Astaxanthin nanocarrier for treating atherosclerosis and preparation method thereof
By preparing phenylboronic acid-modified astaxanthin and dextran self-assembled nanocarriers, the problems of poor water solubility of astaxanthin and inaccurate drug release were solved, achieving efficient treatment of atherosclerosis.
Patent Information
- Application Number
- CN202311024859.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-15
- Publication Date
- 2025-09-12
- Estimated Expiration
- 2043-08-15
AI Technical Summary
In the prior art, astaxanthin has poor water solubility, which affects its application in the treatment of atherosclerosis, and the drug release of nanocarriers at the lesion site is not accurate and efficient enough.
Nanocarriers were prepared by self-assembly of astaxanthin modified with phenylboronic acid and dextran, which achieved targeted enrichment through the CD44 pathway and released astaxanthin in the diseased tissue through the reactive oxygen species responsive structure, achieving anti-inflammatory and delipidation effects.
The utilization rate of astaxanthin is improved, active targeting of atherosclerotic plaques and efficient drug release are achieved, which has a good therapeutic effect and reduces drug leakage and toxicity.
Smart Images
Figure CN116898809B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of drug carriers, and particularly relates to an astaxanthin nanocarrier for treating atherosclerosis and a preparation method thereof. Background Art
[0002] Atherosclerosis is a chronic arterial disease characterized by the formation of plaques in the vascular lining. These plaques, composed of cholesterol, lipids, and other cellular components, gradually enlarge and narrow the vessel lumen. Atherosclerosis is responsible for 90% of peripheral arterial disease, resulting in disability and long-term illness, placing a significant burden on society. Inflammation and lipids, as key factors in the development and progression of early atherosclerotic lesions, have been extensively studied in recent years.
[0003] The wide range of health benefits and multiple biological activities of carotene have made it the focus of industrial and academic research worldwide. Due to its powerful biological activity, especially its ability to protect living systems from reactive oxygen species, it has been proven to be an antioxidant with a series of biological activities. Among them, natural astaxanthin has a variety of biomedical effects, namely antioxidant, anti-inflammatory, wound healing, cardioprotection, liver protection, anti-diabetes, neuroprotection, anti-cancer and bone protection. In some animal models, it was found that astaxanthin can reduce the plaque area of atherosclerotic mice by promoting reverse lipid transport. At the same time, astaxanthin has a strong antioxidant effect and a significant anti-inflammatory effect. However, its extremely poor water solubility greatly affects its application in the medical field.
[0004] In order to reduce adverse reactions and improve therapeutic effects, researchers have developed various nano-drug carriers for efficient drug delivery in recent years. Nano-carriers have been shown to be selectively enriched in atherosclerotic lesions. The CD44 receptor overexpressed by damaged vascular endothelium and foam cells can specifically bind to polysaccharides such as hyaluronic acid and dextran, providing an enrichment pathway for nano-carriers. Considering the accurate release and treatment of drugs, how to efficiently release drugs has become an issue that cannot be ignored in the development of nano-carriers. Along with the inflammatory process of atherosclerosis, researchers have specifically discovered overexpressed reactive oxygen species (ROS) in the lesion tissue, which provides a target for accurate and efficient drug delivery for inflammatory treatment. Therefore, the introduction of a series of ROS-responsive structures into nano-carriers can achieve accurate and efficient release of anti-inflammatory drugs in atherosclerotic tissues.
[0005] Therefore, the development of astaxanthin-based nanocarriers for the treatment of atherosclerosis is of great clinical significance. Summary of the Invention
[0006] To address the aforementioned deficiencies in the prior art, the present invention provides an astaxanthin nanocarrier for the treatment of atherosclerosis and a method for its preparation. The nanocarrier is self-assembled from phenylboronic acid-modified astaxanthin and dextran. The astaxanthin end groups are modified with p-carboxyphenylboronic acid, and the phenylboronic acid reacts with the ortho-dihydroxy groups on the dextran molecule, resulting in a nanocarrier prepared by screening the preparation process. This nanocarrier effectively disperses and dissolves astaxanthin, a poorly water-soluble component, thereby improving its utilization rate. The nanocarrier is responsive to reactive oxygen species (ROS), and the phenylboronic acid structure is sensitively cleavable, allowing the released astaxanthin to exert highly effective anti-inflammatory and delipidating properties. Furthermore, the nanocarrier exhibits excellent dispersibility and stability during intravenous delivery into the bloodstream. The dextran, as a hydrophilic shell, actively targets atherosclerotic plaques via the CD44 pathway. After accumulation in atherosclerotic tissue, the nanocarrier disintegrates under the influence of ROS overexpressed in the inflammatory environment of the diseased tissue, releasing astaxanthin and exerting its anti-inflammatory and delipidating effects. In addition, the low drug leakage and excellent reactive oxygen species-responsive drug release ability make this nanocarrier have a good therapeutic effect on atherosclerosis.
