CN117753376A - A low-density lipoprotein cholesterol-imprinted nanosphere and its preparation method and application - Google Patents

Abstract

The invention discloses a low-density lipoprotein cholesterol imprinting nanosphere and a preparation method and application thereof, wherein a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, an ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, dopamine hydrochloride and a pore-expanding agent are dispersed in a mixed solvent of ethanol and water to form an emulsion, ammonia water is added into the emulsion, and then the emulsion is stirred, and the porous polydopamine nanosphere is obtained through centrifugal separation and washing; dispersing in PBS buffer solution, adding low-density lipoprotein cholesterol, stirring at room temperature for surface anchoring, adding dopamine hydrochloride, stirring for reaction, washing after the reaction is finished to remove template protein, and obtaining the low-density lipoprotein cholesterol imprinted nanospheres. The low-density lipoprotein cholesterol imprinting nanospheres prepared by the invention can efficiently and specifically adsorb target LDL, and solve the problems of high requirements on equipment, complicated process and the like in the separation and purification of LDL.

Description

A low-density lipoprotein cholesterol-imprinted nanosphere and its preparation method and application

Technical field

The invention belongs to the field of biological separation technology, and specifically relates to a low-density lipoprotein cholesterol-imprinted nanosphere and its preparation method and application.

Background technique

Low-Density Lipoprotein Cholesterol (LDL-C), also known as Low Density Lipoprotein (LDL), refers to lipoproteins rich in cholesterol and its esters. When the human body consumes foods containing cholesterol, the cholesterol will be broken down into LDL-C by the liver and distributed throughout the body through blood circulation. LDL-C is a controllable factor in cardiovascular disease. High levels of LDL-C can lead to the deposition and oxidation of cholesterol in blood vessels, forming atherosclerosis, thereby increasing the risk of cardiovascular disease. How to reduce LDL-C levels continues to attract people's attention.

The reduction of LDL-C levels, usually assessed by its concentration, has been considered for many years as the primary therapeutic target for primary and secondary cardiovascular disease prevention. Research shows that relying solely on diet and medication to lower LDL-C levels is not always effective. On the one hand, many patients with hypercholesterolemia due to genetic mutations still fail to reach the reference low-density lipoprotein cholesterol concentration range despite adequate use of LDL-C lowering drugs and appropriate lifestyle changes. On the other hand, long-term use of statins may lead to many serious adverse effects, such as liver damage due to elevated liver enzymes, myotoxicity, gastrointestinal irritation, and rhabdomyolysis. Therefore, pharmacological alternatives are needed to lower LDL-C levels. The in vitro separation of LDL-C refers to the method of using adsorbents to adsorb and separate LDL-C in the blood circulation. There are existing in vitro separation methods of LDL-C, such as plasma exchange, double filtration, immunoadsorption, and dextran. Both the sugar sulfate adsorption method and the heparin-induced in vitro precipitation method need to first separate plasma from whole blood through a plasma separation device, and then separate LDL-C from the plasma. Therefore, the equipment requirements are high and the process is cumbersome.

Contents of the invention

Aiming at the problems that current LDL-C separation and purification methods require high equipment and the separation process is cumbersome, the present invention provides a low-density lipoprotein cholesterol-imprinted nanosphere and its preparation method and application to achieve high selectivity of LDL-C. Separation, simplifying the separation process, reducing separation costs, and improving separation capabilities.

The present invention is realized through the following technical solutions:

A method for preparing low-density lipoprotein cholesterol-imprinted nanospheres, including the following steps:

(1) Preparation of porous polydopamine nanospheres: Polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer Material F127, dopamine hydrochloride, and pore expander are dispersed in a mixed solvent of ethanol and water to form an emulsion. Ammonia water is added to the emulsion and stirred. Porous polydopamine nanospheres are obtained by centrifugation and washing;

(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, stir at room temperature for surface anchoring, and add hydrochloric acid Dopamine, stir the reaction, and after the reaction is completed, wash to remove the template protein to obtain low-density lipoprotein cholesterol-imprinted nanospheres.

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

Further, in step (1), the pore expanding agent is mesitylene, and the added amount of the pore expanding agent and the volume mass of the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 are 1 mg: 15~20 μL; the mass percentage concentration of the ammonia water is 20~30%, and the volume mass ratio of the added amount of ammonia water and the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1mg: 10~20μL.

