Preparation method of molecularly imprinted hydrogel specifically bound with sialic acid

Abstract

The invention provides a preparation method of molecularly imprinted hydrogel specifically bound with sialic acid, and relates to the technical fields of organic chemistry, pharmaceutics, molecular biology and the like. The synthesis method of the molecularly imprinted hydrogel specifically bound with sialic acid comprises the following steps: 1) synthesizing the molecularly imprinted hydrogel taking sialic acid as a target spot; (2) eluting and purifying the molecularly imprinted hydrogel; 3) verifying and characterizing the molecularly imprinted polymer; (4) verifying the in-vitro adsorption of the molecular imprinting hydrogel to the sialic acid; 5) biological function verification of the molecularly imprinted hydrogel; and 6) verifying the molecularly imprinted hydrogel as a drug loading system. The molecularly imprinted hydrogel specifically bound with sialic acid is obtained, the sialic acid is specifically bound and removed, liver cell lipid accumulation is effectively reduced, and the molecularly imprinted hydrogel has profound significance in the field of treatment of metabolic diseases such as non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention involves multiple fields such as organic chemistry, pharmacy, molecular biology, etc. Specifically, it includes the synthesis of molecular imprinted hydrogel with sialic acid as the target, the elution and purification of molecular imprinted hydrogel, and the biological function verification of molecular imprinted hydrogel. Background Art

[0002] An estimated 1.5 billion people worldwide suffer from chronic liver disease, of which 60% suffer from non-alcoholic fatty liver disease (NAFLD). NAFLD is a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes. Lipid metabolism disorders in the body are closely related to the occurrence and development of NAFLD. Lipid lowering is currently the main strategy for the clinical treatment of non-alcoholic fatty liver disease, such as blocking the intestinal absorption of exogenous lipids or using drugs that inhibit endogenous lipid synthesis. However, the drugs used in clinical practice often cause side effects such as increased blood sugar, muscle soreness, rhabdomyolysis, hepato-renal toxicity, and safety issues such as local inflammatory reactions. There is an urgent need for the development of new therapeutic drugs and the discovery of new treatment strategies.

[0003] Metabolic disorders are an important cause of NAFLD. In addition to lipid metabolism disorders, in recent years, many studies have confirmed that disorders in the sialic acid metabolic pathway may also be an important factor affecting the occurrence of metabolic diseases such as NAFLD. Sialic acid is a type of α-keto acid sugar with a 9-carbon main chain. They are widely present in vertebrates and higher invertebrates as the terminal components of cell surface glycoconjugate glycans. Among them, N-acetylneuraminic acid (Neu5Ac) is the most common type of sialic acid, which is usually used as the terminal sugar of cell surface glycans, glycoconjugates, oligosaccharides, lipolytic oligosaccharides and polysaccharides, and has multiple physiological functions. In the human body, Neu5Ac mainly binds to proteins, lipids and oligosaccharides to form sialylated structures. In addition, Neu5Ac also exists in the human body in a free (unbound) form. Sialic acid plays an important role in mediating various biological processes, such as mediating cell-to-cell interactions, regulating immune responses, controlling protein stability, etc. Studies have shown that there is a positive correlation between abnormal increases in sialic acid in the body caused by sialic acid metabolic disorders and liver disease. In patients with NAFLD, serum sialic acid levels are significantly increased. Multivariate logistic regression analysis showed that serum sialic acid levels were significantly positively correlated with the risk of NAFLD. Previous studies by our research group have also confirmed that high concentrations of sialic acid can promote lipid accumulation in hepatocytes, which may be an important risk factor for the development of non-alcoholic fatty liver disease. Therefore, clearing excessive sialic acid may be a new potential strategy for treating metabolic diseases such as non-alcoholic fatty liver disease that are related to sialic acid metabolic disorders.

[0004] Molecular imprinting technology is a technology that uses molecular imprinting polymers to simulate the interaction between enzymes and substrates or antibodies and antigens to specifically identify imprinted molecules. Molecularly imprinted polymers (MIPs) are polymers synthesized by molecular imprinting technology that have the ability to specifically recognize and selectively adsorb specific target molecules and their structural analogs. Hydrogels are a type of hydrophilic three-dimensional mesh structure gel that has high biocompatibility and controllable mechanical and viscoelastic properties. They have been widely used in the pharmaceutical field. We synthesized molecular imprinting hydrogels targeting sialic acid, which can specifically remove sialic acid, thereby achieving a lipid-lowering effect and treating NAFLD. Summary of the invention

