Phospholipid compound for inhibiting beta-amyloid protein fibrosis and preparation method thereof

Abstract

The invention discloses a phospholipid compound for inhibiting beta-amyloid protein aggregation, and the chemical structural formula of the phospholipid compound is as follows: # imgabs0 #, and n is an integer greater than 2. Dioleoyl phosphatidyl ethanolamine molecules are modified by utilizing maleimide alkyl carboxylic acid and glutathione, so that the interaction between a phospholipid compound and amyloid protein can be effectively regulated and controlled, and the aggregation of the amyloid protein is delayed and even inhibited; the preparation method is simple, the reaction energy consumption is low, and the obtained phospholipid compound has good biocompatibility and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a phospholipid compound for inhibiting the fibrillation of β-amyloid protein and a preparation method thereof. Background Art

[0002] Alzheimer's disease is a worldwide medical problem, and there is still no effective therapeutic drug. The toxicity of oligomers and fibers of β-amyloid protein (Aβ) to cells has been confirmed. Currently, clinical strategies for clearing Aβ through immunotherapy or enzyme inhibition show limited clinical efficacy (Nat. Commun. 2025, 16, 2937; J. Controlled Release 2024, 375, 346), which may be due to their inability to regulate the Aβ fibrillation process in the cell membrane environment. Therefore, there is an urgent need for new intervention measures directly targeting the Aβ fibrillation process on the cell membrane.

[0003] Functional phospholipid compounds have good biocompatibility and safety, can improve the absorption and utilization of active ingredients, and have broad application potential in the medical field. A single phospholipid molecule (such as dioleoylphosphatidylethanolamine PE) has been shown to induce the aggregation of Aβ protein to form fibers (Langmuir, 2018, 34, 8408), and usually needs to be chemically modified. A single glutathione (GSH) molecule has a certain effect of inhibiting Aβ fibrillation in solution, but it has redox activity, is unstable in physical and chemical properties in vivo, and has limited pharmacokinetics (Biochim. Biophys. Acta. 2013, 1830, 3350), and its in vivo application is restricted. Summary of the Invention

[0004] The main purpose of the present invention is to provide a phospholipid compound that can effectively inhibit the aggregation of β-amyloid protein, aiming at the problems and deficiencies existing in the prior art, for regulating the interaction between β-amyloid protein and phospholipid membrane, and further inhibiting the fibrillation of β-amyloid protein.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A phospholipid compound for inhibiting the fibrillation of β-amyloid protein, and its chemical structural formula is shown in Formula I;

[0007]

[0008] In the formula, n takes an integer of 2 or more.

[0009] Furthermore, the value of n is 2 to 10; preferably 4 to 6.

[0010] The present invention also discloses a preparation method of the above phospholipid compound, comprising the following steps:

[0011] (1) Activation of maleimide hexanoic acid;

[0012] Dissolve maleimide alkyl carboxylic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in a mixed solvent, and perform heating and stirring activation to obtain a clear and transparent activation solution;

[0013] (2) Synthesis of intermediate;

[0014] Dissolve phosphatidylethanolamine in a mixed solvent to obtain a PE solution; then add it to the obtained activation solution, perform heating and stirring reaction to obtain a clear and transparent reaction solution; perform flocculation precipitation, centrifugal separation, and drying to obtain intermediate powder (PE(C n ));

[0015] (3) Dissolve the intermediate powder in water to prepare a colloid, then add reduced glutathione, perform a reaction at room temperature, and dry to obtain a white granular phospholipid compound.

[0016] In the above scheme, the structural formula of the maleimide alkyl carboxylic acid is shown in Formula II;

[0017]

[0018] In the formula, n takes a value of 2 to 10.

[0019] Furthermore, one or more of maleimide propionic acid, maleimide hexanoic acid, maleimide undecanoic acid, etc. can be selected in the maleimide alkyl carboxylic acid.

[0020] In the above scheme, the molar ratio of the maleimide alkyl carboxylic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(2 - 4):(2 - 4).

[0021] In the above scheme, the mixed solvent includes chloroform and an alcohol solvent.

[0022] Furthermore, the alcohol solvent can be selected from methanol, ethanol, etc.

[0023] Furthermore, the volume ratio of chloroform to methanol is 1:4 to 4:1.

[0024] In the above scheme, the temperature for the heating and stirring activation is 40 - 65 °C, and the time is 4 - 10 h.

[0025] In the above scheme, the heating and stirring activation is carried out under slightly acidic conditions, and the pH value of the reaction solution is 6 - 7 (excluding 7).

