Glucose responsive microcapsule and preparation method thereof
Glucose-responsive microcapsules were prepared by chemically modifying sodium alginate, which solved the environmental responsiveness and anti-enzymatic problems of existing microcapsule drug delivery systems. This enabled dynamic regulation of drug release and intestinal adhesion based on glucose concentration, thereby improving drug delivery efficiency.
Patent Information
- Application Number
- CN202511611802.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-05
- Publication Date
- 2025-12-12
AI Technical Summary
Existing microcapsule drug delivery systems lack environmental responsiveness, cannot dynamically adjust the drug release rate based on the concentration of specific substances in the target microenvironment, have insufficient adhesion, are easily degraded by gastrointestinal enzymes, and are difficult to achieve precise controlled release and efficient drug delivery.
NI-CYS-ALG nanoparticles were prepared by chemically grafting 2-nitroimidazole and L-cysteine hydrochloride onto sodium alginate. These nanoparticles were loaded with glucose oxidase to form glucose-responsive microcapsules. Combined with calcium alginate and chitosan to form a film, the nanoparticles achieved rapid drug release in high glucose environments and slow drug release in low glucose environments, while also enhancing intestinal adhesion and resistance to enzymatic degradation.
It enables dynamic adjustment of drug release rate based on glucose concentration, improves drug adhesion and anti-enzymatic ability in the intestine, enhances oral drug delivery efficiency, prolongs drug residence time in the intestine, and ensures drug stability in the gastrointestinal environment.
Smart Images
Figure CN121102168A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical preparation technology, specifically relating to a glucose-responsive microcapsule and its preparation method. Background Technology
[0002] This invention belongs to the field of biomedical materials and drug delivery technology. Microcapsules have become a research hotspot in this field due to their ability to protect drugs from environmental degradation and achieve controlled drug release, especially in oral drug delivery, where they can reduce drug degradation losses in the stomach and improve drug bioavailability. Among them, polysaccharide-based microcapsules such as sodium alginate and chitosan are widely used in the delivery of oral hypoglycemic agents and antibiotics due to their good biocompatibility and biodegradability. Currently, although microcapsules prepared by chemical methods such as interfacial polymerization or physical methods such as emulsification have achieved drug encapsulation and sustained release, they still have many technical shortcomings and cannot meet the requirements of efficient drug delivery.
[0003] First, most traditional microcapsules lack environmental responsiveness and cannot dynamically adjust the drug release rate based on the concentration of specific substances in the target microenvironment (such as glucose concentration in the intestines of diabetic patients), resulting in insufficient precise drug release. Second, some microcapsules have insufficient adhesion, making it difficult to bind tightly to the intestinal mucosa, resulting in a short intestinal residence time and affecting drug absorption efficiency. Simultaneously, their resistance to enzymatic degradation is weak, making them easily destroyed by degrading enzymes such as pepsin and trypsin in the gastrointestinal tract, leading to premature disintegration. For example, sodium alginate microcapsules are stable under acidic conditions, but they easily disintegrate rapidly after entering the intestines, triggering a burst release of the drug; while chitosan-modified microcapsules can improve tolerance to intestinal enzymes, they are difficult to achieve precise drug release based on target signals.
[0004] Existing drug-loaded microcapsules use carrier materials with limited functionality, failing to simultaneously address multiple requirements such as responsive drug release, mucosal adhesion, and resistance to gastrointestinal degradation. In summary, current microcapsule drug delivery systems exhibit significant technological shortcomings, necessitating the design of novel systems. These systems should address the aforementioned issues through material functionalization and structural optimization, providing more efficient delivery solutions for oral drugs, especially those requiring precise controlled release, thereby improving oral drug delivery efficiency. Summary of the Invention
[0005] The purpose of this invention is to overcome the defects of the prior art and provide a glucose-responsive microcapsule and its preparation method. The microcapsule can dynamically adjust the drug release rate according to the glucose concentration, and at the same time has good intestinal adhesion and anti-gastrointestinal degradation ability, so as to improve the oral delivery efficiency of drugs.