[0005] The present invention provides a method for preparing a molecular imprinted hydrogel that specifically binds to sialic acid, and the specific steps thereof include:

[0006] 1. Synthesis of molecularly imprinted polymers targeting sialic acid: Dissolve the crosslinker methylenebisacrylamide (0.1mmol), the functional monomer acrylamide (0.1mmol), 4-vinylbenzeneboronic acid (0.1mmol), ethylene glycol dimethacrylate (2mmol), the initiator azobisisobutyronitrile (0.08mmol) and the target N-acetylneuraminic acid (0.1mmol) in 20mL dimethyl sulfoxide and mix them thoroughly. Replace the air in the reaction bottle with nitrogen, place the reaction bottle in a constant temperature oil bath reaction pot at 80℃, control the stirring speed at 500rpm, and the polymerization time is 6h. After the reaction is completed, collect the liquid in the bottle and dialyze it in a dialysis bag for 1h until a hydrogel appears. In the same way, without adding the target molecule sialic acid, a non-molecularly imprinted polymer (NIP) is synthesized.

[0007] 2. Elution and purification of molecularly imprinted polymer: Wash the hydrogel with ultrapure water for 3 times, centrifuge at 8000 rpm for 10 minutes, and finally dry the washed and purified molecularly imprinted hydrogel in a vacuum freeze dryer.

[0008] 3. Verification and characterization of molecular imprinted polymers: The morphological characteristics and physical and chemical constants of the products were analyzed by scanning electron microscopy, infrared spectrometer, and Malvern particle size analyzer. The specific steps are as follows:

[0009] (1) Scanning electron microscopy characterization: Take an appropriate amount of freeze-dried powder of MIP and spread it directly on a sample stage with conductive glue, and observe the surface structural characteristics of the particles using a scanning electron microscope.

[0010] (2) Infrared characterization: Weigh 150 mg of spectrally pure potassium bromide powder and 2 mg of MIP sample, mix them evenly and grind them thoroughly, put them into a tablet pressing mold, evacuate and pressurize the tablet press until the pressure rises to 10 MPa, and make the powder into a transparent sheet with a thickness of about 1 mm and a diameter of 10 mm. After the system collects the background, the sheet is mounted on the sample holder, infrared scanning is performed, and the transmittance is checked to see if it exceeds 40%. If it does, the test result is qualified. The detection steps for NIP and sialic acid are the same as above.

[0011] (3) Particle size measurement: Weigh 2 mg of MIP powder, add 2 mL of pure water, mix well by blowing, rinse the sample pool, inject 1 mL of sample solution, push the sample pool into the sample slot until it stops, close the instrument cover, and test on the machine. The steps for zeta potential measurement are the same as above.

[0012] The instruments used for characterization of molecularly imprinted polymers are as follows: scanning electron microscope JSM IT700HR (JEOL, Japan), Fourier transform infrared spectrometer Nicolet iS10 (Thermo fisher, USA), Malvern particle size analyzer ZEN 3600 (Malvern, UK).

[0013] 4. Verification of MIP adsorption of sialic acid in vitro: Take 5 5mL EP tubes, add 1mg MIP / NIP to each tube, and then add 2mL Neu5Ac solution of different concentrations (concentrations are 1mM, 2mM, 3mM, 4mM, 5mM), respectively, and incubate at 37℃, 50rpm, for 24h. Centrifuge and take the supernatant, and measure the sialic acid concentration using SA kit.

[0014] 5. Verification of the biological function of MIP: HepG2 cells growing in logarithmic phase were cultured at 1×10 6 / well were inoculated in 6-well plates and cultured for 24 hours. Sodium oleate + sodium palmitate (200μM + 100μM) were used to treat cells for 24 hours, and MIP (0.5mg / mL) was added to the drug group for intervention. The cells were digested with trypsin, centrifuged, washed once with PBS, and 400μL of PBS solution containing 1μg / mL Bodipy dye was added and incubated in the dark for 15 minutes. Centrifuged, aspirated Bodipy dye, washed once with PBS, filtered through a cell strainer, and the Bodipy fluorescence intensity was detected on a flow cytometer.