[0026] In the above solution, the molar ratio of phosphatidylethanolamine to maleimide alkyl carboxylic acid is 1:(0.75 - 4).

[0027] In the above solution, the temperature for the heating and stirring reaction is 40 - 65°C, and the time is 24 - 56 h.

[0028] In the above solution, acetone or methanol, etc. is used for flocculation precipitation.

[0029] In the above solution, the chemical formula of the intermediate powder PE(C n ) is shown in Formula III;

[0030]

[0031] In the formula, n takes an integer of 2 or more.

[0032] In the above solution, the solid-liquid ratio of the intermediate powder to water in step 3) is 10 mg:10 - 50 mL.

[0033] In the above solution, the molar ratio of reduced glutathione to the intermediate powder is 1:(1 - 4).

[0034] In the above solution, the reaction time at room temperature is 12 - 24 h.

[0035] The present invention also discloses a simulated cell membrane prepared by using the above phospholipid compound. The preparation method includes the following steps: mixing the phospholipid compound with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to prepare a lipid bilayer (simulated cell membrane). The application results show that by introducing the phospholipid compound of the present invention, the interaction between β-amyloid protein and the cell membrane (simulated phospholipid membrane) can be effectively regulated, directly intervening in the interaction between β-amyloid protein and the cell membrane, and effectively inhibiting the fibrillation process of β-amyloid protein.

[0036] Furthermore, the molar ratio of the phospholipid compound to 1,2-dioleoyl-sn-glycero-�-phosphocholine is 1:4 - 10.

[0037] Furthermore, the specific preparation steps of the lipid bilayer include the following steps: dissolving DOPC and the phospholipid compound obtained in the above solution in a buffer solution respectively, mixing the obtained two liposome solutions to obtain a mixed liposome solution; heating in a water bath and performing shaking treatment to obtain a uniform liposome suspension; performing membrane filtration to obtain a vesicle solution; then injecting the obtained vesicle solution into a container provided with a substrate and incubating to form a stable lipid bilayer membrane on the surface of the substrate.

[0038] In the above solution, the temperature for the water bath heating is 55 - 65°C, and the time is 8 - 12 min.

[0039] In the above solution, the pore size of the filter membrane used in the membrane filtration step is 50 - 250 nm.

[0040] Compared with the prior art, the beneficial effects of the present invention include:

[0041] 1) The present invention uses maleimide alkyl carboxylic acid and glutathione to modify dioleoyl phosphatidylethanolamine molecules, so that the obtained phospholipid molecule heads have groups such as maleimide rings, alkyl chain structures, and amphiphilic glutathione structures at the same time, which can effectively regulate the van der Waals and electrostatic interactions with β-amyloid protein, and delay or even inhibit the aggregation of β-amyloid protein on the phospholipid membrane;

[0042] 2) The present invention uses maleimide alkyl carboxylic acid, glutathione, and dioleoyl phosphatidylethanolamine as the main raw materials, and the obtained phospholipid compound has good biocompatibility;

[0043] 3) The preparation method of the phospholipid compound described in the present invention is relatively simple and has low energy consumption, and is suitable for popularization and application. Description of the Drawings

[0044] Figure 1 It is the fibrillization effect diagram of β-amyloid protein (Aβ40) on different DOPC phospholipid membranes. Among them, a is a simple DOPC membrane; b is the lipid bilayer membrane obtained by using the phospholipid compound described in Example 1; c is the lipid bilayer membrane obtained by using the phospholipid compound described in Example 2; d is the lipid bilayer membrane obtained by using the phospholipid compound described in Example 3.

[0045] Figure 2 It is the effect diagram of the simple GSH inhibition of the fibrillization of β-amyloid protein (Aβ40) on the DOPC phospholipid membrane described in Comparative Example 1.

[0046] Figure 3 It is the effect diagram of the fibrillization of β-amyloid protein (Aβ40) on the lipid bilayer obtained in Comparative Example 2. Detailed Embodiments

[0047] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0048] The preparation method of the phospholipid molecule for efficiently inhibiting the aggregation of β-amyloid protein in the present invention, wherein the purity of the dioleoylphosphatidylethanolamine (DOPE) used is 97%, from Macklin Biochemical Inc. (China), and maleimidopropionic acid, maleimidohexanoic acid, and maleimidoundecanoic acid are of analytical grade, from Sigma-Aldrich Inc. (USA), and N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), and reduced glutathione (GSH) are of analytical grade, from Energy-Chemical Inc. (China).

