A lipid droplet-derived nucleic acid drug delivery system, and a preparation method and application thereof

Abstract

The application discloses a lipid droplet-derived nucleic acid drug delivery system and a preparation method and application thereof, wherein the surface of the lipid droplet-derived nucleic acid drug delivery system is coated with lipid droplets as a shell, and siRNA DGAT2 and PEI are used as the inner core. The nucleic acid complex of the lipid droplet-encapsulated siRNA DGAT2 / PEI can inhibit and remove the subcutaneous white adipose tissue of the inguinal, and the lipid droplet drug delivery system in the application uses gene knockout and gene therapy technology, takes DGAT2 as a target, knocks down the expression of DGAT2 in the whole body and local adipose tissue, significantly improves the knockout efficiency of DGAT2 in the white adipose tissue through the similar solubility of adipocytes to lipid droplets, significantly inhibits the weight gain, reduces the fat volume, and thus achieves the purpose of efficiently preventing and treating obesity, provides a safe and efficient fat-reducing method for obese patients, has a good application prospect, and provides a new idea for the treatment of obesity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a lipid droplet-derived nucleic acid drug delivery system, its preparation method, and its application. Background Technology

[0002] With improved living standards and changing lifestyles, obesity has become a significant threat to human health. The main pathological manifestation of obesity is an excessively high percentage of body fat, potentially accompanied by enlarged fat cells and symptoms of glucose and lipid metabolism imbalance, such as hyperglycemia and hyperlipidemia. Current treatments for obesity primarily include medication, surgery, and lifestyle interventions. However, all three methods have drawbacks. Lifestyle interventions mainly aim to increase energy expenditure beyond intake through dietary restrictions and exercise; however, maintaining a regular lifestyle long-term relies heavily on willpower, and for many individuals with weak willpower, the effectiveness is unstable and prone to relapse. Surgical treatment primarily aims to increase satiety by reducing stomach volume or inserting gastric sacs, thereby reducing energy intake and achieving a therapeutic effect. However, it carries significant postoperative risks, potential malnutrition, and a limited pool of patients who can undergo the procedure. Medication treatments currently include drugs such as orlistat and lorcalceline, which offer relatively stable efficacy, but the lack of a clear target leads to significant side effects; for example, orlistat can cause incontinence.

[0003] RNA interference (RNAi) is a highly effective gene silencing tool that utilizes the ability of designed small interfering RNAs (siRNAs) to bind to target mRNAs and cleave them by the cellular RNA-induced silencing complex (RISC). Advances in siRNA design and chemistry, combined with suitable delivery platforms, have overcome many limitations previously associated with unmodified siRNAs. This has led to the immense potential of RNAi therapy. Increased triglycerides within adipocytes are a contributing factor to obesity, and diacylglycerol acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, could serve as a potential target for treating obesity. DGAT2 is a crucial enzyme in the body, its main mechanism being the covalent bonding of diacylglycerol with a fatty acid acyl-CoA to form triacylglycerol. DGAT2 is expressed at the highest levels in tissues with high lipid metabolism (such as the liver and white adipose tissue), indicating its vital role in lipid metabolism. DGAT2 not only plays a crucial role in TAG synthesis but is also essential for animal survival. Based on the benefits of RNAi therapy, studies have shown that a single subcutaneous administration can produce long-term gene silencing effects. Experiments have demonstrated that knocking down DGAT2 expression in mature adipocytes can effectively inhibit fat accumulation. Therefore, we believe that silencing DGAT2 with small interfering double-stranded RNA compounds has the potential to improve the treatment of obesity. We designed a method to target diacylglycerol acyltransferase 2 (DGAT2) with siRNA, thereby inhibiting the final step in the synthesis of triacylglycerols catalyzed by the esterification of fatty acyl-CoA to DAG, thus reducing intracellular triglyceride levels and significantly controlling fat accumulation in obese patients.

