阅读说明：本技术 Alfa-亚麻酸修饰的单甲氧基聚乙二醇-寡壳聚糖及其制备 (Alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan oligosaccharide and preparation thereof ) 是由 冯润良 宋智梅 于 2019-09-05 设计创作，主要内容包括：单甲氧基聚乙二醇对硝基苯基碳酸酯为起始原料,与寡壳聚糖反应得到单甲氧基聚乙二醇-寡壳聚糖。然后,经选择性N-酰化反应,制备得到alfa-亚麻酸修饰单甲氧基聚乙二醇-寡壳聚糖聚合物。反应介质为二甲亚砜,避免了水为反应介质导致的碳酸酯水解以及亚麻酸溶解性偏低等缺陷。(The monomethoxy polyethylene glycol p-nitrophenyl carbonate is used as a starting material and reacts with the chitosan oligosaccharide to obtain the monomethoxy polyethylene glycol-chitosan oligosaccharide. Then, preparing the alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan oligomer through selective N-acylation reaction. The reaction medium is dimethyl sulfoxide, so that the defects of carbonate hydrolysis, low solubility of linolenic acid and the like caused by taking water as the reaction medium are overcome.)

The Alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan amphipathic polymer comprises a hydrophilic chain segment of monomethoxy polyethylene glycol-chitosan oligosaccharide and a hydrophobic chain segment of Alfa-linolenic acid, and has the following structural formula:

wherein m = 4-18;

p=0-18；

the monomethoxy polyethylene glycol has a molecular weight of 2000, 4000, 5000, 10000 or 20000, corresponding to n =44, 90, 113, 226, 454.

The preparation method of Alfa-monomethoxypolyethylene glycol-chitosan oligomer polymer modified by linoleic acid is characterized by comprising the following reaction steps:

(1) mixing activated monomethoxy polyethylene glycol, oligo-chitosan and an alkaline catalyst in an organic reaction medium, heating for reaction, dialyzing, and freeze-drying to obtain monomethoxy polyethylene glycol-oligo-chitosan;

(2) in an organic reaction medium, activating alfa-linolenic acid by N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, adding monomethoxypolyethylene glycol-chitosan oligosaccharide, carrying out selective N-acylation reaction, dialyzing, and freeze-drying to obtain the alfa-linolenic acid modified monomethoxypolyethylene glycol-chitosan amphiphilic polymer.

3. The method of claim 2, wherein: in the step (1), the molar ratio of the activated monomethoxy polyethylene glycol to the chitosan is as follows: 1:1, the molar ratio of the activated monomethoxy polyethylene glycol to the basic catalyst is as follows: 1:1-1: 3; in the step (2), the molar ratio of the N-hydroxysuccinimide to the alfa-linolenic acid is as follows: 1:1-1:2, the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to alfa-is: 1:1-1:2, the molar ratio of N-hydroxysuccinimide to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is: 1:1.

4. The method of claim 2, wherein: in the step (1), the basic catalyst is one of triethylamine, N-methylpiperidine, N-methylpyrrolidine and N-methylmorpholine.

5. The method of claim 2, wherein: in the step (1), the organic reaction medium is dimethyl sulfoxide; in the step (2), the organic reaction medium is dimethyl sulfoxide.

6. The method of claim 2, wherein: in the step (1), the reaction temperature is 50-80 ℃, and the reaction time is 6-24 h; in the step (2), the reaction temperature is 15-35 ℃, and the reaction time is 12-48 h.

Technical Field

The invention mainly relates to an alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan oligosaccharide amphiphilic polymer and a preparation method thereof, belonging to the technical field of chemical synthesis.

Background

The chitosan oligosaccharide is low molecular weight chitosan with monosaccharide number of 6-20, and is a water-soluble polyamino and polyhydroxy chitosan oligomer. It has high solubility which chitosan does not have, and is easy to be absorbed and utilized by organism. Therefore, the method has wide application prospect in the fields of food, cosmetics, medicines and the like.

