Targeted colon-loaded double-drug gel bead as well as preparation method and application thereof
By buried 5-ASA and RES/Hydroxyl-β-β-β-β-cyclone concentrated compounds in gel beads, and coated with shell glycogen, gel beads that targeted colon loaded dual drugs were solved There are technical Chinese medicines that are released in small intestines and the unstable problems of resveratrol, which realize the efficient colon target delivery and release effect.
Patent Information
- Application Number
- CN202411492338.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-01-28
AI Technical Summary
In the prior art, when 5-aminosalicylic acid (5-ASA) is used to treat ulcerative colitis, it is rapidly absorbed in the small intestine after direct oral administration, resulting in insufficient concentration of colorectal drugs, reduced efficacy, and prone to nephrotoxicity; Resveratrol (Res) is not stable in chemical properties and rapidly metabolized, and has a short half-life in the body after oral administration, making it difficult to achieve the effect of treating colorectal cancer and colitis.
The gel beads are used to use sodium alginate, pectin, pectin and calcium chloride, and bury 5-ASA and Res/hydroxyl-β-β-cyclical seizure compounds in their holes. , Gelside beads that targeted colon loading dual drugs.
A targeted colon delivery of 5-ASA and Res, reducing the release of drugs in the stomach and small intestine stage, improving the drug concentration and efficacy of the colon area, and have good drug stability and protection.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug delivery, and in particular relates to a colon-targeted dual-drug loaded gel bead and a preparation method and application thereof. Background Art
[0002] In recent years, ulcerative colitis has become a global public health burden due to its high incidence in developed countries and a substantial increase in its incidence in developing countries. Long-term ulcerative colitis is likely to develop into colorectal cancer, and the basic approach to treating ulcerative colitis has been focused on blocking the production of nonspecific inflammatory mediators through aminosalicylic acid and corticosteroids.
[0003] 5-aminosalicylic acid (5-ASA) is currently the first-line drug for the treatment of ulcerative colitis in clinical practice. It is used to treat mild to moderate ulcerative colitis and has the characteristics of good efficacy and good tolerance. However, after direct oral administration, it is rapidly absorbed by the stomach and small intestine, and only a small amount of the drug can reach the colorectum. At the same time, the drug absorbed into the body is prone to renal toxicity. The therapeutic effect is reduced and the adverse reactions are large.
[0004] Resveratrol (Res) is a hydrophobic polyphenol compound with anti-inflammatory, anti-obesity, antioxidant, anti-cancer, and autoimmune and colon disease inhibitory effects. It has attracted widespread attention for its potential therapeutic activity against colon diseases such as colorectal cancer and colitis. However, the rapid metabolism of Res in the upper gastrointestinal tract results in a short half-life in vivo after oral administration. In addition, Res is easily affected by ultraviolet rays, pH value and temperature, and its chemical properties are unstable. In addition, the low water solubility of Res seriously limits its application. Summary of the invention
[0005] In order to solve the above problems, the purpose of the present invention is to provide a colon-targeted dual-drug loaded gel bead and its preparation method and application. The dual-drug loaded gel bead provided by the present invention has a particle size range of 1 to 3 mm and has colon-targeted delivery and release functions.
[0006] In order to achieve the above object, the present invention adopts the following technical solution:
[0007] A colon-targeted dual-drug gel bead, wherein sodium alginate, pectin and calcium chloride are cross-linked to form a gel bead, and holes exist in the gel bead.
[0008] Resveratrol (Res) is complexed with hydroxypropyl-β-cyclodextrin to form a Res / hydroxypropyl-β-cyclodextrin inclusion complex with a hydrophobic barrel-shaped cavity structure. Resveratrol is included in the hydrophobic cavity.
[0009] Aminosalicylic acid (5-ASA) and / or Res / hydroxypropyl-β-cyclodextrin inclusion complex are embedded in the pores of the gel beads, and chitosan wraps the gel beads.
[0010] The present invention provides a method for preparing colon-targeted dual-drug loaded gel beads, comprising the following steps:
[0011] (1) Dissolve sodium alginate and pectin in deionized water to obtain a precursor solution.
