A method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets

Abstract

The application discloses a method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets, and belongs to the technical fields of biological medicine, cell metabolism regulation and biomaterials. The method comprises the following steps: combining triacetin with a lipase aqueous solution to construct enzyme-loaded biomimetic lipid droplets with relatively concentrated particle size distribution and good stability; in-vitro biological compatibility and cytotoxicity evaluation shows that the biomimetic lipid droplets have excellent safety within a set dose window; the biomimetic lipid droplets can efficiently cross cell membranes and accurately target intracellular endogenous lipid droplets; through physical and chemical diffusion of exogenous lipids and loading and rapid release of lipases, dynamic degradation of endogenous lipid droplets is realized at a cell level. The application provides a brand-new strategy for the treatment of obesity and lipid metabolism disorder related diseases and the development of targeted delivery preparations.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, cell metabolism regulation and biomaterials technology, specifically relating to a method for constructing biomimetic lipid droplets and its application in regulating the degradation of endogenous lipid droplets in cells. Background Technology

[0002] Endogenous lipid droplets (ELDs) are core subcellular organelles responsible for lipid storage and metabolic homeostasis within cells. Excessive accumulation of lipid droplets due to abnormal lipid metabolism is closely related to obesity, fatty liver disease, and cardiovascular disease. Traditional lipid-lowering drugs mostly work through systemic metabolic regulation, lacking subcellular targeting. While lipases are natural core protein molecules capable of hydrolyzing triglycerides, their direct administration is limited by their hydrophilic structure and complex cell membrane barriers, making it difficult to efficiently penetrate the cell membrane and enter the cytoplasm. They are easily lost on the extracellular side or during transport, thus failing to target endogenous lipid droplets. Therefore, developing a targeted regulatory system that possesses high biocompatibility, can effectively carry lipases across the membrane for delivery, and achieves precise degradation of intracellular endogenous lipid droplets has significant clinical and scientific value. Summary of the Invention

[0003] To address the aforementioned problems in the background technology, this invention provides a safe and efficient method for constructing enzyme-loaded biomimetic lipid droplets, which can effectively overcome the transmembrane barrier of macromolecular proteins and precisely target and deliver lipases into the interior of real intracellular lipid droplets, thereby achieving efficient degradation regulation of intracellular triglycerides.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets, the method being as follows:

[0006] Step 1: Mix Lipase aqueous solution with triacetylglycerol at a volume ratio of 20:2~4, shake at 180~220 r / min for 10~30s, and let stand at 35~39℃ for 20~40min to obtain white biomimetic lipid droplets loaded with lipase (Lipase@BLDs).

[0007] Step 2: Co-incubate the enzyme-loaded biomimetic lipid droplets with adipocytes containing endogenous lipid droplets, allowing the biomimetic lipid droplets to come into contact with the adipocytes containing endogenous lipid droplets and mediating the entry of lipase into the interior of the endogenous lipid droplets, thereby achieving the degradation of triglycerides in the endogenous lipid droplets.

[0008] Further, step one specifically involves: adding Lipase aqueous solution and triacetylglycerol in a volume ratio of 20:3 into the same EP tube, mixing, shaking at 200 r / min for 10-30 s, and then letting it stand at 37°C for 20-40 min to obtain white biomimetic lipid droplets (Lipase@BLDs) loaded with lipase at the bottom of the tube.

[0009] Furthermore, in step one, the molecular weight of the Lipase is 33 kDa.

[0010] Furthermore, in step one, the concentration of the Lipase aqueous solution is 5~15 mg / mL.

[0011] Furthermore, in step one, the Lipase aqueous solution is prepared by dissolving Lipase powder in PBS.

[0012] Furthermore, in step two, the co-incubation is carried out in DMEM medium containing 2 wt% BSA. The DMEM containing 2 wt% BSA must be sterilized by filtration through a 0.22 μm filter before use.

[0013] Furthermore, in step two, the co-incubation time is 6–12 hours.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) Efficiently overcomes the transmembrane barrier of macromolecules and achieves precise subcellular-level targeted delivery: The biomimetic lipid droplet carrier constructed in this invention utilizes the physicochemical diffusion characteristics of its exogenous oil phase and biological membrane to successfully overcome the barrier that traditional macromolecular proteins (lipases) have difficulty penetrating the cell membrane. After being loaded by the biomimetic lipid droplets, the lipases are targeted and enriched within all endogenous lipid droplets (ELDs) in the cell, achieving precise subcellular-level targeted localization.

[0016] (2) Excellent in vitro biocompatibility: The present invention has been confirmed by in vitro cytotoxicity evaluation to have excellent cell safety. Within the set dosage window, the cell survival rate can be maintained at more than 90%.

