Platelet-targeted sugar-containing polymer micelle nanoparticles and preparation method thereof

Abstract

The present invention discloses platelet-targeted glycosylated polymer micelle nanoparticles and a method for preparing the same, belonging to the field of polymer materials. The present invention utilizes a RAFT polymerization reaction to prepare an amphiphilic block copolymer: different glycosylated monomers, an initiator, and a chain transfer agent are first reacted in DMF, followed by the addition of N-alkyl acrylamide and an initiator, followed by continued reaction. The resulting glycosylated polymer is dialyzed and freeze-dried, and then dispersed in water to obtain nanomicelles. The glycosylated polymer is simple and controllable to prepare, and the resulting glycosylated polymer micelles have a particle size of less than 200 nm and are uniformly dispersed, exhibiting good affinity for platelets.

Description

Technical Field

[0001] The invention relates to platelet-targeted sugar-containing polymer micelle nanoparticles and a preparation method thereof, and belongs to the field of polymer materials. Background Art

[0002] Tumor metastasis is a major cause of malignant tumors, and hematogenous metastasis is the primary mode of metastasis. Platelets, a major component of blood, can bind to and activate tumor cells. Platelets then adhere to circulating tumor cells (CTCs) through P-selectin, facilitating extravasation of tumor cells to distant organs. P-selectin is a 140 kDa glycoprotein present on the membranes of Weber-Penelope bodies of vascular endothelial cells and platelets. Its high-affinity ligand is P-selectin glycoprotein ligand 1 (PSGL-1). P-selectin is increasingly being studied as a novel target for the treatment of thrombosis, tumors, inflammation, and other diseases. Drugs that mimic the structure of PSGL-1 and competitively bind to P-selectin are primarily classified into three categories: polysaccharides, proteins, and chemically synthesized molecules. However, the preparation of polysaccharide and protein drugs is complex and costly. Chemically synthesized molecules also suffer from short in vivo residence times and weak affinity for P-selectin, limiting the application of these P-selectin-targeting agents. Summary of the Invention

[0003] The present invention designs and synthesizes an amphiphilic sugar-containing polymer micelle. By selecting the type of sugar group, the affinity with the platelet surface is enhanced, thereby achieving platelet targeting. First, N-dodecyl acrylamide and methacryloyl hydrazide-substituted sugar monomers, such as glucose, fucose, and galactose, are block copolymerized in DMF to prepare pH-responsive sugar-containing polymers with different sugar groups (monosaccharides or polysaccharide heteropolymers). The polymers are then dispersed in water to form micelle structures with a particle size between 100 and 200 nm. By characterizing the platelet affinity, sugar-containing polymer micelles with strong binding ability to P-selectin on the platelet surface are obtained.

[0004] The first object of the present invention is to provide a method for preparing sugar-containing polymer micelle nanoparticles, which is to prepare amphiphilic block copolymers through RAFT polymerization, comprising the following steps:

[0005] (1) mixing the sugar monomer, initiator, and chain transfer agent in an organic solvent for uniform reaction;

[0006] (2) Then, N-alkyl acrylamide and an initiator are added to continue the reaction. After the reaction is completed, the reaction is dialyzed and freeze-dried to obtain sugar-containing polymer micelle nanoparticles.

[0007] In one embodiment of the present invention, in step (1), the molar ratio of the glycosyl monomer to the chain transfer agent is (1-2.5): (0.05-0.5).

[0008] In one embodiment of the present invention, in step (1), the molar ratio of the glycosyl monomer to the initiator is (1-2.5): (0.01-0.05).

[0009] In one embodiment of the present invention, in step (1), the amount of the glycosyl monomer relative to the organic solvent is (1-2.5) mmol / mL.

[0010] In one embodiment of the present invention, in step (2), the molar ratio of the saccharide monomer to the N-fatty alkyl acrylamide is (1-2.5):(1.5-2.5), preferably 1:1.5.

[0011] In one embodiment of the present invention, in step (2), the molar ratio of the N-fatty alkyl acrylamide to the initiator is (1.5-2.5): (0.01-0.05).

[0012] In one embodiment of the present invention, the glycosyl monomer includes any one or more of the following: glucose substituted with methacryloylhydrazine, fucose substituted with methacryloylhydrazine, galactose substituted with methacryloylhydrazine, sialic acid substituted with methacryloylhydrazine, and mannose substituted with methacryloylhydrazine.