[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:
[0008] A method for preparing an astaxanthin nanocarrier for treating atherosclerosis, the method comprising the following steps:
[0009] Step 1: dissolving p-carboxyphenylboronic acid, astaxanthin, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a first organic solvent, stirring to fully esterify, and then purifying by dialysis to obtain phenylboronic acid-modified astaxanthin; the molar ratio of astaxanthin, p-carboxyphenylboronic acid, dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 0.5:1:(1-1.5):(0.01-0.1);
[0010] The chemical structural formula of the phenylboronic acid modified astaxanthin is:
[0011]
[0012] Step 2: dissolving the phenylboronic acid-modified astaxanthin in a second organic solvent, dissolving dextran in water, mixing the two solutions, stirring to allow them to fully self-assemble, and dialyzing to remove the organic solvent to prepare astaxanthin nanocarriers for treating atherosclerosis;
[0013] The mass ratio of the phenylboronic acid-modified astaxanthin to dextran is 1-2:0.5-4.
[0014] Furthermore, the stirring time in step 1 is 12-48 hours, and the stirring time in step 2 is 4-24 hours, which allows the components constituting the nanoparticles to fully react with each other and fully self-assemble in water to form nanoparticles.
[0015] Furthermore, the molecular weight of the dextran is 1000-1000000 g / mol, which can efficiently construct nanoparticles and ensure relatively ideal reaction efficiency and yield.
[0016] Furthermore, the first organic solvent in step one is the same as the second organic solvent in step two, and can dissolve the astaxanthin structure and the phenylboronic acid structure at the same time.
[0017] Furthermore, the first organic solvent and the second organic solvent are both dimethyl sulfoxide, which can better dissolve phenylboronic acid-modified astaxanthin and promote efficient reaction.
[0018] Furthermore, the molar ratio of astaxanthin, p-carboxyphenylboronic acid, dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 05:1:1.5:0.01. The phenylboronic acid-modified astaxanthin synthesized at this ratio has a high yield and good purity.
[0019] Furthermore, the mass ratio of the phenylboronic acid-modified astaxanthin to dextran is 1:2. The nanocarrier constructed at this ratio has the most ideal particle size and the best therapeutic effect.
[0020] The beneficial effects of the present invention are:
[0021] (1) The glucan structure in the nanocarrier prepared by the present invention can make astaxanthin, a poorly water-soluble substance, have good monodispersity, thereby promoting its utilization in the body.
[0022] (2) The glucan structure in the nanocarrier prepared by the present invention can actively identify atherosclerotic plaques through the CD44 pathway, thereby achieving active targeting of the nanocarrier to the lesion site. In addition, the low drug leakage and excellent reactive oxygen species responsive drug release ability make the nanocarrier have a good therapeutic effect on atherosclerosis.
[0023] (3) The phenylboronic acid structure in the nanocarrier can undergo sensitive rupture under reactive oxygen conditions, releasing astaxanthin quickly and efficiently, thereby achieving the treatment of atherosclerosis.
[0024] (4) The nanocarriers prepared by the present invention can achieve efficient, low-toxic, and low-leakage astaxanthin delivery at the in vitro cell level and in vivo animal level.