[0017] (3) Pioneering low-lipid toxicity degradation environment, significantly improving the degradation efficiency of intracellular triglycerides: This invention creatively introduces free albumin (BSA) into the degradation system, which effectively binds to and removes free fatty acids generated during triglyceride hydrolysis, eliminating the cytotoxicity caused by lipolysis products. While protecting cell activity, it successfully achieves efficient degradation regulation of intracellular triglyceride content, providing a new and efficient strategy for the treatment of obesity and related lipid metabolism disorders. Attached Figure Description

[0018] Figure 1This is an inverted microscope image of a biomimetic lipid droplet. Scale bar: 50 μm.

[0019] Figure 2 This is a statistical graph showing the activity of biomimetic lipid droplets on differentiated, mature 3T3-L1 adipocytes.

[0020] Figure 3 This is a laser confocal microscope image of a biomimetic lipid droplet loaded with lipase, scale bar 50 μm;

[0021] Figure 4 This is a laser confocal microscope image of the release of lipase by biomimetic lipid droplets, with a scale bar of 50 μm.

[0022] Figure 5 Microscopic images of the degradation of endogenous lipid droplets by biomimetic lipid droplets in different culture media, scale bar 200 μm;

[0023] Figure 6 A diagram illustrating the cellular mechanism by which biomimetic lipid droplets regulate endogenous lipid droplets;

[0024] Figure 7 The image shows a laser confocal microscope (XYZT) image of the degradation of endogenous lipid droplets by biomimetic lipid droplets. The scale bar is 10 μm. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0026] This invention claims protection for a method for regulating the degradation of endogenous lipid droplets based on enzyme-loaded biomimetic lipid droplets, the method comprising the following steps:

[0027] Step 1: Construction and Biocompatibility of Enzyme-Loaded Biomimetic Lipid Droplets

[0028] (1) Construction of enzyme-loaded biomimetic lipid droplets: Lipase aqueous solution and triacetylglycerol were added to the same EP tube at a volume ratio of 20:3 and mixed. The mixture was shaken at 200 r / min for 10-30 s and then allowed to stand at 37℃ for 20-40 min. Finally, white biomimetic lipid droplets loaded with lipase (Lipase@BLDs) were obtained at the bottom of the tube. The molecular weight of Lipase was 33 kDa.

[0029] (2) Aqueous fluorescent labeling of enzyme-loaded biomimetic lipid droplets: The Lipase aqueous solution labeled with RBITC (RBITC-Lipase) and triacetin were added to the same EP tube at a volume ratio of 20:3 and mixed. After shaking at 200r / min for 10~30s, the mixture was allowed to stand at 37℃ for 20~40min. Finally, red biomimetic lipid droplets (RBITC-Lipase@BLDs) were obtained at the bottom of the tube. In (1) and (2), the concentration of Lipase aqueous solution and Lipase aqueous solution labeled with RBITC were both 5~15mg / mL. Lipase aqueous solution is prepared by dissolving Lipase powder in PBS; RBITC-labeled Lipase aqueous solution is prepared by dissolving RBITC-Lipase powder in PBS; Preparation of RBITC-Lipase powder: Rhodamine B isothiocyanate (RBITC), a fluorescent dye used for labeling proteins, is dissolved in dimethyl sulfoxide to prepare a 5 mg / mL RBITC stock solution; 50 mg of lipase is weighed and dissolved in 10-30 mL of 100 mM sodium bicarbonate buffer, centrifuged at 1500 r, and the supernatant is filtered through a 0.22 μm filter. 20-50 μL of 5 mg / mL RBITC stock solution is slowly added and incubated overnight. The solution is then dialyzed and filtered through a 10 kDa dialysis bag, and subsequently freeze-dried to obtain RBITC-Lipase powder;

[0030] (3) Fluorescent labeling of endogenous lipid droplets: Replace the complete culture medium of adipocytes with 1 mL of phenol red-free DMEM, add 0.5~2 μL of 5 mM BODIPY green fluorescent dye solution, gently shake the culture dish to disperse the dye evenly in the phenol red-free DMEM, and after standing in a CO2 cell culture incubator for 30 min, remove the old phenol red-free DMEM and add 500 μL of new PBS to successfully label endogenous lipid droplets (BODIPY-ELDs) in adipocytes; the BODIPY green fluorescent dye solution is a solution with DMSO as solvent and BODIPY as solute.

[0031] (4) Cytotoxicity of biomimetic lipid droplets: Lipase@BLDs from step (1) was added to 96-well cell culture plates according to the possible dosage gradient (5~100μL) for subsequent experiments and incubated in a CO2 cell culture incubator for 5 min. Then, 10μL of CCK-8 was added and incubated for 1 h. The cytotoxicity of biomimetic lipid droplets was analyzed by measuring the UV absorption peak at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader. CCK-8 refers to 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt.

