Lipidated derivative of fucoidin as well as preparation method and application of lipidated derivative

Abstract

The invention discloses a lipid derivative of fucoidin and a preparation method and application of the lipid derivative of fucoidin, and the lipid derivative of fucoidin is characterized in that aminated fucoidin is chemically connected with DSPE-PEG2000-NHS to obtain DSPE-PEG2000-fucoidin. The DSPE-PEG2000-fucoidin can serve as a component to be used for preparing lipid-based nano preparations (including lipid nanoparticles, lipidosome and the like), and modification of the fucoidin on the surface of the lipid nanoparticles or the lipidosome is achieved.

Description

Technical Field

[0001] The present invention relates to a lipidated derivative of fucoidan and a preparation method and application thereof, belonging to the field of drug delivery, especially active targeting nano preparations. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance some understanding of the overall background of the invention and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Fucoidan is a polysaccharide rich in sulfate groups found in brown algae. It is found in the cell walls, intercellular spaces, and mucus secreted by brown algae cells. Fucoidan is also known as brown algae polysaccharide, fucoidan, and fucoidan sulfate. Studies have shown that nanoformulations containing fucoidan components can bind to cell surface selectins and then enter the cell interior through caveolin-mediated entry, achieving efficient nanodrug uptake. Lipid-based nanoformulations (including lipid nanoparticles and liposomes) are one of the most successful solutions in nanodrug delivery systems. They are typically composed of phospholipids and cholesterol, are easy to prepare, have high biocompatibility, and are an ideal and safe form of drug delivery.

[0004] The existing methods of modifying fucoidan on the surface of lipid-based nanoformulations are mainly based on the following principles: (1) using the negatively charged sulfate groups of fucoidan itself to combine it with lipid-based nanoformulations containing cationic lipids (positively charged) components to achieve surface modification through electrostatic interaction. However, this non-covalent binding method is very likely to dissociate in complex environments such as blood circulation in the body, causing the fucoidan modified on the surface of the nanoformulation to fall off; (2) using the hydroxyl groups (-OH) of fucoidan itself to directly bind to DSPE-PEG 2000 The terminal carboxyl group of -COOH undergoes esterification reaction, but the steric hindrance of the hydroxyl group (-OH) of fucoidan is large, the reaction is difficult and the yield is low. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention uses an amination reaction to aminate the hydroxyl groups (-OH) in fucoidan, and then reacts with DSPE-PEG 2000 -NHS reaction to generate DSPE-PEG 2000 -Fucoidan.

[0006] The technical solution adopted in the present invention is as follows: In a first aspect of the present invention, a method for preparing a lipidated derivative of fucoidan is provided, the method comprising the following steps: (1) Fucoidan undergoes a ring-opening addition reaction with epichlorohydrin under alkaline conditions to generate a fucoidan epoxy activated intermediate; (2) The fucoidan epoxy activated intermediate reacts with ammonia to generate fucoidan-NH2; (3) Fucoidan-NH2 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-succinimidyl ester DSPE-PEG 2K -NHS reaction to generate DSPE-PEG 2000 - Fucoidan, which is a lipid derivative of fucoidan.

[0007] In one or some embodiments of the present invention, in step (1), the ratio of fucoidan to epichlorohydrin is (0.8-1.2) g: (3-6) mL; and the ring-opening addition reaction conditions are: nitrogen protective atmosphere, stirring reaction at 30-50°C for 5-7 hours.

[0008] In one or some embodiments of the present invention, in step (1), the alkaline condition is: 0.8-1.2 mol / L NaOH solution; the ratio of fucoidan to NaOH solution is (0.8-1.2) g: (15-30) mL.

[0009] In one or some embodiments of the present invention, in step (1), after the reaction is completed, water is removed by vacuum rotary evaporation and concentration, ultrapure water is added for dispersion, and the product is placed in ice ethanol to precipitate, and anhydrous ethanol is used for solidification several times to remove excess epichlorohydrin; the solid crude product is then dissolved in ultrapure water, and dialyzed with ultrapure water using a 7000 Da dialysis bag for 4-8 hours. After the dialysis is completed, vacuum rotary evaporation is performed to obtain the epoxy activated intermediate of fucoidan.

[0010] In one or some embodiments of the present invention, in step (2), the ratio of fucoidan-NH2 to ammonia water is (0.8-1.2) g: (15-25) mL; and the reaction conditions are: stirring at 30-50°C for 3-6 h.

[0011] In one or some embodiments of the present invention, in step (2), after the reaction is completed, the ammonia water is removed by vacuum rotary evaporation, ultrapure water is added for dispersion, and the product is placed in ice ethanol to precipitate, and then centrifuged and dried to obtain fucoidan-NH2.

