Hydrophobic modification-based algin oligosaccharide micelle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to algin oligosaccharide micelle based on hydrophobic modification, a preparation method and application. According to the algin oligosaccharide micelle provided by the invention, algin oligosaccharide is taken as a raw material, and the algin oligosaccharide micelle is obtained after the algin oligosaccharide reacts with hydrophobic fatty acid. The micelle provided by the invention is nanoscale, and the structure of a hydrophobic core and a hydrophilic shell of the micelle can effectively load fat-soluble substances. Meanwhile, hydrophobic fatty acid is introduced for derivatization modification, on one hand, the amphipathy of the alginate oligosaccharide can be structurally changed through derivatization modification, and the function of embedding hydrophobic bioactive substances of the alginate oligosaccharide is developed; on the other hand, the micelle can be endowed with active targeting or response release ability through derivatization modification of alginate oligosaccharide, and the bioavailability of hydrophobic bioactive substances is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a preparation method and application of oligosaccharide micelles based on hydrophobic modification of alginate. Background Technology

[0002] Alginate, with its non-toxic, stable properties and anti-tumor and immune-enhancing activities, has attracted widespread attention in scientific research and industry in recent years. However, its high viscosity hinders the full realization of many functions in practical applications. After fractionation, alginate is transformed into oligosaccharides, which not only significantly enhances its biological activity but also allows for derivatization modification using the characteristics of multiple hydroxyl groups, opening up a wider range of applications. Gurouronic acid oligosaccharides and mannuronic acid oligosaccharides, as products of fractionation of alginate, are long polymer chains formed by 1,4-glycosidic bonds.

[0003] Currently, liposomes with immunomodulatory activity are formed using guluronic acid oligosaccharides as raw materials. However, these liposomes have poor stability, resulting in poor drug loading, especially for substances with strong bioactivity, such as natural carotenoids. The encapsulation rate of liposomes is low, and natural carotenoids are easily destroyed by the gastrointestinal tract, making them unable to be delivered to the target. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of poor stability of oligosaccharide liposomes in the prior art, thereby providing a method for preparing oligosaccharide micelles based on hydrophobic modification of alginate, and its application.

[0005] Another technical problem to be solved by the present invention is to overcome the defect that the low encapsulation efficiency of oligosaccharide liposomes in the prior art leads to the inability of bioactive substances to be effectively delivered to the target site, thereby providing a method for preparing oligosaccharide micelles based on hydrophobic modification of alginate and its application.

[0006] Micelles, as nanocarriers formed by the self-assembly of amphiphilic surfactant molecules in aqueous solution, possess a unique structure of hydrophobic core and hydrophilic shell, which endows them with the ability to encapsulate lipid-soluble substances. As a novel drug carrier, micelles can enhance drug stability and targeting, improve drug bioavailability, reduce drug toxicity and side effects, and prolong drug circulation time in vivo, achieving the purpose of controlled and sustained drug release. This provides an innovative solution for the efficient delivery of high-cost active ingredients.

[0007] To this end, on the one hand, the present invention provides an alginate oligosaccharide micelle, which is obtained by reacting alginate oligosaccharide with hydrophobic fatty acids.

[0008] In some embodiments, the alginate oligosaccharide includes at least one of alginate mannouronic acid oligosaccharide and alginate guluronic acid oligosaccharide.

[0009] In some embodiments, the hydrophobic fatty acid is a long-chain fatty acid, preferably including at least one of palmitic acid and stearic acid.

[0010] In some embodiments, the alginate oligosaccharide micelles are loaded with fat-soluble nutrients.

[0011] In some embodiments, the method for preparing alginate oligosaccharide micelles loaded with fat-soluble nutrients includes the following steps: mixing a solution containing fat-soluble nutrients with an alginate oligosaccharide micelle solution to form a first oil-in-water mixture; mixing the first mixture with an organic reagent to form a second oil-in-water mixture; removing the organic reagent from the second mixture to obtain uniform alginate oligosaccharide micelles loaded with fat-soluble substances.

[0012] In some embodiments, the organic reagent includes anhydrous ethanol.

[0013] In some embodiments, the fat-soluble nutrient includes at least one of astaxanthin and fucoxanthin, and also includes at least one of curcumin and β-carotene.

[0014] In some embodiments, the mass ratio of alginate oligosaccharide micelles to fat-soluble nutrients is 225-275:0.9-1.

[0015] In some embodiments, the concentration of the fat-soluble nutrient in the solution is 0.18-0.2 mg / mL, and preferably, the solvent in the solution includes acetone.

[0016] In some embodiments, the concentration of alginate oligosaccharide micelles in the alginate oligosaccharide micelle solution is 22.5-27.5 mg / mL.

[0017] In some embodiments, the step of mixing the solution containing fat-soluble nutrients with the alginate oligosaccharide micelle solution includes adding the solution containing fat-soluble nutrients dropwise into the alginate oligosaccharide micelle solution while stirring, with the dropwise addition rate of the solution containing fat-soluble nutrients being 5-6 mL / h and the stirring speed of the alginate oligosaccharide micelle solution being 200-300 rpm.

[0018] In some embodiments, the step of mixing the first mixture with the organic reagent includes adding the first mixture dropwise to the organic reagent while stirring, the dropwise rate of the first mixture being 5-6 mL / h and the stirring speed of the organic reagent being 200-300 rpm.