[0004] Currently, common nucleic acid delivery systems used for gene therapy include cationic liposomes, polymer nanostructures, etc. However, these gene delivery vectors have problems such as high toxicity, low biocompatibility, high immunogenicity, and low transfection efficiency, which have significant defects in intracellular delivery of nucleic acid drugs, and the in vivo delivery effect is also not ideal. Summary of the Invention

[0005] Objective of the Invention: To address the shortcomings and defects of existing technologies, this invention provides a lipid droplet-derived nucleic acid drug delivery system. This system boasts advantages such as low immunogenicity, high adipocyte selectivity, and high transfection efficiency. Furthermore, by using DGAT2 as an RNAi technology target, it regulates the adipogenesis process, effectively treating obesity. The system of this invention effectively solves the problems of low nucleic acid delivery efficiency, low transfection efficiency, high immunogenicity, and high toxicity of delivery vectors in existing gene therapy methods. Moreover, lipid droplets possess the advantage of uniformity and stability, making them a valuable nucleic acid delivery vector.

[0006] This invention also provides a method for preparing and applying a lipid droplet-based nucleic acid drug delivery system.

[0007] Technical solution: In order to achieve the above solution, the present invention provides a lipid droplet-based nucleic acid drug delivery system, wherein the surface of the lipid droplet-based nucleic acid drug delivery system is coated with lipid droplets as a shell, and siRNA DGAT2 and PEI are used as the core. The lipid droplets are formed by adipogenic induction of preadipocytes 3T3-L1.

[0008] The siRNA DGAT2 sequence is as follows:

[0009] 5'-GGUAGAGAAGCAGCUCCAATT-3'

[0010] 3'-UUGGAGCUGCUUCUCUACCTT-5'.

[0011] The preparation method of the lipid droplet-derived nucleic acid drug delivery system of the present invention includes the following steps:

[0012] (1) Lipid droplets were extracted from 3T3-L1 cells after induction;

[0013] (2) The nucleic acid complex of siRNA DGAT2 and PEI was prepared by self-assembly method;

[0014] (3) The lipid droplets and nucleic acid complexes were mixed, co-incubated, and then sonicated to obtain a lipid droplet-derived nucleic acid drug delivery system.

[0015] In step (1), 3T3-L1 cells are induced to become lipids when they are in good growth condition. When the cells increase in volume and a series of lipid droplets appear in the cells, lipid droplets are extracted.

[0016] The specific process of 3T3-L1 cell induction and lipid droplet extraction in step (1) is as follows: After successful induction of lipid droplets from 3T3-L1 cells, the 3T3-L1 adipocytes are digested with trypsin using a lipid droplet separation kit, washed, and then resuspended in buffer A and placed on ice; buffer B is added to the cell suspension and mixed thoroughly, and then incubated on ice; after homogenization of the cells, they are centrifuged, the upper lipid droplets are collected, and then lyophilized and stored.

[0017] Buffer A: An aqueous solution containing 20 mM Tricine and 250 mM sucrose (pH 7.8)

[0018] Buffer B: An aqueous solution containing 20 mM HEPES, 100 mM KCl, and 2 mM MgCl2 (pH 7.4).

[0019] In step (2), PEI and siRNA DGAT2 are incubated for 1-2 hours using a self-assembly method at a ratio of N / P = 5:1-2. This is based on the molar ratio of nitrogen in PEI to phosphorus in nucleic acid.

[0020] As a preferred method, PEI and siRNA DGAT2 are incubated at a ratio of N / P = 5:1 for 1 hour.

[0021] In step (3), each 100 μL lipid droplet is mixed with the nucleic acid complex containing 20-30 μg obtained in step (2), and incubated for 30-40 min, followed by sonication for 5-10 min to obtain a lipid droplet-based nucleic acid drug delivery system.

[0022] Preferably, the preparation method of the lipid droplet-based nucleic acid drug delivery system includes the following steps:

[0023] Step 1: Induction and extraction of lipid droplets

[0024] Preadipocytes (3T3-L1) were induced to adipogenesis when they were in good growth condition. Lipid droplets were extracted when the cells increased in size and formed strings of lipid droplets. Approximately 3 × 10⁶ lipid droplets were separated using a lipid droplet separation kit. 7 Adipogenic cells were digested with trypsin and washed twice with 1×PBS. The cells were then resuspended in 200 μL of buffer A and placed on ice for 10 minutes. 800 μL of 1× buffer B was added to the cell suspension and mixed thoroughly, then incubated on ice for another 10 minutes. The cells were then further homogenized by passing them through a 3 mL syringe with a 1-inch 27-gauge needle, and this process was repeated 5 times. The cell homogenate was centrifuged at 100×g for 5 seconds and carefully separated into layers with 600 μL of 1× buffer B. Finally, the cells were centrifuged at 20,000×g for 3 hours at 4°C, and the supernatant lipid droplets were carefully collected, lyophilized, and stored at -80°C.

