Preparation method of nanoparticles based on biguanide derivative polymer and application of nanoparticles in improvement of lipid deposition

Abstract

The invention discloses a preparation method of nanoparticles based on a biguanide derivative polymer and application of the nanoparticles in improvement of lipid deposition, and relates to the field of biological medicines. Polymeric nanoparticles (Poly-MET-NP) derived from biguanide are prepared by controlled free radical polymerization for use in the treatment of lipid deposition and insulin resistance in non-alcoholic fatty liver disease (NAFLD). In-vitro experiments utilize a HepG2 hepatocyte model to detect the influence of Poly-MET-NP on basic respiration, ATP generation and lipid deposition, and the result shows that the Poly-MET-NP can promote energy metabolism and reduce lipid accumulation. A high fat diet mouse model is adopted in an in-vivo experiment, Poly-MET-NP is injected into the abdominal cavity every week, and the influence of Poly-MET-NP on the body weight, liver lipid accumulation, insulin sensitivity and energy consumption is observed. Results show that the Poly-MET-NP significantly reduces liver lipid deposition, reduces weight gain, improves insulin sensitivity and increases energy consumption. The Poly-MET-NP has good biocompatibility and treatment potential, and is suitable for the treatment of NAFLD (non-alcoholic fatty liver disease).

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation method of biguanide-derived polymer nanoparticles and application thereof in improving lipid deposition in vivo and in vitro. Background Art

[0002] In recent years, with the change of lifestyle and the adjustment of dietary structure, the incidence of metabolic diseases such as obesity, diabetes and non-alcoholic fatty liver disease has increased year by year. These metabolic diseases not only seriously affect human health, but also bring huge burdens to the society and medical system. Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Its main feature is excessive lipid deposition in the liver, which in turn causes a series of health problems, such as insulin resistance, liver fibrosis and cirrhosis.

[0003] In the prevention and treatment of these metabolic diseases, traditional treatment methods mainly rely on drug intervention. For example, metformin and other drugs are widely used to treat type 2 diabetes, which can reduce fat accumulation by increasing glucose uptake in the liver and muscle tissue and reducing glycogen output. However, the efficacy of existing drugs is limited, and drug resistance and side effects may occur during long-term use, so there is an urgent need to develop new treatment strategies.

[0004] In this study, we prepared a biguanide-derived polymer nanoparticle (Poly-MET-NP) by RAFT polymerization, and verified its effectiveness in promoting energy metabolism and reducing lipid deposition through a series of in vitro and in vivo experiments. In cell experiments, Poly-MET-NP significantly increased the basal respiration, maximum respiration, and ATP production of HepG2 cells, indicating that it can promote energy consumption, increase fatty acid oxidation, and reduce lipid accumulation in liver cells. At the same time, Poly-MET-NP showed good safety in cytotoxicity tests and did not cause significant damage to cell viability.

[0005] Therefore, the present invention provides a preparation method based on biguanide-derived polymer nanoparticles and its application in improving lipid deposition, which provides new ideas and strategies for the treatment of non-alcoholic fatty liver disease. Summary of the invention

[0006] The present invention relates to a preparation method based on biguanide-derived polymer nanoparticles and its application in improving lipid deposition, especially regulating lipid metabolism, reducing lipid deposition in vivo and in vitro, and alleviating metabolic disorders such as non-alcoholic fatty liver disease (NAFLD) by increasing energy consumption. The present invention provides a novel biguanide-derived polymer nanoparticle (Poly-MET-NP), which has been verified by in vivo and in vitro experiments to have significant effects in promoting energy metabolism, reducing lipid deposition and improving insulin resistance.

[0007] In a first aspect, the present invention provides a novel biguanide-derived polymer nanoparticle (Poly-MET-NP), wherein the biguanide-derived groups are copolymerized onto polyethylene glycol molecules via a RAFT reaction, thereby improving the therapeutic effect of biguanide drugs on diseases.

[0008] Furthermore, the nanoparticles include: (a) biguanide derivative groups; (b) high molecular polymers; and or (c) connecting molecules, etc.

[0009] Furthermore, the high molecular weight polymer includes polyethylene glycol of any molecular weight.

[0010] Furthermore, the Poly-MET-NP nanoparticles may be polymers or nanoparticles containing polyethylene glycol as a component, or polymers other than polyethylene glycol.