[0012] In one or some embodiments of the present invention, in step (3), the fucoidan-NH2 and DSPE-PEG 2000 The mass ratio of -NHS is (0.4~0.6): (0.1~0.2). During the reaction, N,N-dimethylformamide (DMF) was used to dissolve DSPE-PEG. 2000-NHS. The reaction conditions are: stirring at 20-30°C for 20-40 minutes, using triethylamine (TEA) as the catalyst. The ratio of fucoidan-NH2 to triethylamine is (0.4-0.6) g: (200-500) µL. TEA is used as a catalyst in this invention, as it helps amino groups better attack NHS groups.

[0013] In one or some embodiments of the present invention, in step (3), after the reaction is completed, the product DSPE-PEG is dialyzed using a 7000 Da dialysis bag with ultrapure water for 18 to 36 hours and lyophilized to obtain the product 2000 -Fucoidan.

[0014] In a second aspect of the present invention, a lipidated derivative of fucoidan obtained by the above method is provided.

[0015] In the third aspect of the present invention, there is provided the use of the lipidated derivative of fucoidan in the preparation of lipid nanoformulations.

[0016] In one or some embodiments of the present invention, the lipid nanoformulation is lipid nanoparticles (LNP).

[0017] Compared with the related art known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: The present invention provides a method for preparing a lipid derivative of fucoidan. The method realizes the combination of lipid molecules and fucoidan through covalent bonds, and at the same time utilizes an amination reaction to aminate the hydroxyl groups (—OH) existing in fucoidan itself, which helps to avoid the adverse effects of steric hindrance and makes it easier to react to generate DSPE-PEG. 2000 -Fucoidan. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 : NMR spectrum of fucoidan-NH2 (a); Figure 2 :DSPE-PEG 2K -NMR spectrum of NHS (b); Figure 3 :DSPE-PEG 2000 -NMR spectrum of fucoidan (c); Figure 4 : Effects of different endocytic pathway inhibitors on cellular uptake of F-LNP or LNP. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.

[0022] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0023] Example 1 Dissolve 1.0 g of fucoidan (fucoidan is a naturally occurring macromolecule commercially available) in 20 mL of 1 mol / L NaOH solution. Add 5 mL of epichlorohydrin and stir at 40°C under nitrogen for 6 h. Remove water by rotary evaporation, briefly disperse in ultrapure water, and precipitate the product in icy ethanol. Solidify the product three times with anhydrous ethanol to remove excess epichlorohydrin. Dissolve the crude solid in ultrapure water and dialyze it against ultrapure water using a 7000 Da dialysis bag for 6 h.

[0024] The dialyzed solution was placed in a flask, evaporated under reduced pressure, and 20 mL of ammonia water was added to completely dissolve it. The reaction was stirred at 40 °C for 4 h, and the ammonia water was removed by rotary evaporation under reduced pressure. Ultrapure water was added to slightly disperse it, and the product was placed in ice ethanol to precipitate. Fucoidan-NH2 was obtained by centrifugation and drying. It was determined that 500 mg of fucoidan-NH2 contained approximately 49.36 μmol of -NH2.

[0025] Take 0.5 g of fucoidan-NH2, place it in a flask, add 15 mL of ultrapure water to completely dissolve it, and then add 300 μL of TEA, 173 mg of DSPE-PEG dissolved in DMF to the flask. 2K -NHS (molecular weight approximately 2914.537Da). Stir the reaction at 25°C for 30 minutes, dialyze with ultrapure water using a 7000 Da dialysis bag for 24 hours, and lyophilize to obtain the product DSPE-PEG. 2000 - Fucoidan, NMR spectrum such as Figure 1 、 Figure 2 and Figure 3 shown.

[0026] Example 2 Microfluidic technology was used to prepare lipid nanoparticles to verify the DSPE-PEG 2000 -Modification of fucoidan can promote the entry of lipid nanoparticles into cells through the caveolin-mediated pathway.

[0027] Dissolve dimyristoylphosphatidylcholine, cholesterol, and hydrophobic fluorescent dye coumarin 6 in 1 mL of anhydrous ethanol to obtain an organic phase. 2000 Fucoidan was dissolved in 4 mL of ultrapure water and sonicated to obtain the aqueous phase. Microfluidic lipid nanoparticle preparation was used at a total flow rate of 20 mL / min and a flow rate ratio of 4:1 between the aqueous phase and the organic phase. The mixture was then stirred at room temperature under magnetic stirring at 400 rpm for 1 h to evaporate the ethanol and remove the alcohol odor. Free drug was removed using a 0.22 μm microporous filter membrane to obtain DSPE-PEG. 2000 - Fucoidan modified lipid nanoparticles F-LNP. Remove DSPE-PEG from the above preparation formula. 2000 - Fucoidan, lipid nanoparticles LNP were prepared in the same way.