[0025] Step 2: Prepare a nucleic acid complex of siRNA DGAT2 and PEI using a self-assembly method. siRNA DGAT2 is negatively charged while PEI is positively charged. The mixture of PEI and siRNA DGAT2 is N / P = 5:1 and the mixture is incubated for 1 h.

[0026] Step 3: Mix the lipid droplets obtained in Step 1 and the nucleic acid complex obtained in Step 2, incubate them together for 40 minutes, and then sonicate to obtain nucleic acid complex nanoparticles encapsulated by the lipid droplets.

[0027] The application of the obesity treatment drug described in this invention in the preparation of the obesity treatment drug.

[0028] The application of the obesity treatment drug is specifically in the preparation of the siRNA DGAT2 gene therapy drug for obesity.

[0029] In this invention, lipid droplets on adipocytes exert a "homing" effect on adipocytes, thereby significantly improving the efficiency of nucleic acid drugs entering cells. This invention designs a lipid droplet-derived nucleic acid complex delivery vector, in which the nucleic acid complex is coated with a lipid droplet membrane, to deliver DGAT2 siRNA to subcutaneous and visceral white adipose tissue. This achieves the effects of knocking down DGAT2 expression, inhibiting the synthesis and excessive accumulation of adipose tissue, reducing fat volume, and reducing weight and fat accumulation.

[0030] In this invention, siRNA DGAT2 and PEI are co-incubated in a certain ratio to form a nucleic acid complex. This nucleic acid complex is positively charged and self-assembles with negatively charged lipid droplets through positive and negative charge interactions to form a nanoscale delivery carrier. The lipid droplets in the system prepared in this invention encapsulate the siRNA DGAT2 / PEI nucleic acid complex, inhibiting and clearing subcutaneous white adipose tissue in the groin. By establishing a high-fat diet-induced obesity model in mice, the lipid droplet delivery system of this invention utilizes gene knockout and gene therapy technologies, targeting DGAT2, to knock down DGAT2 expression in systemic and local adipose tissue. Through the similarity-compatibility of lipid droplets with adipocytes, the knockout efficiency of DGAT2 in white adipose tissue is significantly improved, significantly inhibiting weight gain and reducing fat volume, thereby achieving the goal of effectively preventing and treating obesity. This provides a safe and efficient fat reduction method for obese patients, showing good application prospects and offering a new approach to the treatment of obesity.

[0031] This invention relates to a lipid droplet-derived nucleic acid complex delivery system, which internally encapsulates a complex of siRNA DGAT2 and PEI. Lipid droplets, serving as the shell of the nucleic acid delivery system, possess excellent properties such as uniformity and stability, low immunogenicity, high biocompatibility, and high delivery efficiency, significantly knocking down DGAT2 expression in the groin and visceral white fat tissue. This invention established a high-fat diet-induced obesity model in mice and used RNAi technology to knock down DGAT2 expression in the groin and visceral adipose tissue. Results showed that under high-fat dietary stimulation, the treatment group mice exhibited significantly reduced DGAT2 expression and significantly inhibited weight gain, significantly suppressed the growth and accumulation of white fat cells, and reduced fat volume. Therefore, the lipid droplet-derived nucleic acid delivery vector has excellent efficacy in delivering siRNA DGAT2, enabling the model group of obese mice to achieve highly effective treatment of obesity. This invention is the first to propose the concept of a lipid droplet-derived nucleic acid delivery vector, and also verifies that the system has advantages such as low immunogenicity, high selectivity for adipocytes, and high transfection efficiency. Furthermore, it selects DGAT2, a key enzyme, as the target of RNAi technology to regulate the adipogenesis process and effectively treat obese mice.