[0012] (2) 5-aminosalicylic acid (5-ASA) and Res / HP-β-CD (Res / hydroxypropyl-β-cyclodextrin) inclusion complex are added to the precursor solution and completely homogenized to obtain a mixed solution.
[0013] (3) Use a syringe to drop the mixed solution into the calcium chloride solution to form gel beads after cross-linking.
[0014] (4) The gel beads are rinsed with distilled water and placed in a chitosan solution for stirring, and then rinsed with distilled water again. After drying, the gel beads loaded with dual drugs are obtained.
[0015] The Res / HP-β-CD inclusion complex is prepared by the following method: 5-20 mmol·L -1 Res(Res) ethanol solution was slowly dripped into 10~20mmol·L at a volume ratio of 1:5~10 -1 A mixed solution was obtained from an aqueous solution of HP-β-CD (hydroxypropyl-β-cyclodextrin), and the mixed solution was incubated in the dark for 3 to 6 hours and then placed in a refrigerator at 4°C overnight. The mixed solution after cold precipitation was then centrifuged (4000 to 6000 rpm, 10 to 15 minutes), and the precipitate was washed three times with anhydrous ethanol, and the washed precipitate was placed in a fume hood to slowly evaporate the ethanol, and finally the Res / HP-β-CD inclusion complex was obtained.
[0016] Furthermore, in the precursor solution, the mass concentration of sodium alginate is 2-5%, and the mass concentration of pectin is 1-3%.
[0017] Furthermore, the mass concentration of 5-ASA in the mixed solution is 0.1-0.5%, and the mass concentration of Res / HP-β-CD inclusion compound in the mixed solution is 0.3-1%.
[0018] Furthermore, the CaCl 2 The solution concentration is 2-5%, the cross-linking time is 3-4 hours, and CaCl is added by syringe. 2 The flow rate in the solution was 2 mL / min.
[0019] Furthermore, the mass concentration of the chitosan solution is 1-3%, and the stirring time is 20-40 minutes.
[0020] The present invention also provides the use of the colon-targeted dual-drug loaded gel beads in colon-targeted delivery and drug release.
[0021] The beneficial effects of the present invention are:
[0022] The present invention cross-links sodium alginate and pectin with calcium chloride to form gel beads, and then places the beads in a chitosan solution for coating through electrostatic interaction, thereby solving the problem of large-scale drug release in the small intestine stage of general gel beads, and improving the efficiency of colon-targeted drug delivery; the gel beads prepared by the present invention have a good protective effect on the stability of drugs targeting the colon, and release less drugs in the stomach and small intestine digestion stages, and release a large amount in the colon, so they have colon-targeted delivery characteristics. The present invention achieves co-loading of 5-ASA and Res by directly adding 5-ASA and Res / HP-β-CD to the gel beads, which helps the drugs to exert a synergistic effect. The preparation method has mild conditions, is non-toxic and environmentally friendly, and is economical and efficient. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the external morphology of the dual-drug loaded gel beads prepared in Example 1.
[0024] Figure 2 This is the SEM detection spectrum of the dual-drug loaded gel beads prepared in Example 1.
[0025] Figure 3 The infrared absorption spectra of the dual-drug loaded gel beads prepared in Example 1 and the non-drug loaded gel beads in the comparative example.
[0026] Figure 4 This is the in vitro drug release diagram of the dual-drug loaded gel beads prepared in Example 1.
[0027] Figure 5 These are the results of the colon length of each group of experimental mice measured in Example 5. DETAILED DESCRIPTION
[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0029] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.
[0030] The Res / HP-β-CD inclusion complex used in the following examples was prepared by the following method: 5-20 mmol·L -1Res(Res) ethanol solution was slowly dripped into 10~20mmol·L at a volume ratio of 1:5~10 -1 A mixed solution was obtained from an aqueous solution of HP-β-CD (hydroxypropyl-β-cyclodextrin), and the mixed solution was incubated in the dark for 3 to 6 hours and then placed in a refrigerator at 4°C overnight. The mixed solution after cold precipitation was then centrifuged (4000 to 6000 rpm, 10 to 15 minutes), and the precipitate was washed three times with anhydrous ethanol, and the washed precipitate was placed in a fume hood to slowly evaporate the ethanol, and finally the Res / HP-β-CD inclusion complex was obtained.