[0013] In one embodiment of the present invention, the number of carbon atoms in the alkyl group of N-fatty alkyl acrylamide is 6-18, and specifically 12.

[0014] In one embodiment of the present invention, the organic solvent includes any one or more of the following: dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and oxirane.

[0015] In one embodiment of the present invention, the chain transfer agent includes any one or more of the following: 4-cyano-4-(thiobenzoyl)valeric acid, 2-cyano-2-propyldodecyl trithiocarbonate, and 2-cyano-2-propyl-4-cyanophenyl dithiocarbonate.

[0016] In one embodiment of the present invention, the initiator is selected from any one or more of the following: azo initiator, peroxide initiator.

[0017] In one embodiment of the present invention, the azo initiator is selected from azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile; and the peroxide initiator is selected from dibenzoyl peroxide and potassium persulfate.

[0018] In one embodiment of the present invention, the reaction time and reaction temperature vary depending on the selected initiator. For example, when AIBN is used as the initiator, the reaction needs to be carried out at 70° C.-80° C. for 24 hours.

[0019] In one embodiment of the present invention, different sugar-based monomers, an initiator, and a chain transfer agent are first mixed in an organic solvent and reacted for 24 hours; then N-fatty alkyl acrylamide and an initiator are added and the reaction is continued for 24 hours.

[0020] In one embodiment of the present invention, the reaction process is as follows:

[0021]

[0022] Wherein, R is glucose, fucosyl, sialic acid, galactosyl, mannosyl, etc.; n is 20-50, m is 30-50, and k is 4-16.

[0023] The present invention provides a sugar-containing polymer micelle nanoparticle prepared based on the above method.

[0024] In one embodiment of the present invention, the sugar-containing polymer micelle nanoparticles are dispersed in an aqueous solution system to prepare nanomicelles:

[0025] In one embodiment of the present invention, in order to improve the platelet targeting of the sugar-containing polymer micelles, the activated platelets are adhered to the above-mentioned sugar-containing polymer micelle nanoparticles for 30 minutes, the non-adherent sugar-containing polymer micelle nanoparticles are washed away, and the adhesion of the activated platelets to the sugar-containing polymer is observed and measured.

[0026] The third object of the present invention is to apply the above-mentioned sugar-containing polymer micelles to the preparation of cancer cell detection devices and drug delivery carriers.

[0027] Advantages and effects of the present invention:

[0028] The sugar-containing polymers of the present invention are simple and controllable to prepare, resulting in uniformly dispersed, glycopolymer micelles with a particle size below 200 nm. Certain sugar-based polymers or polysaccharide-based nanomicelles exhibit a stronger affinity for platelets and can be used for targeted detection and treatment of inflammation and tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Graph showing the particle size and dispersion of the glucose-containing polymer micelle nanoparticle bundles in Example 1.

[0030] Figure 2 This is a flow fluorescence comparison diagram of the glucose-containing polymer micelle nanoparticles in Example 1.

[0031] Figure 3Graph showing the particle size and dispersion of the fucose-containing polymer micelle nanoparticles in Example 2.

[0032] Figure 4 This is a flow fluorescence comparison diagram of the fucose-containing polymer micelle nanoparticle bundle in Example 2.

[0033] Figure 5 Graph showing the particle size and dispersion of the sialic acid-containing polymer micelle nanoparticles in Example 2.

[0034] Figure 6 This is a flow fluorescence comparison diagram of the sialic acid-containing polymer micelle nanoparticles in Example 2. DETAILED DESCRIPTION

[0035] The technical solution of the invention is described in detail below with reference to the accompanying drawings:

[0036] Example 1: Preparation of Sugar-Containing Polymer Micellar Nanoparticles

[0037] Dissolve 1 g of glucose in freshly prepared acetic acid / ammonium acetate buffer (pH 5), add 750 mg of methacryloyl hydrazide, 10 μL of aniline as a catalyst, and 7.8 mg of hydroquinone as a polymerization inhibitor, and react at 50°C for 24 hours. The reaction solution is then freeze-dried, dissolved in dichloromethane, centrifuged, and the solid fraction is collected and dried to obtain a pale yellow powder of methacryloyl hydrazide glucose.

