Low density lipoprotein cholesterol imprinting nanospheres, and preparation method and application thereof

Abstract

The application discloses low-density lipoprotein cholesterol imprinting nanospheres as well as a preparation method and application thereof. Polyethylene oxide-polypropylene oxide-polyethylene oxide three-block copolymer P123, ethylene glycol-polypropylene glycol-polyethylene glycol three-block copolymer F127, dopamine hydrochloride and a pore expander are dispersed in a mixed solvent of ethanol and water to form an emulsion, ammonia water is added to the emulsion and then stirring is carried out, porous polydopamine nanospheres are obtained through centrifugal separation and washing; the porous polydopamine nanospheres are dispersed in a PBS buffer solution, low-density lipoprotein cholesterol is added, surface anchoring is carried out at room temperature through stirring, dopamine hydrochloride is added, stirring reaction is carried out, and the template protein is removed through washing after the reaction is completed, thereby obtaining low-density lipoprotein cholesterol imprinting nanospheres. The low-density lipoprotein cholesterol imprinting nanospheres prepared by the application can efficiently and specifically adsorb target LDL, and problems such as high requirement for equipment and complicated process in LDL separation and purification are solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological separation, and particularly relates to low-density lipoprotein cholesterol imprinting nanospheres, a preparation method thereof and application thereof. BACKGROUND

[0002] Low-density lipoprotein cholesterol (LDL-C) is also called low-density lipoprotein (LDL), which refers to lipoprotein rich in cholesterol and its ester. When the human body ingests food containing cholesterol, the cholesterol is decomposed into LDL-C by the liver and distributed to the whole body through blood circulation. LDL-C is a controllable factor of cardiovascular disease, and high levels of LDL-C can cause cholesterol deposition and oxidation in blood vessels, forming atherosclerosis, thereby increasing the risk of cardiovascular disease. How to reduce the level of LDL-C has been a concern.

[0003] The reduction of LDL-C level is usually evaluated by its concentration, which has been considered as the primary treatment target for primary and secondary cardiovascular disease prevention for many years. Studies have shown that simply relying on diet and drugs to reduce LDL-C levels is not always effective. On the one hand, many patients with hypercholesterolemia due to genetic mutations have not reached the reference low-density lipoprotein cholesterol concentration range despite the full use of LDL-C reducing drugs and appropriate lifestyle changes. On the other hand, long-term use of statins can cause many serious adverse reactions, such as liver damage caused by elevated liver enzymes, muscle toxicity, gastrointestinal irritation, and rhabdomyolysis. Therefore, it is necessary to reduce the level of LDL-C by drug alternatives. In vitro separation of LDL-C refers to a method of separating LDL-C in blood circulation by using an adsorbent. Existing methods for in vitro separation of LDL-C, such as plasma exchange, double filtration, immunoadsorption, dextran sulfate adsorption, and heparin-induced in vitro precipitation, all require the separation of plasma from whole blood by a plasma separation device, and then the separation of LDL-C from the plasma. Therefore, the equipment requirements are high, and the process is complicated. SUMMARY

[0004] In view of the problems of high equipment requirements and complicated separation process in the current LDL-C separation and purification method, the application provides low-density lipoprotein cholesterol imprinting nanospheres, a preparation method thereof and application thereof, which realize high selectivity separation of LDL-C, simplify the separation process, reduce the separation cost, and improve the separation capacity.

[0005] The application is achieved by the following technical solutions:

[0006] A preparation method of low-density lipoprotein cholesterol imprinting nanospheres, comprising the following steps:

[0007] (1) Preparation of porous polydopamine nanospheres: disperse polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, dopamine hydrochloride, pore expander in a mixed solvent of ethanol and water to form an emulsion, add ammonia water to the emulsion after stirring, and obtain porous polydopamine nanospheres by centrifugal separation and washing;

[0008] (2) Preparation of low-density lipoprotein cholesterol imprinted nanospheres: disperse the porous polydopamine nanospheres prepared in step (1) in PBS buffer, add low-density lipoprotein cholesterol, anchor the surface at room temperature, add dopamine hydrochloride, stir and react, wash away the template protein after the reaction is completed, and obtain low-density lipoprotein cholesterol imprinted nanospheres.