[0032] This invention utilizes lipid droplets as a nucleic acid delivery carrier. Lipid droplet-based nucleic acid delivery carriers have advantages over other carriers in terms of biosafety and delivery efficiency. Furthermore, lipid droplet-based nucleic acid delivery carriers can be selectively taken up by adipocytes in the body. This is because lipid droplets originate from adipocytes, thus exhibiting a "homing" effect on adipocytes. This design principle effectively supports experimental results with high delivery efficiency.

[0033] This invention is the first to use lipid droplets as a delivery carrier for nucleic acids, loading nucleic acid complexes onto lipid droplets, which are then selectively taken up by adipocytes in the body and achieve therapeutic effects with low immunogenicity.

[0034] Beneficial results: Compared with the prior art, the present invention has the following advantages:

[0035] This invention utilizes biomimetic technology to prepare a lipid droplet-derived nucleic acid delivery system. The lipid droplets used have a negative charge and spontaneously assemble and load onto a positively charged siRNA DGAT2 / PEI complex via electrostatic interactions, forming capsule-like nanoparticles. As a homogeneous and stable source of nanostructures, the lipid droplets, when used to coat the nucleic acid complex, enhance the uptake of the nucleic acid complex by adipocytes and significantly improve transfection efficiency.

[0036] The lipid droplet-derived nucleic acid delivery system of the present invention not only has higher safety and lower immunogenicity, reducing the toxic side effects of transfection reagents, but also significantly improves drug delivery efficiency, enabling siRNA DGAT2 to achieve better knockdown efficiency, and provides a more effective delivery vector for the delivery of more genes from adipose tissue, with very broad application prospects. Attached Figure Description

[0037] Figure 1 The particle size and zeta potential of lipid droplets (LDs) and their derived vector (LDs@siRNADGAT2);

[0038] Figure 2 This study compares the cytotoxicity of LDs@siRNADGAT2 with Lipo@siRNA DGAT2.

[0039] Figure 3 This experiment compares the cellular uptake rates of LDs@siRNA DGAT2 and Lipo@siRNA DGAT2.

[0040] Figure 4 This is a comparison of food intake between the control group and the treatment group of mice;

[0041] Figure 5 The changes in body weight of mice in the control and treatment groups;

[0042] Figure 6 The changes in body size were observed in mice in the control and treatment groups.

[0043] Figure 7 To verify the knockdown of DGAT2 expression after LDs@siRNADGAT2 was injected into the groin area using immunofluorescence experiments;

[0044] Figure 8 To verify the knockdown of DGAT2 expression after LDs@siRNADGAT2 was injected into the groin area in a Western blot experiment. Detailed Implementation

[0045] To make the present invention easier to understand, the present invention will be further described below with reference to specific embodiments. These embodiments are not intended to limit the present invention in any way. They are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any modifications or changes to the present invention that are easily implemented by those skilled in the art without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

[0046] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0047] 3T3-L1 cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0048] siRNADGAT2 was synthesized by Shanghai Kelei Company. Two sequences were mixed in equimolar amounts and used simultaneously: 5'-GGUAGAGAAGCAGCUCCAATT-3'; 3'-UUGGAGCUGCUUCUCUAC CTT-5'.

[0049] PEI was purchased from Yuan Ye, catalog number: S22706. Liposomes can be purchased or synthesized according to existing methods; in this invention, they were synthesized according to the method described in the literature "Mitochondrion-specific dendritic lipopeptide liposomes for targeted sub-cellular delivery".

[0050] Example 1

[0051] A method for preparing a lipid droplet-derived nucleic acid complex delivery system includes the following steps:

[0052] 1. Induction and extraction of lipid droplets

[0053] (1) Culture of the 3T3-LI cell line:

[0054] 3T3-LI is a mouse pre-adipocyte fibroblast. Following standard adherent cell culture methods, cells were placed in DMEM high-glucose medium containing 10% FBS and cultured at 37°C, 5% CO2, and saturated humidity. Cells were passaged and divided into flasks, with each flask containing approximately 1 × 10⁻⁶ cells. 5 Change the solution every 48 hours.