[0049] Example 1

[0050] A phospholipid compound for inhibiting the fibrillation of β-amyloid protein (denoted as phospholipid compound 1);

[0051]

[0052] Its preparation method includes the following steps:

[0053] (1) Activate the carboxylic acid group of maleimidohexanoic acid

[0054] First, prepare a mixed solution of 4 mL of chloroform and 1 mL of methanol for standby. Weigh 40 mg of maleimidohexanoic acid and 45 mg of NHS respectively with an analytical balance, quickly weigh 75 mg of EDC·HCl, and dissolve them in the mixed solvent respectively. Under the conditions of heating and stirring at 60 °C, activate the carboxylic acid group of maleimidohexanoic acid for 8 h to obtain a clear and transparent activation solution. The whole reaction system is carried out under slightly acidic conditions (pH = 6 - 7, and 7 is not included);

[0055] (2) Synthesize the intermediate PE(C5) powder

[0056] Weigh 175 mg of PE and dissolve it in another 5 mL of chloroform:methanol = 4:1 mixed solvent. Add the obtained PE solution to the activation solution obtained in step 1). Under the conditions of heating and stirring at 60 °C, react for 48 h to obtain a colorless and clear PE(C5) solution. Divide 10 mL of the above solution into 10 5 mL centrifuge tubes, add 4 mL of acetone to each tube. At this time, it is observed that PE(C5) precipitates as a flocculent. Centrifuge in a high-speed centrifuge for several minutes, and set the rotation speed to 1000 rpm; filter and separate the precipitate, and repeat the above operation for the supernatant. The precipitates obtained twice are placed in a vacuum drying oven and dried for more than 24 h to collect the PE(C5) powder. Prepare a sufficient amount of PE(C5) powder in batches for standby;

[0057] (3) Synthesize phospholipid compound 1

[0058] The prepared 10 mg of PE(C5) powder was dissolved in 10 mL of pure water to obtain a colloidal dispersion. 2 mg of GSH was added. PE(C5) self-assembled into micelles in water, with the hydrophilic head groups exposed outside. It reacted with the sulfhydryl group of GSH, and the reaction was carried out at room temperature for 24 hours. The water solvent was removed by freeze-drying for 3 days to obtain a white granular phospholipid compound IV, which was stored at 4°C.

[0059] Example 2

[0060] A phospholipid compound that inhibits the fibrillation of β-amyloid protein (denoted as phospholipid compound 2);

[0061]

[0062] Its preparation method includes the following steps:

[0063] (1) Activate the carboxylic acid group of maleimidopropionic acid

[0064] First, prepare 4 mL of a chloroform and 1 mL of methanol mixed solution for standby. Weigh 30 mg of maleimidopropionic acid and 45 mg of NHS separately with an analytical balance, and quickly weigh 75 mg of EDC·HCl, and dissolve them in the mixed solvent respectively. Under the condition of heating and stirring at 55°C, activate for 7 h to obtain a clear and transparent activation solution. The whole reaction system is carried out under slightly acidic conditions (pH = 6 - 7, and 7 is not included);

[0065] (2) Synthesize the intermediate PE(C2) powder

[0066] Weigh 175 mg of PE and dissolve it in another 5 mL of chloroform:methanol = 4:1 mixed solvent. Add the obtained PE solution to the activation solution obtained in step 1). Under the condition of 55°C, heat and stir for 40 h to obtain a colorless and clear PE(C2) solution. Divide 10 mL of the above solution into 10 5-mL centrifuge tubes, and add 4 mL of acetone to each tube. At this time, it is observed that PE(C2) precipitates as an oily substance. Centrifuge in a high-speed centrifuge for several minutes, and set the rotation speed to 5000 rpm; filter and separate the precipitate, and repeat the above operation for the supernatant. The precipitates obtained twice are placed in a vacuum drying oven and dried for more than 24 h, and the PE(C2) powder is collected;

[0067] (3) Synthesize phospholipid compound 2

[0068] The prepared 9.4 mg of PE(C2) powder was dissolved in 10 mL of pure water to obtain a colloidal dispersion. 2 mg of GSH was added. PE(C2) self-assembled into micelles in water, with the hydrophilic head groups exposed outside. It reacted with the sulfhydryl group of GSH, and the reaction was carried out at room temperature for 24 hours. The water solvent was removed by freeze-drying for 3 days to obtain a white granular phospholipid compound V, which was stored at 4°C.