[0019] In some embodiments, the step of removing organic reagents from the second mixture includes rotary evaporation at a temperature of 45-50°C and a rotation speed of 90-100 rpm.

[0020] On the other hand, the present invention provides a method for preparing alginate oligosaccharide micelles, comprising the following steps: mixing and reacting alginate oligosaccharides with hydrophobic fatty acids to obtain the micelles.

[0021] In some embodiments, the alginic acid oligosaccharide micelles can be prepared by stirring alginic acid oligosaccharide and stearic acid to induce an esterification reaction.

[0022] In some embodiments, the specific steps of reacting alginate oligosaccharides with hydrophobic fatty acids include the following steps: S1, reacting alginate oligosaccharides with cystamine in the presence of a catalyst to form an oligosaccharide-cystamine bridge; S2, mixing the hydrophobic fatty acids and the alginate oligosaccharide-cystamine bridge formed in step S1 in the presence of a catalyst, and heat-treating to obtain oligosaccharide micelles.

[0023] In some embodiments, the catalyst in step S1 and / or step S2 comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0024] In some embodiments, in step S1, the mass ratio of the alginate oligosaccharide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 85-115:10:6.

[0025] In some embodiments, the mass ratio of the alginate oligosaccharide to cystamine is 8-12:1.

[0026] In some embodiments, in step S2, the mass ratio of stearic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 555-585:197:115.

[0027] In some embodiments, the mass ratio of oligosaccharide-cystamine bridge to stearic acid is 475-525:555-585.

[0028] In some embodiments, the heat treatment temperature is 75-85°C.

[0029] In some embodiments, a solution containing alginate oligosaccharides is reacted with a catalyst under stirring to obtain an intermediate. The intermediate is then reacted with cystamine and purified to obtain an oligosaccharide-cystamine bridge. The stirring speed is 450-550 rpm, the reaction time of the solution containing alginate oligosaccharides with the catalyst is 1.9-2.1 h, the reaction time of the intermediate with cystamine is 24-25 h, and the concentration of alginate oligosaccharides in the solution is 21.25-28.75 mg / mL.

[0030] In some embodiments, a solution containing hydrophobic fatty acids is reacted with a catalyst to generate activated fatty acids while the solution is stirred. The stirring speed is 450-550 rpm, the reaction time is 1-1.5 h, and the concentration of hydrophobic fatty acids in the solution is 22.2-23.4 mg / mL.

[0031] In some embodiments, the activated fatty acid and the solution containing the oligosaccharide-cystamine bridge are reacted under stirring at a speed of 200-300 rpm for a reaction time of 6-6.5 h, and the concentration of the oligosaccharide-cystamine bridge in the solution is 19-21 mg / mL.

[0032] In some embodiments, step S1, after reacting the alginate oligosaccharide with the catalyst and cystamine in sequence, also includes a step of purifying the obtained oligosaccharide-cystamine bridge, using a 1000 Da dialysis bag for purification.

[0033] In some embodiments, step S2 further includes a step of purifying the heat-treated oligosaccharide micelles using a 1000 Da dialysis bag.

[0034] Meanwhile, the alginate oligosaccharide micelles provided by this invention, or the alginate oligosaccharide micelles prepared by the above-mentioned method, can simulate free movement in intestinal mucus, thus improving the bioavailability of nutrients. Furthermore, the alginate oligosaccharide micelles provided by this invention have the characteristic that, upon contact with macrophages, they can be endocytosed into macrophages primarily through a caveolin-dependent endocytosis pathway, and partially through macropinocytosis, within a short period of time. Therefore, they can be used as delivery carriers for nutrients and / or drugs. Through corresponding experimental verification, the alginate oligosaccharide micelles provided by this invention have anti-inflammatory effects and can also enhance immune activity or prepare products that enhance immune activity.

[0035] The technical solution of this invention has the following advantages:

[0036] 1. This invention provides an alginate oligosaccharide micelle, using alginate oligosaccharide as a raw material. The alginate oligosaccharide micelle is obtained by reacting alginate oligosaccharide with hydrophobic fatty acids. The micelles provided by this invention are nanoscale, and their structure of a hydrophobic core and a hydrophilic shell can effectively load lipid-soluble nutrients. Simultaneously, by introducing hydrophobic fatty acids for derivatization modification, on the one hand, derivatization modification can structurally alter the amphiphilicity of alginate oligosaccharide, developing its function of encapsulating hydrophobic bioactive substances; on the other hand, derivatization modification of alginate oligosaccharide can endow the micelles with active targeting or responsive release capabilities, significantly improving the bioavailability of hydrophobic bioactive substances.

[0037] 2. This invention provides an alginate oligosaccharide micelle loaded with lipid-soluble nutrients. The oligosaccharide micelles provided by this invention can effectively load lipid-soluble substances and improve their encapsulation efficiency. The oligosaccharide micelles loaded with lipid-soluble substances exhibit good temperature and pH stability, a certain degree of salt tolerance, and can respond to glutathione, promoting cell activity, enhancing the stability and targeting of lipid-soluble materials, improving drug bioavailability, reducing drug toxicity, and prolonging drug circulation time in vivo, achieving the purpose of controlled and sustained drug release. This provides an innovative solution for the efficient delivery of high-cost active ingredients. In particular, even in low pH environments, the lipid-soluble substances loaded with the alginate oligosaccharide micelles provided by this invention still show a relatively high retention rate, indicating that the alginate oligosaccharide micelles provided by this invention have gastric clearance capabilities.

