Preparation method and application of amphiphilic azo polymer and micelle thereof
By synthesizing the intermediate 4-((4-aminophenyl)diazalyl)benzoic acid and Lys(z)-NCA ring-opening polymerization method, amphiphilic azo polymer polymer was prepared by polyethylene glycol lipidation reaction, and azo polymer micelles were prepared by organic solvent evaporation, which solved the problems of micelles aggregation in blood circulation, complex synthesis process and biosafety, and achieved structural stability, simplification of synthesis and biosafety improvement.
Patent Information
- Application Number
- CN202411718847.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-03-04
AI Technical Summary
Amphilic azo polymer micelles may aggregate and deform during the blood circulation due to external fluid mechanical interference or protein adsorption in the body, affecting the efficiency of drug transportation; their synthesis process is complex, with high requirements for experimental instruments and operating accuracy, and production efficiency and quality stability need to be optimized; some metabolites may have potential toxicity or trigger immune responses, affecting biosafety.
The intermediate 4-((4-aminophenyl)diazalyl)benzoic acid was synthesized, and a hydrophobic end azo polymer was obtained by the Lys(z)-NCA ring-opening polymerization method was used to prepare amphiphilic azo polymers in combination with polyethylene glycol lipidation reaction, and azo polymer micelles were prepared by organic solvent evaporation.
It improves the structural stability of micelles, simplifies synthesis technology, enhances dispersion and morphological regularity of drug carriers, reduces the toxic side effects of drugs on normal cells, and improves biosafety.
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Figure CN119552371A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of polymer self-assembly, and in particular relates to a preparation method and application of an amphiphilic azo polymer and micelles thereof. Background Art
[0002] Amphiphilic azo polymer micelles are a type of nanoscale drug carrier formed by self-assembly of amphiphilic polymers with hydrophilic and hydrophobic properties. This type of micelle encapsulates poorly soluble drugs in the core through hydrophobic interaction, while providing good water dispersibility through the hydrophilic shell, achieving efficient drug loading and targeted delivery. The introduction of azo groups gives the micelles unique response characteristics, which can achieve precise drug release through stimuli such as light, heat, pH or biological enzymes, significantly improve the therapeutic efficiency of anticancer drugs, and reduce toxic side effects on normal cells. This versatility makes amphiphilic azo polymer micelles an important research hotspot in the field of modern tumor treatment.
[0003] The azo group is a special functional group that can undergo controllable cleavage under external stimuli, thus providing a reliable mechanism for targeted drug release. For example, the azo group can undergo photoisomerization under light conditions and can be reduced to amine compounds by specific degradation enzymes under the action of biological enzymes. This property makes amphiphilic azo polymer micelles show significant advantages in colon-targeted drug delivery: colon microorganisms can secrete azoreductase to degrade the azo groups in the core of the micelles and release the encapsulated drugs. Compared with traditional chemotherapy, this stimulus-responsive release mechanism not only improves the accuracy of drug delivery, but also significantly reduces the impact of drug doses on other normal tissues.
[0004] Although amphiphilic azo polymer micelles have broad application prospects, their practical application still faces many technical difficulties. On the one hand, micelles may aggregate and deform in the blood circulation due to external fluid mechanics interference or protein adsorption in the body, thereby affecting the transport efficiency of drugs; on the other hand, the synthesis process of azo polymers is complicated, and high requirements are placed on experimental instruments and operation precision. The production efficiency and polymer quality stability still need to be further optimized. In addition, some metabolites of azo polymers may be potentially toxic or induce immune responses, and in-depth evaluation of their biosafety remains a key research direction. Therefore, future development should focus on improving the structural stability of micelles, optimizing synthesis technology, and conducting toxicity and immunology research to promote their clinical application. Summary of the invention
[0005] The purpose of the present invention is to overcome the defects of the prior art and provide an amphiphilic azo polymer, a polymer micelle and a preparation method and application thereof. The amphiphilic azo polymer is obtained by synthesizing an intermediate 4-((4-aminophenyl)diazenyl)benzoic acid and using a Lys(z)-NCA ring-opening polymerization method to obtain a hydrophobic end azo polymer and a polyethylene glycol esterification reaction; and an azo polymer micelle is prepared by an organic solvent evaporation method.