[0069] Example 3

[0070] A phospholipid compound that inhibits β-amyloid protein fibrillization (denoted as phospholipid compound 3)

[0071]

[0072] The preparation method comprises the following steps:

[0073] (1) Activation of the carboxylic acid group of maleimide undecanoic acid

[0074] First, prepare a mixed solution of 4 mL of chloroform and 1 mL of methanol. Use an analytical balance to weigh 50 mg of maleimide undecanoic acid and 45 mg of NHS, then quickly weigh 75 mg of EDC·HCl. Dissolve each in the mixed solvent and heat at 65°C with stirring for 10 h to obtain a clear, transparent activated solution. The entire reaction system is controlled under slightly acidic conditions (pH = 6-7, excluding 7).

[0075] (2) Synthetic intermediate PE(C 10 )powder

[0076] Weigh 175 mg of PE and dissolve it in another 5 mL of a mixed solvent of chloroform: methanol = 4:1. Add the resulting PE solution to the activation solution obtained in step 1) and heat and stir at 65°C for 56 hours to obtain colorless and clear PE (C 10 ) solution, 10 mL of the above solution was divided into 10 5 mL centrifuge tubes, and 4 mL of acetone was added to each tube. At this time, PE (C 10 ) was precipitated as flocculent matter and centrifuged in a high-speed centrifuge for several minutes at 1000 rpm; the precipitate was separated by filtration, and the supernatant was subjected to the above operation again. The precipitate obtained twice was placed in a vacuum drying oven and dried for more than 24 hours to obtain PE(C 10 ) powder; prepare sufficient PE (C 10 ) Powder for later use;

[0077] (3) Synthesis of compound 3

[0078] The obtained 12mg PE(C 10 ) powder was dissolved in 10 mL of pure water to obtain a colloidal dispersion, and 2 mg of GSH, PE (C 10 ) self-assembled into micelles in water, with the hydrophilic group on the head exposed, and reacted with the thiol group of GSH. The reaction was carried out at room temperature for 24 hours, and the water solvent was removed by freeze-drying for 3 days to obtain white granular phospholipid compound 3, which was stored at 4°C.

[0079] The phospholipid compounds obtained in Examples 1 to 3 were respectively complexed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to prepare mixed supported lipid bilayers for simulating cell membranes, and the effect of the phospholipid compounds on inhibiting the fibrillation of β-amyloid protein was observed. The specific steps are as follows:

[0080] (1) Pretreat the mica sheet

[0081] Use transparent tape to stick and tear off the mica surface layer to obtain a mica sheet with a fresh and clean surface, and then cut it according to the size of a 48-well plate to obtain multiple small mica sheets of the same size and square shape;

[0082] (2) Prepare phosphate buffer solution

[0083] Weigh 0.8 g of sodium chloride, 0.024 g of potassium chloride, 0.3628 g of disodium hydrogen phosphate dodecahydrate, and 0.024 g of potassium dihydrogen phosphate in sequence, put them into a washed beaker, add 100 mL of pure water to dissolve, and obtain phosphate buffer solution (PBS). After preparation, use a pH meter to measure, and the pH value is about 7.4. If it is too high or too low, adjust it with hydrochloric acid and sodium hydroxide solutions; seal the cup mouth with plastic wrap for later use.

[0084] (3) Prepare lipid bilayer membrane

[0085] Use the newly prepared PBS buffer solution to dissolve DOPC and one of the phospholipid compounds 1 to 3 respectively, prepare liposome solutions with concentrations of 1 mg / mL respectively, and place them in an environment of -20 °C for later use; according to the requirement that the total volume is 100 μL and the volume ratio is DOPC:(phospholipid compound 1 or 2 or 3)=8.5:1.5 (mol), mix the two liposome solutions to obtain a mixed liposome solution;

[0086] Add 900 μL of the newly prepared PBS buffer solution to the obtained mixed liposome solution to keep the total volume of the solution 1 mL; heat in a water bath for 10 min, keep the water bath temperature at 60 °C, and shake and suspend with a vortex shaker to obtain a uniformly distributed liposome suspension; then use a liposome extruder to push the above liposome suspension back and forth through the filter membrane 20 times to obtain a vesicle solution with a diameter of about 200 nm;

[0087] Place the processed clean square mica sheet flat at the bottom of each well, inject 400 μL of the above vesicle solution into each small hole, and incubate for 2 h to form a stable lipid bilayer membrane on the mica sheet surface.