[0038] 3. This invention provides a method for preparing alginate oligosaccharide micelles, which involves mixing and reacting alginate oligosaccharides with hydrophobic fatty acids. The method for preparing alginate oligosaccharide micelles according to this invention is simple and convenient.

[0039] 4. This invention provides a method for preparing alginate oligosaccharide micelles, comprising the following steps: S1, reacting alginate oligosaccharides with cystamine in the presence of a catalyst to form an oligosaccharide-cystamine bridge; S2, mixing hydrophobic fatty acids and the alginate oligosaccharide-cystamine bridge formed in step S1 in the presence of a catalyst, followed by heat treatment to obtain oligosaccharide micelles. In the presence of a catalyst, this invention utilizes cystamine to bridge alginate oligosaccharides, and then connects them with hydrophobic fatty acids through heat treatment to form alginate oligosaccharide micelles, resulting in a more stable alginate oligosaccharide micelle structure. Furthermore, this invention uses cystopeptide as a cross-linking agent. Cystopeptide is a small molecule compound containing disulfide bonds, which typically breaks under high concentrations of glutathione stimulation. Therefore, it ensures that after cystamine is phagocytosed by inflammatory macrophages, the high concentration of glutathione within the macrophages responsively opens the micelles, thereby releasing lipid-soluble nutrients and exerting their biological activity. Attached Figure Description

[0040] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 This is a standard curve of absorbance for astaxanthin content in Experimental Example 1 of the present invention;

[0042] Figure 2 This is a particle size distribution diagram of guluronic acid oligosaccharide micelles loaded with astaxanthin in Experimental Example 2 of the present invention.

[0043] Figure 3 This is a scanning electron microscope image of guluronic acid oligosaccharide micelles loaded with astaxanthin in Experimental Example 3 of the present invention.

[0044] Figure 4 The results show the temperature stability of the astaxanthin-loaded guluronic acid oligosaccharide micelles in Experimental Example 4 of this invention.

[0045] Figure 5 The acid-base stability results of astaxanthin-loaded guluronic acid oligosaccharide micelles in Experimental Example 5 of this invention.

[0046] Figure 6 The results show the ionic stability of the astaxanthin-loaded guluronic acid oligosaccharide micelles in Experimental Example 6 of this invention.

[0047] Figure 7 The results of glutathione-responsive release from astaxanthin-loaded guluronic acid oligosaccharide micelles in Experimental Example 7 of this invention.

[0048] Figure 8 The movement of astaxanthin-loaded guluronic acid oligosaccharide micelles in simulated intestinal fluid is shown in Experimental Example 8 of the present invention.

[0049] Figure 9 The effect of astaxanthin-loaded guluronic acid oligosaccharide micelles on the cell viability of mouse mononuclear macrophages (RAW264.7) in Experimental Example 9 of the present invention;

[0050] Figure 10 The image shows the results of macrophages (RAW264.7) phagocytosing the astaxanthin-loaded guluronic acid oligosaccharide micelles of the present invention in Experiment Example 10 of the present invention.

[0051] Figure 11 This is a diagram showing the endocytic pathway of macrophages (RAW264.7) engulfing the astaxanthin-loaded guluronic acid oligosaccharide micelles of the present invention in Experiment Example 11 of the present invention.

[0052] Figure 12 The figure shows the NO release results of mouse monocytes / macrophages (RAW264.7) cells in Experiment 12 of this invention, which were relieved by astaxanthin-loaded guluronic acid oligosaccharide micelles to alleviate LPS stimulation.

[0053] Figure 13 The figure shows the TNF-α release results of mouse mononuclear macrophages (RAW264.7) cells in Experiment Example 13 of the present invention, which were relieved by astaxanthin-loaded guluronic acid oligosaccharide micelles to alleviate LPS stimulation.

[0054] Figure 14 The image shows the IL-6 release results of mouse mononuclear macrophages (RAW264.7) cells in Experiment 13 of this invention, which were relieved by astaxanthin-loaded guluronic acid oligosaccharide micelles to alleviate LPS stimulation. Detailed Implementation

[0055] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0056] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0057] In the embodiments and application examples of this invention, stearic acid and astaxanthin were purchased from Shanghai Maclean Biochemical Co., Ltd.; cystamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 2-hydroxyethylcellulose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; anhydrous ethanol was purchased from Guangdong Guanghua Technology Co., Ltd.; mouse mononuclear macrophages (RAW264.7) were purchased from the American Type Culture Collection; high-glucose DMEM medium, penicillin, streptomycin, fetal bovine serum and lipopolysaccharide (LPS) were purchased from Gibco.

[0058] The molecular weight of guluronic acid in the embodiments and application examples of this invention is 100 Da.

[0059] Example 1

[0060] This embodiment provides a method for preparing guluronic acid oligosaccharide micelles, and the specific steps and parameters are as follows:

[0061] (1) Weigh 1g of guluronic acid oligosaccharide and dissolve it in 40mL of deionized water. After the guluronic acid oligosaccharide is completely dissolved, add 100mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 60mg of N-hydroxysuccinimide (NHS). Activate the above materials at 25°C and 500rpm for 2h by stirring. Then add 100mg of cystamine and continue stirring at 25°C and 500rpm for 24h. Dialyze the reaction product using a 1000Da dialysis bag for 48h. Then vacuum dry at 1Pa and -80°C to obtain cystamine-bridged guluronic acid oligosaccharide powder.

