Preparation method of benzaldehyde modified oligosaccharide-based self-assembling material

Abstract

The application discloses a preparation method of a benzaldehyde modified oligosaccharide self-assembly material. The method is that an oligosaccharide is subjected to etherification reaction with p-hydroxybenzaldehyde to obtain a benzaldehyde modified oligosaccharide etherification product, and then the product is dissolved in an aqueous solution to form a self-assembly system. The self-assembly system formed by the benzaldehyde modified oligosaccharide etherification product in the aqueous solution comprises micelles with a size of 20-100 nm and vesicles with a size of 100-1000 nm. The self-assembly system can be used in the field of drug delivery in the biomedical field.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medical materials, and particularly relates to a preparation method of benzaldehyde modified oligosaccharide-based self-assembled material. BACKGROUND

[0002] In order to enter the interior of cells, nanomaterials must avoid traditional clathrin-mediated endocytosis and need to enter cells through lipid raft / caveolae-mediated or direct membrane penetration, which requires that the surface of the material is usually modified with hydrophilic or hydrophobic groups and ligand molecules combined with membrane receptors. For nanometer entity particles, this not only further increases the difficulty of controlling the size of the material, but also requires smaller size (preferably <10 nm) if direct penetration of the cell membrane is to be achieved, so that the function of nanometer entity particles at the cell level is strictly limited by size, shape, surface charge and multivalency. In contrast, deformable carriers such as micelles, vesicles, nanocapsules are not subject to such restrictions. They have high deformability and mechanical flexibility, and can change size and shape at any time; at the same time, their flexible structure of amphiphilicity is closer to the cell membrane, and is easy to penetrate the cell membrane through lipid raft or caveolae, and relies on diversified transport pathways to promote cell uptake, so as to more easily achieve efficient drug targeted delivery.

[0003] Synthetic polymer micelles / vesicles are the most widely used micelles / vesicles, which are usually prepared by grafting or block copolymerization to introduce hydrophobic structures into hydrophilic chains to form amphiphilic molecules, or by non-covalent interactions such as aromatic π-π stacking, electrostatic interaction or hydrophobic interaction to form self-assemblies. They have low critical micelle concentration (CMC) and high stability, high efficiency of single administration, low cost and easy synthesis, but poor biocompatibility, and the degradation products and residues (such as monomers, initiators and crosslinking agents) in the polymerization reaction can easily cause drug accumulation and toxicity of metabolic products, which has a high risk in vivo application. In addition, they have no specific recognition effect on cells and lack active targeting, which needs to be linked with monoclonal antibodies, proteins or sugar receptors for targeting cells. Biological vesicles have high safety and are the most approved drug carriers for clinical use and testing by the US Food and Drug Administration (FDA), such as liposomes and exosomes. However, liposomes also lack active targeting to cells and need additional chemical modification, which are easily degraded by lipases; the extraction method of exosomes is complex, and the vesicle surface carries a complete set of nucleic acids, receptor proteins and lipids of living organisms, some of which cannot be removed and may help tumor immune tolerance and escape. Compared with synthetic polymer materials, natural polymer materials have lower cytotoxicity, abundant cell membrane targeting epitopes, good biocompatibility and biodegradability, non-toxic degradation products and easy internalization by the body, and the corresponding receptors exist on the cell membrane surface, which can be used as ligands to target cells, making them ideal self-assembly matrices. Among the self-assemblies based on natural polymer materials, natural proteins, artificial polypeptides and lipid self-assemblies are more common. These self-assemblies are easy to synthesize and have lower prices than monoclonal antibodies, but their conformation is unstable and easy to inactivate, and their binding force to receptors is not as strong as that of monoclonal antibodies, and they are easily degraded by proteases and lipases in the body. Compared with proteins and lipids, natural polysaccharides have strong enzyme resistance, and the types of antigens in nature are far more than proteins and polypeptides, with low immunogenicity, lower extracellular degradation rate than proteins and lipids, higher stability, recognition by various membrane proteins of pattern recognition receptors (PRRs), and easy degradation by lysosomal enzymes (including glycosidases, esterases and proteases) after endocytosis, which have both targeting and immunomodulatory activities, making them ideal carriers for precise targeting. Therefore, the preparation of micelles / vesicles based on sugar-based ordered and flexible nanocarriers is a potential research direction in the field of drug carriers.