[0011] In a second aspect, the present invention provides an application of the Poly-MET-NP nanoparticles according to any one of the first aspects, the application comprising:

[0012] Used to treat or prevent metabolic diseases in humans or other mammals, including but not limited to obesity, non-alcoholic fatty liver disease, hyperlipidemia, etc.

[0013] Used to increase cellular basal respiration and ATP production, enhance cellular energy metabolism, and reduce intracellular lipid deposition.

[0014] Used to control the increase in body weight and liver weight caused by a high-fat diet and reduce the effect of hepatic lipid deposition.

[0015] Used to relieve insulin resistance caused by a high-fat diet.

[0016] Used to effectively increase energy expenditure in the body without affecting food intake, motor activity and respiratory exchange rate.

[0017] The present invention provides a novel biguanide-derived polymer nanoparticle (Poly-MET-NP), and demonstrates its outstanding effects in improving lipid metabolism, increasing energy consumption and alleviating insulin resistance. Its main advantages and beneficial effects include:

[0018] Enhance energy metabolism: Poly-MET-NP promotes cellular energy metabolism and reduces lipid accumulation by significantly increasing basal respiration and ATP production, especially showing good therapeutic effects in lipid metabolism disorders.

[0019] Reduce lipid deposition: Poly-MET-NP can significantly reduce lipid deposition in in vivo and in vitro experiments, especially in the non-alcoholic fatty liver disease model caused by a high-fat diet. It can effectively reduce the number of liver fat droplets and reduce the lipid burden of the liver.

[0020] Relieve insulin resistance: In in vivo experiments, Poly-MET-NP significantly improved the insulin sensitivity of mice and alleviated insulin resistance caused by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 NMR spectrum of biguanide-derived polymer

[0022] Figure 2 Transmission electron microscopy image of biguanide-derived polymer nanoparticles

[0023] Figure 3 Changes in HepG2 cell viability after treatment with different concentrations of biguanide-derived polymers

[0024] Figure 4 The changes in oxygen consumption rate and energy metabolism of HepG2 cells after treatment with biguanide-derived polymers. A. Changes in oxygen consumption rate. B. Cell non-mitochondrial respiration. C. Cell maximum respiration. D. Cell basal respiration. E. Cell ATP production.

[0025] Figure 5 The changes in lipid metabolism of HepG2 cells after treatment with biguanide-derived polymers. A. Laser confocal microscopy of BODIPY staining to detect lipid droplet content. B. Fluorescence quantification of BODIPY staining to detect lipid droplets.

[0026] Figure 6 The changes in lipid deposition in HepG2 cells after treatment with biguanide-derived polymers. A. Oil Red O staining of HepG2 cells after treatment with biguanide-derived polymers. B. Quantification of lipid droplets in HepG2 cells stained with Oil Red O. C. Triglyceride (TG) levels in HepG2 cells.

[0027] Figure 7 Treatment of non-alcoholic fatty liver model mice with biguanide-derived polymers improves lipid metabolism. A. Changes in mouse body weight. B. Differences in mouse liver weight. C. Dietary intake of mice. D. Oil red O staining of mouse liver sections. E. Triglyceride levels in mouse liver. F. Free fatty acid levels in mouse liver.

[0028] Figure 8Biguanide-derived polymer nanoparticles improve insulin resistance in mice with non-alcoholic fatty liver disease. A. Blood glucose change curve of insulin tolerance test (ITT). B. Total blood glucose area under the curve (AUC) of insulin tolerance test. C. Blood glucose change curve of glucose tolerance test (ITT). D. Total blood glucose area under the curve (AUC) of glucose tolerance test.

[0029] Fig. 9 The effect of biguanide-derived polymer nanoparticles on energy consumption. A. Curve of energy consumption changes in mice within 24 hours. B. Area under the curve of energy consumption changes in mice within 24 hours. C. Curve of motor activity level changes in mice within 24 hours. D. Area under the curve of motor activity level changes in mice within 24 hours. E. Curve of respiratory exchange rate (RER) changes in mice within 24 hours. F. Area under the curve of respiratory exchange rate (RER) changes in mice within 24 hours.

[0030] Fig.10 It is the serum index of mice after treatment with biguanide-derived polymers. DETAILED DESCRIPTION

[0031] The present invention relates to a preparation method based on biguanide-derived polymer nanoparticles and its application in improving lipid deposition, and specifically describes the preparation of biguanide-derived polymer nanoparticles Poly-MET-NP and the effect of improving lipid deposition in cells and animal models. The technical scheme in the embodiments of the present invention is clearly and completely described below. Obviously, the described examples are only part of the embodiments of the present invention, not all of the embodiments. Based on the implementation examples in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention.

