Preparation process of sodium azulene sulfonate
By using uric acid-modified chitosan and sodium azulene sulfonate to form nanomicelles, the problems of stability and low transdermal absorption of sodium azulene sulfonate formulations were solved, achieving efficient targeted drug release and anti-inflammatory repair effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG HUASU PHARM CO LTD
- Filing Date
- 2026-04-28
- Publication Date
- 2026-06-09
AI Technical Summary
Sodium azulene sulfonate has poor stability in formulations, low transdermal absorption, and lacks targeted release properties, resulting in limited alleviation effects with existing technologies.
Uric acid-modified chitosan and sodium azulene sulfonate were used to form nanomicelles through self-assembly. The electrostatic complexing and hydrophobic self-assembly of uric acid-modified chitosan were utilized, combined with the protonated charge repulsion of the imidazole ring in slightly acidic inflammatory tissue, to achieve targeted release.
It significantly improves the physicochemical stability of sodium azulene sulfonate, extends shelf life, and enables precise drug release in deep inflamed tissues, exhibiting synergistic anti-inflammatory and mucosal repair effects.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical formulation technology, and specifically to a preparation process for sodium azulene sulfonate. Background Technology
[0002] Sodium azulene sulfonate is a water-soluble azulene derivative with excellent anti-inflammatory, anti-allergic and mucosal repair effects, and is widely used in oral care, ophthalmic and topical dermatological preparations.
[0003] However, sodium azulene sulfonate faces significant technical bottlenecks in its formulation applications. Firstly, the conjugated double bond structure in the sodium azulene sulfonate molecule makes it highly sensitive to light, heat, and oxygen. During storage, especially in aqueous solution, it is highly susceptible to oxidative degradation, causing the characteristic blue color of the formulation to fade rapidly and the effective content to decrease. This not only affects efficacy but also significantly limits the product's shelf life.
[0004] Secondly, as a water-soluble salt compound, sodium azulene sulfonate has poor penetration through the skin or mucous membranes, making it difficult to reach deep inflamed tissues, which limits its efficacy in treating deep inflammation or in scenarios requiring high-concentration local administration.
[0005] Existing technologies primarily address stability issues by adding antioxidants or using light-shielding packaging, but with limited effectiveness. While traditional liposome carriers can improve encapsulation to some extent, they often suffer from low encapsulation rates and easy leakage during storage, especially for highly water-soluble sodium azulene sulfonate. Based on the above, this invention proposes a preparation process for sodium azulene sulfonate. Summary of the Invention
[0006] To address the technical problems of poor stability, low transdermal absorption rate, and lack of targeted release characteristics in existing sodium azulene sulfonate formulations, this invention proposes a preparation process for sodium azulene sulfonate.
[0007] This invention provides a preparation process for sodium azulene sulfonate, employing the following technical solution: A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: Sodium azulene sulfonate and modified polysaccharide derivatives are added to a solvent and stirred until completely dissolved to form a homogeneous organic phase solution; (2) Self-assembly micellization: The organic phase solution obtained in step (1) is added dropwise to deionized water under stirring to induce the self-assembly of the modified polysaccharide derivative and form a nano micelle dispersion. (3) Post-processing: The nano micelle dispersion obtained in step (2) is dialyzed to remove the solvent and unencapsulated free drug to obtain a nano micelle solution; a lyophilization protectant is added to the nano micelle solution, and the solution is freeze-dried to obtain a lyophilized powder injection loaded with sodium azulene sulfonate.
[0008] Preferably, in step (1), the mass ratio of sodium azulene sulfonate, modified polysaccharide derivative and solvent is 1:(3-5):(80-100).
[0009] Preferably, in step (1), the modified polysaccharide derivative is uric acid-modified chitosan.
[0010] Preferably, the uric acid-modified chitosan is prepared by the following method: Chitosan was dissolved in a weakly acidic aqueous solution, and uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added and stirred. After mixing, an amidation coupling reaction was carried out. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain uric acid-modified chitosan.
