CN100441225C - Amino Acid Modified Chitosan Nucleophilic NO Donor and Its Synthetic Method - Google Patents

Abstract

The present invention relates to an amino-acid modified chitosan nucleophilic NO donor and a synthesis method thereof, which belongs to the technical field of medicine engineering. In the present invention, chitosan is used as base material, amino acid is used for modifying the chitosan so as to increase the nucleophilic sites (NH groups) of the chitosan, and the modified chitosan reacts with NO so as to generate the nucleophilic NO donor containing N(O)NO functional groups, wherein the molecular structure of the donor is disclosed in the specification. The new nucleophilic NO donor has different NO releasing speed and half life and can overcome difficulties existing in the clinical application of NO donors at present, namely the cytotoxicity of a nucleophilic reagent (polyamine) and the generation of a carcinogenic side product-ammonium nitrite. Therefore, the present invention has important clinical application value and application prospects.

Description

Amino Acid Modified Chitosan Nucleophilic NO Donor and Its Synthetic Method

technical field

The invention relates to a medicine in the technical field of medical engineering and a synthesis method thereof, in particular to an amino acid modified chitosan nucleophilic NO donor and a synthesis method thereof.

Background technique

In the last ten years, a lot of researches have been done abroad on controlled-release materials based on NO donors containing [N(O)NO] ^-functional groups. The initial research focused on dispersing the ionic nucleophilic NO donor NONOate in various hydrophobic polymers to enhance the hemocompatibility of these materials through the release of NO. Although these studies have been fairly successful, it has been found that hydrophilic NO donors leach from the polymer to form carcinogenic N-nitrosamine species in the blood. To apply this kind of material in clinic, the following points must also be considered: biocompatibility and biodegradability as polymer matrix materials. Cytotoxicity and biocompatibility of nucleophiles. Some [N(O)NO] ^-group carriers such as diethylenetriamine were found to be harmful to the body; polyethyleneimine (PEI) induced extensive cell death and was poorly biocompatible. Therefore, choosing a non-toxic NO carrier with good biocompatibility has attracted people's attention. One of the solutions is to use nucleophilic carriers with secondary amino groups that are harmless to the body, such as polyamino acids and small molecule polypeptides.

Amino acids are bifunctional life small molecule ligands that exist in large quantities in organisms and have both amino and carboxyl groups. Amino acids are raw materials for the synthesis of human proteins, hormones, enzymes and antibodies, and participate in normal metabolism and physiological activities in the human body. Moreover, amino acids are important living substances in organisms and the basic units of enzymes and proteins. As small molecules, amino acids play an extremely important role in the activity and physiological functions of biomacromolecules; as ligands, they can coordinate with various metal ions, providing information for the study of anti-tumor and anti-cancer drugs. Various amino acids have different biological functions in organisms. For example, tryptophan in organisms is closely related to the normal metabolism of the brain, and L-cysteine can enhance the disease resistance of organisms. Therefore, the application of polyamino acid materials in the controlled release of drugs has attracted widespread attention. Amino acids participate in metabolism through the action of enzymes in the human body, nutrients are absorbed by the body, and metabolites are small inorganic molecules that are excreted by the excretory system, so they have good biocompatibility.

Found through literature search to prior art, Bohl etc. published " Nitric oxide-generating polymers reduceplatelet adhesion and smooth muscle cell proliferation " (NO produces polymerization substances to reduce platelet adhesion and smooth muscle cell proliferation), this paper proposed the use of amino acids to modify optically active polyethylene glycol (PEG) and polyvinyl alcohol (PVA) to have nucleophilic NH groups. After reacting with NO, a series of NO-releasing gel materials with different half-lives were obtained. Its disadvantage is that the base material used has poor biocompatibility, and films and coatings with good mechanical properties cannot be prepared due to the water solubility of the base material.

USP6261594 (Chitosan-based nitric oxide donor compositions) and USP6451337 (Chitosan-based nitric oxide donor compositions) propose to use modified chitosan as a nucleophilic NO carrier to synthesize a series of chitosan-NO adducts. The specific modification of chitosan is divided into two categories: (1) modification of hydrophilic groups: N-carboxybutyl chitosan, N-carboxyethyl chitosan, N-carboxymethyl chitosan. (2) Hydrophobic group modification: N-propyl chitosan, N-carboxyethyl chitosan methyl ester. The disadvantage is that the biocompatibility of modified chitosan is poor, and the loading degree of NO is low.