[0031] Example 1
[0032] This embodiment provides a method for preparing dual-drug loaded gel beads, comprising the following steps:
[0033] First, 3g of sodium alginate and 1g of pectin were dissolved in deionized water and fixed to 100mL to obtain a precursor solution. Then, 0.2g of 5-ASA and 0.6g of Res / HP-β-CD (Res / hydroxypropyl-β-cyclodextrin) inclusion compound were added to the precursor solution and completely homogenized to form a mixed solution. Then, a 3% calcium chloride solution was dripped into the mixed solution with a syringe, and the gel beads were taken out after cross-linking for 4h. The obtained gel beads were rinsed with distilled water and placed in a 2.5% chitosan solution with a mass concentration and stirred for 1h, then rinsed with distilled water, and dried to obtain gel beads loaded with dual drugs.
[0034] The encapsulation efficiency and drug loading of the prepared dual-drug loaded gel beads were measured. The results showed that the encapsulation efficiency and drug loading of the gel beads for 5-ASA were 61.8% and 3.7%, respectively, and the encapsulation efficiency and drug loading of resveratrol (Res) were 58.6% and 2.9%, respectively.
[0035] The external morphology of the gel beads prepared in Example 1 is as follows Figure 1 As shown in the figure, the gel beads are nearly spherical, uniform in size, brownish yellow, and about 1-2 mm in diameter. The particle size can be adjusted by adjusting the sodium alginate, pectin, CaCl 2 The concentration ratio, syringe needle size and drop rate are controlled.
[0036] The gel beads prepared in Example 1 were scanned by SEM (see Figure 2 ). It can be seen that there are a lot of holes in the gel beads, which can provide enough space for drug embedding.
[0037] The preparation process of the dual-drug loaded gel beads was the same as that in Example 1, except that 5-ASA and Res / HP-β-CD (Res / hydroxypropyl-β-cyclodextrin) inclusion complex were not added, and the prepared gel beads were non-drug loaded. The infrared spectroscopy scanning of the dual-drug loaded gel beads and non-drug loaded gel beads prepared in Example 1 was performed, and the results are shown in FIG. Figure 3 .
[0038] Example 2
[0039] This embodiment provides a method for preparing dual-drug loaded gel beads, comprising the following steps:
[0040] First, 2g of sodium alginate and 2g of pectin were dissolved in deionized water until the solution was 100mL to obtain a precursor solution. Then, 0.2g of 5-ASA and 0.6g of Res / HP-β-CD (Res / hydroxypropyl-β-cyclodextrin) inclusion complex were added to the precursor solution and completely homogenized to form a mixed solution. Then, a syringe was used to drop the mixed solution into a calcium chloride solution with a mass concentration of 3%, and the gel beads were taken out after cross-linking for 4 hours. The obtained gel beads were rinsed with distilled water and placed in a chitosan solution with a mass concentration of 3% and stirred for 1 hour, then rinsed with distilled water, and dried to obtain gel beads loaded with dual drugs.
[0041] The encapsulation efficiency and drug loading of the prepared dual-drug loaded gel beads were measured. The results showed that the encapsulation efficiency and drug loading of the gel beads for 5-ASA were 59.1% and 3.2%, respectively, and the encapsulation efficiency and drug loading of the gel beads for resveratrol (Res) were 57.4% and 2.8%, respectively.