[0055] (2) Induction of the 3T3-LI cell line:

[0056] Preadipocyte induction needs to be performed when cells are in good growth condition. After 2 days of contact inhibition, the culture medium is replaced with DMEM containing 10% FBS and 0.5 mM IBMX, 0.25 μM dexamethasone, 1 μg / ml insulin, and 2 μM rosiglitazone, and cultured for 48 h. Then, it is replaced with 10% FBSDMEM containing 1 mg / ml insulin and cultured for 48 h. Finally, the culture medium is replaced with fresh DMEM containing 10% FBS every 48 h for 7 consecutive days. When the cells increase in volume and strings of lipid droplets appear in the cells, lipid droplet induction is successful, and adipocytes are obtained.

[0057] (3) Extraction of lipid droplets from mature adipocytes

[0058] The lipid droplet separation kit (Abcam, cat no. ab242290) was used, with some modifications. Approximately 3 × 10⁻⁶ droplets cultured in step (2) were... 7 Adipogenic cells were digested with 1-2 ml of trypsin and washed twice with 1 ml of 1×PBS (pH 7.3). The cells were then resuspended in 200 μL of buffer A and placed on ice for 10 minutes. 800 μL of 1× buffer B was added to the cell suspension and mixed thoroughly, then incubated on ice for another 10 minutes. The cells were then further homogenized by passing them through a 3 mL syringe with a 1-inch 27-gauge needle, and this process was repeated 5 times. The cell homogenate was centrifuged at 100×g for 5 seconds and carefully separated into layers with 600 μL of 1× buffer B. Finally, the cells were centrifuged at 20,000×g for 3 hours at 4°C, and the supernatant lipid droplets were carefully collected, lyophilized, and stored at -80°C.

[0059] Buffer A: An aqueous solution containing 20 mM Tricine and 250 mM sucrose (pH 7.8)

[0060] Buffer B: An aqueous solution (pH 7.4) containing 20 mM HEPES, 100 mM KCl, and 2 mM MgCl2.

[0061] 2. Preparation of the core of the nucleic acid complex:

[0062] A nucleic acid complex of siRNA DGAT2 and PEI was prepared using a self-assembly method. siRNA DGAT2 carries a negative charge, while PEI carries a positive charge. The two bind to each other through electrostatic interactions under co-incubation conditions to form the nucleic acid complex. Specifically, PEI and two equimolar amounts of siRNA DGAT2 were mixed at a N / P ratio of 5:1 and allowed to stand at room temperature for 1 hour to form the nucleic acid complex.

[0063] Step 3: Mix 100 μL of lipid droplets obtained in Step 1 with the nucleic acid complex containing 20 μg obtained in Step 2, incubate for 40 min, and then sonicate for 5 min to obtain the nucleic acid complex nanoparticles encapsulated by the lipid droplets, i.e., the lipid droplet-derived nucleic acid complex delivery system.

[0064] Example 2

[0065] Characterization of particle size and potential of lipid droplet-derived nucleic acid complex delivery systems

[0066] The particle size and potential of the lipid droplets prepared in Example 1 and the lipid droplet-derived nucleic acid complex delivery system were measured using a particle size and potential analyzer (Litesizer 500, Anton Paar, Austria). Figure 1 As shown, the extracted lipid droplets have a particle size of approximately 225 nm and a potential of -11.7 mV. The lipid droplet-derived vector (lipid droplet-derived nucleic acid complex delivery system) loaded with nucleic acid complexes has a particle size of approximately 294 nm and a potential of 17.94 mV.

[0067] Example 3

[0068] The cytotoxicity MTT assay of lipid droplet nucleic acid delivery vectors was used to evaluate their safety and verify their efficacy.

