

Claims (6)

1. A lipid drop inducing and imaging reagent is characterized in that the reagent is a liposome formed by negatively charged unsaturated glycerophospholipid and tail-labeled fluorescent phospholipid; the negatively charged unsaturated glycerophospholipid is a negatively charged unsaturated glycerophospholipid of which the head group is phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid or diphosphatidylglycerol; the negative electricity unsaturated glycerophospholipid is a single-chain or multi-chain negative electricity unsaturated glycerophospholipid, the alkane chain length of which is 14-18 carbon atoms and contains at least one unsaturated cis double bond; the head group of the phospholipid with the tail part marked with fluorescence is phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or phosphatidic acid; the fluorophore contained in the phospholipid with tail labeled fluorescence is 7-nitrobenzo-2-oxa-1, 3-diazolyl (NBD), pyrene, dipyrromethene boron Difluoride (DPMB) or Diphenylhexatriene (DPH).

2. The lipid droplet inducer and imaging agent of claim 1, wherein the negatively charged unsaturated glycerophospholipid is a single phospholipid or a mixture of phospholipids in any ratio.

3. The lipid droplet induction and imaging agent of claim 1, wherein the mass ratio of the tail labeled fluorescent phospholipid to the negatively charged unsaturated glycerophospholipid is 1:20 to 1: 500.

4. A method of preparing a lipid droplet inducer and imaging agent according to any of claims 1 to 3, comprising the steps of:

uniformly mixing phospholipid with tail part marked fluorescence and negatively charged unsaturated glycerophospholipid in chloroform or methanol, drying the mixture in vacuum after drying a solvent, adding phosphate buffer solution, oscillating and hydrating, and ultrasonically preparing liposome at room temperature by using a probe, namely the lipid drop inducing and imaging reagent.

5. Use of the lipid droplet inducer and imaging agent of claim 1 for lipid droplet induction.

6. Use of the lipid droplet inducer and imaging agent of claim 1 in lipid droplet specific fluorescence imaging.

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