[0042] Example 3
[0043] This embodiment provides a method for preparing dual-drug loaded gel beads, comprising the following steps:
[0044] First, 1.5g sodium alginate and 2.5g pectin were dissolved in deionized water and fixed to 100mL to obtain a precursor solution. Then, 0.2g 5-ASA and 0.6g Res / HP-β-CD (Res / hydroxypropyl-β-cyclodextrin) inclusion compound were added to the precursor solution and completely homogenized to form a mixed solution. Then, a 3% calcium chloride solution was added dropwise to the mixed solution with a syringe, and the gel beads were taken out after cross-linking for 4h. The obtained gel beads were rinsed with distilled water and placed in a 2% chitosan solution with a mass concentration and stirred for 1h, then rinsed with distilled water, and dried to obtain gel beads loaded with dual drugs.
[0045] The encapsulation efficiency and drug loading of the prepared dual-drug loaded gel beads were measured, and the results showed that the encapsulation efficiency and drug loading of the gel beads for 5-ASA were 52.9% and 3.0%, respectively, and the encapsulation efficiency and drug loading of the gel beads for resveratrol (Res) were 53.6% and 2.1%, respectively.
[0046] Example 4
[0047] In vitro drug release experiment was performed on the gel beads loaded with dual drugs in Example 1
[0048] Weigh 0.3 g of the double-drug loaded gel beads in Example 1 and put them into a conical flask, then add 50 mL of simulated gastric juice at pH 1.2, and place it in a 37°C water bath shaker and oscillate at 150 rpm for 2 h. Remove the simulated gastric juice at pH 1.2, add simulated small intestinal juice at pH 6.8, and oscillate at 150 rpm in a 37°C water bath shaker for 2 h. Remove the simulated small intestinal juice at pH 6.8 and add simulated colonic juice at pH 7.4, and continue to release for 10 h. Each group of in vitro release experiments was performed 3 times. Samples (3 mL) were taken from the medium at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 14 h, and the corresponding volume of medium was supplemented at the same time, and the light absorption value of the sample was determined according to the calibration curve using a UV-visible spectrophotometer to calculate the drug release rate.
[0049] The results of the test are as follows Figure 4 As described, after 2 hours of simulated release under pH 1.2 conditions, the release amounts of 5-ASA and Res were only 8.4% and 9.5%; under pH 6.8 conditions, the total release amounts of 5-ASA and Res were 27.2% and 31.1% respectively after 2 hours, and the total release amounts of 5-ASA and Res were 93.6% and 91.4% respectively after 10 hours under pH 7.4 conditions. The above data show that in the strong acidity of simulated gastric fluid or the weak acidity of small intestinal fluid buffer environment, the gel beads have good stability and only a small amount of drugs are released; while in the colon, the total release amounts of the two drugs are both over 60%, indicating that the gel beads prepared by the present invention can be used as carriers to achieve the co-targeted delivery and release of the two drugs in the colon.
[0050] Example 5
[0051] The dual-drug loaded gel beads in Example 1 were tested for the therapeutic effect on colitis in mice
[0052] After 7 days of adaptive feeding, the experimental mice were randomly divided into 5 groups (blank group, model group, low-dose group, medium-dose group and high-dose group). The blank group mice were fed with standard animal diet, and the other mice drank 2.5% DSS aqueous solution prepared with distilled water for 7 consecutive days to prepare ulcerative colitis mouse model. Subsequently, each group of mice was given oral gavage. Among them, the blank group and the model group were given normal saline, and the low-dose group, medium-dose group and high-dose group of drug-loaded gel beads were given 400 mg / kg·d, 600 mg / kg·d and 800 mg / kg·d, respectively. Each group was gavaged for 7 consecutive days, once a day. The weight changes, fecal properties and occult blood conditions of all mice were recorded daily, and then the mice were euthanized. Under sterile conditions, the colon tissue and blood of the mice were collected, and the blood was centrifuged (3000r / min, 20min) to obtain the supernatant. Serum.
[0053] The results of measuring the colon length of each group of mice are as follows Figure 5 As shown in the figure, compared with the blank group, "*" indicates p<0.05, "**" indicates p<0.01, and "***" indicates p<0.001; compared with the model group, "#" indicates p<0.05, "##" indicates p<0.01, and "###" indicates p<0.001. It can be seen that compared with the blank group, the colon length of the model group mice was significantly reduced (p<0.001), and compared with the model group, the colon length of the drug-loaded gel beads group was significantly increased, indicating that the dual-drug loaded gel beads prepared in Example 1 of the present invention can improve DSS-induced colitis.