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

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

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

Further, the centrifuged product in step (1) is washed several times with water and ethanol; the method of washing to remove the template protein in step (2) is to wash with acetic acid multiple times to remove the template protein until the UV-visible spectrophotometer detects the temperature at 280 nm. No absorption peaks could be detected at the left and right wavelengths, and then washed three times with water and ethanol to remove residual acetic acid.

Further, the acetic acid is an ethanol aqueous solution with a volume ratio of 15 to 30%.

In the present invention, the low-density lipoprotein cholesterol-imprinted nanospheres are prepared by the preparation method.

In the present invention, the low-density lipoprotein cholesterol-imprinted nanospheres are used in the separation of low-density lipoprotein cholesterol.

The beneficial effects of the present invention are:

In the present invention, triblock polymer P123, triblock copolymer F127, dopamine hydrochloride and pore expander are polymerized under certain conditions to form porous polydopamine nanospheres as the imprinting substrate. Two similar block copolymers ( P123 and F127) with hydrophilic chains of different lengths as soft templates, a dual soft template strategy was used to prepare mesoporous polydopamine nanoparticles; then LDL was added, and based on protein anchoring technology, the template molecule LDL passed through π-π interactions, multiple hydrogen Bonds and hydrophobic interactions attach to the surface of PPDA to form a PPDA-LDL complex; then dopamine hydrochloride is added, and a polymerization reaction occurs under certain conditions to form a copolymer. This copolymer is then eluted, and it will be on the surface of the imprinted polymer. An imprinted cavity complementary to the shape, functional group and spatial size of the template molecule is formed. Due to the shape memory effect of the imprinted cavity in recognition selectivity, PPDA-MIPs exhibit excellent selectivity for LDL. The low-density lipoprotein cholesterol-imprinted nanospheres prepared by the invention can efficiently and specifically adsorb the target LDL, which solves the problems of high equipment requirements and cumbersome processes in the separation and purification of LDL.

Description of drawings

Figure 1 shows 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);

Figure 2 shows the infrared spectra of PPDA, PPDA-MIPs and PPDA-NIPs (a) and the thermogravimetric curves of PPDA and PPDA-MIPs (b);

Figure 3 shows the pH-dependent adsorption behavior of LDL on the surface of PPDA, PPDA-NIPs and PPDA-MIPs (a) and the relationship between LDL adsorption efficiency and ionic strength in the concentration range of 0.0-3.0 mol L ^-1 NaCl (b );

Figure 4 shows the adsorption isotherm (a) of PPDA-NIPs and PPDA-MIPs on LDL and the curve of 1/Q _e versus 1/C _e (b);

Figure 5 shows the reusability of PPDA-MIPs in LDL separation (a) and SDS-PAGE analysis results (b);

Detailed ways

The technical solution of the present invention will be further described below in conjunction with the accompanying drawings, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the present invention. within the scope of protection.

Example 1

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

(2) Preparation of low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs): Disperse 100 mg PPDA in 50 mL, pH 8.5 PBS buffer (PBS buffer concentration is 0.01 mol L ^-1 ), and then add 5 mg of LDL. After stirring for 50 min at room temperature for surface anchoring, add 50 mg of dopamine hydrochloride to the above solution, stir for 16 h, and then wash with acetic acid (20%, V/V) multiple times to remove the template protein until the UV-visible spectrophotometer reaches 280 No absorption peak could be detected at wavelengths around nm, and then washed three times with ultrapure water and ethanol to remove residual acetic acid to obtain low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs).

Comparative example 1

(1) Same as step (1) in Example 1;

(2) Preparation of low-density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs): Disperse 100 mg PPDA in 50 mL, pH 8.5 PBS buffer (PBS buffer concentration is 0.01 mol L ^-1 ). After stirring for 50 min at room temperature, add 50 mg of dopamine hydrochloride to the above solution, continue stirring for 16 h, and then wash with acetic acid (20%, V/V) several times, and then wash with ultrapure water and ethanol three times to remove residues of acetic acid to obtain low-density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs).