Contents of the invention

The object of the present invention is to provide an amino acid modified chitosan nucleophilic NO donor and a synthesis method thereof for the deficiencies in the prior art. The present invention uses amino acids to modify chitosan, thereby providing more nucleophilic sites and increasing the loading capacity of NO. It is helpful to solve the problems existing in clinical application of such NO donor drugs.

The present invention is achieved through the following technical solutions:

The amino acid modified chitosan nucleophilic NO donor of the present invention uses chitosan as a matrix material, adopts amino acid modification to increase its nucleophilic site (NH group), and reacts with NO to produce [N(O)NO ] ^- functional group, the molecular structure is as follows:

Wherein [N(O)NO] ^-functional group is that NH group reacts with NO to form, and NH group comes from the amino _NH of amino acid or the amino _NH in chitosan molecular structure Modification reaction; R is linear or branched chain of alkyl groups.

When the amino acid reacts at the C6 position of chitosan, the amino _NH2 on the amino acid molecule reacts with the COOH on the carboxylated chitosan molecule at the C6 position to form an NH site, which reacts with NO to form [N(O)NO ] ^- group.

When the amino acid reacts at the C2 position of chitosan, the carboxyl group on the amino acid molecule reacts with the amino _NH2 in the chitosan molecule to form an NH site, which reacts with NO to form a [N(O)NO] ^-group .

Amino acid modified chitosan nucleophilic NO donor synthesis method of the present invention can adopt any one of the following two kinds:

Method (1): introducing amino acid at C6 site. The carboxyl group COOH of C ₆ -O carboxylated chitosan is first activated with N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) or EDC, and then with amino acid amino group (NH ₂ ) Reaction, C6 amino acid modified chitosan can be prepared. The modified product is reacted with NO gas molecules in a methanol solution of sodium methoxide under high pressure (Na ⁺ /NH≥3), the pressure is 5-10atm, and the room temperature is reacted for 3-7 days to generate [N(O)NO ^] -radical group.

Method (2): introducing an amino acid at the C2 site. The carboxyl group of the amino acid is first activated with N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) or EDC, and then reacted with the amino group (NH ₂ ) on the chitosan molecule to obtain C2 Site amino acid modification of chitosan. The modified product is reacted with NO gas molecules in a methanol solution of sodium methoxide under high pressure (Na ⁺ /NH≥3), the pressure is 5-10atm, and the room temperature is reacted for 3-7 days to generate [N(O)NO ^] -radical group.

The chitosan used has a viscosity-average molecular weight of 1-1 million and a deacetylation degree of 50-100%.

The used carboxylated chitosan molecule has a carboxylation degree of substitution of 1.1, mainly C ₆ -O carboxymethyl chitosan.

The amino acids used include glycine, lysine, alanine, cysteine, arginine, aspartic acid, glutamic acid and other common acidic, basic and neutral amino acids.

The amino acid-modified chitosan nucleophilic NO donor prepared by the present invention can be prepared into drug films, microspheres, and nanospheres capable of releasing NO molecules, and can be widely used in the treatment of cardiovascular system diseases, pulmonary hypertension, and promoting wound healing. Heal, effectively prevent restenosis after plastic surgery, and improve the antithrombotic performance of medical devices.

Detailed ways

The synthetic route of synthetic method of the present invention is as follows:

Method (1): Introducing an amino acid at the C6 site (taking glycine (neutral) GLY and lysine (basic) LYS as examples)

carboxyl activation

Glycine modification

Lysine modification

react with NO

Method (2): Introducing an amino acid at the C2 site

Because there are also carboxyl groups in amino acids, first activate the carboxyl groups in the amino acids, and then react with -NH ₂ at the No. 2 carbon position in chitosan.

Embodiments are provided below in conjunction with the technical content of the present invention:

Example 1: Introduction of glycine at the C6 site

Preparation of carboxymethyl chitosan

Add 15g of chitosan and 200ml of isopropanol into a three-necked bottle, stir to disperse chitosan evenly in isopropanol, divide 50ml of 10mol/L NaOH solution into 5 parts, add one part every 30min, and stir at room temperature After 1 hour, a slurry of the reactant was obtained. Dissolve 24g of solid chloroacetic acid in 70ml of isopropanol and add it to the reactant slurry in 4 portions. After the addition, the reactant was heated to 70° C. with a water bath, and the reflux condensing device was turned on under temperature control, and stirred for 2 hours. After stopping, adjust the pH value to 7-8 with glacial acetic acid, and a large amount of light yellow flocculent precipitates precipitated. Vacuum filtration gave a yellow solid, which was washed with methanol three times, and the obtained yellow solid was vacuum-dried for 1 day at 60°C.