[0054] The levels of cytokines TGF-β, IL-6, IL-10 and TNF-α in the serum of each group of mice were determined, and the results are shown in Table 1. It can be seen that compared with the model group, the levels of pro-inflammatory cytokines IL-6 and TNF-α in the serum of mice in the drug-loaded gel beads group were significantly reduced, and the levels of anti-inflammatory cytokines IL-10 and TGF-β were significantly increased, indicating that the dual-drug loaded gel beads prepared in the embodiment of the present invention can be used for the effective treatment of colitis and improve the inhibitory effect on colitis.
[0055] Table 1 Results of cytokine detection in mouse serum
[0056]
[0057] It should be noted that although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A colon-targeted dual-drug gel bead, characterized in that Sodium alginate, pectin and calcium chloride are cross-linked to form gel beads, which contain a large number of holes. Resveratrol (Res) is complexed with hydroxypropyl-β-cyclodextrin to form a Res / hydroxypropyl-β-cyclodextrin inclusion complex with a hydrophobic barrel-shaped cavity structure. Resveratrol is included in the hydrophobic cavity. Aminosalicylic acid (5-ASA) and / or Res / hydroxypropyl-β-cyclodextrin inclusion complex are embedded in the pores of the gel beads, and chitosan wraps the gel beads.
2. A method for preparing colon-targeted dual-drug gel beads, characterized in that The following steps are involved: (1) Dissolving sodium alginate and pectin in deionized water to obtain a precursor solution; (2) adding 5-aminosalicylic acid 5-ASA and Res / hydroxypropyl-β-cyclodextrin inclusion complex to the precursor solution and completely homogenizing to obtain a mixed solution; (3) using a syringe to drop the mixed solution into a calcium chloride solution to form gel beads after cross-linking; (4) The gel beads are rinsed with distilled water and placed in a chitosan solution for stirring, and then rinsed with distilled water and dried to obtain colon-targeted dual-drug loaded gel beads; Wherein, in step (2), the Res / hydroxypropyl-β-cyclodextrin inclusion compound is prepared by the following method: 5-20 mmol·L -1 Resveratrol (Res) ethanol solution was slowly dripped into 10~20mmol·L at a volume ratio of 1:5~10 -1 A mixed solution was obtained from an aqueous solution of HP-β-CD (hydroxypropyl-β-cyclodextrin), and the mixed solution was incubated in the dark for 3 to 6 hours and then placed in a refrigerator at 4°C overnight; the mixed solution after cold precipitation was then centrifuged at a speed of 4000 to 6000 rpm for 10 to 15 minutes, and the precipitate was washed three times with anhydrous ethanol, and the washed precipitate was placed in a fume hood to slowly evaporate the ethanol, and finally the Res / HP-β-CD inclusion complex was obtained.
3. The method for preparing gel beads according to claim 2, characterized in that: In the step (1), in the precursor solution, the mass concentration of sodium alginate is 2-5%, and the mass concentration of pectin is 1-3%.
4. The method for preparing gel beads according to claim 2, characterized in that: In the step (2), the mass concentration of 5-aminosalicylic acid 5-ASA in the mixed solution is 0.1-0.5%, and the mass concentration of Res / hydroxypropyl-β-cyclodextrin inclusion compound in the mixed solution is 0.3-1%.
5. The method for preparing gel beads according to claim 2, characterized in that: In the step (3), the mass concentration of the calcium chloride solution is 3-4%, the cross-linking time is 3-4 hours, and the flow rate of dripping into the CaCl2 solution by syringe is 2 mL / min.
6. The method for preparing gel beads according to claim 2, characterized in that: In the step (4), the mass concentration of the chitosan solution is 1-3%, and the stirring time is 20-40 min.
7. Use of the colon-targeted dual-drug loaded gel beads as claimed in claim 1 in preparing drugs for ulcerative colitis.