[0062] (2) Dissolve 500 mg of cystamine-bridged guluronic acid oligosaccharide powder in 25 mL of deionized water to form a cystamine-bridged guluronic acid oligosaccharide solution.

[0063] 568 mg of stearic acid was dissolved in 25 mL of anhydrous ethanol, and then 197 mg of EDC and 115 mg of NHS were added. The mixture was stirred at 500 rpm for 1 h at 25 °C. Then, a cystamine-bridged guluronic acid oligosaccharide solution was added. The mixture was stirred at 200 rpm for 6 h in an 80 °C water bath. The reaction product was dialyzed for 48 h using a 1000 Da dialysis bag and then vacuum dried at 1 Pa and -80 °C to obtain guluronic acid oligosaccharide micelles.

[0064] Example 2

[0065] This embodiment provides a method for preparing guluronic acid oligosaccharide micelles, and the specific steps and parameters are as follows:

[0066] (1) Weigh 0.85g of guluronic acid oligosaccharide and dissolve it in 40mL of deionized water. After the guluronic acid oligosaccharide is completely dissolved, add 100mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 60mg of N-hydroxysuccinimide (NHS). Activate the above materials at 25℃ and 450rpm for 2.1h by stirring. Then add 72mg of cystamine and continue stirring at 25℃ and 550rpm for 25h. Dialyze the reaction product using a 1000Da dialysis bag for 48h. Then vacuum dry at 1Pa and -80℃ to obtain cystamine-bridged guluronic acid oligosaccharide powder.

[0067] (2) Dissolve 525 mg of cystamine-bridged guluronic acid oligosaccharide powder in 25 mL of deionized water to form a cystamine-bridged guluronic acid oligosaccharide solution.

[0068] 555 mg of stearic acid was dissolved in 25 mL of anhydrous ethanol, and then 197 mg of EDC and 115 mg of NHS were added. The mixture was stirred at 550 rpm for 1 h at 25 °C. Then, a cystamine-bridged guluronic acid oligosaccharide solution was added. The mixture was stirred at 300 rpm for 6 h in a water bath at 75 °C. The reaction product was dialyzed for 48 h using a 1000 Da dialysis bag and then vacuum dried at 1 Pa and -80 °C to obtain guluronic acid oligosaccharide micelles.

[0069] Example 3

[0070] This embodiment provides a method for preparing guluronic acid oligosaccharide micelles, and the specific steps and parameters are as follows:

[0071] (1) Weigh 115g of guluronic acid oligosaccharide and dissolve it in 40mL of deionized water. After the guluronic acid oligosaccharide is completely dissolved, add 100mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 60mg of N-hydroxysuccinimide (NHS). Activate the above materials at 25℃ and 550rpm for 1.9h by stirring. Then add 140mg of cystamine and continue stirring at 25℃ and 450rpm for 24h. Dialyze the reaction product using a 1000Da dialysis bag for 48h. Then vacuum dry at 1Pa and -80℃ to obtain cystamine-bridged guluronic acid oligosaccharide powder.

[0072] (2) Dissolve 475 mg of cystamine-bridged guluronic acid oligosaccharide powder in 25 mL of deionized water to form a cystamine-bridged guluronic acid oligosaccharide solution.

[0073] 585 mg of stearic acid was dissolved in 25 mL of anhydrous ethanol, and then 197 mg of EDC and 115 mg of NHS were added. The mixture was stirred at 450 rpm for 1.5 h at 25 °C. Then, a cystamine-bridged guluronic acid oligosaccharide solution was added. The mixture was stirred at 200 rpm for 6.5 h in a water bath at 85 °C. The reaction product was dialyzed through a 1000 Da dialysis bag for 48 h and then vacuum dried at 1 Pa and -80 °C to obtain guluronic acid oligosaccharide micelles.

[0074] Application Example 1

[0075] This application example provides a method for encapsulating astaxanthin in guluronic acid oligosaccharide micelles. The specific steps and parameters are as follows:

[0076] (1) Weigh 250 mg of guluronic acid oligosaccharide micelles and dissolve them in 10 mL of deionized water to form a micelle solution;

[0077] Weigh 1 mg of astaxanthin and dissolve it in 5 mL of acetone. Add micelle solution dropwise at a rate of 5 mL / h at 300 rpm to obtain the oil-in-water system product.

[0078] (2) At a speed of 300 rpm, the water-in-oil system product was added to 100 mL of anhydrous ethanol at a dropping rate of 5 mL / h to obtain the oil-in-water-in-oil system product.

[0079] (3) The product of the oil-in-water-in-oil system was transferred to a round-bottom flask and placed on a rotary evaporator. The organic reagent was removed at 45°C to obtain guluronic acid oligosaccharide micelles loaded with astaxanthin.

[0080] Application Example 2

[0081] This application example provides a method for encapsulating astaxanthin in guluronic acid oligosaccharide micelles. The specific steps and parameters are as follows:

[0082] (1) Weigh 225 mg of guluronic acid oligosaccharide micelles and dissolve them in 10 mL of deionized water to form a micelle solution;

[0083] Weigh 1 mg of astaxanthin and dissolve it in 5 mL of acetone. Add micelle solution dropwise at a rate of 6 mL / h at 200 rpm to obtain the oil-in-water system product.