[0011] Preferably, the mass ratio of chitosan, weakly acidic aqueous solution, uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 10:(400-600):(2-3):(2-4):(1.5-2.5).
[0012] Preferably, the weakly acidic aqueous solution is an acetic acid aqueous solution with a mass concentration of 0.8-1.5%.
[0013] Preferably, the stirring speed is 300-500 rpm, the reaction temperature is 20-25℃, and the reaction time is 12-14 h.
[0014] Preferably, the grafting rate of the uric acid-modified chitosan is 15-25%.
[0015] Preferably, in step (1), the solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, methanol, and ethanol.
[0016] Preferably, in step (2), the mass ratio of the organic phase solution to deionized water is 1:(10-12).
[0017] Preferably, in step (2), the dripping condition is to drip at a constant rate and complete the dripping within 0.5-1h.
[0018] Preferably, in step (2), the stirring rate is 600-800 rpm.
[0019] Preferably, in step (2), the self-assembly process is carried out under light-protected conditions and the temperature is controlled at 20-40℃.
[0020] Preferably, in step (3), dialysis refers to: placing the nanomicelle dispersion in a dialysis bag with a molecular weight cutoff of 5000-8000 Da, and dialyzing with deionized water for 12-24 hours under light-protected conditions, with the deionized water being replaced every 4-6 hours during the process.
[0021] Preferably, in step (3), the amount of freeze-drying protectant added is 3-5% of the mass of the nano micelle solution.
[0022] Preferably, in step (3), the freeze-drying protectant is trehalose or mannitol.
[0023] Preferably, in step (3), the freeze-drying conditions are: first pre-freezing at -40 to -50℃ for 4-6 hours, and then drying at -70 to -80℃ for 24-36 hours.
[0024] In summary, the present invention has the following beneficial effects: 1. This invention solves the technical problem of low encapsulation efficiency and easy leakage of water-soluble drugs in conventional liposomes by forming a strong electrostatic complexation between the amino group in chitosan modified with uric acid and the sulfonate group of sodium azulene sulfonate, supplemented by the hydrophobic self-assembly of uric acid, thus achieving effective encapsulation of highly water-soluble sodium azulene sulfonate.
[0025] 2. This invention utilizes the dense polymer shell of nanomicelles to greatly block the damage of conjugated double bonds by light, heat and oxygen, significantly improving the physicochemical stability of sodium azulene sulfonate and extending the product's shelf life.
[0026] 3. The imidazole ring in the uric acid structure introduced in this invention can remain stable in a physiologically neutral environment; when it enters slightly acidic inflammatory tissue, the imidazole ring is protonated, generating charge repulsion, which leads to micelle swelling and disintegration, achieving targeted release and endowing sodium azulene sulfonate preparations with precise pH-responsive release characteristics in the inflammatory microenvironment.
[0027] 4. The chitosan backbone introduced in this invention possesses natural antibacterial and tissue repair-promoting functions. When delivered in combination with sodium azulene sulfonate, it can exert a synergistic therapeutic effect at deep inflammatory lesions, thereby achieving unexpected synergistic anti-inflammatory and mucosal repair effects. The preparation process of this invention is simple, and the raw materials used are natural, inexpensive, and biocompatible, making it easy to scale up industrial production. Detailed Implementation
[0028] The present invention will be further described in detail below with reference to the embodiments.
[0029] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.
[0030] Among them, chitosan: CAS No. 9012-76-4, degree of deacetylation 85%, brand name Yuanye, product number S24914, purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0031] Preparation Examples 1-3 and Comparative Preparation Examples 1-2 provide methods for preparing uric acid-modified chitosan.
[0032] Preparation Example 1 Uric acid-modified chitosan is prepared by the following method: The mass ratio of chitosan, aqueous acetic acid, uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was controlled at 10:500:2.5:3:2. Chitosan was dissolved in a 1.2% (w / w) aqueous acetic acid solution. Uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were then added. The mixture was stirred at 400 rpm and subjected to an amidation coupling reaction at 22°C for 13 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 3 days. The dialysate was then freeze-dried at -60°C for 18 h to obtain uric acid-modified chitosan. The grafting rate of the uric acid-modified chitosan was determined to be 20.1% by 1H NMR spectroscopy.