[0025] (5) The nanocarriers prepared by the present invention can achieve efficient treatment of atherosclerosis through anti-inflammatory and lipid-removing pathways. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the H NMR spectrum of phenylboronic acid-modified astaxanthin;
[0027] Figure 2 The particle size distribution diagram and transmission electron microscopy image of the nanocarrier;
[0028] Figure 3 Figure 2 is a graph showing the particle size changes of nanocarriers in PBS and under reactive oxygen conditions;
[0029] Figure 4 This is a graph showing the results of the nanocarrier's inhibition of foam cell formation in vitro;
[0030] Figure 5 This is a Western Blot result showing that nanocarriers regulate foam cell inflammation and lipid pathways in vitro;
[0031] Figure 6 This figure shows the results of nanocarrier imaging and treatment of atherosclerosis in animal experiments. DETAILED DESCRIPTION
[0032] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.
[0033] Example 1
[0034] Dissolve p-carboxyphenylboronic acid (0.83 g, 0.01 mol), astaxanthin (5.97 g, 0.005 mol), dicyclohexylcarbodiimide (3.09 g, 0.015 mol) and 4-dimethylaminopyridine (12.2 mg, 0.0001 mol) in dimethyl sulfoxide, stir at room temperature for 48 h, and dialysis to obtain phenylboronic acid-modified astaxanthin.
[0035] The H NMR spectrum of the phenylboronic acid modified astaxanthin prepared above is shown in Figure 1 , from its peak position and integral ratio, it can be seen that the molecule was successfully synthesized.
[0036] Phenylboronic acid-modified astaxanthin (10 mg) was dissolved in dimethyl sulfoxide, and dextran (molecular weight 50,000, 10 mg) was dissolved in deionized water. The two solutions were mixed and stirred for 24 h. The dimethyl sulfoxide was removed by dialysis to prepare a nanocarrier solution.
[0037] The nanocarrier particle size diagram and transmission electron microscopy image are shown in Table 1 and Figure 2, Figure 2 A is a nanocarrier diameter image, and B is a transmission electron microscope image. It can be seen from the figure that the self-assembled nanocarriers are uniform particles with nanometer size.
[0038] Example 2
[0039] Dissolve p-carboxyphenylboronic acid (0.83 g, 0.01 mol), astaxanthin (5.97 g, 0.005 mol), dicyclohexylcarbodiimide (2.06 g, 0.01 mol) and 4-dimethylaminopyridine (12.2 mg, 0.0001 mol) in dimethyl sulfoxide, stir at room temperature for 24 h, and dialysis to obtain phenylboronic acid-modified astaxanthin.
[0040] Phenylboronic acid-modified astaxanthin (10 mg) was dissolved in dimethyl sulfoxide, and dextran (molecular weight 50,000, 20 mg) was dissolved in deionized water. The two solutions were mixed and stirred for 24 h. The dimethyl sulfoxide was removed by dialysis to prepare a nanocarrier solution.
[0041] The particle size diagram and transmission electron microscopy image of the nanocarrier are shown in Table 1.
[0042] Example 3
[0043] Dissolve p-carboxyphenylboronic acid (0.83 g, 0.01 mol), astaxanthin (5.97 g, 0.005 mol), dicyclohexylcarbodiimide (3.09 g, 0.015 mol) and 4-dimethylaminopyridine (122 mg, 0.001 mol) in dimethyl sulfoxide, stir at room temperature for 12 h, and dialysis to obtain phenylboronic acid-modified astaxanthin.
[0044] Phenylboronic acid-modified astaxanthin (10 mg) was dissolved in dimethyl sulfoxide, and dextran (molecular weight 50,000, 5 mg) was dissolved in deionized water. The two solutions were mixed and stirred for 24 h. The dimethyl sulfoxide was removed by dialysis to prepare a nanocarrier solution.
[0045] The particle size diagram and transmission electron microscopy image of the nanocarrier are shown in Table 1.
[0046] Example 4
[0047] Dissolve p-carboxyphenylboronic acid (0.83 g, 0.01 mol), astaxanthin (5.97 g, 0.005 mol), dicyclohexylcarbodiimide (3.09 g, 0.015 mol) and 4-dimethylaminopyridine (12.2 mg, 0.0001 mol) in dimethyl sulfoxide, stir at room temperature for 48 h, and dialysis to obtain phenylboronic acid-modified astaxanthin.
[0048] Phenylboronic acid-modified astaxanthin (10 mg) was dissolved in dimethyl sulfoxide, and dextran (molecular weight 1000, 10 mg) was dissolved in deionized water. The two solutions were mixed, stirred for 24 h, and dialyzed to remove dimethyl sulfoxide to prepare a nanocarrier solution.