[0009] Further, the ratio of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127 and dopamine hydrochloride in step (1) is 1:2-4:4-7; the volume ratio of water and ethanol in the mixed solvent is 1:0.5-2.

[0010] Further, the pore expander in step (1) is mesitylene, and the volume mass ratio of the pore expander to the polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1 mg:15-20 μL; the mass percentage concentration of ammonia water is 20-30%, and the volume mass ratio of ammonia water to polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1 mg:10-20 μL.

[0011] Further, in step (2), the mass ratio of porous polydopamine nanospheres to low-density lipoprotein cholesterol is 15-25:1, and the mass ratio of porous polydopamine nanospheres to dopamine hydrochloride is 1.5-3:1.

[0012] Further, in step (1), the stirring time after adding ammonia water is 1.5-3 h; in step (2), the stirring time at room temperature is 30-60 min, and the stirring reaction time is 14-20 h.

[0013] Further, in step (2), the concentration of PBS buffer is 0.01 mol / L, and the pH is 8.5. -1

[0014] ​Further, the product after centrifugal separation in step (1) is washed several times with water and ethanol; and the method for removing the template protein in step (2) is to remove the template protein by washing with acetic acid for multiple times until no absorption peak can be detected at a wavelength of about 280 nm by using a UV-visible spectrophotometer, and then washing with water and ethanol for three times to remove the residual acetic acid.

[0015] Further, the acetic acid is an ethanol aqueous solution with a volume ratio of 15-30%.

[0016] In the application, the low-density lipoprotein cholesterol imprinted nanospheres are prepared by the preparation method.

[0017] In the application, the low-density lipoprotein cholesterol imprinted nanospheres are used in the separation of low-density lipoprotein cholesterol.

[0018] The application has the following beneficial effects:

[0019] In the application, the porous polydopamine nanospheres are formed as imprinting substrates by polymerization of the triblock polymer P123, the triblock copolymer F127, dopamine hydrochloride and a pore-expanding agent under certain conditions; the two similar block copolymers (P123 and F127) have hydrophilic chains with different lengths as soft templates, and the mesoporous polydopamine nanoparticles are prepared by using a double-soft-template strategy; then, LDL is added, and based on the protein anchoring technology, the template molecule LDL is attached to the surface of the PPDA through π-π interaction, multiple hydrogen bonds and hydrophobic interaction to form a PPDA-LDL complex; then, dopamine hydrochloride is added, and a polymerization reaction occurs under certain conditions to form a copolymer, and then the copolymer is eluted to form an imprinting cavity on the surface of the imprinting polymer, which is complementary to the shape, functional groups and space size of the template molecule; due to the shape memory effect of the imprinting cavity in the recognition selectivity, the PPDA-MIPs exhibit excellent selectivity to LDL. The low-density lipoprotein cholesterol imprinted nanospheres prepared by the application can efficiently and specifically adsorb target LDL, and solve the problems of high requirement for equipment and complicated process in the separation and purification of LDL. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The scanning electron microscope images of PPDA (a), PPDA-NIPs (b) and PPDA-MIPs (c); the transmission electron microscope images of PPDA (d), PPDA-NIPs (e) and PPDA-MIPs (f);

[0021] Figure 2 The infrared spectra of PPDA, PPDA-MIPs and PPDA-NIPs (a) and the thermogravimetric curve of PPDA and PPDA-MIPs (b);

[0022] Figure 3 Fig. 7 LDL adsorption behavior on the surface of PPDA, PPDA-NIPs and PPDA-MIPs (a) and the relationship between LDL adsorption efficiency and ionic strength in the range of 0.0-3.0 mol L -1 NaCl (b);

[0023] Figure 4 Fig. 8 LDL adsorption isotherm of PPDA-NIPs and PPDA-MIPs (a) and the curve of 1 / Q e versus C e (b);

[0024] Figure 5 Fig. 9 Reusability of PPDA-MIPs in LDL separation (a) and SDS-PAGE analysis results (b); DETAILED DESCRIPTION

[0025] The technical solutions of the present application are further described below in conjunction with the accompanying drawings, but are not limited thereto, and any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application shall be encompassed in the protection scope of the present application.