[0033] Preparation Example 2 Uric acid-modified chitosan is prepared by the following method: The mass ratio of chitosan, aqueous acetic acid, uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was controlled at 10:400:2:2:1.5. Chitosan was dissolved in a 0.8% (w / w) aqueous acetic acid solution. Uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were added, and the mixture was stirred at 300 rpm. An amidation coupling reaction was then carried out at 20°C for 14 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 3 days. The dialysate was then freeze-dried at -60°C for 18 h to obtain uric acid-modified chitosan. The grafting rate of the uric acid-modified chitosan was determined to be 15.3% by 1H NMR spectroscopy.
[0034] Preparation Example 3 Uric acid-modified chitosan is prepared by the following method: The mass ratio of chitosan, aqueous acetic acid, uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was controlled at 10:600:3:4:2.5. Chitosan was dissolved in a 1.5% (w / w) aqueous acetic acid solution. Uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were then added. The mixture was stirred at 500 rpm and subjected to an amidation coupling reaction at 25°C for 12 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 3 days. The dialysate was then freeze-dried at -60°C for 18 h to obtain uric acid-modified chitosan. The grafting rate of the uric acid-modified chitosan was determined to be 24.8% by 1H NMR spectroscopy.
[0035] Comparative Preparation Example 1 Uric acid-modified chitosan is prepared by the following method: The mass ratio of chitosan, aqueous acetic acid, uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was controlled at 10:500:0.5:0.8:0.5. Chitosan was dissolved in a 1.2% (w / w) aqueous acetic acid solution. Uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were added, and the mixture was stirred at 400 rpm. An amidation coupling reaction was then carried out at 22°C for 13 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 3 days. The dialysate was then freeze-dried at -60°C for 18 h to obtain uric acid-modified chitosan. The grafting rate of the uric acid-modified chitosan was determined to be 8.5% by 1H NMR spectroscopy.
[0036] Comparative Preparation Example 2 Uric acid-modified chitosan is prepared by the following method: The mass ratio of chitosan, aqueous acetic acid, uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was controlled at 10:500:5:6:5. Chitosan was dissolved in a 1.2% (w / w) aqueous acetic acid solution. Uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were then added. The mixture was stirred at 400 rpm and subjected to an amidation coupling reaction at 22°C for 13 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 3 days. The dialysate was then freeze-dried at -60°C for 18 h to obtain uric acid-modified chitosan. The grafting rate of the uric acid-modified chitosan was determined to be 32.6% by 1H NMR spectroscopy.
[0037] Examples 1-3 provide a process for preparing sodium azulene sulfonate.
[0038] Example 1 A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, uric acid modified chitosan and N,N-dimethylformamide was controlled to be 1:4:90. Sodium azulene sulfonate and uric acid modified chitosan (obtained from Preparation Example 1) were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Self-assembly micellization: The mass ratio of organic phase solution to deionized water is controlled at 1:11. Under light-protected conditions, the temperature is 30℃ and the stirring speed is 700rpm. The organic phase solution obtained in step (1) is added dropwise to the deionized water under stirring at a constant rate and controlled to be completed within 0.8h. This induces the self-assembly of the modified polysaccharide derivative to form a nano micelle dispersion. (3) Post-processing: The nano micelle dispersion obtained in step (2) was placed in a dialysis bag with a molecular weight cutoff of 6500 Da. Under light-protected conditions, it was dialyzed with deionized water for 18 hours, and the deionized water was replaced every 5 hours to remove the solvent and unencapsulated free drug, so as to obtain a nano micelle solution. 4% of mannitol by mass was added to the nano micelle solution, and after stirring and mixing evenly, it was pre-frozen at -45℃ for 5 hours and then dried at -75℃ for 30 hours to obtain a lyophilized powder injection loaded with sodium azulenate.