The chitosan oligosaccharide contains functional groups such as hydroxyl, amino and the like, and can be modified to form various chitosan oligosaccharide derivatives to expand the application field of the chitosan oligosaccharide derivatives. Mono-methoxy polyethylene glycol-chitosan oligosaccharide is a water-soluble polyamino chitosan oligosaccharide polyelectronic derivative. On one hand, the electric medicine can be coated and carried through electrostatic action and is used for preventing and treating diseases. On the other hand, lipophilic molecules can be introduced through amino and hydroxyl modification ways to form a novel amphiphilic polymer which is used for entrapment of lipophilic drugs, and the goals of solubilization, targeted delivery, slow release, pharmacokinetic property, cellular uptake improvement and the like of the drugs are achieved. In the early research, activated monomethoxy polyethylene glycol (i.e., monomethoxy polyethylene glycol p-nitrophenyl carbonate) is reacted with chitosan in water by using sodium hydroxide and the like as alkaline catalysts to prepare monomethoxy polyethylene glycol-chitosan polymer (Progress in Polymer science, 2012, 37 (5), 659-. However, carbonates are themselves sensitive to alkaline environments and are susceptible to hydrolysis, thereby affecting polymer synthesis.

Alfa-linolenic acid is a natural lipophilic polyunsaturated fatty acid and has a variety of pharmacological activities such as antibacterial, antioxidant, anti-inflammatory, anticancer, cardioprotection and neuroprotection (Food and Chemical Toxicology, 2014, 70, 163-178). Therefore, the introduction of alfa-linolenic acid into monomethoxypolyethylene glycol-chitosan oligosaccharide will result in an amphiphilic polymer. Micelles, nanoparticles and the like formed by the self-assembly of the polymer can be used for encapsulating anticancer, antibacterial, anti-inflammatory and other drugs so as to improve the release characteristics, activity, stability or pharmacokinetic properties and the like of the drugs.

Disclosure of Invention

The invention provides an alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan oligosaccharide amphiphilic polymer, wherein the hydrophilic chain segment is monomethoxy polyethylene glycol-chitosan oligosaccharide, the hydrophobic chain segment is alfa-linolenic acid, and the structural formula is as follows:

wherein m = 4-18;

p=0-18；

the monomethoxy polyethylene glycol has a molecular weight of 2000, 4000, 5000, 10000 or 20000, corresponding to n =44, 90, 113, 226, 454.

The preparation method of the alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan amphipathic polymer is characterized by comprising the following reaction steps:

the specific reaction mainly comprises the following steps:

(1) in an organic reaction medium, activated monomethoxy polyethylene glycol prepared in reference (Gene Therapy, 2004, 11, 194-203) was mixed with an oligo-chitosan, basic catalyst, and reacted with heating. Dialyzing, and freeze-drying to obtain monomethoxy polyethylene glycol-chitosan oligosaccharide;

(2) in an organic reaction medium, activating alfa-linolenic acid by N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, adding monomethoxypolyethylene glycol-chitosan oligosaccharide, carrying out selective N-acylation reaction, dialyzing, and freeze-drying to obtain the alfa-linolenic acid modified monomethoxypolyethylene glycol-chitosan amphiphilic polymer.

In the step (1), the molar ratio of the activated monomethoxy polyethylene glycol to the chitosan is as follows: 1:1, the molar ratio of the activated monomethoxy polyethylene glycol to the basic catalyst is as follows: 1:1-1:3.

In the step (1), the basic catalyst is one of triethylamine, N-methylpiperidine, N-methylpyrrolidine and N-methylmorpholine.

In the step (1), the organic reaction medium is dimethyl sulfoxide.

In the step (1), the reaction temperature is 50-80 ℃, and the reaction time is 6-24 h.

In the step (2), the molar ratio of the N-hydroxysuccinimide to the alfa-linolenic acid is as follows: 1:1-1:2, the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to alfa-is: 1:1-1:2, wherein the molar ratio of the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is as follows: 1:1.

In the step (2), the organic reaction medium is dimethyl sulfoxide.

In the step (2), the reaction temperature is 15-35 ℃, and the reaction time is 12-48 h.

The invention provides an alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan oligosaccharide polymer and a preparation method for preparing the alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan oligosaccharide polymer by taking activated monomethoxy polyethylene glycol as a starting material. Firstly, activated monomethoxy polyethylene glycol reacts with oligo-chitosan to obtain monomethoxy polyethylene glycol-oligo-chitosan, and then the monomethoxy polyethylene glycol-oligo-chitosan undergoes alfa-linolenic acid N-acylation reaction in the presence of N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and the like to obtain alfa-linolenic acid modified monomethoxy polyethylene glycol-oligo-chitosan polymer. The reaction medium is dimethyl sulfoxide, so that the defects of carbonate hydrolysis, poor solubility of linolenic acid and the like when water is used as the reaction medium can be avoided.

The alfa-linolenic acid modified monomethoxy polyethylene glycol-oligo-chitosan polymer obtained by the invention is formed by introducing alfa-linolenic acid at one end of oligo-chitosan, so that an amphiphilic polymer is formed, the polymer is used for lipophilic drug entrapment, and the improvement of the drug activity, stability, slow release property, cell uptake or pharmacokinetic property and the like is realized.