[0088] The inhibition experiments of β-amyloid (Aβ40) were carried out on the simple DOPC membrane and the DOPC / phospholipid compound 1 lipid bilayer membrane, DOPC / phospholipid compound 2 lipid bilayer membrane, and DOPC / phospholipid compound 3 lipid bilayer membrane obtained by the above-mentioned scheme respectively. The specific steps are as follows:

[0089] 1) At 37 °C and a solution concentration of 1 μM of Aβ40 in PBS, mica sheets loaded with DOPC: (phospholipid compound 1 or 2 or 3) mixed lipid bilayer membranes and mica sheets with pure DOPC phospholipid bilayers (prepared in the same way as in the application example, except that phospholipid compound 1 or 2 or 3 is not introduced) were used for incubation for 72 h on the surface;

[0090] 2) Atomic force microscopy was used to characterize the morphology of β-amyloid on different surfaces after incubation ( Figure 1 ). The comparison results with Figure a show that after introducing phospholipid compound 1, phospholipid compound 2, and phospholipid compound 3 molecules, the fibrillation of Aβ can be inhibited, and among them, the effect of compound 1 is the best.

[0091] Comparative Example 1

[0092] A DOPC phospholipid membrane was prepared (prepared in the same way as above, without introducing the phospholipid compounds described in the present invention), and then GSH molecules were added to carry out the β-amyloid (Aβ40) fibrillation experiment. The specific steps are as follows:

[0093] 1) At 37 °C and a solution concentration of 1 μM of Aβ40 in PBS, it was incubated for 72 h on the surface of the mica sheet loaded with the DOPC bilayer in the application example;

[0094] 2) Atomic force microscopy was used to characterize the morphology of β-amyloid on the surface after incubation ( Figure 2 ). The results show that the introduced GSH molecules cannot inhibit the formation of Aβ fibers on the DOPC surface.

[0095] Comparative Example 2

[0096] A phospholipid compound (phospholipid compound 4), the chemical structural formula of which is as follows:

[0097]

[0098] The phospholipid compound 4 was compounded with DOPC to prepare a lipid bilayer (prepared in the same way as above), and then the β-amyloid (Aβ40) fibrillation experiment was carried out. The specific steps are as follows:

[0099] 1) Under the conditions of 37 °C and PBS buffer solution (solution concentration of 1 μM) of Aβ40, it was added to the surface of the mica sheet loaded with the lipid bilayer for incubation for 72 h.

[0100] 2) Characterize the morphology of amyloid-β after incubation on the surface using atomic force microscopy (see Figure 3 ), and the results show that the obtained phospholipid compound 4 cannot effectively inhibit the formation of Aβ fibrils on the DOPC surface.

[0101] It is easy for those skilled in the art to understand that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A phospholipid compound that inhibits the aggregation of β-amyloid protein, characterized in that, Its chemical structural formula is shown in Formula I; In the formula, n is an integer greater than or equal to 2.

2. The phospholipid compound according to claim 1, characterized in that, n ranges from 2 to 10.

3. A method for preparing a phospholipid compound that inhibits the aggregation of β-amyloid protein according to claim 1 or 2, characterized in that, It includes the following steps: 1) Dissolve maleimide alkyl carboxylic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in a solvent, heat and stir for activation to obtain a clear and transparent activation solution; 2) Dissolve phosphatidylethanolamine in a mixed solvent to obtain a PE solution; then add it to the obtained activation solution, heat and stir for reaction to obtain a clear and transparent reaction solution; perform flocculation precipitation, centrifugal separation, and drying to obtain an intermediate powder; 3) Dissolve the obtained intermediate powder in water to prepare a colloid, then add reduced glutathione, react at room temperature, and dry to obtain a white granular phospholipid compound.

4. The preparation method according to claim 3, wherein The structural formula of the maleimide alkyl carboxylic acid is shown in Formula II; In the formula, n ranges from 2 to 10.

5. The preparation method according to claim 3, characterized in that, The molar ratio of the maleimide alkyl carboxylic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(2-4):(2-4).

6. The preparation method according to claim 3, characterized in that, The temperature used in the heating and stirring activation step is 40-65 °C, and the time is 4-10 h.

7. The preparation method according to claim 3, characterized in that, The molar ratio of the phosphatidylethanolamine to the maleimide alkyl carboxylic acid is 1:(0.75-4).

8. The preparation method according to claim 3, characterized in that, The temperature used in the heating and stirring reaction is 40-65 °C, and the time is 24-56 h.

9. The preparation method according to claim 3, characterized in that, The chemical formula of the intermediate powder is shown in Formula III; In the formula, n ranges from 2 to 10.

10. The preparation method according to claim 3, characterized in that, The molar ratio of the reduced glutathione to the intermediate powder is 1:(1-4).

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