Activation of carboxymethyl chitosan

Add carboxymethyl chitosan (CMCS) into the reactor, then add solvent (water, acetonitrile, alcohol), stir to fully dissolve carboxymethyl chitosan. Dissolved N-hydroxysuccinimide (N-Hydroxysuccinimide NHS) aqueous solution and dicyclohexylcarbodiimide (Dicuclohexylcarbodiimide DCC) alcohol solution are added to the above solution respectively. In the above reaction system, the molar ratio of CMCS:NHS:DCC It is 1:1.5:1. Stir at room temperature for 24h to obtain milky yellow cloudy liquid. After filtering, the white solid is fully washed with an organic solvent and then vacuum-dried, and then the reactants in the filtrate are precipitated out again, washed, and vacuum-dried for later use.

Glycine modified carboxymethyl chitosan

Weigh glycine with a molar ratio of 1:1 and activated carboxymethyl chitosan into 0.05 mol/L NaHCO ₃ buffer solution, stir and react for 1 day to obtain a light yellow solution. After the resulting solution was concentrated, it was dialyzed for three days and freeze-dried to obtain a white fibrous substance.

Glycine modified chitosan FTIR: 1650-1655cm ^-1 (amide I), 1560cm ^-1 (amide II), 1600cm ^-1 (-COONa), ¹³ C NMR: 176.0ppm, -COOH; 43.0ppm, -CH ₂ -, ¹ H NMR: 4.22(1H), 2.52(2H), 3.91(3H), 3.0-3.3(4H, 5H, 6H), 1.42(7H), 4.33(8H), 3.62(9H)

Example 2: Introduction of lysine at the C6 site

Weigh the lysine with a molar ratio of 1:1 and the activated carboxymethyl chitosan in Example 1 and add it to 0.05 mol/L NaHCO ₃ buffer solution, stir and react for 1 day to obtain a light yellow solution. After the resulting solution was concentrated, it was dialyzed for three days and freeze-dried to obtain a white fibrous substance.

Lysine modified chitosan FTIR: 1730cm ^-1 (-COOH), 1630cm ^-1 and 1514cm ^-1 (NH ₃ ⁺ ), 1415cm ^-1 (-CH ₂ and -CH ₃ ), 2928cm ^-1 (-CH ₂ ). ¹ H NMR: 4.33(8H), 3.41(9H), 1.05(10H), 0.88(11H), 2.35(12H), 3.60(13H).

Example 3: Introduction of amino acids at the C2 site

Because there are also carboxyl groups in amino acids, first activate the carboxyl groups in amino acids, and then react with -NH ₂ at the No. 2 carbon position in chitosan. Dissolve amino acid in 100ml of water alcohol (2:1), add N-hydroxysuccinimide (NHS), stir for 1 hour, add dicyclohexylcarbodiimide (DCC) alcohol solution, and react at room temperature for 24 hours. In the above reaction system, the molar ratio of amino acid:NHS:DCC is 1:1.5:1. Take a quantitative amount of chitosan (amino acid: chitosan molar ratio 1:1) dissolved in 0.1M acetic acid solution, add dropwise to the above solution under stirring, react at room temperature for 24 hours, concentrate the resulting solution, dialyze for three days, freeze-dry A white fibrous substance was obtained.

FTIR: 1650cm ^-1 (amide I), 1560cm ^-1 (amide II), 1310cm ^-1 (amide III); ¹ H NMR: 3.53(8H)

Embodiment 4: Amino acid modified chitosan loaded NO reaction

Dissolve CH ₃ ONa (1.5g, 13.9mmol) in 50ml methanol to prepare a sodium methoxide solution, and then suspend amino acid-modified chitosan (1g, 4.5mmol) in the solution, which needs to satisfy Na ⁺ /NH≥ 3. Place it in a high-pressure reactor, react under a NO pressure of 5 atm, and pass NO for 60 min every day. After reacting for 3 days, filter and wash the product with ether, dry it in vacuum at room temperature, and store it in a closed desiccator at a temperature of -20°C.