[0026] Example 1

[0027] (1) Preparation of porous polydopamine nanospheres (PPDA): 50 mg of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, 150 mg of ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, 300 mg of dopamine hydrochloride, 800 μL of mesitylene TMB dispersant were dispersed in a mixture of 10 mL of ultrapure water and 10 mL of ethanol, and dispersed in an ultrasonic reactor for 5 min to form an emulsion, 750 μL of ammonia water was added to the emulsion, and stirred at room temperature for 2 h, and then centrifuged and washed with ultrapure water and ethanol for several times to obtain porous polydopamine nanospheres (PPDA);

[0028] (2) Preparation of low-density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs): 100 mg of PPDA was dispersed in 50 mL of PBS buffer solution (pH 8.5) with a concentration of 0.01 mol L -1) and then 5 mg of LDL was added. After surface anchoring for 50 min at room temperature, 50 mg of dopamine hydrochloride was added to the above solution and stirred for 16 h. The template protein was removed by multiple washing with acetic acid (20%, V / V) until no absorption peak was detected at 280 nm by UV-visible spectrophotometer, followed by washing with ultrapure water and ethanol for three times, respectively, to remove the residual acetic acid, to obtain the low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs).

[0029] Comparative Example 1

[0030] (1) The same as step (1) of Example 1;

[0031] (2) Preparation of low density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs): 100 mg of PPDA was dispersed in 50 mL of PBS buffer (PBS buffer concentration was 0.01 mol / L, pH 8.5). After stirring for 50 min at room temperature, 50 mg of dopamine hydrochloride was added to the above solution and stirred for 16 h. The template protein was removed by multiple washing with acetic acid (20%, V / V), followed by washing with ultrapure water and ethanol for three times, respectively, to remove the residual acetic acid, to obtain the low density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs). -1 ) and then 5 mg of LDL was added. After surface anchoring for 50 min at room temperature, 50 mg of dopamine hydrochloride was added to the above solution and stirred for 16 h. The template protein was removed by multiple washing with acetic acid (20%, V / V) until no absorption peak was detected at 280 nm by UV-visible spectrophotometer, followed by washing with ultrapure water and ethanol for three times, respectively, to remove the residual acetic acid, to obtain the low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs).

[0032] The scanning electron microscope images and transmission electron microscope images of the porous polydopamine nanospheres (PPDA), the low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in Example 1 and the low density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) prepared in Comparative Example 1 are shown in Figure 1 ; wherein (a) is the scanning electron microscope image of PPDA, (b) is the scanning electron microscope image of PPDA-MIPs, (c) is the scanning electron microscope image of PPDA-NIPs, (d) is the transmission electron microscope image of PPDA, (e) is the scanning electron microscope image of PPDA-MIPs, and (f) is the transmission electron microscope image of PPDA-NIPs. Figure 1 (a) clearly shows that the prepared PPDA is a uniform nanosphere with a dense mesoporous structure, with a diameter of about 270 nm, and (d) TEM image further characterizes the rich mesoporous structure of PPDA, which provides sufficient specific surface area. In addition, it can be seen from the scanning electron microscope (SEM) Figure 1 b and Figure 1 c) and transmission electron microscope (TEM) Figure 1 e and Figure 1 f) that the polymerization of dopamine results in PPDA-NIPs and PPDA-MIPs having a relatively rough surface structure and nanomorphology.

[0033] The FTIR spectra of porous polydopamine nanospheres (PPDA), low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in Example 1 and low density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) prepared in Comparative Example 1 are shown in Figure Figure 2 (a); the spectrum of PPDA has a broad band at about 3420 cm -1 , which can be attributed to the O-H stretching of intermolecular hydrogen bonds; there is a set of distinguishable bands at 1617, 1450 and 1114 cm -1 , which can be attributed to the aromatic ring stretching vibration of polyindole structure25; in addition, the band at 1496 cm -1 corresponds to the stretching vibration of N-H, and the typical peaks at 1350 and 1292 cm -1 are attributed to the bending and stretching vibrations of C-O-H on the benzene ring, respectively; it is worth noting that the FTIR spectra of PPDA-NIPs and PPDA-MIPs are similar to that of PPDA, which confirms the successful formation of polydopamine-based nanomaterials.