[0049] The particle size diagram and transmission electron microscopy image of the nanocarrier are shown in Table 1.
[0050] Example 5
[0051] Dissolve p-carboxyphenylboronic acid (0.83 g, 0.01 mol), astaxanthin (5.97 g, 0.005 mol), dicyclohexylcarbodiimide (3.09 g, 0.015 mol) and 4-dimethylaminopyridine (12.2 mg, 0.0001 mol) in dimethyl sulfoxide, stir at room temperature for 48 h, and dialysis to obtain phenylboronic acid-modified astaxanthin.
[0052] Phenylboronic acid-modified astaxanthin (10 mg) was dissolved in dimethyl sulfoxide, and dextran (molecular weight 100,000, 10 mg) was dissolved in deionized water. The two solutions were mixed and stirred for 24 h. The dimethyl sulfoxide was removed by dialysis to prepare a nanocarrier solution.
[0053] The particle size diagram and transmission electron microscopy image of the nanocarrier are shown in Table 1.
[0054] Example 6
[0055] Dissolve p-carboxyphenylboronic acid (0.83 g, 0.01 mol), astaxanthin (5.97 g, 0.005 mol), dicyclohexylcarbodiimide (3.09 g, 0.015 mol) and 4-dimethylaminopyridine (12.2 mg, 0.0001 mol) in dimethyl sulfoxide, stir at room temperature for 48 h, and dialysis to obtain phenylboronic acid-modified astaxanthin.
[0056] Phenylboronic acid-modified astaxanthin (10 mg) was dissolved in dimethyl sulfoxide, and dextran (molecular weight 50,000, 10 mg) was dissolved in deionized water. The two solutions were mixed, stirred for 4 h, and dialyzed to remove dimethyl sulfoxide to prepare a nanocarrier solution.
[0057] The particle size diagram and transmission electron microscopy image of the nanocarrier are shown in Table 1.
[0058] Example 7
[0059] Dissolve p-carboxyphenylboronic acid (0.83 g, 0.01 mol), astaxanthin (5.97 g, 0.005 mol), dicyclohexylcarbodiimide (3.09 g, 0.015 mol) and 4-dimethylaminopyridine (12.2 mg, 0.0001 mol) in dimethyl sulfoxide, stir at room temperature for 48 h, and dialysis to obtain phenylboronic acid-modified astaxanthin.
[0060] Phenylboronic acid-modified astaxanthin (10 mg) was dissolved in dimethyl sulfoxide, and dextran (molecular weight 50,000, 10 mg) was dissolved in deionized water. The two solutions were mixed, stirred for 12 h, and dialyzed to remove dimethyl sulfoxide to prepare a nanocarrier solution.
[0061] The particle size diagram and transmission electron microscopy image of the nanocarrier are shown in Table 1.
[0062] Table 1 shows the particle size of nanocarriers obtained by different preparation processes
[0063]
[0064] Experimental Example 1 Study on the stability and sensitivity of nanocarriers
[0065] The micelles prepared in Example 1 were placed at 37°C and a hydrogen peroxide concentration of 0.1 mM, and the particle size change was measured using a dynamic light scattering instrument DLS at regular intervals. The particle size change results are shown in FIG. Figure 3 .
[0066] Depend on Figure 3 It can be seen that the nanocarrier prepared in the present invention exhibits good stability in the absence of hydrogen peroxide, but can expand rapidly under hydrogen peroxide conditions and its uniformity deteriorates, indicating that it disintegrates.
[0067] Experimental Example 2 Inhibitory Effect of Nanocarriers on Foam Cell Formation
[0068] The nanocarriers prepared in Example 1 were co-cultured with macrophages activated by lipopolysaccharide and supplemented with oxidized low-density lipoprotein for 24 hours. The cells were stained with Oil Red O and observed under an optical microscope.
[0069] Depend on Figure 4 It can be seen that, compared with the free drug, the nanocarrier prepared by the present invention can achieve an effective inhibitory effect on the formation of foam cells, and has a concentration-dependent effect.
