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CN-121758652-B - Nitric oxide donor based on heparin sodium and preparation method and application thereof

CN121758652BCN 121758652 BCN121758652 BCN 121758652BCN-121758652-B

Abstract

The invention relates to a heparin sodium-based nitric oxide donor and a preparation method and application thereof, wherein the method comprises the following steps of (1) dissolving heparin sodium in a 2- (N-morpholino) ethanesulfonic acid buffer solution, adding an activating agent for reaction to obtain a reaction solution A, (2) adding beta-mercaptoethylamine into the reaction solution A for continuous reaction to obtain a reaction solution B, (3) dialyzing the reaction solution B to obtain a solution C, (4) adding tert-butyl nitrite into the solution C for reaction to obtain the heparin sodium-based nitric oxide donor, wherein in the step (1), the activating agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or hydrochloride thereof. The nitric oxide donor based on heparin sodium prepared by the method prolongs the time of nitric oxide release and solves the problem of short half-life of nitric oxide.

Inventors

  • SHEN JIAN
  • YUAN JIANG
  • ZHANG XINYU

Assignees

  • 南京师范大学

Dates

Publication Date
20260512
Application Date
20260304

Claims (10)

  1. 1. A method of preparing a heparin sodium based nitric oxide donor, the method comprising the steps of: (1) Dissolving heparin sodium in a 2- (N-morpholino) ethanesulfonic acid buffer solution, adding an activating agent, and reacting to obtain a reaction solution A; (2) Adding beta-mercaptoethylamine into the reaction liquid A, and continuing to react to obtain a reaction liquid B; (3) Dialyzing the reaction solution B to obtain a solution C; (4) Adding tert-butyl nitrite into the solution C, and reacting to obtain the nitric oxide donor based on heparin sodium; wherein in step (1), the activator is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or its hydrochloride; the molar ratio of the heparin sodium to the beta-mercaptoethylamine is 1 (10-20); In the step (4), before tert-butyl nitrite is added into the solution C, the mercapto content of the solution C is measured, and the adding molar quantity of the tert-butyl nitrite is 5-7 times of the mercapto content.
  2. 2. The method according to claim 1, wherein in the step (1), the concentration of the 2- (N-morpholino) ethanesulfonic acid buffer solution is 0.05-0.2 m, and the ph value is 5.0-6.0.
  3. 3. The method according to claim 1, wherein in the step (1), the reaction temperature is 20 to 30 ℃ and the reaction time is 0.5 to 1 hour.
  4. 4. The method according to claim 1, wherein in the step (2), the reaction temperature is 20 to 30 ℃ and the reaction time is 18 to 24 hours.
  5. 5. The method of claim 1, wherein in step (3), the dialysis is performed using a dialysis bag for 48-60 hours, and the molecular weight cut-off is 3500Da.
  6. 6. The method according to claim 1, wherein in step (4), the pH of the reaction is adjusted to 6 to 7; The pH regulator used for regulating the pH value is sodium hydroxide solution or potassium hydroxide solution.
  7. 7. The method according to claim 1, wherein in the step (4), the reaction is performed under the protection of inert gas and/or in the absence of light, and the reaction time is 8-12 hours.
  8. 8. The method of claim 1, wherein in step (4), the post-reaction further comprises a post-treatment step; the post-treatment step includes dialysis and/or lyophilization; the dialysis adopts a dialysis bag for dialysis for 48-60 hours, and the molecular weight cut-off is 3500Da; The freeze-drying temperature is-20 to-50 ℃ and the freeze-drying time is 48-72 hours.
  9. 9. A heparin sodium-based nitric oxide donor prepared by the method of any one of claims 1-8.
  10. 10. Use of the method according to any one of claims 1 to 8 or the heparin sodium based nitric oxide donor according to claim 9 for the preparation of a biomedical material, characterized in that the biomedical material is selected from a wound dressing, a vascular stent or a vascular prosthesis.