Scanning electron microscope images 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 And the transmission electron microscope image is shown in Figure 1; among them, (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, (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 approximately 270 nm. 1(d) TEM image further characterizes the rich mesoporous structure of PPDA. These mesoporous structures are The pore structure provides sufficient specific surface area. In addition, it can be seen through scanning electron microscopy (Figure 1b and Figure 1c) and transmission electron microscopy (Figure 1e and Figure 1f) that the polymerization of dopamine results in PPDA-NIPs and PPDA-MIPs with rougher surface structures and nanomorphology.

Infrared 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 The image is shown in Figure 2(a); the spectrum of PPDA has a plate-like band at about 3420 cm ^-1 , which can be attributed to the OH stretching of intermolecular hydrogen bonds; there are two bands at 1617, 1450 and 1114 cm ^- 1 A group of distinguishable bands can be attributed to the aromatic ring stretching vibration of the polybenzazole structure25; in addition, the band at 1496 cm ^-1 corresponds to the stretching vibration of NH, with typical peaks at 1350 and 1292 cm ^-1 They are attributed to the bending vibration and stretching vibration of COH on the benzene ring respectively; it is worth noting that the FTIR spectra of PPDA-NIPs and PPDA-MIPs are similar to PPDA, confirming the successful formation of polydopamine-based nanomaterials.

The thermogravimetric analysis curves of porous polydopamine nanospheres (PPDA) and low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs) in Example 1 are shown in Figure 2(b); for PPDA and PPDA-MIPs, at 25 The first weight loss that occurs at -150°C is in response to physically adsorbed water. For PPDA, a second weight loss of 45.4% can be noticed, 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. This is related to the surface coating of polydopamine on PPDA.

Application examples

1. Under pH 3-9 conditions, study the porous polydopamine nanospheres (PPDA), low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs) in Example 1 and the low-density lipoprotein cholesterol-free nanospheres in Comparative Example 1. The adsorption behavior of imprinted nanospheres (PPDA-NIPs) on LDL (15 μg mL ^-1 , 300 μL) under different pH conditions. The results are shown in Figure 3(a). Under pH 3-9 conditions, LDL on the surface of PPDA-MIPs The adsorption efficiency is always higher than those of unimprinted materials. At the same time, the adsorption efficiency of LDL increases with the increase of pH, reaching a maximum at its isoelectric point (pH 5.5), while continuing to increase the pH will reduce the adsorption efficiency of LDL. When the pH is 5.5, the optimal adsorption efficiencies of PPDA, PPDA-NIPs and PPDA-MIPs for LDL are 40.0%, 51.5% and 92.0% respectively. This adsorption behavior can be well explained by hydrogen bonding interactions between polydopamine nanospheres and LDL. 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 catechols, amines, and imines, which can serve as hydrogen bond donors/acceptors. Based on these, hydrogen bonding interactions lead to maximal adsorption at pH values near the pI of LDL.

2. The effect of low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs) on LDL adsorption at different NaCl concentrations in Example 1 was studied. The results are shown in Figure 3(b). The figure shows that in a wider range Within the range (0 ~ 3 mol L ^-1 ), changes in ionic strength have no effect on the adsorption of LDL, indicating that electrostatic interactions do not contribute to the adsorption of LDL, proving that low-density lipoprotein cholesterol-imprinted nanospheres usually encounter relatively Practicality in real biological samples at higher ionic strengths.

3. Study the low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs) in Example 1 and the low-density lipoprotein cholesterol non-imprinted nanospheres in Comparative Example 1 within the initial concentration range of 1 ~ 100 μg mL ^-1 (PPDA-NIPs)’s adsorption capacity for LDL, its adsorption isotherm is shown in Figure 4(a). The binding amount of LDL to PPDA-NIPs and PPDA-MIPs increases rapidly with the increase of the initial LDL concentration and reaches adsorption equilibrium. . Under the same conditions, the maximum adsorption capacity of PPDA-NIPs is 250.4 g mg ^-1 , while the maximum adsorption capacity of PPDA-MIPs is 550.3 g mg ^-1 . It can be seen that the binding of LDL to PPDA-MIPs is mainly due to the specific binding to the recognition cavity. In contrast, the adsorption of LDL on PPDA-NIPs is mainly a non-specific effect. 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):

Formula 1)

C _e (μg mL ^-1 ) is the LDL equilibrium concentration; Q _max (μg mg ^-1 ) and Q _e (μg mg ^-1 ) are the maximum theoretical adsorption capacity and equilibrium adsorption capacity respectively. The linear relationship curve is shown in Figure 4(b) ), the Langmuir model is suitable for the adsorption behavior of PPDA-NIPs and PPDA-MIPs, and the correlation coefficients (R2) are 0.9900 and 0.9906 respectively. Therefore, it is inferred that the adsorption of LDL on the material surface is a single layer.