[0039] Example 2 A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, uric acid modified chitosan and N,N-dimethylformamide was controlled to be 1:3:80. Sodium azulene sulfonate and uric acid modified chitosan (obtained from preparation example 2) were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Self-assembly micellization: The mass ratio of organic phase solution to deionized water is controlled at 1:10. Under light-protected conditions, the temperature is 20℃ and the stirring speed is 600rpm. The organic phase solution obtained in step (1) is added dropwise to the deionized water under stirring at a constant rate and the addition is completed within 1h. This induces the self-assembly of the modified polysaccharide derivative to form a nano micelle dispersion. (3) Post-processing: The nano micelle dispersion obtained in step (2) was placed in a dialysis bag with a molecular weight cutoff of 5000 Da. Under light-protected conditions, it was dialyzed with deionized water for 24 hours. During this period, the deionized water was replaced every 6 hours to remove the solvent and unencapsulated free drug, and a nano micelle solution was obtained. 3% of mannitol by mass was added to the nano micelle solution. After stirring and mixing evenly, it was pre-frozen at -40℃ for 6 hours and then dried at -70℃ for 36 hours to obtain a lyophilized powder injection loaded with sodium azulenate.
[0040] Example 3 A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, uric acid modified chitosan and N,N-dimethylformamide was controlled to be 1:5:100. Sodium azulene sulfonate and uric acid modified chitosan (obtained from preparation example 3) were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Self-assembly micellization: The mass ratio of organic phase solution to deionized water is controlled at 1:12. Under light-protected conditions, the temperature is 40℃ and the stirring rate is 800rpm. The organic phase solution obtained in step (1) is added dropwise to the deionized water under stirring at a constant rate and the addition is completed within 0.5h. This induces the self-assembly of the modified polysaccharide derivative to form a nano micelle dispersion. (3) Post-processing: The nano micelle dispersion obtained in step (2) was placed in a dialysis bag with a molecular weight cutoff of 8000 Da. Under light-protected conditions, it was dialyzed with deionized water for 12 hours. During this period, the deionized water was replaced every 4 hours to remove the solvent and unencapsulated free drug, and a nano micelle solution was obtained. 5% of mannitol by mass was added to the nano micelle solution. After stirring and mixing evenly, it was pre-frozen at -50℃ for 4 hours and then dried at -80℃ for 24 hours to obtain a lyophilized powder injection loaded with sodium azulenate.
[0041] To verify the comprehensive performance of the sodium azulene sulfonate prepared by the method of the present invention, comparative examples 1-5 were set up, wherein: Comparative Example 1 Comparative Example 1 is the same as Example 1, except that the uric acid-modified chitosan was prepared from Comparative Preparation Example 1. Details are as follows: A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, uric acid modified chitosan and N,N-dimethylformamide was controlled to be 1:4:90. Sodium azulene sulfonate and uric acid modified chitosan (prepared from Comparative Preparation Example 1) were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Self-assembly micellization: The mass ratio of organic phase solution to deionized water is controlled at 1:11. Under light-protected conditions, the temperature is 30℃ and the stirring speed is 700rpm. The organic phase solution obtained in step (1) is added dropwise to the deionized water under stirring at a constant rate and controlled to be completed within 0.8h. This induces the self-assembly of the modified polysaccharide derivative to form a nano micelle dispersion. (3) Post-processing: The nano micelle dispersion obtained in step (2) was placed in a dialysis bag with a molecular weight cutoff of 6500 Da. Under light-protected conditions, it was dialyzed with deionized water for 18 hours, and the deionized water was replaced every 5 hours to remove the solvent and unencapsulated free drug, so as to obtain a nano micelle solution. 4% of mannitol by mass was added to the nano micelle solution, and after stirring and mixing evenly, it was pre-frozen at -45℃ for 5 hours and then dried at -75℃ for 30 hours to obtain a lyophilized powder injection loaded with sodium azulenate.