[0070] Experimental Example 3: Effects of Nanocarriers on Regulating Foam Cell Inflammation and Lipid Pathways in Vitro
[0071] The nanocarrier prepared in Example 1 was co-cultured with macrophages activated by lipopolysaccharide and supplemented with oxidized low-density lipoprotein for 24 hours. The cells were collected to extract cell proteins, and the expression of proteins such as NF-κB, CD36, and Sortilin in the cells was detected.
[0072] Depend on Figure 5 As shown in the immunoblotting test results, the nanocarrier can significantly inhibit the expression of inflammation-related protein NF-κB, and inhibit the expression of lipid endocytosis pathway proteins CD36 and Sortilin, thereby achieving the treatment of atherosclerosis through anti-inflammatory and anti-lipid pathways.
[0073] Experimental Example 4: Animal Experiment Verification of the Therapeutic Effect of Nanocarriers on Atherosclerosis
[0074] Nanocarriers with known drug loading were freeze-dried and reconstituted with saline to prepare a stock solution of a certain concentration. A non-targeted nanocarrier stock solution with the same drug concentration was also prepared. A mouse model of atherosclerosis was established. Free drug, nanocarriers, or an equal volume of saline were injected via the tail vein. The astaxanthin dosage was 10 mg / kg mouse. After administration, the mouse aorta was isolated, sectioned, and stained with Oil Red O. The experimental results are shown in Figure 2. Figure 6 As shown in the figure, compared with the control group and the non-targeted nanocarrier, the mice administered with the nanocarrier prepared in Example 2 had fewer Oil Red O-stained plaques (black spots) on the inner wall of the aorta, demonstrating that the nanocarrier has a better effect in inhibiting the formation of atherosclerosis.
[0075] Those skilled in the art will understand that the foregoing descriptions are merely preferred embodiments of the invention and are not intended to limit the invention. Although the invention has been described in detail with reference to the foregoing examples, those skilled in the art will still be able to modify the technical solutions described in the foregoing examples or substitute equivalents for some of the technical features therein. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the invention shall be included within the scope of protection of the invention.
Claims
1. A method for preparing astaxanthin nanocarriers for treating atherosclerosis, characterized in that: The method comprises the following steps: Step 1: dissolving p-carboxyphenylboronic acid, astaxanthin, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a first organic solvent, stirring to fully esterify, and then purifying by dialysis to obtain phenylboronic acid-modified astaxanthin; the molar ratio of astaxanthin, p-carboxyphenylboronic acid, dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 0.5:1:(1-1.5):(0.01-0.1); The chemical structural formula of the phenylboronic acid modified astaxanthin is: ； Step 2: dissolving the phenylboronic acid-modified astaxanthin in a second organic solvent, dissolving dextran in water, mixing the two solutions, stirring to allow them to fully self-assemble, and dialyzing to remove the organic solvent to prepare astaxanthin nanocarriers for treating atherosclerosis; The mass ratio of the phenylboronic acid-modified astaxanthin to dextran is 1-2:0.5-4.
2. The method for preparing astaxanthin nanocarrier for treating atherosclerosis according to claim 1, characterized in that: The stirring time in step 1 is 12-48 hours, and the stirring time in step 2 is 4-24 hours.
3. The method for preparing astaxanthin nanocarrier for treating atherosclerosis according to claim 1, characterized in that: The molecular weight of the dextran is 1000-1000000 g / mol.
4. The method for preparing astaxanthin nanocarrier for treating atherosclerosis according to claim 1, characterized in that: The first organic solvent in step 1 is the same as the second organic solvent in step 2.
5. The method for preparing astaxanthin nanocarrier for treating atherosclerosis according to claim 4, characterized in that: The first organic solvent and the second organic solvent are both dimethyl sulfoxide.
6. The method for preparing astaxanthin nanocarrier for treating atherosclerosis according to claim 1, characterized in that: The molar ratio of astaxanthin, p-carboxyphenylboronic acid, dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 0.5:1:1.5:0.
01.
7. The method for preparing astaxanthin nanocarrier for treating atherosclerosis according to claim 1, characterized in that: The mass ratio of the phenylboronic acid-modified astaxanthin to dextran is 1:
2.
8. An astaxanthin nanocarrier for treating atherosclerosis prepared by the preparation method according to any one of claims 1 to 7.