[0006] This invention modifies sodium alginate (ALG) with 2-nitroimidazole (NI) and L-cysteine hydrochloride (CYS) through chemical grafting to obtain NI-CYS-ALG. Drug delivery and glucose oxidase (GOx) are loaded into NI-CYS-ALG using an ultrasonic self-assembly method to obtain glucose-responsive nanoparticles. The nanoparticles are then dispersed in a sodium alginate solution, and calcium chloride solution is added to form calcium alginate beads. These beads are then immersed in a chitosan solution to form a film, which is washed, freeze-dried, and then microcapsules are obtained. This system utilizes the GOx in the nanoparticles to catalyze the oxidation of glucose in a high-glucose environment, consuming oxygen and creating a localized hypoxic environment. This causes NI to change from hydrophobic to hydrophilic ("nanoswitch" turned on) for rapid insulin release. In low-glucose concentration solutions, the "nanoswitch" is turned off for slow drug release. Simultaneously, the sulfhydryl groups of CYS enhance intestinal adhesion, and the microcapsule shell resists gastrointestinal enzymatic degradation and acidic damage, achieving highly efficient oral drug delivery.
[0007] To achieve the above objectives, one of the technical solutions of the present invention is: a method for preparing glucose-responsive microcapsules, comprising the following steps:
[0008] (1) Sodium alginate was dissolved in water to obtain sodium alginate solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC・HCl) and N-hydroxysuccinimide (NHS) were added. After adjusting the pH, the solution was activated in the dark. L-cysteine hydrochloride was then added. After adjusting the pH, the solution was stirred and reacted. After dialysis and freeze-drying, CYS-ALG was obtained.
[0009] (2) Dissolve the CYS-ALG obtained in step (1) in PBS to obtain a PBS solution of CYS-ALG, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC・HCl) and NHS, stir for the first time in the dark, add a solution of 2-nitroimidazolium hexylamine (NI-NH2) in N,N-dimethylformamide (DMF), stir for the second time in the dark, dialyze, and freeze dry to obtain NI-CYS-ALG;
[0010] (3) The NI-CYS-ALG obtained in step (2) was dissolved in PBS by ultrasonic self-assembly and ultrasonically treated to obtain a PBS solution of NI-CYS-ALG. The loaded drug and glucose oxidase were added and ultrasonically treated again. After centrifugation, washing and drying, GR-NPs were obtained.
[0011] (4) Disperse the GR-NPs obtained in step (3) in sodium alginate solution and stir to obtain a mixed solution. Add calcium chloride solution to the mixed solution through a high voltage electrostatic device to obtain calcium alginate beads. Immerse the calcium alginate beads in chitosan solution to form a film. After washing, freeze dry to obtain GR-NEMs glucose-responsive microcapsules.
[0012] In a preferred embodiment of the present invention, the concentration of sodium alginate in the sodium alginate solution in step (1) is 0.005-0.02 g / mL, and the mass ratio of sodium alginate, EDC·HCl, NHS and L-cysteine hydrochloride is (8-12):(3-6):(2-4):(18-22).
[0013] In a preferred embodiment of the present invention, in step (1), the pH is adjusted to 3.5-6.5, the activation time is 30-60 min, the stirring reaction temperature is 70-90℃, and the stirring reaction time is 4-6 h.
[0014] In a preferred embodiment of the present invention, the molecular weight cutoff for dialysis in steps (1) and (2) is 3000-4000.
[0015] In a preferred embodiment of the present invention, in step (2), the mass ratio of CYS-ALG, EDC・HCl, NHS and NI-NH2 is (18-22):(4-7):(2-4):(18-22), the concentration of CYS-ALG in the PBS solution of CYS-ALG is 0.01-0.05 g / mL, and the content of 2-nitroimidazolylamine (NI-NH2) in the N,N-dimethylformamide (DMF) solution of 2-nitroimidazolylamine (NI-NH2) is 0.01-0.05 g / mL.
[0016] More preferably, the pH of the PBS solution is 7-8.
[0017] In a preferred embodiment of the present invention, the first stirring time in the dark in step (2) is 20-40 min, and the second stirring time in the dark is 20-28 h.