[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0007] An amphiphilic azo polymer is based on 4-((4-aminophenyl)diazenyl)benzoic acid as an intermediate, a hydrophobic end of which is poly-N6-Cbz-L-lysine, and a hydrophilic end of which is polyethylene glycol.
[0008] Furthermore, the preparation method of the above-mentioned amphiphilic azo polymer comprises the following steps:
[0009] 1) Synthesis of 4-((4-aminophenyl)diazenyl)benzoic acid: dissolve p-aminobenzoic acid in deionized water, add concentrated hydrochloric acid and stir for 20 minutes, and cool in an ice bath after stirring; dissolve sodium nitrite in deionized water, and cool the dissolved sodium nitrite solution in an ice bath, maintaining the temperature at 0-5°C; slowly drop the cooled sodium nitrite solution into the p-aminobenzoic acid solution in the ice bath, and react for 15 minutes; filter the solution after the reaction, retain the filtrate and store it in an ice bath; take another large beaker, dissolve sodium acetate and aniline sulfite in deionized water, maintain the solution temperature at 0-5°C, then drop the filtrate into the large beaker, stir for 1 hour, adjust the pH to maintain acidity, filter and dry; after drying, react with sodium hydroxide solution at 90°C for 3 hours, finally adjust the solution pH to 6 in an ice bath, filter and dry, and obtain 4-((4-aminophenyl)diazenyl)benzoic acid;
[0010] 2) Synthesis of hydrophobic azo polymer: N6-Cbz-L-lysine dissolved in THF was added to a round-bottom flask and stirred for 15 minutes. Triphosgene dissolved in THF was slowly added dropwise to the round-bottom flask for reaction for 2 hours. The solution after the reaction was added to n-hexane and refrigerated overnight. The white solid Lys(z)-NCA was obtained by suction filtration and vacuum drying. The dried white solid Lys(z)-NCA and 4-((4-aminophenyl)diazenyl)benzoic acid obtained in step 1) were dissolved in DMF and stirred at room temperature for 36 hours to form a yellow solution. Anhydrous ethanol was then added to the yellow solution and stirred. The yellow solid obtained by suction filtration and drying was the hydrophobic azo polymer.
[0011] 3) Synthesis of amphiphilic azo polymer: The hydrophobic azo polymer obtained in step 2) is dissolved in DMF, polyethylene glycol is added to the solution, and the mixture is stirred at room temperature for 30 minutes. After the solid is dissolved, catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are added to react at 35° C. for 48 hours. After the reaction is completed, the solution is dialyzed three times, and finally lyophilized to obtain a yellow solid, which is the amphiphilic azo polymer.
[0012] Furthermore, in the above preparation method, in step 1), the amount of p-aminobenzoic acid used is 13.7 g, the amount of concentrated hydrochloric acid used is 33.6 mL, the amount of sodium nitrite used is 6.9 g, the amount of sodium acetate used is 60 g, the amount of aniline sulfite used is 21.1 g, and the amount of sodium hydroxide used is 15 g.
[0013] Furthermore, in the above preparation method, in step 2), the amount of N6-Cbz-L-lysine used is 6 g, the amount of triphosgene used is 2 g, the amount of Lys(z)-NCA used is 5.2 g, and the amount of 4-((4-aminophenyl)diazenyl)benzoic acid solution used is 0.18 g.
[0014] Furthermore, in the above preparation method, in step 2), the volume of n-hexane is 500 mL.
[0015] Furthermore, in the above preparation method, in step 3), the amount of hydrophobic azo polymer used is 3.6 g, the amount of polyethylene glycol used is 1.766 g, and the molecular weight of polyethylene glycol is 2000 Mv; the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine used is 0.5 g each.