[0038] 278 mg of methacryloyl hydrazide glucose (molar weight 1 mmol), 13.9 mg of 4-cyano-4-(thiobenzoyl)valeric acid (molar weight 0.05 mmol) and 1.64 mg of azobisisobutyronitrile (molar weight 0.01 mmol) were dissolved in 1 mL of dimethyl sulfoxide (molar weight 0.01 mmol). After nitrogen and liquid nitrogen were replaced several times, the reaction was carried out at 80°C for 24 h. The reaction was then stopped with liquid nitrogen, and 159.5 mg of N-dodecyl acrylamide (molar weight 1.5 mmol) and 1.64 mg of azobisisobutyronitrile (molar weight 0.01 mmol) were added. After nitrogen and liquid nitrogen were replaced several times, the reaction was carried out at 80°C for 24 h. The reaction solution was dialyzed with pure water for 72 h and freeze-dried to obtain white solid glucose-containing polymer micelle nanoparticles.

[0039] Particle size and dispersion were measured using a Malvern particle size analyzer. For the specific test, the sugar-containing polymer was dissolved in pure water and sonicated for 10 minutes. The particle size and dispersion were then measured using a particle size cup at a concentration of 1 mg / mL.

[0040] Flow cytometry was used to quantitatively measure the adhesion of micelles to platelets. Blood was collected from the orbits of C57 mice and anticoagulated with 0.38% trisodium citrate. The whole blood was centrifuged at 200g for 12 minutes at room temperature, and the upper platelet-rich plasma was taken out; the plasma was then centrifuged at 1200g for 6 minutes, and the upper plasma was discarded. The white precipitate at the bottom was the platelets. The platelets were resuspended in PBS to 108 / mL, 10U / mL of thrombin was added and incubated at 37°C for 30 minutes to activate them. Thereafter, the sugar-containing polymer in this example was added to adhere to the activated platelets at 37°C for 1 hour. The non-adherent sugar-containing polymer was washed away by centrifugation at 1200g for 6 minutes, and the precipitated platelets were resuspended and the fluorescence intensity was measured by flow cytometry.

[0041] Figure 1 Graph showing the particle size and dispersion of the glucose-containing polymer micelle nanoparticles in this example.

[0042] Figure 2 This is a flow cytometry fluorescence comparison diagram of the glucose-containing polymer micelle nanoparticles in this example.

[0043] Depend on Figure 1 It can be seen that the particle size of the prepared glucose-containing polymer micelle nanoparticles is about 156 nm and the PDI is 0.176 as measured by a Malvern particle size analyzer.

[0044] Depend on Figure 2 It can be seen that the platelets adhered to the glucose-containing polymer micelle nanoparticles have a stronger fluorescence intensity than the blank platelets as determined by flow cytometry, indicating that the micelle nanoparticles have a certain ability to adhere to platelets, with a fluorescence intensity of 14815.

[0045] Example 2 Effect of different hydrophobic chain ratios on micelle nanoparticle size

[0046] Referring to Example 1, the amount of N-dodecyl acrylamide was changed while other factors remained unchanged to prepare the corresponding sugar-containing polymer.

[0047] The particle size of the obtained sugar-containing polymer was tested in the same manner. The results are shown in Table 1.

[0048] Table 1

[0049]

[0050] Glycopolymer micelles self-assemble into nanoparticles in water. Nanoparticles of varying sizes can be produced by controlling the ratio of hydrophilic segments (glycosylated blocks) to hydrophobic segments (N-dodecyl acrylamide). A 1:2 molar ratio of glycosylated monomer to N-alkyl acrylamide resulted in excessive hydrophobic segments, resulting in particle sizes exceeding 2 μm and preventing the formation of suitable nanoparticles. A 1:1 or 1:1.5 molar ratio of glycosylated monomer to N-alkyl acrylamide resulted in uniform nanoparticle size (PDI < 0.2). However, a 1:1 molar ratio resulted in excessively large particle sizes, which may limit the nanoparticles' ability to enter cells. Therefore, a 1:1.5 molar ratio was selected for subsequent polymerization and platelet adhesion experiments.