[0069] First, adipose tissue cells 3T3-LI were subjected to a 1×10⁻⁶ solution. 5 Cells were seeded at a density of 1,000 cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. When the cells adhered and reached a cell density of 50%, 100 μL of PBS (pH 7.3) and the lipid droplet-derived nucleic acid complex delivery system prepared in Example 1 (6 replicates per group) were added to each well, and incubation continued for another 24 h under the cell culture conditions. 15 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for another 4 h. The 96-well plate was inverted, residual liquid was tapped out, and 150 μL of DMSO solution was added to each well. After shaking on a shaker at room temperature for 15 min, the OD value of each well was measured at 490 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability = OD value of drug-treated group / OD value of blank group × 100%. Results are as follows: Figure 2As shown, the lipid droplet-derived nucleic acid delivery vector did not cause significant damage to cells, and the cell survival rate was not significantly different from that of the control group.

[0070] Example 4

[0071] Validation of the delivery efficiency of the lipid droplet-derived nucleic acid complex delivery system and investigation of its uptake by adipocytes.

[0072] Lipid droplets derived from mature adipocytes exhibit extremely high affinity for adipocytes, significantly enhancing the uptake efficiency of gene delivery systems. To obtain fluorescently labeled lipid droplets (LDs-FITC), 100 μL of a lipid droplet-nucleic acid complex (the lipid droplet-derived nucleic acid complex delivery system prepared in Example 1) was labeled with 20 mM fluorescein FITC. After reacting at room temperature for 2 hours, the mixture was centrifuged at 20000 × g for 1 hour, and the upper layer of fluorescently labeled lipid droplets was carefully collected. Lipo-FITC staining was performed using the same method: 100 μL of a liposome-nucleic acid complex (liposomes were replaced with lipid droplets according to the preparation method in Example 1) was labeled with 20 mM fluorescein FITC, centrifuged at 2000 × g for 1 hour in a 100 kDa ultrafiltration tube to obtain fluorescently labeled liposomes. For cellular uptake experiments, 1 × 10⁻⁶ LDs were used to label the liposomes. 5 3T3-L1 cells were incubated with 50 μL of LDs-FITC and Lipo-FITC for 4 hours, respectively. Then, 2 μg / mL DAPI was added for staining for 10 min to label the cell nuclei. The cells were washed three times with PBS to remove excess dye. Finally, 50 μL of anti-fluorescence quenching mounting buffer was added, and qualitative observation was performed using a confocal laser scanning microscope. Under confocal microscopy, the fluorescence intensity of the intracellular fluorescently labeled carriers reflected the uptake efficiency of the two carriers by adipocytes.

[0073] like Figure 3 As shown, the intracellular fluorescence intensity of the LDs-FITC group was higher than that of the Lipo-FITC group, indicating that the adipocytes took up significantly more LDs-FITC. The greater the uptake, the better the delivery efficiency, indicating that the lipid droplet-derived nucleic acid drug delivery system has a higher affinity for adipocytes and can be taken up by more adipocytes.

[0074] Example 5

[0075] Establishment of an obese mouse model

[0076] C57 mice aged 6-8 weeks were fed a high-fat diet containing 60% kcal of fat (purchased from Nanjing Hemiao Biotechnology Co., Ltd.). The diet was added to the cages every three days, and the mice's drinking water was changed. This free-range feeding was maintained for eight weeks, with cages changed weekly, to establish an obese mouse model. After eight weeks, the successfully established obesity model mice were randomly divided into a control group and a treatment group. Mice in the treatment group received a subcutaneous injection of a lipid droplet nucleic acid vector carrying siRNA DGAT2 (prepared in Example 1) in the groin. Mice in the control group received an equal volume of PBS (pH 7.3) as a control. The specific injection steps are as follows:

[0077] 1) Grasp the mouse: Gently pull the mouse by the tail with your right hand, and grasp the fur behind the ear on the back of the mouse with your left thumb and forefinger. After grasping, clamp the tail between your left little finger and ring finger to fix the entire body of the mouse. After holding it firmly, make the mouse face up and head down.

[0078] 2) Injection: Gently wipe the skin of the mouse groin area with an alcohol swab, lift the skin at the injection site to create a subcutaneous space, insert the syringe into the space, slowly inject the carrier solution, and then gently withdraw the syringe. Inject 50 μL of the lipid droplet-derived nucleic acid complex delivery system prepared in Example 1 into each mouse's bilateral femoral groin area, for a total injection volume of 100 μL per mouse. Injections were given every other day for a total of one week. The knockdown of DGAT2 was verified by tissue immunofluorescence and Western blotting experiments.