[0016] Furthermore, in the above preparation method, in step 3), the dialysis uses a dialysis bag with a molecular weight cutoff of 3000Da.
[0017] A amphiphilic azo polymer micelle, the preparation method of which comprises the following steps: dissolving 0.5 g of the above-mentioned amphiphilic azo polymer in 10 mL of acetone solution, magnetically stirring for 30 minutes, slowly dropping 250 mL of deionized water, stirring for overnight reaction, centrifuging at 10000 rpm for 5 minutes after the reaction is completed, and vacuum freeze-drying to obtain micelle powder.
[0018] Application of the above-mentioned amphiphilic azo polymer micelles in drug carriers.
[0019] Furthermore, the above application is as follows: 5 mg of doxorubicin hydrochloride is dissolved in 10 mL of acetone solution, and then 20 mg of the amphiphilic azo polymer is dissolved in the above acetone solution, and after magnetic stirring, 250 mL of deionized water is slowly added dropwise, and the reaction is stirred overnight. After the acetone evaporates, the solution is placed in a dialysis bag with a molecular weight cutoff of 1000 Da for dialysis to remove excess doxorubicin hydrochloride; then centrifuged at 10000 rpm for 5 minutes, and freeze-dried to obtain the drug-loaded amphiphilic azo polymer.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] 1. The amphiphilic azo polymer of the present invention utilizes N6-Cbz-L-lysine as the hydrophobic end and synthesizes the hydrophilic end with hydroxyl group through lipidation reaction, and the preparation method is simple.
[0022] 2. The amphiphilic azo polymer micelles of the present invention are in the form of regular spheres with uniform size and good dispersibility. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 1 is a structural diagram of the amphiphilic azo polymer prepared in Example 1.
[0024] Figure 2 These are infrared absorption spectra of (a) 4-((4-aminophenyl)diazenyl)benzoic acid, (b) hydrophobic azo polymer, and (c) amphiphilic azo polymer obtained in Example 1. DETAILED DESCRIPTION
[0025] In order to further understand the present invention, the preferred experimental scheme of the present invention is described below in conjunction with embodiments, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the requirements of the present invention.
[0026] Example 1 Preparation of amphiphilic azo polymer
[0027] 1) Synthesis of 4-((4-aminophenyl)diazenyl)benzoic acid: 13.7 g of p-aminobenzoic acid was dissolved in 30 mL of deionized water, and 33.6 mL of concentrated hydrochloric acid was added and stirred for 20 minutes. After stirring, the mixture was cooled in an ice bath. 6.9 g of sodium nitrite was weighed and dissolved in 10 mL of deionized water. The dissolved sodium nitrite solution was cooled in an ice bath and the temperature was maintained at 0-5°C. The cooled sodium nitrite solution was slowly added dropwise to the p-aminobenzoic acid solution in the ice bath and reacted for 15 minutes. The solution after the reaction was extracted and Filter, retain the filtrate and store it in an ice bath; take another large beaker, dissolve 60g of sodium acetate and 21.1g of aniline sulfite in 25mL of deionized water, keep the solution temperature at 0-5°C, then add the filtrate dropwise to the large beaker and stir for 1 hour, adjust the pH to maintain acidity, filter and dry; after drying, react with sodium hydroxide solution (15g of sodium hydroxide dissolved in 50mL of deionized water) at 90°C for 3 hours, finally adjust the solution pH to 6 in an ice bath, filter and dry to obtain 4-((4-aminophenyl)diazenyl)benzoic acid.