[0004] Currently, the role of polysaccharides in targeted drug carriers is mainly as a targeting ligand to modify the surface of other nanoparticles. When self-assembled nanoparticles are formed by electrostatic interaction, long-chain fatty acid / alcohol hydrophobic modification, graft copolymerization or block copolymerization with other materials, the introduced block polymers will cause the material to be non-degradable and biotoxic, and the long fatty chain will also make the material easily degraded by lipase during in vivo circulation, leading to the disintegration of the self-assembly. Self-assembled nanomaterials based on a single polysaccharide are very rare, mainly focusing on hydrophobic modification such as formylation, acetylation, and fatty chain carboxyl esterification. In our previous work, we obtained a class of glucan nanocapsules by formylating β-1, 3-D-glucan, and used them for tumor-targeted immunotherapy, which is one of the few polysaccharide nanomaterials obtained by hydrophobic substitution of small molecular groups.

[0005] Similar to amphiphilic block polymers, the morphology of self-assembled amphiphilic polysaccharides mainly depends on the properties of the hydrophobic and hydrophilic moieties (the type of hydrophobic groups, the degree of substitution of hydrophobic groups, etc.) and the self-assembly conditions (such as the concentration of amphiphilic polysaccharides, the properties of solvents used to dissolve and disperse amphiphilic polysaccharides, etc.). Currently, the research on polysaccharide-based micelles / vesicles is relatively rare, mainly due to two reasons: (1) Generally, amphiphilic block polymers continuously transform from spherical micelles to rod-like or worm-like micelles and then to vesicles as the concentration of amphiphilic precursors increases or the mass fraction of the hydrophilic moiety decreases. However, due to the presence of abundant hydroxyl groups on the sugar ring of polysaccharides, strong hydrogen bonding interactions between molecular chains are easily formed, and multiple hydrophobic microdomains rather than a single hydrophobic core are formed after self-assembly of amphiphilic polysaccharides. These microdomains are prone to form nanogels through physical cross-linking. Therefore, the self-assembly morphology of polysaccharides usually presents a flip-flop change, i.e., spherical micelles are formed when the concentration of amphiphilic polysaccharides (usually <0.5wt%) or the proportion of hydrophobic groups is below a certain critical point, and solid nanoparticles are formed above the critical point, without a continuous intermediate state. This results in a narrow range of concentrations or hydrophobic group proportions for the formation of micelles, and it is difficult to form advanced non-cross-linked ordered aggregation structures similar to vesicles. (2) The classic self-assembly process usually requires the amphiphilic polymer to be pre-dissolved in an organic solvent, while most sugars can only be dissolved in a few highly polar organic solvents such as DMSO, DMF, and pyridine, which cannot be dissolved in the same solvent as the hydrophobic moiety, thereby hindering the realization of self-assembly. In order to obtain a micelle / vesicle system, some studies have adopted the method of polymerization-induced self-assembly (PISA). This method can prepare high-concentration (>30wt%) polysaccharide nano self-assemblies in aqueous or other organic systems, but this method introduces a polymerization reaction, and the prepared micelles / vesicles may contain chemical residues from the polymerization. Another feasible method is to use small molecule hydrophobic modified oligosaccharides, to reduce the hydrogen bonding interactions and other supramolecular interactions between sugar chains, and to adjust the degree of substitution of hydrophobic groups to control the water solubility of amphiphilic oligosaccharides. Some studies have synthesized a series of oligosaccharides substituted with 9-fluorenylmethoxycarbonyl (Fmoc) protecting groups, and found that oligosaccharides with different molecular weights, glycosidic bond connection modes, and Fmoc substitution degrees can self-assemble into nanostructures with different morphologies, but no micelles / vesicles structures have been found. There are many factors that affect the self-assembly of small molecule hydrophobic modified oligosaccharides (such as molecular weight, degree of substitution of hydrophobic groups, type of hydrophobic groups, concentration of oligosaccharides, etc.), and there is currently no report on the research of obtaining micelles / vesicles self-assemblies through rational design. SUMMARY

[0006] In view of the above reasons, the present application provides a preparation method of a benzaldehyde-modified oligosaccharide-based self-assembly material. The self-assembly material is obtained by etherification of oligosaccharide and p-hydroxybenzaldehyde (PHB) to obtain a benzaldehyde-modified oligosaccharide etherate, and then dissolving the benzaldehyde-modified oligosaccharide etherate in an aqueous solution to form a self-assembly system. The self-assembly system is composed of micelles with a size of 20-100 nm and vesicles with a size of 100-1000 nm.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The present application provides a benzaldehyde-modified oligosaccharide-based self-assembly system, which is obtained by etherification of oligosaccharide and p-hydroxybenzaldehyde to obtain a benzaldehyde-modified oligosaccharide etherate, and then dispersing the benzaldehyde-modified oligosaccharide etherate in an aqueous solution to form a self-assembly system.