[0042] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that the uric acid-modified chitosan was prepared from Comparative Example 2. Details are as follows: A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, uric acid modified chitosan and N,N-dimethylformamide was controlled to be 1:4:90. Sodium azulene sulfonate and uric acid modified chitosan (obtained from Comparative Preparation Example 2) were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Self-assembly micellization: The mass ratio of organic phase solution to deionized water is controlled at 1:11. Under light-protected conditions, the temperature is 30℃ and the stirring speed is 700rpm. The organic phase solution obtained in step (1) is added dropwise to the deionized water under stirring at a constant rate and controlled to be completed within 0.8h. This induces the self-assembly of the modified polysaccharide derivative to form a nano micelle dispersion. (3) Post-processing: The nano micelle dispersion obtained in step (2) was placed in a dialysis bag with a molecular weight cutoff of 6500 Da. Under light-protected conditions, it was dialyzed with deionized water for 18 hours, and the deionized water was replaced every 5 hours to remove the solvent and unencapsulated free drug, so as to obtain a nano micelle solution. 4% of mannitol by mass was added to the nano micelle solution, and after stirring and mixing evenly, it was pre-frozen at -45℃ for 5 hours and then dried at -75℃ for 30 hours to obtain a lyophilized powder injection loaded with sodium azulenate.
[0043] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that the uric acid-modified chitosan is replaced with chitosan in equal quantities. Details are as follows: A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, chitosan and N,N-dimethylformamide was controlled to be 1:4:90. Sodium azulene sulfonate and chitosan were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Mixing and dispersing: The mass ratio of organic phase solution to deionized water is controlled at 1:11. Under light-protected conditions, the temperature is 30℃ and the stirring speed is 700rpm. The organic phase solution obtained in step (1) is added dropwise to the deionized water under stirring at a constant rate and controlled to be completed within 0.8h, so that sodium azulene sulfonate and chitosan are mixed and precipitated to form a mixed dispersion. (3) Post-processing: The mixed dispersion obtained in step (2) was placed in a dialysis bag with a molecular weight cutoff of 6500 Da. Under light-protected conditions, it was dialyzed with deionized water for 18 hours. During this period, the deionized water was replaced every 5 hours to remove the solvent and unencapsulated free drug, and a mixed solution was obtained. 4% of mannitol by mass was added to the mixed solution. After stirring and mixing evenly, it was pre-frozen at -45℃ for 5 hours and then dried at -75℃ for 30 hours to obtain a mixed lyophilized powder injection.
[0044] Comparative Example 4 Comparative Example 4 is the same as Example 1, except that the self-assembly micellization operation in step (2) and the dialysis treatment in step (3) are omitted. Specifically: A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, uric acid modified chitosan and N,N-dimethylformamide was controlled to be 1:4:90. Sodium azulene sulfonate and uric acid modified chitosan (obtained from Preparation Example 1) were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Post-processing: Add 4% of mannitol by mass to the organic phase solution obtained in step (1), stir and mix evenly, pre-freeze at -45℃ for 5h, and then dry at -75℃ for 30h to obtain lyophilized powder injection loaded with sodium azulene sulfonate.
[0045] Comparative Example 5 Comparative Example 5 is the same as Example 1, except that the uric acid-modified chitosan in step (1) is replaced by a conventional liposome carrier (prepared by mixing soybean lecithin and cholesterol in a mass ratio of 4:1). Specifically: A process for preparing sodium azulene sulfonate includes the following steps: (1) Organic phase preparation: The mass ratio of sodium azulene sulfonate, conventional liposome carrier and N,N-dimethylformamide was controlled to be 1:4:90. Sodium azulene sulfonate and conventional liposome carrier (prepared by mixing soybean lecithin and cholesterol at a mass ratio of 4:1) were added to N,N-dimethylformamide and stirred until completely dissolved to form a homogeneous organic phase solution. (2) Liposome self-assembly: The mass ratio of organic phase solution to deionized water is controlled at 1:11. Under light-protected conditions, the temperature is 30℃ and the stirring speed is 700rpm. The organic phase solution obtained in step (1) is added dropwise to the deionized water under stirring at a constant rate and controlled to be completed within 0.8h. This induces the self-assembly of liposome carriers to form a liposome dispersion. (3) Post-processing: The liposome dispersion obtained in step (2) was placed in a dialysis bag with a molecular weight cutoff of 6500 Da. Under light-protected conditions, it was dialyzed with deionized water for 18 hours, and the deionized water was replaced every 5 hours to remove the solvent and unencapsulated free drug to obtain a liposome solution. 4% of mannitol by mass was added to the liposome solution, and after stirring and mixing evenly, it was pre-frozen at -45℃ for 5 hours and then dried at -75℃ for 30 hours to obtain a lyophilized powder injection loaded with sodium azulenate.