[0028] 2) Synthesis of hydrophobic azo polymer: 6 g of N6-Cbz-L-lysine dissolved in 50 mL of THF was added to a round-bottom flask and stirred for 15 minutes. 2 g of triphosgene dissolved in 50 mL of THF was slowly added dropwise to the round-bottom flask and reacted for 2 hours. The reaction solution was added to 500 mL of n-hexane and refrigerated overnight. Lys(z)-NCA was obtained by suction filtration and vacuum drying. 5.2 g of the dried white solid Lys(z)-NCA and 0.18 g of 4-((4-aminophenyl)diazenyl)benzoic acid obtained in step 1) were dissolved in DMF and stirred at room temperature for 36 hours to form a yellow solution. Anhydrous ethanol was then added to the yellow solution and stirred. The yellow solid obtained by suction filtration and drying was the hydrophobic azo polymer.
[0029] 3) Synthesis of amphiphilic azo polymer: 3.6 g of the hydrophobic azo polymer obtained in step 2) was dissolved in 50 mL of DMF, and 1.776 g of polyethylene glycol (molecular weight 2000 Mv) was added to the solution, and stirred at room temperature for 30 minutes. After the solid was dissolved, 0.5 g of catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added, and the mixture was reacted at 35° C. for 48 hours. After the reaction was completed, the solution was dialyzed three times (using a dialysis bag with a molecular weight cutoff of 3000 Da), and finally freeze-dried to obtain a yellow solid, which was an amphiphilic azo polymer (whose structural formula is as follows: Figure 1 ).
[0030] Figure 2 The infrared absorption spectra of (a) 4-((4-aminophenyl)diazenyl)benzoic acid, (b) hydrophobic azo polymer, and (c) amphiphilic azo polymer prepared in Example 1 are shown in FIG. Figure 2It can be seen that the CH2 absorption peak appears in the (b) curve, indicating that the hydrophobic end has been connected, and the ether bond absorption peak appears in (c), indicating that the polyethylene glycol is successfully connected.
[0031] Example 2 Preparation of amphiphilic azo polymer micelles
[0032] Weigh 0.5 g of the amphiphilic azo polymer and dissolve it in 10 mL of acetone solution, stir thoroughly until dissolved; stir magnetically for 30 minutes, slowly add 250 mL of deionized water, and stir to react overnight; after the reaction is completed, place the reaction solution in a centrifuge at 10,000 rpm for 5 minutes, and finally place it in a freeze dryer for freeze drying.
[0033] Example 3 Preparation of drug-loaded amphiphilic azo polymer
[0034] 5 mg of doxorubicin hydrochloride was dissolved in 10 mL of acetone solution, and then 20 mg of the amphiphilic azo polymer was weighed and dissolved in the above acetone solution. After magnetic stirring, 250 mL of deionized water was slowly added dropwise, and the reaction was stirred overnight. After the acetone evaporated, the solution was dialyzed in a dialysis bag with a molecular weight of 1000 Da to remove excess doxorubicin hydrochloride; then centrifuged at 10000 rpm for 5 minutes, and finally freeze-dried to obtain the drug-loaded amphiphilic azo polymer.
[0035] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. An amphiphilic azo polymer, characterized in that The amphiphilic azo polymer is based on 4-((4-aminophenyl)diazenyl)benzoic acid as an intermediate, the hydrophobic end of which is poly-N6-Cbz-L-lysine, and the hydrophilic end of which is polyethylene glycol.