[0032] Step 2: Regulation of lipase loading and release by biomimetic lipid droplets

[0033] (1) Loading of lipase by biomimetic lipid droplets: After shaking, take 2~10μL of RBITC-Lipase@BLDs from step 1 (2) into a confocal culture dish and observe the loading of lipase by RBITC-Lipase@BLDs in situ under a confocal microscope; the biomimetic lipid droplets will sink to the bottom after preparation, and the purpose of shaking is to ensure that they are added evenly.

[0034] (2) Release of lipase by biomimetic lipid droplets: After preparing RBITC-Lipase@BLDs in step 1 (2), the RBITC-Lipase@BLDs were allowed to stand at 37℃ for 20-40 min. 2-10 μL of the solution was placed in a confocal culture dish. The release of lipase by RBITC-Lipase@BLDs after the addition of 24-60 μL of deionized water was observed in situ using a confocal microscope. To avoid disturbing the distribution of BLDs by the addition of deionized water, the same volume of deionized water was added in four directions to balance and counteract the forces in each direction, ensuring that the position of BLDs remained unchanged and facilitating in-situ observation of the lipase release process. The purpose of adding deionized water was to demonstrate the lipase release process during the experiment.

[0035] Step 3: Regulation of the degradation of endogenous lipid droplets by biomimetic lipid droplets

[0036] (1) Selection of culture medium: Differentiated mature 3T3-L1 adipocytes were placed in 500 μL PBS and DMEM containing 2wt% BSA, respectively. To better compare the degradation effect, 10~100 μL of Lipase@BLDs were added and incubated for 6~12 h. The degradation effect was photographed by an inverted microscope and compared. It can be seen that the degradation effect of DMEM containing 2wt% BSA is better. The reason is that BSA can bind to fatty acids generated during lipid degradation and stabilize the pH change in the environment to a certain extent, thereby improving cell activity and lipase activity. DMEM containing 2wt% BSA needs to be filtered through a 0.22μm filter for sterilization before use.

[0037] (2) Degradation of endogenous lipid droplets by biomimetic lipid droplets: To better observe the lipid degradation behavior induced by BLDs in adipocytes, 20-100 μL of Lipase@BLDs from step one (1) were co-incubated with adipocytes containing labeled endogenous lipid droplets from step one (5). The xyzt lipid degradation process of intracellular ELDs under the action of BLDs was fully recorded using a confocal microscope. The adipocyte culture medium was DMEM containing 2 wt% BSA.

[0038] Example 1: Construction and biocompatibility of biomimetic lipid droplets:

[0039] (1) Construction of enzyme-loaded biomimetic lipid droplets: Lipase aqueous solution and triacetin were added to the same EP tube at a volume ratio of 20:3 and mixed. The mixture was shaken at 200 r / min for 30 s and then incubated at 37℃ for 30 min. Finally, white biomimetic lipid droplets loaded with lipase (Lipase@BLDs) were obtained at the bottom of the tube. Figure 1 The molecular weight of the Lipase is 33 kDa. The Lipase aqueous solution is prepared by dissolving Lipase powder in PBS, with a concentration of 10 mg / mL.

[0040] (2) Aqueous fluorescent labeling of enzyme-loaded biomimetic lipid droplets: Aqueous solution of RBITC-labeled Lipase (RBITC-Lipase) and triacetin were added to the same EP tube at a volume ratio of 20:3. The mixture was shaken at 200 rpm for 30 seconds and then allowed to stand at 37°C for 30 minutes. Red biomimetic lipid droplets (RBITC-Lipase@BLDs) were finally obtained at the bottom of the tube. The molecular weight of the Lipase was 33 kDa. The RBITC-labeled Lipase aqueous solution was prepared by dissolving RBITC-Lipase powder in PBS, with a concentration of 10 mg / mL.

[0041] (3) Cytotoxicity of biomimetic lipid droplets: The dosage gradient of Lipase@BLDs from step (1) was set according to the possible dosage in subsequent experiments (5, 10, 20, 30, 40, 50 μL). Different doses of Lipase@BLDs were dissolved in 1 mL of complete culture medium, shaken to mix, and then added in 100 μL per well to a 96-well cell culture plate (6 replicate wells per group). The plates were incubated in a CO2 cell culture incubator for 5 min, the culture medium was removed, washed with PBS, and then 10 μL of CCK-8 was added and incubated for 1 h. The UV absorption peak at 450 nm was measured using a microplate reader. Figure 2 As shown, the results indicate that Lipase@BLDs has no significant toxicity to adipocytes, demonstrating its good biocompatibility.