[0015] 6. Verification of MIP as a drug delivery system: In addition to being able to adsorb sialic acid and exert certain biological functions, MIP also has drug loading and release functions due to its porous structure, and can be used as a drug delivery system. Taking the small molecule drug Cepharanthine as an example, its drug loading and release capabilities were verified. Weigh 10 mg of Cepharanthine (CEP) and dissolve it in 1 ml of ethanol, sonicate, and then add 10 mg of MIP and swell for 30 minutes. Add PBS to 10 ml to form an emulsion, centrifuge at 8000 rpm for 10 minutes, discard the supernatant, and put the precipitate into a freeze dryer for freeze drying overnight to obtain MIP-CEP. Weigh 4 mg of freeze-dried MIP-CEP powder in 4 mL of PBS, shake at 37°C, 50 rpm, take 100 μL of liquid at 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h (replenish 100 μL PBS after taking), dilute with 4900 μL ethanol, detect on a UV spectrophotometer, and calculate the released CEP concentration based on the standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Scanning electron microscopy image of MIP. Molecularly imprinted hydrogel MIPs presents a distinct network structure.

[0017] Figure 2 Fourier transform infrared spectra of MIP, NIP and sialic acid. The characteristic peak of the carbonyl group in the SA structure at 1650cm can be detected simultaneously in the infrared absorption peak of MIP, proving that the molecular imprinted polymer MIP is successfully imprinted with sialic acid.

[0018] Figure 3 The particle size statistics of MIP and NIP are evenly distributed at 460 and 295 nm, respectively.

[0019] Figure 4 The Zeta potential statistics of MIP and NIP are both negative potentials.

[0020] Figure 5 The adsorption curves of MIP and NIP on sialic acid show that the adsorption of MIP on sialic acid is significantly stronger than that of NIP. Figure 6 The effect of MIP on reducing intracellular lipids. Compared with the positive control group, MIP intervention can significantly reduce the intracellular lipid level.

[0021] Figure 7 The drug release effect diagram of MIP. MIP can successfully load the drug cephalothin and has a good drug release effect.

Claims

1. A method for preparing a molecularly imprinted hydrogel that specifically binds to sialic acid, comprising the steps of: (1) Synthesis of molecularly imprinted hydrogels (MIPs) targeting sialic acid; (2) Elution and purification of MIP; (3) Validation and characterization of MIPs; (4) Verification of MIP binding to sialic acid in vitro; (5) Verification of the biological function of MIP; (6) Validation of MIP as a drug delivery system.

2. The method according to claim 1, the synthesis step of the molecular imprinting hydrogel (MIP) with sialic acid as the target: the cross-linking agent methylene bisacrylamide (0.1mmol), the functional monomer acrylamide (0.1mmol), 4-vinylbenzene boronic acid (0.1mmol), ethylene glycol dimethacrylate (2mmol), the initiator azobisisobutyronitrile (0.08mmol) and the target N-acetylneuraminic acid (0.1mmol) are dissolved in 20mL dimethyl sulfoxide to make them fully mixed. The air in the reaction bottle is replaced by nitrogen, and the reaction bottle is placed in a constant temperature oil bath reaction pot at 80°C, the stirring speed is controlled at 500 rpm, and the polymerization time is 6 hours. After the reaction is completed, the liquid in the collection bottle is dialyzed in a dialysis bag for 1h until a hydrogel-like product appears.

3. The method according to claim 1, wherein the molecularly imprinted hydrogel is eluted and purified by washing the hydrogel with ultrapure water for 3 times, centrifuging at 8000 rpm for 10 minutes, and finally drying the washed and purified molecularly imprinted hydrogel in a vacuum freeze dryer.

4. Characterization of molecularly imprinted hydrogels, comprising the following steps: The surface morphology of MIP was observed by scanning electron microscopy, the particle size and potential of MIP and non-molecularly imprinted hydrogel (NIP) were measured by Malvern particle size analyzer, and the successful imprinting of sialic acid on MIP was verified by Fourier transform infrared spectroscopy.

5. Verification of MIP adsorption of sialic acid in vitro, comprising the following steps: MIP / NIP was incubated with sialic acid solutions of different concentrations for 24 h, and the concentration of sialic acid in the supernatant was measured using a sialic acid kit and compared with the initial concentration.

6. The biological function verification of MIP includes the following steps: The product described in claim 3 is used to intervene in an in vitro cell model, and the intracellular lipid content is detected by Bodipy staining and flow cytometry.

7. Validation of MIP as a drug delivery system, which includes the following steps: The drug-loaded MIP-CEP freeze-dried powder was dispersed in PBS solution, and the supernatant was taken at different time points, diluted with anhydrous ethanol, and the released drug concentration was measured by UV spectrophotometer.

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