4. Elution and reuse of molecularly imprinted nanospheres

Reusability is an important indicator for evaluating the performance of molecularly imprinted materials. In the present invention, a desorption study was conducted for 30 minutes 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, six adsorption/desorption cycles were repeated using the same imprinting material (low-density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in Example 1) under optimal parameters, and the results are shown in Figure 5(a) , after completing 6 adsorption/desorption cycles, the adsorption efficiency of PPDA-MIPs only dropped by 11.51%. This was due to the fact that 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 can still reach 83.09%, indicating that PPDA-MIPs has good reuse prospects.

5. Molecularly imprinted nanospheres are used to separate and purify LDL from goat serum samples.

The low-density lipoprotein cholesterol-imprinted nanospheres (PPDA-MIPs) in Example 1 were used for the selective separation of LDL in goat serum samples. After diluting goat serum 20 times with 4.0 mmol L ^-1 BR buffer at pH 5.5, using MeOH:HAc (4:1) solution as the eluent, LDL was collected from PPDA-MIPs using dodecyl sulfate. Sodium-polyacrylamide gel electrophoresis (SDS-PAGE) technology was used for analysis. The SDS-PAGE analysis results are shown in Figure 5(b). From the figure, it can be seen that there are several main components in the diluted goat serum sample. Bands. The molecular weight of LDL is approximately 2.7x10 ³ -3.3x10 ³ kDa. The LDL standard solution is located above the standard protein band and has a molecular weight of 200 kda. Identified at the same position as the LDL standard solution (lane 5). After adsorption of PPDA-MIPs (lane 3), the color of the LDL strip faded significantly. In lane 4, the PPDA-MIPs eluate showed a strong band above 200kDa, and there were no other protein bands in the eluate, indicating that PPDA-MIPs had good selectivity. The above results indicate that the prepared PPDA-MIPs can selectively recognize LDL in the presence of other proteins in goat serum samples.

Claims (10)

1. The preparation method of the low-density lipoprotein cholesterol imprinting nanospheres is characterized by comprising the following steps of:

(1) Preparation of porous polydopamine nanospheres: dispersing a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, an ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, dopamine hydrochloride and a pore-expanding agent in a mixed solvent of ethanol and water to form an emulsion, adding ammonia water into the emulsion, stirring, and carrying out centrifugal separation and washing to obtain porous polydopamine nanospheres;

(2) Preparation of low-density lipoprotein cholesterol imprinted nanospheres: dispersing the porous polydopamine nanospheres prepared in the step (1) in PBS buffer solution, adding low-density lipoprotein cholesterol, stirring at room temperature for surface anchoring, adding dopamine hydrochloride, stirring for reaction, and washing to remove template proteins after the reaction is finished to obtain the low-density lipoprotein cholesterol imprinted nanospheres.

2. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein 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 to ethanol in the mixed solvent is 1: 0.5-2.

3. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein in the step (1), the pore-expanding agent is mesitylene, and the addition amount of the pore-expanding agent and the volume mass of the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 are 1mg: 15-20 mu L; the mass percentage concentration of the ammonia water is 20-30%, and the volume mass ratio of the added amount of the ammonia water to the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1mg:10 to 20 mu L.

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

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

6. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein the concentration of the PBS buffer in the step (2) is 0.01 mol L ^-1 The pH was 8.5.

7. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein the product after centrifugal separation in the step (1) is washed with water and ethanol for several times; the method of washing to remove the template protein in the step (2) is to wash with acetic acid a plurality of times to remove the template protein until the absorption peak cannot be detected by the ultraviolet-visible spectrophotometer at a wavelength of about 280 nm, and then wash with water and ethanol three times to remove the residual acetic acid.

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

9. A low-density lipoprotein cholesterol imprinted nanosphere prepared by the preparation method of any one of claims 1 to 8.

10. Use of the low-density lipoprotein cholesterol imprinted nanospheres of claim 9 for separating low-density lipoprotein cholesterol.