[0084] (2) At a speed of 200 rpm, the water-in-oil system product was added to 100 mL of anhydrous ethanol at a dropping rate of 6 mL / h to obtain the oil-in-water-in-oil system product.

[0085] (3) The product of the oil-in-water-in-oil system was transferred to a round-bottom flask and placed on a rotary evaporator. The organic reagent was removed at 50°C to obtain guluronic acid oligosaccharide micelles loaded with astaxanthin.

[0086] Application Example 3

[0087] This application example provides a method for encapsulating astaxanthin in guluronic acid oligosaccharide micelles. The specific steps and parameters are as follows:

[0088] (1) Weigh 275 mg of guluronic acid oligosaccharide micelles and dissolve them in 10 mL of deionized water to form a micelle solution;

[0089] Weigh 0.9 mg of astaxanthin and dissolve it in 5 mL of acetone. Add micelle solution dropwise at a rate of 6 mL / h at 200 rpm to obtain the oil-in-water product.

[0090] (2) At a speed of 200 rpm, the water-in-oil system product was added to 100 mL of anhydrous ethanol at a dropping rate of 6 mL / h to obtain the oil-in-water-in-oil system product.

[0091] (3) The product of the oil-in-water-in-oil system was transferred to a round-bottom flask and placed on a rotary evaporator. The organic reagent was removed at 50°C to obtain guluronic acid oligosaccharide micelles loaded with astaxanthin.

[0092] Application Example 4

[0093] This application example provides a method for embedding fucoxanthin in guluronic acid oligosaccharide micelles. The specific steps and parameters are the same as in application example 1, except that an equal mass of fucoxanthin is used to replace astaxanthin in step (1).

[0094] Experimental Example 1

[0095] Weigh 2 mg of astaxanthin and dilute to 100 mL with anhydrous ethanol to prepare a standard solution. Take eight test tubes and number them. Add 1 mL of anhydrous ethanol to tubes 1 through 7. Add 2 mL of the standard solution to tube 8, and then add 1 mL of the solution from tube 8 to tube 7. Repeat this process for tube 7, adding 1 mL of the solution to tube 6, and so on. Tube 1 contains anhydrous ethanol. Measure the absorbance at 475 nm. Plot the absorbance on the x-axis and the astaxanthin concentration on the y-axis to create a standard curve. The standard curve is shown below. Figure 1 As shown.

[0096] 100 μL of astaxanthin-loaded guluronic acid oligosaccharide micelles (Application Example 1) were placed in a centrifuge tube and centrifuged at 3000 rpm. The supernatant was discarded, and 500 μL of anhydrous ethanol was added. The mixture was then sonicated at room temperature for 10 min (ultrasonic frequency 25 kHz), centrifuged again at 3000 rpm, and the supernatant was collected. The absorbance was measured at a wavelength of 475 nm. Figure 1 The standard curve plot was used to calculate the astaxanthin concentration (W). 内 W 内 (This indicates the concentration of astaxanthin encapsulated in the micelles).

[0097] 100 μL of astaxanthin-loaded guluronic acid oligosaccharide micelles were placed in a centrifuge tube, and 400 μL of anhydrous ethanol was added. The tube was sonicated at room temperature for 10 min (ultrasonic frequency 25 kHz), centrifuged at 3000 rpm, and the supernatant was collected. The absorbance was measured at 475 nm. Figure 1 The standard curve plot was used to calculate the astaxanthin concentration (W). 总 W 总 This indicates the medium concentration of astaxanthin in micelles, including both encapsulated and free astaxanthin.

[0098] Encapsulation efficiency (%) = W 内 / W 总 ×100%;

[0099] This experiment was repeated 5 times.

[0100] Calculations show that the encapsulation efficiency of astaxanthin by the guluronic acid oligosaccharide micelles prepared in Example 1 of this invention is 89.06 ± 2.00%.

[0101] Experiment Example 2

[0102] 100 μL of astaxanthin-loaded guluronic acid oligosaccharide micelles (Application Example 1) were diluted 20 times with deionized water, and their particle size, polydispersity index (PDI) and potential were determined using a nanolaser particle size analyzer.

[0103] The particle size distribution of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention is shown in the figure below. Figure 2 As shown. The particle size of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared by this invention is 254.50±8.30 nm, that is, the micelles prepared by this invention are nanoscale.

[0104] Experimental Example 3

[0105] The prepared astaxanthin-loaded guluronic acid oligosaccharide micelles (Application Example 1) were diluted 10-fold and dropped onto a copper grid. After air drying, the morphology of the micelles was observed using a scanning electron microscope. The results are shown in the figure. Figure 3 .

[0106] Experiment Example 4

[0107] Take 1 mL of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention (Application Example 1), and place them in aqueous solutions at 4℃, 20℃, 37℃, 50℃, and 80℃, respectively. The volume of the aqueous solution is 50 mL. Heat in a water bath at 100 rpm for 2 h. According to the method for determining the concentration of astaxanthin encapsulated in the micelles in Experiment Example 1, determine the initial concentration of astaxanthin encapsulated in the micelles and the concentration of astaxanthin in the micelles after treatment at a certain temperature to obtain the retention rate of the micelles. The retention rate is calculated as follows: (Concentration of astaxanthin encapsulated in the astaxanthin-loaded guluronic acid oligosaccharide micelles - Concentration of astaxanthin in the micelles after treatment at a certain temperature) / Astaxanthin encapsulated in the astaxanthin-loaded guluronic acid oligosaccharide micelles × 100%.