Detailed Description

The names of monomethoxypolyethylene glycol, oligochitosan, etc. are described below:

the same holds for activated monomethoxy polyethylene glycol with molecular weight of 2000 as activated monomethoxy polyethylene glycol 2000, and for chitosan oligosaccharide with molecular weight of 1000 as chitosan oligosaccharide 1000.

The present invention will be further described with reference to the following examples, which are provided for the purpose of illustrating the present invention and are not intended to limit the scope thereof.

Synthesis of monomethoxy polyethylene glycol-chitosan oligomer polymer

The method comprises the following steps: in a dry reaction flask, activated monomethoxypolyethylene glycol 2000 (2.167 g,1 mmol), oligo-chitosan 1000 (1 g,1 mmol), triethylamine (202 mg, 2 mmol) and dimethyl sulfoxide were added in this order, and dissolved by heating at 60 ℃. Reacting at the same temperature for 24 hours. Dialyzing, and freeze-drying to obtain 2.42 g of light yellow solid with the yield of 80%;

the second method comprises the following steps: in a dry reaction flask, activated monomethoxypolyethylene glycol 5000 (5.167 g,1 mmol), oligo-chitosan 3000 (3 g,1 mmol), N-methylpiperidine (297 mg, 3 mmol) and dimethylsulfoxide were added in this order and dissolved by heating at 80 ℃. Reacting at the same temperature for 24 hours. Dialyzing, and freeze-drying to obtain 6.5 g of light yellow solid with the yield of 81 percent;

the third method comprises the following steps: activated monomethoxy polyethylene glycol 10000 (10.167 g,1 mmol), chitosan oligosaccharide 2000 (2 g,1 mmol), N-methylmorpholine (202 mg, 2 mmol) and dimethyl sulfoxide are sequentially added into a drying reaction bottle, and heated and dissolved at 70 ℃. Reacting at the same temperature for 12 h. Dialyzed and lyophilized to obtain 9.38 g of light yellow solid with the yield of 78%.

Synthesis of linolenic acid modified monomethoxy polyethylene glycol-chitosan oligomer

The method comprises the following steps: in a dry reaction flask, N-hydroxysuccinimide (276 mg, 2.4 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (460 mg, 2.4 mmol) and alfa-linolenic acid (557 mg, 2 mmol) were mixed with dimethyl sulfoxide and dissolved, followed by reaction at room temperature for 4 hours. Monomethoxy polyethylene glycol 2000-chitosan oligosaccharide 1000 (3.026 g,1 mmol) was added thereto, and the reaction was continued at the same temperature for 24 h. Dialyzing, and freeze-drying to obtain 2.68 g of yellow solid with the yield of 82%;

the second method comprises the following steps: in a dry reaction flask, N-hydroxysuccinimide (828 mg, 7.2 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1380 mg, 7.2 mmol) and alfa-linolenic acid (1670 mg, 6 mmol) were dissolved in dimethyl sulfoxide and reacted at room temperature for 4 hours. Monomethoxypolyethylene glycol 5000-Chitosan 3000 (8.026 g,1 mmol) was added and the reaction was continued for 24 h at 35 ℃. Dialyzing, and freeze-drying to obtain 7.57 g of yellow solid with the yield of 79%;

the third method comprises the following steps: in a dry reaction flask, N-hydroxysuccinimide (1.15 g, 10 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.92 g, 10 mmol) and alfa-linolenic acid (1.95 g,7 mmol) were dissolved in dimethyl sulfoxide, and then reacted at room temperature for 4 hours. Monomethoxypolyethylene glycol 10000-Chitosan 3000 (6.5 g, 0.5 mmol) was added and the reaction was continued for 24 h at 35 ℃. Dialyzing, and freeze-drying to obtain 6.75 g of yellow solid with the yield of 81 percent;

the method four comprises the following steps: in a dry reaction flask, N-hydroxysuccinimide (805 mg, 7 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.34 g,7 mmol) and alfa-linolenic acid (1.39 g, 5 mmol) were dissolved in dimethyl sulfoxide, and the mixture was reacted at room temperature for 4 hours. Monomethoxypolyethylene glycol 5000-Chitosan 2000 (7.026 g, 0.5 mmol) was added and the reaction was continued for 24 h at 35 ℃. Dialyzed and lyophilized to obtain 8.05 g of yellow solid with the yield of 83.6%.