[0051] Example 3 Effects of different sugar groups on the properties of the obtained micellar nanoparticles

[0052] Referring to Example 1, glucose was replaced with equimolar amounts of fucose and sialic acid (as shown in Table 2) to obtain the corresponding methacryloyl hydrazide fucose and sialic acid; then, the corresponding fucose-containing polymer micelle nanoparticles and sialic acid-containing polymer nanomicelles were obtained according to the same method, and their PDI values ​​were all below 0.2.

[0053] The affinity of the resulting micelle particles for platelets was tested using the same method as in Example 1. The specific results are shown in Table 1. The adhesion of the fucose polymer micelles to platelets was significantly enhanced compared to that of the glucose polymer micelles, with their fluorescence intensity being twice that of the glucose polymer micelles. The adhesion of the sialic acid polymer micelles to platelets was also slightly stronger than that of glucose, with the fluorescence intensity increasing by approximately 11%.

[0054] Table 2

[0055] Different sugar groups Particle size Flow fluorescence intensity glucose 156nm 14815 Fucose 102nm 28205 Sialic acid 124nm 16456

[0056] Figure 3 Graph showing the particle size and dispersion of the fucose-containing polymer micelle nanoparticles in Example 2.

[0057] Figure 4 This is a flow fluorescence comparison diagram of the fucose-containing polymer micelle nanoparticles in Example 2.

[0058] Figure 5 Graph showing the particle size and dispersion of the sialic acid-containing polymer micelle nanoparticles in Example 2.

[0059] Figure 6 This is a flow fluorescence comparison diagram of the sialic acid-containing polymer micelle nanoparticles in Example 2.

[0060] Depend on Figure 3 It can be seen that the particle size of the prepared fucose-containing polymer micelle nanoparticles was about 102 nm and the PDI was 0.180 as measured by a Malvern particle size analyzer.

[0061] Depend on Figure 4 It can be seen from the flow cytometer that the platelets attached to the fucose-containing polymer micelle nanoparticles have a stronger fluorescence intensity than the platelets attached to the glucose polymer micelles, which is about twice as strong as the glucose micelles, showing a higher platelet affinity.

[0062] Depend on Figure 5 It can be seen that the particle size of the prepared sialic acid-containing polymer micelle nanoparticles was about 140 nm and the PDI was 0.110 as measured by a Malvern particle size analyzer.

[0063] Depend on Figure 6 It can be seen from the flow cytometer that the fluorescence intensity of platelets attached to sialic acid polymer micelle nanoparticles is slightly stronger than that of platelets attached to glucose polymer micelles, indicating that the affinity of sialic acid polymer micelles for platelets is slightly stronger than that of glucose.

Claims

1. A method for preparing sugar-containing polymer micelle nanoparticles used as drug delivery carriers, characterized in that: The amphiphilic block copolymer is prepared by RAFT polymerization, which includes the following steps: (1) mixing the sugar monomer, initiator, and chain transfer agent in an organic solvent for uniform reaction; (2) then adding N-alkyl acrylamide and initiator to continue the reaction, and after the reaction is completed, dialyzing and freeze-drying are performed to obtain sugar-containing polymer micelle nanoparticles; The sugar monomer is fucose substituted with methacryloylhydrazide, and the number of carbon atoms of the alkyl group in the N-fatty alkyl acrylamide is 6-18; The molar ratio of the sugar-based monomer to the N-fatty alkyl acrylamide is 1:1.

5.

2. The method according to claim 1, characterized in that In step (1), the molar ratio of the glycosyl monomer to the chain transfer agent is (1-2.5): (0.05-0.5).

3. The method according to claim 1, characterized in that In step (1), the molar ratio of the glycosyl monomer to the initiator is (1-2.5): (0.01-0.05).

4. The method according to claim 1, wherein In step (1), the amount of the glycosyl monomer relative to the organic solvent is (1-2.5) mmol / mL.

5. The method according to claim 1, wherein In step (2), the molar ratio of the N-fatty alkyl acrylamide to the initiator is (1.5-2.5): (0.01-0.05).

6. A sugar-containing polymer micellar nanoparticle prepared by the method according to any one of claims 1 to 5.

7. Use of the sugar-containing polymer micelle nanoparticles according to claim 6 in the preparation of cancer cell detection devices and drug delivery carriers.

Images