[0042] Example 2: Loading and release of lipase by biomimetic lipid droplets:

[0043] (1) Loading of lipase by biomimetic lipid droplets: After shaking, 10 μL of RBITC-Lipase@BLDs from Example 1(2) was placed in a confocal culture dish. The loading of lipase by RBITC-Lipase@BLDs was observed in situ under a confocal microscope over 30 min. Figure 3 As shown, the biomimetic lipid droplets successfully loaded lipase;

[0044] (2) Release of lipase by biomimetic lipid droplets: After preparing RBITC-Lipase@BLDs from Example 1(2), the samples were allowed to stand at 37°C for 30 min. 10 μL of each droplet was placed in a confocal culture dish, and the release of lipase by RBITC-Lipase@BLDs after the addition of 40 μL of deionized water was observed in situ using a confocal microscope. Figure 4 As shown, biomimetic lipid droplets enable the rapid release of lipase.

[0045] Example 3: Regulation of endogenous lipid droplet degradation by biomimetic lipid droplets

[0046] (1) Culture medium selection: Differentiated mature 3T3-L1 adipocytes were placed in 500 μL of PBS ( Figure 5 c) and DMEM containing 2wt% BSA Figure 5 In the culture medium environment of (a), to better compare the degradation effect, 50 μL of Lipase@BLDs (a) was added to each culture medium. Figure 5 b, Figure 5 d) After incubation for 12 hours, the degradation effect was photographed using an inverted microscope, as shown. Figure 5 As shown, DMEM containing 2wt% BSA ( Figure 5 b) has a much higher degradation capacity in the culture medium environment than PBS. Figure 5 d) The culture medium environment was used, so the subsequent Lipase@BLDs-induced ELDs degradation was achieved based on the DMEM (containing 2wt% BSA) culture medium environment;

[0047] (2) Fluorescent labeling of endogenous lipid droplets: Replace the complete culture medium for adipocytes with 1 mL of phenol red-free DMEM, add 1 μL of 5 mM BODIPY green fluorescent dye solution, gently shake the culture dish to evenly disperse the dye in the phenol red-free DMEM, let it stand for 10 min, then remove the old phenol red-free DMEM and add 500 μL of fresh PBS. Figure 7 We successfully labeled endogenous lipid droplets (BODIPY-ELDs) in adipocytes.

[0048] (3) Degradation of endogenous lipid droplets by biomimetic lipid droplets: such as Figure 6 As shown, this illustrates the Lipase@BLDs-induced ELDs degradation process. To observe the lipid degradation behavior of adipocytes induced by Lipase@BLDs, 50 μL of Example 1(1) Lipase@BLDs was added to a 20 mm culture dish containing labeled endogenous lipid droplets and co-incubated. The xyzt lipid degradation process of intracellular ELDs under the action of Lipase@BLDs was fully recorded using a confocal microscope. Figure 7 As shown, the biomimetic lipid droplets demonstrate that they achieve the degradation of endogenous lipid droplets in adipocytes.

Claims

1. A method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets, characterized in that: The method is as follows: Step 1: Mix Lipase aqueous solution with triacetylglycerol at a volume ratio of 20:2~4, shake at 180~220 r / min for 10~30s, and let stand at 35~39℃ for 20~40min to obtain biomimetic lipid droplets loaded with lipase (Lipase@BLDs). Step 2: Co-incubate the enzyme-loaded biomimetic lipid droplets with adipocytes containing endogenous lipid droplets, allowing the biomimetic lipid droplets to come into contact with the adipocytes containing endogenous lipid droplets and mediating the entry of lipase into the interior of the endogenous lipid droplets, thereby achieving the degradation of triglycerides in the endogenous lipid droplets.

2. The method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets according to claim 1, characterized in that: Step one specifically involves adding Lipase aqueous solution and triacetin in a volume ratio of 20:3 to the same EP tube, mixing, shaking at 200 r / min for 10-30 s, and then letting it stand at 37°C for 20-40 min to obtain white biomimetic lipid droplets (Lipase@BLDs) loaded with lipase at the bottom of the tube.

3. A method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets according to claim 1 or 2, characterized in that: In step one, the molecular weight of the Lipase is 33 kDa.

4. A method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets according to claim 1 or 2, characterized in that: In step one, the concentration of the Lipase aqueous solution is 5~15 mg / mL.

5. A method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets according to claim 1 or 2, characterized in that: In step one, the Lipase aqueous solution is prepared by dissolving Lipase powder in PBS.

6. The method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets according to claim 1, characterized in that: In step two, the co-incubation is carried out in DMEM medium containing 2 wt% BSA.

7. The method for regulating endogenous lipid droplet degradation based on enzyme-loaded biomimetic lipid droplets according to claim 1, characterized in that: In step two, the co-incubation time is 6 to 12 hours.

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