[0028] Monocytes were preincubated with different endocytic inhibitors to assess the extent to which these inhibitors affect different endocytic pathways, thereby comparing the primary endocytic pathways involved in cellular uptake of F-LNP or LNP. Methyl-β-cyclodextrin (1 mg / mL), chlorpromazine (10 μg / mL), cytochalasin D (10 μg / mL), and Z-Phe-Phe-Phe-OH (50 μg / mL) were added to the cells or preincubated at 4°C for 30 min. Subsequently, F-LNP or LNP (quantified by coumarin 6 concentration, 200 ng / mL) was added to each well while maintaining the inhibitor concentration or at 4°C. After an additional 2 h of incubation, cells were washed twice with 1× PBS buffer, harvested, and coumarin 6 fluorescence intensity was determined by flow cytometry.

[0029] The results of flow cytometry showed that Figure 4 , the uptake of F-LNP and LNP was completely blocked at 4°C, indicating that the uptake of F-LNP and LNP by monocytes was through an energy-dependent endocytic pathway. The inhibitor of the caveolin-mediated pathway (methyl-β-cyclodextrin) can significantly inhibit the cellular uptake of F-LNP, while the inhibitor of the clathrin-mediated pathway (chlorpromazine) or the inhibitor of the macropinocytosis pathway (cytochalasin D) or the inhibitor of the membrane fusion pathway (Z-Phe-Phe-Phe-Phe-OH) has a weak effect on the cellular uptake of F-LNP, indicating that F-LNP is mainly taken up by cells through the caveolin-mediated pathway, which is significantly different from the effect of the caveolin-mediated pathway inhibitor on the entry of LNP into cells. In summary, DSPE-PEG2000 -The modification of fucoidan enables F-LNP to be taken up by cells mainly through the caveolin-mediated pathway.

[0030] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a lipid derivative of fucoidan, characterized in that: The method comprises the following steps: (1) Fucoidan undergoes a ring-opening addition reaction with epichlorohydrin under alkaline conditions to generate a fucoidan epoxy activated intermediate; (2) The fucoidan epoxy activated intermediate reacts with ammonia to generate fucoidan-NH2; (3) Fucoidan-NH2 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-succinimidyl ester DSPE-PEG 2K -NHS reaction to generate DSPE-PEG 2000 - Fucoidan, which is a lipid derivative of fucoidan.

2. The method for preparing the lipidated derivative of fucoidan according to claim 1, wherein: In step (1), the ratio of fucoidan to epichlorohydrin is (0.8-1.2) g: (3-6) mL; the ring-opening addition reaction conditions are: nitrogen protective atmosphere, stirring reaction at 30-50°C for 5-7 hours.

3. The method for preparing the lipidated derivative of fucoidan according to claim 1, wherein: In step (1), the alkaline condition is: 0.8~1.2 mol / L NaOH solution; the ratio of fucoidan to NaOH solution is (0.8~1.2) g: (15~30) mL.

4. The method for preparing the lipidated derivative of fucoidan according to claim 1, wherein: In step (1), after the reaction is completed, water is removed by vacuum rotary evaporation, ultrapure water is added for dispersion, and the product is placed in ice ethanol to precipitate. Anhydrous ethanol is used for solidification several times to remove excess epichlorohydrin; ultrapure water is then used to dissolve the solid crude product, and the product is dialyzed with ultrapure water using a 7000 Da dialysis bag for 4 to 8 hours. After the dialysis is completed, vacuum rotary evaporation is performed to obtain the epoxy activated intermediate of fucoidan.

5. The method for preparing the lipidated derivative of fucoidan according to claim 1, wherein: In step (2), the ratio of fucoidan-NH2 and ammonia water is (0.8-1.2 g): (15-25) mL; the reaction conditions are: stirring at 30-50 °C for 3-6 h.

6. The method for preparing the lipidated derivative of fucoidan according to claim 1, wherein: In step (2), after the reaction is completed, the ammonia water is removed by rotary evaporation under reduced pressure, ultrapure water is added for dispersion, and the product is placed in ice ethanol to precipitate, and then centrifuged and dried to obtain fucoidan-NH2.

7. The method for preparing the lipidated derivative of fucoidan according to claim 1, wherein: In step (3), the fucoidan-NH2 and DSPE-PEG 2000 The mass ratio of -NHS is (0.4~0.6): (0.1~0.2); during the reaction, N,N-dimethylformamide (DMF) is used to dissolve DSPE-PEG 2000 -NHS; reaction conditions: stirring at 20~30℃ for 20~40min.

8. The method for preparing the lipidated derivative of fucoidan according to claim 1, wherein: In step (3), after the reaction is completed, the product DSPE-PEG is dialyzed with ultrapure water using a 7000 Da dialysis bag for 18 to 36 hours and freeze-dried to obtain the product. 2000 -Fucoidan.

9. A lipidated derivative of fucoidan prepared by the method according to any one of claims 1 to 8.

10. Use of the lipidated derivative of fucoidan according to claim 9 in the preparation of lipid nanoformulations.

Images