[0079] Weight measurement: The mice were weighed weekly and the weight changes were recorded.

[0080] Feed consumption measurement: Each day, the remaining high-fat feed of each cage of mice was taken out, weighed and recorded. The difference between the weights was used to obtain the feed consumption of the mice. The feed consumption was recorded.

[0081] Figure 4 The results showed that the control and treatment groups had similar feed consumption, but Figure 5 The weight change curves of mice showed that, under the same dietary conditions, the weight of mice in the control group increased significantly, while the weight change of mice in the treatment group was relatively stable, with no obvious increasing trend. Figure 6 The mouse body size chart also shows that the control group mice (left) showed a significant increase in body size, but the mice injected with the lipid droplet-derived nucleic acid complex delivery system (right) showed no significant change in body size. Therefore, high-fat feeding did not significantly increase the weight and body size of DGAT2 knockdown mice. Thus, under high-fat feeding conditions, DGAT2 knockdown still significantly inhibited weight gain in mice. Under the same high-fat diet, the control group mice without the delivery system still gained weight, but the mice with the delivery system clearly showed a weight-controlling effect.

[0082] Furthermore, in vivo injection of simple nucleic acid complexes has almost no effect; the control group injected with PBS can be compared. This further demonstrates the significant effectiveness of the delivery system of this invention.

[0083] This embodiment demonstrates that untreated mice fed a high-fat diet still experience weight gain, while mice treated while also consuming a high-fat diet maintain a stable weight without significant increase. This indicates that the system of the present invention has a significant effect on inhibiting fat production, thereby controlling weight.

[0084] Example 6

[0085] Paraffin sections, DGAT2 immunofluorescence staining, and Western blotting were performed on the inguinal adipose tissue of mice treated for 6 weeks in Example 5.

[0086] Paraffin sections of inguinal adipose tissue:

[0087] 1) Fixation: After rinsing with PBS, fix with PFA.

[0088] 2) Dehydration: First, soak in 70% ethanol at 4°C overnight, then soak in 95% ethanol for 2 hours, then transfer to another 95% ethanol for 2 hours, then transfer to 100% ethanol for 4 hours, and finally transfer to another 100% ethanol for overnight.

[0089] 3) Embedding: Immerse the tissue in xylene substitute at room temperature for 2 hours. After turning on the embedding machine, let the tissue soak in paraffin in a 62°C water bath for 2.5 hours. Turn on the cooling stage 30 minutes before embedding, and perform a short cooling after sectioning. Place a large beaker filled with water in a 55°C water bath and immerse the sections in the water to keep them warm.

[0090] 4) Dewaxing: Immerse the sections in xylene for 15 minutes to dewax, and repeat this step once.

[0091] 5) Rehydration: Wash with 100%, 95%, and 70% ethanol for 3 minutes each.

[0092] 6) Staining: Perform staining according to the Solarbio IE staining kit instructions.

[0093] 7) Dehydration: Soak the stained adipose tissue paraffin sections in 70% ethanol for 1 min, 95% ethanol for 1 min, and finally in 100% ethanol for 2 min.

[0094] 8) Mounting: Soak the sections twice with xylene substitute, 5 minutes each time, and then mount with neutral resin to obtain fatty paraffin sections.

[0095] 9) Observation: Place the slide under an optical microscope to observe the cell morphology and take pictures.

[0096] Figure 7 The results showed that the expression level of DGAT2 in the adipose tissue of mice injected with the lipid droplet-derived nucleic acid complex delivery system was significantly reduced compared with the control group.

[0097] Western-Blot detection method:

[0098] 1) Select a suitable size adhesive sheet that matches the sample loading amount. Clean the adhesive sheet and blow-dry it until it is completely dry, then fix it to the clamp.

[0099] 2) Prepare stacking gel and separating gel of appropriate concentration, and add about 20 μg of protein sample to each lane.

[0100] 3) Adjust the voltage to 80V, and increase it to 120V after the markers are completely separated.

[0101] 4) After electrophoresis is completed, the target protein is transferred to the PVDF membrane using a constant current of 220mA.