2. The method for preparing an amphiphilic azo polymer according to claim 1, characterized in that: The steps include: 1) Synthesis of 4-((4-aminophenyl)diazenyl)benzoic acid: dissolve p-aminobenzoic acid in deionized water, add concentrated hydrochloric acid and stir for 20 minutes, and cool in an ice bath after stirring; dissolve sodium nitrite in deionized water, and cool the dissolved sodium nitrite solution in an ice bath, maintaining the temperature at 0-5°C; slowly drop the cooled sodium nitrite solution into the p-aminobenzoic acid solution in the ice bath, and react for 15 minutes; filter the solution after the reaction, retain the filtrate and store it in an ice bath; take another large beaker, dissolve sodium acetate and aniline sulfite in deionized water, maintain the solution temperature at 0-5°C, then drop the filtrate into the large beaker, stir for 1 hour, adjust the pH to maintain acidity, filter and dry; after drying, react with sodium hydroxide solution at 90°C for 3 hours, finally adjust the solution pH to 6 in an ice bath, filter and dry, and obtain 4-((4-aminophenyl)diazenyl)benzoic acid; 2) Synthesis of hydrophobic azo polymer: N6-Cbz-L-lysine dissolved in THF was added to a round-bottom flask and stirred for 15 minutes. Triphosgene dissolved in THF was slowly added dropwise to the round-bottom flask for reaction for 2 hours. The solution after the reaction was added to n-hexane and refrigerated overnight. The white solid Lys(z)-NCA was obtained by suction filtration and vacuum drying. The dried white solid Lys(z)-NCA and 4-((4-aminophenyl)diazenyl)benzoic acid obtained in step 1) were dissolved in DMF and stirred at room temperature for 36 hours to form a yellow solution. Anhydrous ethanol was then added to the yellow solution and stirred. The yellow solid obtained by suction filtration and drying was the hydrophobic azo polymer. 3) Synthesis of amphiphilic azo polymer: The hydrophobic azo polymer obtained in step 2) is dissolved in DMF, polyethylene glycol is added to the solution, and the mixture is stirred at room temperature for 30 minutes. After the solid is dissolved, catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are added to react at 35° C. for 48 hours. After the reaction is completed, the solution is dialyzed three times, and finally lyophilized to obtain a yellow solid, which is the amphiphilic azo polymer.
3. The preparation method according to claim 2, characterized in that: In step 1), the amount of p-aminobenzoic acid used is 13.7 g, the amount of concentrated hydrochloric acid used is 33.6 mL, the amount of sodium nitrite used is 6.9 g, the amount of sodium acetate used is 60 g, the amount of aniline sulfite used is 21.1 g, and the amount of sodium hydroxide used is 15 g.
4. The preparation method according to claim 2, characterized in that: In step 2), the amount of N6-Cbz-L-lysine used is 6 g, the amount of triphosgene used is 2 g, the amount of Lys(z)-NCA used is 5.2 g, and the amount of 4-((4-aminophenyl)diazenyl)benzoic acid solution used is 0.18 g.
5. The preparation method according to claim 2, characterized in that: In step 2), the volume of n-hexane is 500 mL.
6. The preparation method according to claim 2, characterized in that: In step 3), the amount of the hydrophobic azo polymer used is 3.6 g, the amount of polyethylene glycol used is 1.766 g, and the molecular weight of the polyethylene glycol is 2000 Mv; the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine used is 0.5 g each.
7. The preparation method according to claim 2, characterized in that: In step 3), the dialysis uses a dialysis bag with a molecular weight cutoff of 3000Da.
8. An amphiphilic azo polymer micelle, characterized in that The preparation method comprises the following steps: dissolving 0.5 g of the amphiphilic azo polymer according to claim 1 in 10 mL of acetone solution, stirring magnetically for 30 minutes, slowly dropping 250 mL of deionized water, stirring and reacting overnight, centrifuging at 10,000 rpm for 5 minutes after the reaction is completed, and vacuum freeze-drying to obtain micelle powder.
9. Use of the amphiphilic azo polymer micelle according to claim 8 in a drug carrier.
10. The use according to claim 9, characterized in that: The method is as follows: 5 mg of doxorubicin hydrochloride is dissolved in 10 mL of acetone solution, and then 20 mg of an amphiphilic azo polymer is dissolved in the above acetone solution. After magnetic stirring, 250 mL of deionized water is slowly added dropwise, and the reaction is stirred overnight. After the acetone evaporates, the solution is placed in a dialysis bag with a molecular weight cutoff of 1000 Da for dialyzing to remove excess doxorubicin hydrochloride; then, the solution is centrifuged at 10,000 rpm for 5 minutes, and the drug-loaded amphiphilic azo polymer is obtained by freeze drying.