[0032] Example 1 Preparation of Biguanide-derived Polymer Nanoparticles Poly-MET-NP

[0033] The biguanide-derived polymer nanoparticles Poly-MET-NP of the present invention are prepared by RAFT (reversible addition-fragmentation chain transfer) polymerization. First, under nitrogen protection, a monomer containing biguanide derivatives (N-(2-(3-carbamoylguanidino)ethyl)methacrylamide) and 4-cyano-4-(phenylthiocarbamoylthio)pentanoic acid (PEG-CPADN) are copolymerized in anhydrous dimethyl sulfoxide, and the reaction is heated for 24 hours. After the reaction is completed, the product is purified by dialysis and freeze-drying to obtain a biguanide-derived modified PEG copolymer. The nuclear magnetic resonance results are as follows: Figure 1 The copolymer self-assembles in water to form nanoparticles Poly-MET-NP. Transmission electron microscopy observation shows that Figure 2 As shown, the size of the nanoparticles is about 50-100 nanometers, with good stability and uniform dispersion.

[0034] Example 2 Cell Viability Test

[0035] First, HepG2 cells were seeded in a 96-well plate and cultured for 24 hours to ensure cell adhesion. Then, different concentrations of Poly-MET-NP (such as 0μg / mL, 2μg / mL, 10μg / mL, 50μg / mL, 100μg / mL, etc.) were added and incubated for another 24 hours. After the incubation, 10μL of CCK8 reagent was added to each well. After incubation for 2 hours, the 450nm absorbance (OD value) of each well was measured on a microplate reader. Based on the blank control group, the cell viability of each group was calculated, and the effect of Poly-MET-NP on cell proliferation or toxicity was evaluated by statistical analysis. Figure 3 As shown, Poly-MET-NP has no obvious toxicity to cell viability within a certain concentration range, but has a certain promoting effect.

[0036] Example 3 Oxygen Consumption Rate and Energy Metabolism

[0037] In this experiment, the oxygen consumption rate (OCR) and energy metabolism parameters of HepG2 cells after treatment with biguanide-derived polymer nanoparticles Poly-MET-NP were measured in detail by Seahorse XF24 analyzer. First, HepG2 cells were seeded into Seahorse XF24-specific cell culture plates at a density of 2×10 ~5×10 cells / well and cultured for 24 hours to ensure that the cells adhered to the wall and grew stably. Subsequently, Poly-MET-NP was added to the culture medium to treat the cells for 24-48 hours to ensure that the effect of Poly-MET-NP on cell energy metabolism was apparent.

[0038] After the treatment, the cells were replaced with carbonate-free Seahorse medium (containing glucose, pyruvate, and glutamine) and balanced for 1 hour in a CO-free environment to adapt the cells to the experimental conditions. In the Seahorse XF24 analyzer, the basal respiration of the cells was first measured, reflecting the oxygen consumption rate of the cells in the absence of drug interference. Subsequently, mitochondrial ATP production was inhibited by adding oligomycin (inhibiting ATP synthase), and the oxygen consumption associated with ATP synthesis was measured to calculate ATP production. Next, FCCP (mitochondrial uncoupler) was added to put the mitochondria in a maximal respiratory state, and the maximum respiratory capacity was measured to evaluate the oxygen consumption capacity of the cells under extreme conditions. Finally, antimycin A and rotundine were added to block the mitochondrial electron transport chain and non-mitochondrial respiration was measured.

[0039] The whole process was recorded in real time by Seahorse XF24 analyzer. Each set of experiments was repeated at least three times, and the results were analyzed using Seahorse Wave software. The data included basal respiration, ATP generation, maximum respiration, and non-mitochondrial respiration. Figure 4 As shown, Poly-MET-NP treatment can significantly increase the basal respiration, ATP generation and maximum respiration of cells, indicating that Poly-MET-NP improves the energy metabolism efficiency of cells by enhancing mitochondrial function and promoting fatty acid oxidation.

[0040] Example 4 Effect of Poly-MET-NP on Lipid Deposition

[0041] In order to evaluate the effect of Poly-MET-NP on cellular lipid metabolism, the present invention uses a variety of detection methods to quantify and observe lipid accumulation in cells.