[0102] 5) Immerse the membrane completely in the 5% BSA solution prepared by TBST and seal it on a shaker at room temperature for 1 hour.

[0103] 6) Prepare antibody dilution solution and dilute the primary antibody to make antibody reaction bags.

[0104] 7) Place the membrane in an antibody reaction bag and incubate overnight at 4°C.

[0105] 8) Wash the membrane with TBST at room temperature for 5 minutes each time, for a total of 6 washes, while keeping it on a decolorizing shaker throughout the process.

[0106] 9) Dilute the horseradish peroxidase-labeled secondary antibody to the required concentration with TBST, place the membrane and secondary antibody into the antibody reaction bag at the same time, and incubate at room temperature on a horizontal shaker for 1 hour.

[0107] 10) Wash the membrane with TBST at room temperature for 5 minutes each time, for a total of 6 washes. Keep the membrane on a decolorizing shaker throughout the process.

[0108] 11) Prepare chemiluminescent substrates using the ECL Western Blot kit and expose them using an exposure unit.

[0109] 12) Observe and save the images, and analyze the changes in the target protein at the corresponding molecular weight.

[0110] Figure 8 Western blot results showed that, compared with the control group, the expression of DGAT2 in the inguinal adipose tissue of mice treated with the lipid droplet-derived nucleic acid complex delivery system was significantly reduced, indicating that the nucleic acid delivery system can significantly silence DGAT2 in adipocytes, and that the weight loss effect is due to DGAT2 knockdown.

Claims

1. A lipid droplet-derived nucleic acid drug delivery system, characterized in that, The lipid droplet-derived nucleic acid drug delivery system has lipid droplets as its outer shell and siRNA DGAT2 and polyethyleneimine (PEI) as its core. The system uses a self-assembly method to form a nucleic acid complex with PEI and siRNA DGAT2 at an N / P ratio of 5:1-2. The sequence of the siRNA DGAT2 is as follows: 5'-GGUAGAGAAGCAGCUCCAATT-3' 3'- UUGGAGCUGCUUCUCUACCTT- 5'; The lipid droplets are formed by adipogenic induction of preadipocytes 3T3-L1.

2. A method for preparing the lipid droplet-derived nucleic acid drug delivery system according to claim 1, characterized by, Includes the following steps: (1) Lipid droplets were extracted from 3T3-L1 cells after induction; (2) The nucleic acid complex of siRNA DGAT2 and PEI was prepared by self-assembly; (3) The lipid droplets and nucleic acid complexes were mixed, co-incubated, and then sonicated to obtain a lipid droplet-derived nucleic acid drug delivery system.

3. The production method according to claim 2, characterized by, In step (1), 3T3-L1 cells were induced to become lipids when they were in good growth condition. When the cells increased in size and a string of lipid droplets appeared in the cells, the lipid droplets were extracted.

4. The preparation method according to claim 2, characterized in that, The specific process of 3T3-L1 cell induction and lipid droplet extraction in step (1) is as follows: After successful induction of lipid droplets from 3T3-L1 cells, the 3T3-L1 adipocytes were digested with trypsin using a lipid droplet separation kit, washed, and then resuspended in buffer solution and placed on ice; buffer solution was added to the cell suspension and mixed thoroughly, and then incubated on ice; after homogenization of the cells, they were centrifuged, and the upper lipid droplets were collected by centrifugation, lyophilized and stored.

5. The preparation method according to claim 2, characterized in that, In step (2), PEI and siRNA DGAT2 are incubated for 1-2 hours using a self-assembly method at a ratio of N / P=5:1-2.

6. The preparation method according to claim 2, characterized in that, In step (3), lipid droplets and nucleic acid complexes are mixed, co-incubated for 30-40 min, and then sonicated to obtain a nucleic acid drug delivery system based on lipid droplets.

7. The use of the lipid droplet-derived nucleic acid drug delivery system of claim 1 in the preparation of an obesity treatment drug.

8. Use according to claim 7, characterized in that, The application of the lipid droplet-derived nucleic acid drug delivery system in the preparation of siRNA DGAT2 gene therapy drugs for obesity.

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