[0046] The comprehensive properties of sodium azulene sulfonate prepared in Examples 1-3 and Comparative Examples 1-5 of this invention were tested respectively.
[0047] 1. Encapsulation efficiency test: Accurately weigh 10 mg of each group of lyophilized powder, add 10 mL of deionized water to reconstitute, and prepare a homogeneous reconstituted solution. Take 1 mL of the reconstituted solution and place it in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000 Da. Centrifuge at 4000 rpm for 15 min to separate the free sodium azulene sulfonate, and collect the lower filtrate. Determine the free drug amount using high performance liquid chromatography (HPLC). Encapsulation efficiency (%) = (Total drug amount - Free drug amount) / Total drug amount × 100%.
[0048] 2. Photostability test: Each group of lyophilized powders was reconstituted with deionized water to prepare an aqueous solution containing 100 μg / mL sodium azulene sulfonate, and then exposed in a light chamber with a light intensity of 4500 lx for 48 h. The sodium azulene sulfonate content before and after exposure was determined by HPLC, and the retention rate (%) was calculated as (content after exposure / initial content) × 100%.
[0049] 3. pH-responsive release test of the inflammatory microenvironment: Using dynamic dialysis, 5 mL of reconstituted solution (containing 100 μg / mL sodium azulene sulfonate) was precisely measured and placed in a dialysis bag (molecular weight cutoff 3500 Da). The dialysis bag was completely immersed in 50 mL of phosphate buffer solution at pH 7.4 (simulating normal physiological environment) and pH 5.5 (simulating inflammatory microenvironment), respectively. The mixture was incubated for 12 h in a constant temperature shaker at 37 °C and 100 rpm. The release rate (%) was measured at regular intervals, and an equal volume of fresh buffer solution was added after each sampling.
[0050] The test results are shown in Table 1: Table 1: Comprehensive performance test data of sodium azulene sulfonate in Examples 1-3 and Comparative Examples 1-5 As shown in Table 1, the sodium azulene sulfonate prepared in Examples 1-3 of this invention all exhibited excellent comprehensive performance, achieving high encapsulation efficiency, excellent photostability, and significant pH-responsive targeted release characteristics. Their comprehensive performance was significantly better than that of Comparative Examples 1-5.
[0051] As can be seen from Example 1 and Comparative Example 1, when the grafting rate of uric acid-modified chitosan is too low, the hydrophobicity of the carrier is insufficient, resulting in a loose nanomicelle structure formed by self-assembly. Consequently, the water-soluble sodium azulene sulfonate is difficult to be effectively encapsulated, and the encapsulation rate drops significantly. At the same time, the loose structure cannot effectively block light, resulting in a decrease in photostability retention. Furthermore, due to the non-dense hydrophobic core, drug leakage is very likely to occur in a neutral environment of pH 7.4.
[0052] As can be seen from Example 1 and Comparative Example 2, when the grafting rate of uric acid-modified chitosan is too high, the polymer is too hydrophobic. Although it can maintain a high encapsulation rate to a certain extent, in the simulated inflammatory microenvironment at pH 5.5, the charge repulsion generated by the protonation of the imidazole ring is insufficient to overcome the excessive hydrophobic interaction, making it difficult for the micelles to swell and disintegrate. This significantly reduces the drug release rate in the inflammatory microenvironment and results in the loss of targeted release characteristics.