[0042] BODIPY 493 / 503 fluorescent dye was used to label lipid droplets in HepG2 cells, and the effect of Poly-MET-NP treatment on lipid deposition was detected by laser confocal microscopy (CLSM). First, HepG2 cells were seeded on glass slides or glass-bottomed culture dishes. After the cells adhered to the wall, biguanide-derived polymer nanoparticles Poly-MET-NP were added for treatment for 24 hours. After the treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde for 10-15 minutes, and washed again with PBS. Subsequently, 1μg / mL BODIPY dye was used for incubation in the dark for 30 minutes, and DAPI was added to stain the nucleus after washing and incubated for 5-10 minutes. Cell fluorescence images were taken by laser confocal microscopy to record the distribution and number of lipid droplets. The results showed that Poly-MET-NP treatment significantly reduced the number and size of intracellular lipid droplets, indicating that it has a potential role in reducing lipid deposition.

[0043] Oil Red O staining was used to evaluate the improvement of lipid deposition in HepG2 cells by biguanide-derived polymer nanoparticles (Poly-MET-NP). First, HepG2 cells were seeded into 6-well plates with approximately 1×10 cells per well and incubated for 24 hours to ensure cell adhesion. Poly-MET-NP was then added to treat the cells for 24 hours. After treatment, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10-15 minutes, and washed again to remove the fixative. Subsequently, the cells were pretreated with 60% isopropanol for 5 minutes, followed by the addition of Oil Red O staining solution, and stained at room temperature in the dark for 15-30 minutes. After staining, the cells were washed three times with distilled water, and the distribution of cellular lipid droplets was observed under a microscope. The lipid droplets were dyed red to visualize lipid deposition. The results showed that lipid deposition was significantly reduced in the Poly-MET-NP treatment group, indicating that it has the potential to improve lipid accumulation in HepG2 cells.

[0044] Determination of triglycerides: This experiment aims to evaluate the effect of biguanide-derived polymer nanoparticles (Poly-MET-NP) treatment on lipid metabolism by detecting the level of triglycerides (TG) in HepG2 cells. First, HepG2 cells were seeded into 6-well plates at a density of approximately 1×10 cells / well and cultured for 24 hours to ensure cell adherence and growth. Poly-MET-NP was then added to treat the cells for 24 hours. After the treatment, the cells were washed three times with PBS, isopropanol was added to lyse the cells, and triglycerides in the supernatant were extracted. The TG content was detected by a triglyceride quantitative detection kit. If the experimental results show that the triglyceride level in the Poly-MET-NP treatment group is significantly lower than that in the control group, it indicates that Poly-MET-NP can effectively reduce lipid deposition in HepG2 cells and improve lipid metabolism.

[0045] Example 5 In vivo study on the effect of biguanide-derived polymer nanoparticles on lipid deposition in mice fed a high-fat diet (HFD)

[0046] The mice with non-alcoholic fatty liver model were induced by high-fat diet. The animals were randomly divided into model group and Poly-MET-NP treatment group. The treatment group was treated with intraperitoneal injection of 2 mg / kg Poly-MET-NP twice a week for 16 consecutive weeks. Body weight and food intake were recorded regularly. After the treatment, the experimental animals were killed and liver tissue samples were collected for subsequent analysis. Oil red O staining was first used to evaluate lipid deposition in the liver. To this end, frozen embedded liver tissue sections (10 μm thick) were equilibrated with 60% isopropanol, then stained in Oil Red O staining solution, rinsed and lightly counterstained with hematoxylin for nuclei, and finally observed under a microscope for red staining areas to reflect the degree of lipid deposition in the liver. In order to further quantify the improvement of lipid metabolism, the triglyceride (TG) and free fatty acid (FFA) contents in the liver were determined. First, the liver tissue was homogenized, centrifuged after homogenization, and the supernatant was collected. Then, commercial TG and FFA detection kits were used to perform enzymatic reactions according to the instructions to detect the TG and FFA contents in the liver tissue.