[0108] The temperature stability results of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention are as follows: Figure 4 As shown, the retention rate of astaxanthin in micelles is above 80% when the temperature is between 4-80℃, indicating that guluronic acid oligosaccharide micelles can efficiently retain astaxanthin at different temperatures. The astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention have good temperature stability.

[0109] Experimental Example 5

[0110] Take 1 mL of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in Example 1 of this invention, and place them in 0.01 mol / L PBS (150 mM NaCl) buffer at pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. The buffer volume is 1 mL, and the mixture is allowed to stand at room temperature for 2 h. The encapsulation efficiency of the astaxanthin-loaded guluronic acid oligosaccharide micelles is then determined. The method for determining the encapsulation efficiency is the same as in Experimental Example 1. The pH stability results of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in Example 1 of this invention are as follows: Figure 5 As shown.

[0111] according to Figure 5 It can be seen that in an environment with a pH value of 3-9, the guluronic acid oligosaccharide micelles prepared in Example 1 of this invention retain more than 60% of astaxanthin, indicating that the guluronic acid oligosaccharide micelles can efficiently retain astaxanthin under different acid and alkali conditions, and that the guluronic acid oligosaccharide micelles still have a high retention rate of astaxanthin at a lower pH, indicating that the astaxanthin-loaded guluronic acid oligosaccharide micelles of this invention have potential gastric clearance ability.

[0112] Experimental Example 6

[0113] Take 1 mL of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in Example 1 of this invention, and place them in NaCl aqueous solutions with concentrations of 0.0, 62.5, 125.0, 250.0, and 500.0 mM / mL, respectively. The volume of the NaCl aqueous solution is 1 mL. Let stand at room temperature for 2 h, and determine the encapsulation efficiency of the astaxanthin-loaded guluronic acid oligosaccharide micelles. The method for determining the encapsulation efficiency is the same as in Experimental Example 1. The ionic stability results of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in Example 1 of this invention are as follows: Figure 6 As shown.

[0114] Depend on Figure 6 It was found that the astaxanthin-loaded guluronic acid oligosaccharide micelles maintained an astaxanthin retention rate exceeding 70% in a 500 mM / mL NaCl aqueous solution. Specifically, the retention rate increased when the NaCl concentration was between 0.0 and 125.0 mM / mL, and decreased slightly when the NaCl concentration was between 125.0 and 500.0 mM / mL. This indicates that the guluronic acid oligosaccharide micelles prepared in this invention can efficiently retain astaxanthin even in high-salt environments.

[0115] Experimental Example 7

[0116] Five mL of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in Application Example 1 of this invention were placed in 1.0, 5.0, and 10.0 mmol / L glutathione solutions, respectively. The volume of the glutathione solution was 5 mL. The astaxanthin release was measured after treatment in glutathione solutions of different concentrations for 0, 1, 2, 6, 10, 24, and 48 h. The response characteristics of the astaxanthin-loaded guluronic acid oligosaccharide derivatization prepared in this invention to glutathione are as follows: Figure 7 As shown, with increasing glutathione concentration and longer response time to micelle stimulation, guluronic acid oligosaccharides can release more astaxanthin from the micelles.

[0117] Experimental Example 8

[0118] Take 1 mL of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in Application Example 1 of this invention and add them to 9 mL of PBS solution to form a micelle dilution.

[0119] Hydroxyethyl cellulose was dissolved in PBS solution to prepare a simulated intestinal fluid (hydroxyethyl cellulose concentration was 8 μg / mL);

[0120] 1 mL of the micelle dilution was dispersed in simulated intestinal fluid, and its motion was tracked using a nanoparticle tracking analyzer. The motion of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention in simulated intestinal fluid is as follows: Figure 8 As shown, the guluronic acid oligosaccharide micelles loaded with astaxanthin exhibited irregular movement within 1 second in simulated intestinal fluid, indicating that the nanomicelles undergo Brownian motion. Furthermore, the mean square displacement increases with time, suggesting that the nanomicelles are not affected by other factors during diffusion.

[0121] Experimental Example 9

[0122] Following the method for encapsulating astaxanthin in guluronic acid oligosaccharide micelles provided in Application Example 1, guluronic acid oligosaccharide micelles loaded with astaxanthin with astaxanthin concentrations of 2.0, 5.0, and 10.0 μg / mL were prepared, wherein the astaxanthin concentration in the guluronic acid oligosaccharide micelles loaded with astaxanthin prepared in Application Example 1 was 100 μg / mL.

[0123] RAW264.7 macrophages (from the Shanghai Cell Bank of the Chinese Academy of Sciences) were seeded in 96-well plates at a density of 2 × 10⁶ cells per well. 5After adhering to the culture vessel and culturing for 5 hours, the supernatant was discarded. 100 μL of serum-free medium containing astaxanthin-loaded guluronic acid oligosaccharide micelles at concentrations of 2.0, 5.0, and 10.0 μg / mL were added to each well as the test group; 100 μL of serum-free medium containing 100 μg / mL lipopolysaccharide was added as the control group; and 100 μL of PBS buffer was added as the blank control group. After culturing for 24 hours, 10 μL of LCK-8 reagent was added to each well, and after incubation for 1 hour, the absorbance was measured at 450 nm using a microplate reader. The effect of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention on the viability of mouse mononuclear macrophages (RAW264.7) is as follows: Figure 9 As shown, compared with the control group, the astaxanthin-loaded guluronic acid oligosaccharide micelles can not only prevent lipopolysaccharide-induced cell death, but also promote the viability of RAW264.7 cells. At the same time, this experimental example also proves that the astaxanthin-loaded guluronic acid oligosaccharide micelles provided by the present invention are non-toxic to cells.