[0034] The thermogravimetric analysis curves of porous polydopamine nanospheres (PPDA), low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in Example 1 are shown in Figure Figure 2 (b); for PPDA and PPDA-MIPs, the first weight loss occurring at 25-150°C is in response to the physically adsorbed water. For PPDA, it can be noted that the second weight loss is 45.4%, which is due to the carbonization process of PPDA. The thermal behavior of PPDA-MIPs is similar to that of PPDA, but the second stage weight loss rate is as high as 48.8%, which is higher than that of PPDA, which is related to the coating of polydopamine on the surface of PPDA.

[0035] Application Examples

[0036] 1. The adsorption behavior of porous polydopamine nanospheres (PPDA), low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in Example 1 and low density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) in Comparative Example 1 to LDL (15 μg mL -1 , 300 μL) under different pH conditions was studied at pH 3-9, and the results are shown in Figure Figure 3(a) As shown in Figure (a), the adsorption efficiency of LDL on the surface of PPDA-MIPs was always higher than those without imprinting materials under the condition of pH 3-9. Meanwhile, the adsorption efficiency of LDL increased with the increase of pH, and reached the maximum at its isoelectric point (pH 5.5), while the further increase of pH would decrease the adsorption efficiency of LDL. The optimal adsorption efficiency of LDL on PPDA, PPDA-NIPs and PPDA-MIPs was 40.0%, 51.5% and 92.0% at pH 5.5, respectively. The hydrogen bond interaction between polydopamine nanospheres and low density lipoprotein can well explain this adsorption behavior. When the pH value is close to the pi of LDL, LDL is neutral and the electrostatic repulsion is minimal. The chemical structure of polydopamine includes a large number of hydrophilic groups, including catechol, amine and imine, which can act as hydrogen bond donors / acceptors. Based on these, hydrogen bond interaction leads to the maximum adsorption at pH values near the pi of LDL.

[0037] 2. The effect of different NaCl concentrations on the adsorption of LDL by low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in Example 1 was studied, and the results are shown in Figure 3 (b) As shown in Figure (b), the change of ionic strength had no effect on the adsorption of LDL in a wide range (0 ~ 3 mol L -1 ), indicating that electrostatic interaction has no contribution to the adsorption of LDL, which proves the practicability of low density lipoprotein cholesterol imprinted nanospheres in dealing with real biological samples which usually encounter relatively high ionic strength.

[0038] 3. The adsorption capacity of low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in Example 1 and low density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) in Comparative Example 1 for LDL was studied in the initial concentration range of 1 ~ 100 μg mL -1 , and the adsorption isotherms are shown in Figure 4 (a) As shown in Figure (a), the binding amount of LDL to PPDA-NIPs and PPDA-MIPs increased rapidly with the increase of initial LDL concentration and reached adsorption equilibrium. Under the same conditions, the maximum adsorption amount of PPDA-NIPs was 250.4 g mg -1 , while the maximum adsorption amount of PPDA-MIPs was 550.3 g mg -1 . It can be seen that the binding of LDL to PPDA-MIPs is mainly due to specific binding with the recognition cavity. On the contrary, the adsorption of LDL on PPDA-NIPs is mainly non-specific. In order to further analyze the adsorption behavior of PPDA-NIPs and PPDA-MIPs, the Langmuir isotherm model was applied to the equilibrium data. The equation is shown in equation (1):

[0039] Formula (1)

[0040] C e (μg mL) -1 Q represents the equilibrium concentration of LDL. max (μg mg) -1 ) and Q e (μg mg) -1 The maximum theoretical adsorption capacity and the equilibrium adsorption capacity are shown in the figure below. Their linear relationship curves are as follows: Figure 4 As shown in (b), the Langmuir model is applicable to the adsorption behavior of PPDA-NIPs and PPDA-MIPs, with correlation coefficients (R²) of 0.9900 and 0.9906, respectively. Therefore, it is inferred that the adsorption of LDL on the material surface is monolayer.

[0041] 4. Elution and reuse of molecularly imprinted nanospheres

[0042] Reusability is an important indicator for evaluating the performance of molecularly imprinted materials. In this invention, a 30-minute desorption study was conducted using MeOH:HAc (80:20) as the eluent. In the first adsorption / desorption cycle, 77.77% of the adsorbed LDL was desorbed by PPDA-MIPs. Then, under optimal parameters, six adsorption / desorption cycles were repeated using the same imprinted material (low-density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) from Example 1), and the results are as follows. Figure 5 As shown in (a), the adsorption efficiency of PPDA-MIPs decreased by only 11.51% after 6 adsorption / desorption cycles. This is because some imprinted sites were blocked by residual LDL, and a few binding sites were destroyed under acidic conditions. After 6 adsorption / desorption cycles, the adsorption efficiency still reached 83.09%, indicating that PPDA-MIPs have good prospects for reuse.