[0009] Preferably, the oligosaccharide is selected from agar oligosaccharide (AO).

[0010] Preferably, the molecular weight distribution of the oligosaccharide is in the range of 1000-5000 Da.

[0011] Preferably, the benzaldehyde-modified oligosaccharide etherate is obtained by adding N,N-dicyclohexyl carbodiimide, 4-dimethylaminopyridine and p-hydroxybenzaldehyde into DMSO, and then adding the oligosaccharide into the reaction, and after the reaction is completed, first removing dicyclohexyl urea by filtration, then adding anhydrous ethanol into the filtrate to precipitate the product, and finally centrifuging and drying the product to obtain the oligosaccharide etherate.

[0012] Preferably, the molar ratio of N,N-dicyclohexyl carbodiimide, 4-dimethylaminopyridine and oligosaccharide is 1.5:(0.25-0.4):1; and the molar ratio of oligosaccharide and p-hydroxybenzaldehyde is 1:(0.5-2.5).

[0013] Preferably, the reaction conditions are: room temperature and N2 atmosphere for 24 h.

[0014] Preferably, the degree of substitution of the oligosaccharide etherate is greater than 4%, and the oligosaccharide etherate is dissolved in pure water at a concentration of 0.5 g / 100 mL to form a self-assembly system after standing overnight.

[0015] The present application has the following beneficial effects:

[0016] The present application uses agar oligosaccharide as raw material, which is safe and non-toxic, has excellent biocompatibility and degradability, and the prepared self-assembly system contains micelles with a size of 20-100 nm and vesicles with a size of 100-1000 nm. The self-assembly system has the potential to be used in the field of drug delivery in the biomedical field.

[0017] The present application has the following beneficial effects: The raw materials of the present application are simple and easy to obtain, the preparation process is simple, the reaction is mild, the preparation cost is low, and the industrialized production is convenient. Attached Figure Description

[0018] Figure 1 AO ethers prepared for different AO to PHB ratios (AO / PCB = 1:0.5, 1:1, 1:2.5) 1 H nuclear magnetic resonance spectrum.

[0019] Figure 2 Transmission electron micrographs of AO ether (degree of substitution 4.8%, aqueous solution concentration 0.5%) (top: magnification 46,000; bottom: magnification 230,000).

[0020] Figure 3 The particle size distribution of the AO etherified compound (4.8% degree of substitution, 0.5% concentration in aqueous solution) self-assembled system is shown in the diagram (top: light intensity distribution; bottom: number distribution). Detailed Implementation

[0021] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without a specified manufacturer are all commercially available conventional products.

[0022] Example 1: Preparation of AO-based self-assembly system

[0023] The specific operation steps of the method described in this invention are as follows:

[0024] 1. Preparation of AO: AO was prepared by acid degradation.

[0025] Agarose was dissolved to obtain a 2% (m / vg / mL) agarose aqueous solution; after cooling, HCl solution was added for degradation at 70℃, with a final HCl concentration of 0.1 mol / L and a degradation time of 1 h. The degradation product was dialyzed with pure water (dialysis bag molecular weight cutoff of 1000 Da) for 3 days, and the dialysate was centrifuged at 6000 rpm to obtain the supernatant; the supernatant was freeze-dried to obtain AO lyophilized powder.

[0026] 2. Preparation of benzaldehyde-substituted AO ethers:

[0027] N,N-dicyclohexyl carbodiimide (DCC, 1.97 mmol), 4-dimethylaminopyridine (DMAP, 0.33 mmol or 0.52 mmol) and different amounts of p-hydroxybenzaldehyde (PHB) (AO / PHB = 1:0.5, 1:1, 1:2.5, molar ratio) were added into DMSO (4 mL) respectively and mixed for 30 min; meanwhile, AO powder (1.31 mmol) in Example 1 was dissolved in DMSO (6 mL) and added into the above system, and the reaction was carried out at room temperature under N2 atmosphere for 24 h. After the reaction was completed, dicyclohexyl urea (DCU) was removed by filtration, and anhydrous ethanol (100 mL) was added into the filtrate to precipitate the product. The product was centrifuged (7000 rpm, 5 min) and freeze-dried to obtain the benzaldehyde-substituted AO etherate in powder form.