For functional groups containing [NONO] ^- , the most effective characterization method is the characteristic ultraviolet absorption: Take a small amount of product and dissolve it in 0.1M NaOH solution, and measure the characteristic absorption of [NONO ^] -functional group at 256nm.

Research on NO release performance

Amino acid-modified chitosan products have better water solubility because of the introduction of carboxyl groups. Experimental studies have shown that they have better solubility in both acidic and alkaline solutions. Put it into a dialysis bag to measure its NO release process. Function fitting was carried out on the release curve, and the release half-life was calculated. For C6GLYCS/NO, the total amount released is 423nmol/mg, and the half-life is 0.143h; for C6LYSCS/NO, the total amount released is 327nmol/mg, and the half-life is 0.223h; for C2GLYCS/NO, the total amount released is 310nmol/mg, and the half-life is O. 311h.

From the above data, it can be seen that the release amount of the NO nucleophilic donor of chitosan modified at the C6 site is greater than that of the NO nucleophilic donor of chitosan modified at the C2 site, but the half-life is significantly shorter than that of the chitosan modified at the C2 site. Sugar NO nucleophilic donor. The release of NO from glycine-modified chitosan at the C6 site was greater than that of lysine-modified chitosan, and the half-life of lysine-modified chitosan was greater than that of glycine-modified chitosan.

Claims (8)

1, a kind of amino acid modified chitosan nucleophilic NO donor, it is characterized in that: chitosan is used as base material, adopts glycine or lysine modification to increase its NH nucleophilic site, reacts with NO to produce [N( O)NO] ^-functional group, the molecular structure is as follows:

Wherein [N(O)NO] ^-functional group is that NH group reacts with NO to form, and NH group comes from the amino NH of glycine or lysine ₂ or _the amino NH in chitosan molecular structure Modification reaction; R is _CH2 or ( _{CH2 ) 4NH2CH} group. 2. The amino acid modified chitosan nucleophilic NO donor according to claim 1 is characterized in that: when glycine or lysine reacts at the C6 position of chitosan, on the glycine or lysine molecule The amino group _NH2 reacts with COOH on the carboxylated chitosan molecule at the C6 position to form an NH site, which reacts with NO to form a [N(O)NO ^] -group. 3. The amino acid modified chitosan nucleophilic NO donor according to claim 1 is characterized in that: when glycine or lysine reacts at the C2 site of chitosan, the glycine or lysine molecule The carboxyl group reacts with the amino NH ₂ in the chitosan molecule to form an NH site, which reacts with NO to form a [N(O)NO] ^-group . 4. A method for synthesizing an amino acid modified chitosan nucleophilic NO donor as claimed in claim 1, characterized in that: NH on the C6 site or C2 site glycine or lysine modified chitosan molecule Group reacts with NO gas molecule in the methanol solution of sodium methoxide, wherein Na ⁺ /NH≥3, the pressure is 5atm, reacts at room temperature for 3 days, produces [N(O)NO ^] -group, obtains containing [N( O)NO] ^-group nucleophilic NO donor. 5. The amino acid modified chitosan nucleophilic NO donor synthesis method according to claim 4, characterized in that the NH group is derived from the amino group of chitosan and the carboxyl group of carboxylated chitosan and glycine or lysine It is formed after the carboxyl or amino group of the acid reacts. 6. The synthesis method of amino acid modified chitosan nucleophilic NO donor according to claim 4, characterized in that, the preparation of modified chitosan with glycine or lysine at C6 position is to carboxylate chitosan with C6 position The COOH of the sugar is first carried out with N-hydroxysuccinimide NHS and dicyclohexylcarbodiimide DCC or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride EDC Activated and then reacted with amino NH ₂ of glycine or lysine. 7. The amino acid modified chitosan nucleophilic NO donor synthesis method according to claim 4, characterized in that the preparation of glycine or lysine modified chitosan at the C2 site is the preparation of glycine or lysine Carboxyl COOH is first activated with N-hydroxysuccinimide NHS and dicyclohexylcarbodiimide DCC or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride EDC Then react with the amino NH ₂ of chitosan to prepare. 8. The method for synthesizing nucleophilic NO donors of amino acid modified chitosan according to claim 6, characterized in that chloroacetic acid and chitosan are used for carboxymethyl reaction, and the product is C ₆ -O carboxymethyl shell Polysaccharide-based.