[0043] 5. Molecularly imprinted nanospheres for the separation and purification of LDL in goat serum samples.

[0044] The low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs) from Example 1 were used for the selective separation of LDL in goat serum samples. 4.0 mmol L⁻¹ -1 Goat serum was diluted 20-fold with BR buffer at pH 5.5, and LDL was collected from PPDA-MIPs using MeOH:HAc (4:1) solution as the elution buffer. The LDL was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE results are shown below. Figure 5(b) As shown, several major bands were observed in the diluted goat serum sample. The molecular weight of LDL is about 2.7 x 10 3 -3.3 x 10 3 kDa, and the position of the LDL standard solution is above the standard protein band with a molecular weight of 200 kDa. The same position was identified as the LDL standard solution (lane 5). The color of the LDL band was obviously faded after the adsorption of the PPDA-MIPs (lane 3). In lane 4, a strong band appeared above 200 kDa in the eluate of the PPDA-MIPs, and there were no other protein bands in the eluate, indicating that the PPDA-MIPs had good selectivity. The above results show that the prepared PPDA-MIPs can selectively recognize LDL in the presence of other proteins in the goat serum sample.

Claims

1. A method for preparing low density lipoprotein cholesterol imprinted nanospheres, characterized by, The preparation method comprises the following steps: (1) Preparation of porous polydopamine nanospheres: dispersing polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, dopamine hydrochloride and pore expander in a mixed solvent of ethanol and water to form an emulsion, stirring after adding ammonia water to the emulsion, and obtaining the porous polydopamine nanospheres through centrifugal separation and washing; (2) Preparation of low-density lipoprotein cholesterol imprinted nanospheres: dispersing the porous polydopamine nanospheres prepared in step (1) in PBS buffer, adding low-density lipoprotein cholesterol, anchoring the surface at room temperature, adding dopamine hydrochloride, stirring and reacting, and obtaining the low-density lipoprotein cholesterol imprinted nanospheres after removing the template protein through washing.

2. The method for preparing low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, characterized in that, In step (1), the ratio of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127 and dopamine hydrochloride is 1:2-4:4-7; the volume ratio of water to ethanol in the mixed solvent is 1:0.5-2.

3. The method for preparing low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, characterized in that, In step (1), the pore expander is mesitylene, the volume-to-mass ratio of the pore expander to polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1 mg:15-20 μL, the mass percentage of the ammonia water is 20-30%, and the volume-to-mass ratio of the ammonia water to polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1 mg:10-20 μL.

4. The method for preparing low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, characterized in that, In step (2), the mass ratio of the porous polydopamine nanospheres to low-density lipoprotein cholesterol is 15-25:1, and the mass ratio of the porous polydopamine nanospheres to dopamine hydrochloride is 1.5-3:

1.

5. The method for preparing low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, characterized in that, In step (1), the stirring time after adding ammonia water is 1.5-3 h; in step (2), the stirring time at room temperature is 30-60 min, and the stirring reaction time is 14-20 h.

6. The method for preparing low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, characterized in that, The PBS buffer concentration in step (2) is 0.01 mol L -1 , and pH is 8.

5.

7. The method for preparing low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, characterized in that, In step (1), the product after centrifugal separation is washed with water and ethanol for several times; in step (2), the method for removing the template protein through washing is removing the template protein through multiple times of acetic acid washing until no absorption peak can be detected at a wavelength of about 280 nm by ultraviolet-visible spectrophotometry, and then washing with water and ethanol for three times to remove residual acetic acid.

8. The method for preparing low-density lipoprotein cholesterol imprinted nanospheres according to claim 7, characterized in that, The acetic acid is a 15-30% volume ratio of ethanol aqueous solution.

9. Low-density lipoprotein cholesterol imprinted nanospheres prepared by the preparation method in any one of claims 1-8.

10. Application of the low-density lipoprotein cholesterol imprinted nanospheres in claim 9 in separation of low-density lipoprotein cholesterol.

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