[0047] Example 6 Biguanide-derived polymer nanoparticles improve non-alcoholic fatty liver disease

[0048] The experiment used 6-8 week old male C57BL / 6 mice, and established the NAFLD model by feeding a high-fat diet for 8 consecutive weeks. At the same time, 2 mg / kg of Poly-MET-NP was intraperitoneally injected twice a week. Insulin tolerance test (ITT) and glucose tolerance test (GTT) were performed at the 8th week. In ITT, mice were intraperitoneally injected with 0.75 U / kg of insulin after 6 hours of fasting, and then blood glucose levels were measured at 0, 15, 30, 60 and 120 minutes to evaluate insulin sensitivity; in GTT experiment, mice were intraperitoneally injected with glucose solution (2 g / kg) after 12 hours of fasting, and blood glucose levels were also measured at multiple time points, and the total blood glucose area under the curve (AUC) was calculated to evaluate glucose tolerance. The results showed that Poly-MET-NP treatment can significantly improve the insulin sensitivity of mice, reduce blood glucose levels, improve insulin resistance symptoms, and show its potential advantages in the treatment of non-alcoholic fatty liver disease.

[0049] Example 7 Effect of biguanide-derived polymer nanoparticles on energy consumption

[0050] The experiment used 6-8 week old male C57BL / 6 mice, which were divided into NAFLD model group and NAFLD+Poly-MET-NP treatment group. The NAFLD model was induced by high-fat diet, and the mice in the Poly-MET-NP treatment group were intraperitoneally injected with 2 mg / kg of Poly-MET-NP every day for 4 weeks. At the end of the 4th week, the mice were placed in the small animal energy metabolism detection system, and energy consumption, food intake, exercise activity level and respiratory exchange ratio (RER) were monitored for 24 hours. The experimental results showed that the energy consumption of mice in the Poly-MET-NP treatment group increased significantly, especially during the active period at night, but there was no significant change in food intake, exercise activity level and RER, indicating that Poly-MET-NP can improve its energy metabolism without affecting the behavior and metabolic balance of mice. Through data analysis, the enhancing effect of Poly-MET-NP treatment on the energy metabolism of mice was confirmed, which provides a basis for its potential application in metabolic abnormalities related to non-alcoholic fatty liver disease.

[0051] Example 8 Toxicity Evaluation of Biguanide-Derived Polymer Nanoparticles

[0052] After the Poly-MET-NP treatment, the mice were killed and the serum was taken to detect the ALT, AST, BUN, and S-Cr levels. The results showed that there was no significant difference between the serum indicators of the mice in the Poly-MET-NP treatment group and those in normal mice, indicating that the Poly-MET-NP treatment did not produce obvious toxic effects, further supporting its good biosafety.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements based on different inventive methods. These improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing biguanide-derived polymer nanoparticles, characterized in that The method comprises the following steps: using azobisisobutyronitrile (AIBN) as an initiator, copolymerizing a biguanide-derived functional monomer with polyethylene glycol-modified 4-cyano-4-(phenylthiocarboxymethylthio) valeric acid (PEG-CPADN) through a reversible addition-fragmentation chain transfer (RAFT) polymerization reaction to generate a polymer with a biguanide-derived functional group.

2. A method for preparing biguanide-derived polymer nanoparticles, characterized in that: In water, the interaction between hydrophilic and hydrophobic components is used to self-assemble into biguanide-derived polymer nanoparticles with a particle size of 50-100 nanometers ( Poly-MET-NP), the nanoparticles have stable structure and are evenly dispersed.

3. The method for preparing biguanide-derived polymer nanoparticles according to claim 1, characterized in that: The biguanide derivative monomer is N-(2-(3-carbamoylguanidinyl)ethyl)methacrylamide; the PEG chain length of the PEG-modified 4-cyano-4-(phenylthiocarbamoylthio)pentanoic acid (PEG-CPADN) is 4000-6000 Daltons, which ensures the water solubility and stability of the nanoparticles.

4. The use of biguanide-derived polymer nanoparticles in improving lipid deposition as claimed in claim 3, characterized in that: Poly-MET-NP demonstrated the ability to promote basal respiration and ATP production in HepG2 cells through in vitro cell experiments, and significantly reduce the number of intracellular lipid droplets.

5. The use of biguanide-derived polymer nanoparticles in improving lipid deposition as claimed in claim 3 or 4, characterized in that: Poly-MET-NPs significantly reduced hepatic lipid accumulation, improved insulin resistance, and increased energy expenditure in a high-fat diet-fed mouse model in vivo.

6. The use of biguanide-derived polymer nanoparticles in improving lipid deposition according to any one of claims 3 to 5, characterized in that: Poly-MET-NP injected intraperitoneally at 2 mg / kg weekly for 16 weeks in a high-fat diet mouse model significantly reduced the number and size of liver lipid droplets, improved cell structure and reduced inflammatory response.

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