Description

Nitric oxide donor based on heparin sodium and preparation method and application thereof Technical Field The invention relates to the field of biological medicine, in particular to a nitric oxide donor based on heparin sodium, and a preparation method and application thereof. Background Nitric Oxide (NO) is an important class of endogenous messenger molecules and has been the focus of research widely focused by the scientific and medical community. NO is widely involved in the regulation of physiological functions and pathological process interventions in the body's multisystem, playing a central role in cell signaling, tissue protection and maintenance of homeostasis. In the cardiovascular system, NO can promote cyclic guanosine monophosphate (cGMP) production by activating guanylate cyclase of vascular smooth muscle cells, mediate vasodilation to regulate vascular tension, inhibit platelet activation aggregation and thrombosis, protect vascular endothelial structural integrity, and delay lesion processes such as atherosclerosis. In the immune system, NO generated by immune cells such as macrophages can play an antibacterial and antiviral role, and participate in immune response regulation by regulating and controlling inflammatory factor release. In the nervous system, NO is involved in nerve signal transmission as a neurotransmitter, and has important significance for learning and memory, pain perception and nerve cell protection. In addition, NO can promote local angiogenesis and tissue repair of wounds, inhibit smooth muscle cell hyperproliferation and play a key role in physiological metabolism and tissue regeneration. However, the half-life of NO is extremely short (only 1-5 seconds), the property is unstable, the effective concentration is difficult to maintain at the action part, the physiological effects such as vasodilation, antithrombotic and the like cannot be continuously exerted, and the direct application of NO is greatly limited. Heparin sodium is a linear polymer which is mainly extracted from the mucous membrane of the small intestine of a cow lung or a pig and is formed by alternately connecting D-glucosamine, L-iduronic acid or D-glucuronic acid through 1-4 glycosidic bonds, and the molecular chain of the heparin sodium is provided with a large amount of negative charges. Because heparin sodium has better anticoagulation activity, good biocompatibility, adjustable anticoagulation strength and a certain anti-inflammatory effect, the heparin sodium has wide exploration and intensive research on the application in the aspects of anticoagulation treatment of cardiovascular diseases (such as thrombus formation prevention), interventional operation auxiliary anticoagulation, hemodialysis extracorporeal circulation anticoagulation, drug carrier modification (such as targeting enhancement) and the like. However, there is no report concerning the use of heparin sodium as a nitric oxide donor. Disclosure of Invention Based on the defects of the prior art, the invention provides a heparin sodium-based nitric oxide donor (HMA), a preparation method and application thereof, and the heparin sodium-based nitric oxide donor prepared by the invention prolongs the time of nitric oxide release and solves the problem of short half-life of nitric oxide. The invention provides a method for preparing a nitric oxide donor based on heparin sodium, which comprises the following steps: (1) Dissolving heparin sodium in a 2- (N-morpholino) ethanesulfonic acid buffer solution, adding an activating agent, and reacting to obtain a reaction solution A; (2) Adding beta-mercaptoethylamine into the reaction liquid A, and continuing to react to obtain a reaction liquid B; (3) Dialyzing the reaction solution B to obtain a solution C; (4) Adding tert-butyl nitrite into the solution C, and reacting to obtain the nitric oxide donor based on heparin sodium; Wherein in the step (1), the activator is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or its hydrochloride. Preferably, the molar ratio of the heparin sodium to the beta-mercaptoethylamine is 1 (10-20). Preferably, in the step (1), the concentration of the 2- (N-morpholino) ethanesulfonic acid buffer solution is 0.05-0.2M, and the pH value is 5.0-6.0; and/or the reaction temperature of the reaction is 20-30 ℃ and the reaction time is 0.5-1 hour. Preferably, in the step (2), the reaction temperature of the reaction is 20-30 ℃ and the reaction time is 18-24 hours. Preferably, in the step (3), the dialysis is performed for 48-60 hours by using a dialysis bag, and the molecular weight cut-off is 3500Da. Preferably, in the step (4), before tert-butyl nitrite is added into the solution C, the mercapto content of the solution C is measured, and the molar quantity of the tert-butyl nitrite added is 5-7 times of the mercapto content. Preferably, in the step (4), the pH value of the reaction is adjusted to 6-7; and/or the pH regulator used for regulating the pH value is selected