[0124] Experimental Example 10

[0125] RAW264.7 cells were seeded in confocal culture dishes at a density of 4 × 10⁶ cells per dish. 5 After adhering to the culture vessel and culturing for 5 hours, the supernatant was discarded, and 2 mL of serum-free medium containing 10.0 μg / mL astaxanthin-loaded guluronic acid oligosaccharide micelles (astaxanthin-loaded guluronic acid oligosaccharide micelles were labeled with Nile blue at a concentration of 1 mg / mL, in excess; the preparation method for the corresponding concentration of astaxanthin-loaded guluronic acid oligosaccharide micelles is described in Experimental Example 9) was added. After incubation at room temperature for 0.5 hours, the supernatant was discarded, and the micelles were fixed with 4% paraformaldehyde for 0.5 hours. The micelles were washed three times with PBS, stained with DAPI for 10 minutes, and then washed again with PBS.

[0126] The phagocytosis of astaxanthin-loaded guluronic acid oligosaccharide micelles by RAW264.7 macrophages was observed under a confocal microscope. The results are shown in [Figure number missing]. Figure 10 It can be seen that RAW264.7 cells can effectively internalize guluronic acid oligosaccharide micelles loaded with astaxanthin within 0.5 h.

[0127] Experimental Example 11

[0128] RAW264.7 cells were seeded in 96-well plates at a density of 4 × 10⁶ cells per well. 5After adhering to the culture vessel for 5 hours, the supernatant was discarded, and 2 mL of serum-free culture medium containing inhibitors of the endocytosis pathway was added to each group (PBS buffer was used for test group 1, and the inhibitors of the endocytosis pathway included amiloride, methyl-β-cyclodextrin, quercetin, cytochalasin, and chlorpromazine; 20 μg / mL quercetin was used for test group 2, 10 μg / mL chlorpromazine for test group 3, 10 μg / mL cytochalasin B for test group 4, 1 mM amiloride for test group 5, and 10 mM methyl-β-cyclodextrin for test group 6). After incubation at room temperature for 0.5 hours, fluorescent astaxanthin-loaded guluronic acid oligosaccharide micelle solution (astaxanthin concentration in micelles was 10 μg / mL, fluorescence was Nile blue; see Experimental Example 10 for details) was added to each test group. After incubation at room temperature for 3 hours, the cells were collected.

[0129] The phagocytosis of astaxanthin-loaded guluronic acid oligosaccharide micelles by RAW264.7 was detected by flow cytometry. The results are shown in [Figure number missing]. Figure 11 As shown in the figure, quercetin (an inhibitor of endocytosis pathway independent of clathrin and caveolin), chlorpromazine (an inhibitor of clathrin-dependent endocytosis pathway), and cytochalasin-B (an inhibitor of phagocytosis-dependent endocytosis pathway) do not affect the entry of astaxanthin-loaded guluronic acid oligosaccharide micelles into RAW264.7 cells. However, amiloride (an inhibitor of macropinocytosis-dependent endocytosis pathway) can partially affect the entry of astaxanthin-loaded guluronic acid oligosaccharide micelles into RAW264.7 cells, while methyl-β-cyclodextrin (an inhibitor of caveolin-dependent endocytosis pathway) can significantly affect the entry of astaxanthin-loaded guluronic acid oligosaccharide micelles into RAW264.7 cells. This indicates that astaxanthin-loaded guluronic acid oligosaccharide micelles are mainly endocytosed into macrophages via the caveolin-dependent endocytosis pathway, with some endocytosis via macropinocytosis.

[0130] Experimental Example 12

[0131] RAW264.7 cells were seeded in 96-well plates at a density of 2 × 10⁶ cells per well. 5 After adhering to the culture vessel and culturing for 5 hours, the supernatant was discarded. 100 μL of medium containing guluronic acid oligosaccharide micelles with astaxanthin concentrations of 2.0, 5.0, and 10.0 μg / mL were added as test groups. 100 μL of serum-free medium containing 100 μg / mL lipopolysaccharide was added as the control group, and 100 μL of PBS buffer was added as the blank control group. After culturing each group for 2 hours, 10 ng of lipopolysaccharide (LPS) was added and incubated for 24 hours.

[0132] Detection of NO2 in cell culture medium using the Griess method -The content of NO can be indirectly obtained by measuring the amount of NO produced. The specific operation is as follows: Take 50 μL of cell suspension from the supernatant of each well of culture medium, add 50 μL of sulfonamide reagent and react for 5 min, then add 50 μL of naphthylvinyldiamine reagent, incubate in the dark for 5 min, and then use a microplate reader to detect the absorbance value at a wavelength of 545 nm.

[0133] The effect of astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention on NO secretion in RAW264.7 cells that alleviate LPS stimulation is as follows: Figure 12 As shown, astaxanthin-loaded guluronic acid oligosaccharide micelles can promote NO secretion in RAW264.7 cells in a concentration-dependent manner.