[0028] 3. Preparation of AO-based self-assembly system

[0029] The benzaldehyde-substituted AO etherate (benzaldehyde substitution degree 4.8% or 7.5%, initial concentration 0.5% (m / v g / mL)) was added into pure water, heated to complete dissolution in a 50°C constant temperature water bath, and left overnight to obtain the self-assembly system.

[0030] Example 2: Physicochemical performance characterization of benzaldehyde-modified AO etherate and its self-assembly system

[0031] 1. 1 H nuclear magnetic resonance (NMR) spectrum was used to determine the structure and substitution degree of the AO etherate

[0032] The structure and substitution degree of the AO etherate in Example 1 were determined by 1 H NMR. 15-20 mg of AO and AO etherate samples prepared under different conditions were dissolved in 0.55 mL of DMSO-d6 for 1 H spectrum analysis. The substitution degree was calculated from the integral area ratio of the aldehyde hydrogen on PHB (9.8 ppm) to the anomeric carbon hydrogen of 3,6-anhydrogalactose (A ring) in AO unit (A1, 5.2 ppm). Figure 1 When the AO / PHB ratio was 1:0.5, 1:1 and 1:2.5, the benzaldehyde substitution degree was 0.4%, 4.8% and 7.5% respectively, and the AO etherate was soluble in water.

[0033] 2. Observation of the self-assembly system of AO etherate by high-resolution transmission electron microscopy (TEM)

[0034] 10 μL of the aqueous solution of AO etherate (degree of substitution 4.8%) self-assembly system was dropped on the carbon supporting film of copper mesh, and 10 μL of phosphotungstic acid staining solution was dropped on the same position of the copper mesh after 30 s. After natural air-drying, high resolution TEM observation was performed. The acceleration voltage of high resolution TEM was 200 kV, and the resolution was 0.1 nm. It can be seen from Figure 2 that the AO etherate formed a self-assembly system in water, which was composed of micelles (lower) with a size of 20-100 nm and vesicles (upper) with a size of 100-1000 nm. Figure 2 Figure 2

[0035] 3. Dynamic laser scattering measurement of the particle size distribution of AO etherate self-assembly system

[0036] The prepared AO etherate (degree of substitution 4.8%) self-assembly system was poured into a quartz cuvette, and the particle size and particle size distribution were measured on a dynamic laser scattering instrument. The measurement temperature was 25°C. It can be seen from Figure 3 that the AO etherate self-assembly system has two relatively concentrated particle size distributions, which are 40-110 nm and 110-900 nm (upper), respectively. It can be seen from Figure 3 that the micelles with a size of 40-110 nm are dominant. This is basically consistent with the particle size distribution in the TEM photograph. Figure 3 Figure 2

[0037] The above is only a preferred embodiment of the present application, and it should be noted that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined in the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.​​​​

Claims

1. A method for preparing a benzaldehyde-modified oligosaccharide-based self-assembly material, characterized in that, The oligosaccharide is etherified with p-hydroxybenzaldehyde to obtain benzaldehyde-modified oligosaccharide etherate, and then the benzaldehyde-modified oligosaccharide etherate is dissolved in an aqueous solution to form a self-assembly system; the oligosaccharide is selected from agar oligosaccharide; the benzaldehyde-modified oligosaccharide etherate is obtained by adding N, N-dicyclohexyl carbodiimide, 4-dimethylaminopyridine and p-hydroxybenzaldehyde into DMSO, and adding the oligosaccharide into the reaction at the same time; after the reaction is completed, dicyclohexylurea is removed by filtration, anhydrous ethanol is added into the filtrate, the product is precipitated, and the oligosaccharide etherate is obtained by centrifugation and drying; the molar ratio of N, N-dicyclohexyl carbodiimide, 4-dimethylaminopyridine and the oligosaccharide is 1.5:(0.25-0.4):1; and the molar ratio of the oligosaccharide and p-hydroxybenzaldehyde is 1:(0.5-2.5).

2. The production method according to claim 1, characterized by, The molecular weight distribution of the oligosaccharide ranges from 1000 to 5000 Da.

3. A benzaldehyde-modified oligosaccharide-based aqueous solution self-assembly system prepared by the preparation method according to claim 1 or 2.

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