[0053] As can be seen from Example 1 and Comparative Example 3, when uric acid-modified chitosan is replaced with unmodified chitosan, the system cannot undergo amphiphilic self-assembly and form a dense micelle core-shell structure due to the lack of hydrophobic driving force provided by uric acid groups. As a result, highly water-soluble drugs can hardly be encapsulated, the drugs exist in a free state, have extremely poor photostability, and completely lose their pH-responsive properties.
[0054] As can be seen from Example 1 and Comparative Example 4: by omitting the self-assembly micellization step and directly mixing and lyophilizing the organic phase with the drug, only a physical mixture is obtained, and no nano-drug-loaded system is formed, with a drug encapsulation rate of 0; the drug is completely exposed and not only rapidly photodegrades, but also exhibits rapid physical dissolution in any pH buffer solution.
[0055] As can be seen from Example 1 and Comparative Example 5, when conventional liposomes are used as carriers, due to the extremely high water solubility of sodium azulene sulfonate, the drug is very easy to leak from the hydrophilic core of the liposome bilayer, resulting in an encapsulation rate of only 38.5%. At the same time, conventional liposomes lack environmental responsive groups and cannot adjust the release rate according to changes in environmental pH, thus they do not have the function of targeted release from the inflammatory microenvironment.
[0056] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.
Claims
1. A process for preparing sodium azulene sulfonate, characterized in that, Includes the following steps: (1) Organic phase preparation: Sodium azulene sulfonate and modified polysaccharide derivatives are added to a solvent and stirred until completely dissolved to form a homogeneous organic phase solution; (2) Self-assembly micellization: The organic phase solution obtained in step (1) is added dropwise to deionized water under stirring to induce the self-assembly of the modified polysaccharide derivative and form a nano micelle dispersion. (3) Post-processing: The nano micelle dispersion obtained in step (2) is dialyzed to remove the solvent and unencapsulated free drug to obtain a nano micelle solution; a lyophilization protectant is added to the nano micelle solution, and the solution is freeze-dried to obtain a lyophilized powder injection loaded with sodium azulene sulfonate. In step (1), the modified polysaccharide derivative is uric acid-modified chitosan.
2. The preparation process of sodium azulene sulfonate according to claim 1, characterized in that, In step (1), the mass ratio of sodium azulene sulfonate, modified polysaccharide derivative and solvent is 1:(3-5):(80-100).
3. The preparation process of sodium azulene sulfonate according to claim 1, characterized in that, In step (1), the uric acid-modified chitosan is prepared by the following method: Chitosan was dissolved in a weakly acidic aqueous solution, and uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added and stirred. After mixing, an amidation coupling reaction was carried out. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain uric acid-modified chitosan.
4. The preparation process of sodium azulene sulfonate according to claim 3, characterized in that, The mass ratio of chitosan, weakly acidic aqueous solution, uric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 10:(400-600):(2-3):(2-4):(1.5-2.5).
5. The preparation process of sodium azulene sulfonate according to claim 3, characterized in that, The grafting rate of the uric acid-modified chitosan is 15-25%.
6. The preparation process of sodium azulene sulfonate according to claim 1, characterized in that, In step (2), the mass ratio of the organic phase solution to deionized water is 1:(10-12).
7. The preparation process of sodium azulene sulfonate according to claim 1, characterized in that, In step (2), the dripping condition is to drip at a constant rate and complete the dripping within 0.5-1h.
8. The preparation process of sodium azulene sulfonate according to claim 1, characterized in that, In step (2), the self-assembly process is carried out under light-protected conditions and the temperature is controlled at 20-40℃.
9. The preparation process of sodium azulene sulfonate according to claim 1, characterized in that, In step (3), the amount of freeze-drying protectant added is 3-5% of the mass of the nano micelle solution; the freeze-drying protectant is trehalose or mannitol.
10. The preparation process of sodium azulene sulfonate according to claim 1, characterized in that, In step (3), the freeze-drying conditions are: first, pre-freeze at -40 to -50℃ for 4-6 hours, and then dry at -70 to -80℃ for 24-36 hours.