[0134] Example 13

[0135] RAW264.7 cells were seeded in 96-well plates at a density of 2 × 10⁶ cells per well. 5 After adhering to the culture vessel and culturing for 5 hours, the supernatant was discarded. 100 μL of medium containing guluronic acid oligosaccharide micelles at concentrations of 2.0, 5.0, and 10.0 μg / mL were added as test groups. 100 μL of serum-free medium containing 100 μg / mL lipopolysaccharide was added as the control group, and 100 μL of PBS buffer was added as the blank control group. After culturing each group for 2 hours, 10 ng LPS was added and incubated for 24 hours.

[0136] Following the instructions of the ELISA kit, the effect of the astaxanthin-loaded guluronic acid oligosaccharide micelles prepared in this invention on the secretion of TNF-α and interleukin-6 in RAW264.7 cells that have been relieved of LPS stimulation was tested. The results are shown in [Figure number missing]. Figure 13 , Figure 14 As shown, astaxanthin-loaded guluronic acid oligosaccharide micelles can inhibit the secretion of TNF-α and interleukin-6 by RAW264.7 cells in a concentration-dependent manner.

[0137] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An alginate oligosaccharide micelle, characterized in that, The alginate oligosaccharide micelles are obtained by reacting alginate oligosaccharide with hydrophobic fatty acids, using alginate oligosaccharide as the raw material.

2. The alginate oligosaccharide micelles according to claim 1, characterized in that, The alginate oligosaccharides include at least one of alginate mannuronic acid oligosaccharides and alginate guluronic acid oligosaccharides; and / or Hydrophobic fatty acids include at least one of palmitic acid and stearic acid; and / or, The alginate oligosaccharide micelles are loaded with fat-soluble nutrients.

3. The alginate oligosaccharide micelles according to claim 2, characterized in that, The preparation method of the alginate oligosaccharide micelles loaded with fat-soluble nutrients includes the following steps. A solution containing fat-soluble nutrients is mixed with a solution of alginate oligosaccharides to form a first oil-in-water mixture; The first mixture is mixed with an organic reagent to form a second oil-in-water-in-oil mixture; The organic reagents in the second mixture were removed to obtain homogeneous alginate oligosaccharide micelles loaded with lipid-soluble substances.

4. The alginate oligosaccharide micelles according to claim 3, characterized in that, The fat-soluble nutrient includes at least one of astaxanthin or fucoxanthin; and / or, The mass ratio of alginate oligosaccharide micelles to fat-soluble nutrients is 225-275:0.9-1; and / or, The concentration of fat-soluble nutrients in the solution is 0.18-0.2 mg / mL; and / or, The concentration of alginate oligosaccharide micelles in the alginate oligosaccharide micelle solution was 22.5-27.5 mg / mL.

5. A method for preparing alginate oligosaccharide micelles according to any one of claims 1-4, characterized in that, Includes the following steps, It is obtained by reacting alginate oligosaccharides with hydrophobic fatty acids.

6. The method for preparing alginate oligosaccharide micelles according to claim 5, characterized in that, The specific steps for reacting alginate oligosaccharides with hydrophobic fatty acids include the following: S1, in the presence of a catalyst, alginate oligosaccharides react with cystamine to form oligosaccharide-cystamine bridges; S2, in the presence of a catalyst, hydrophobic fatty acids and the alginate oligosaccharide-cystamine bridge formed in step S1 are mixed and heat-treated to obtain oligosaccharide micelles.

7. The method for preparing alginate oligosaccharide micelles according to claim 6, characterized in that, The catalysts in step S1 and / or step S2 include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

8. The method for preparing oligosaccharide micelles according to claim 7, characterized in that, In step S1, the mass ratio of the alginate oligosaccharide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 85-115:10:6; and / or, In step S2, the mass ratio of stearic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 555-585:197:115; and / or, The mass ratio of oligosaccharide-cystamine bridge to stearic acid is 475-525:555-585.

9. The method for preparing oligosaccharide micelles according to claim 8, characterized in that, The heat treatment temperature is 75-85℃; and / or, Under stirring, a solution containing alginate oligosaccharides is reacted with a catalyst to obtain an intermediate. This intermediate is then reacted with cystamine and purified to obtain an oligosaccharide-cystamine bridge. The stirring speed is 450-550 rpm, the reaction time between the alginate oligosaccharide solution and the catalyst is 1.9-2.1 h, and the reaction time between the intermediate and cystamine is 24-25 h. The concentration of alginate oligosaccharides in the solution is 21.25-28.75 mg / mL; and / or, Under stirring, a solution containing hydrophobic fatty acids is reacted with a catalyst to generate activated fatty acids. The stirring speed is 450-550 rpm, the reaction time is 1-1.5 h, and the concentration of hydrophobic fatty acids in the solution is 22.2-23.4 mg / mL; and / or, The activated fatty acids and the solution containing the oligosaccharide-cystamine bridge were reacted under stirring at a speed of 200-300 rpm for 6-6.5 h. The concentration of the oligosaccharide-cystamine bridge in the solution was 19-21 mg / mL.

10. An alginate oligosaccharide micelle according to any one of claims 1-4, or an alginate oligosaccharide micelle prepared by the method according to any one of claims 5-9, has any of the following uses: (a) Improve the bioavailability of nutrients; (ii) Delivery carriers for nutrients and / or drugs; (III) Anti-inflammatory effects; (iv) Enhance immune activity or prepare products that enhance immune activity.

Images