[0018] In a preferred embodiment of the present invention, the mass ratio of NI-CYS-ALG, the loaded drug and glucose oxidase in step (3) is (6-10):(4-6):(0.5-1.5).
[0019] In a preferred embodiment of the present invention, the concentration of NI-CYS-ALG in the PBS solution of NI-CYS-ALG in step (3) is 0.005-0.015 g / mL.
[0020] In a preferred embodiment of the present invention, the drug contained in step (3) is one or more of a small molecule drug and a protein polypeptide drug.
[0021] In a preferred embodiment of the present invention, the ultrasonic self-assembly conditions in step (3) are an ice bath, a power of 80-120 W, an ultrasonic interval of 2 seconds after 5 seconds, and a total ultrasonic time of 10-20 minutes.
[0022] In a preferred embodiment of the present invention, in step (4), the voltage of the high-voltage electrostatic device is 8-12kV, the addition rate is 0.5-2 mL / min, the pH of the chitosan solution is 3-5, and the film formation time is 10-30 min.
[0023] In a preferred embodiment of the present invention, the centrifugation speed in step (4) is 10,000-15,000 rpm and the centrifugation time is 10-30 min.
[0024] In a preferred embodiment of the present invention, the volume ratio of the mixed solution to the calcium chloride solution in step (4) is:
[0025] (3-5):(100-200), the concentration of GR-NPs in the mixed solution is 0.01-0.05 g / mL, and the mass fraction of calcium chloride in the calcium chloride solution is 1-2 wt%.
[0026] In a preferred embodiment of the present invention, the pH of the chitosan solution in step (4) is 3-6 and the mass fraction is 0.1-0.3 wt%.
[0027] To achieve the above objectives, the second technical solution of the present invention is: a glucose-responsive microcapsule prepared by the above preparation method.
[0028] To achieve the above objectives, the third technical solution of the present invention is: the application of a glucose-responsive microcapsule prepared by the above preparation method.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] 1. The glucose-responsive microcapsules prepared by this invention have intelligent responsiveness and can dynamically regulate drug release according to glucose concentration. When glucose is high, the "nano switch" is turned on to achieve rapid drug release, and when glucose is low, it remains closed to release the drug slowly.
[0031] 2. The glucose-responsive microcapsules prepared by this invention have anti-degradation and targeted delivery capabilities, good anti-enzymatic activity, and can prevent drugs from being attacked by proteases. At the same time, they can protect the loaded drugs from being destroyed in the extremely acidic environment of the stomach. Furthermore, the nanoparticles enhance intestinal adhesion due to the thiol groups of CYS, prolonging the residence time and promoting cellular uptake. Attached Figure Description
[0032] Figure 1This is an optical microscope image of the glucose-responsive microcapsules prepared in Example 1 of the present invention. Detailed Implementation
[0033] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.
[0034] A method for preparing glucose-responsive microcapsules includes the following steps:
[0035] (1) Sodium alginate was dissolved in water to obtain sodium alginate solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC・HCl) and N-hydroxysuccinimide (NHS) were added. After adjusting the pH, the solution was activated in the dark. L-cysteine hydrochloride was then added. After adjusting the pH, the solution was stirred and reacted. After dialysis and freeze-drying, CYS-ALG was obtained.
[0036] (2) Dissolve the CYS-ALG obtained in step (1) in PBS to obtain a PBS solution of CYS-ALG, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC・HCl) and NHS, stir for the first time in the dark, add a solution of 2-nitroimidazolium hexylamine (NI-NH2) in N,N-dimethylformamide (DMF), stir for the second time in the dark, dialyze, and freeze dry to obtain NI-CYS-ALG;
[0037] (3) The NI-CYS-ALG obtained in step (2) was dissolved in PBS by ultrasonic self-assembly and ultrasonically treated to obtain a PBS solution of NI-CYS-ALG. The loaded drug and glucose oxidase were added and ultrasonically treated again. After centrifugation, washing and drying, GR-NPs were obtained.
[0038] (4) Disperse the GR-NPs obtained in step (3) in sodium alginate solution and stir to obtain a mixed solution. Add calcium chloride solution to the mixed solution through a high voltage electrostatic device to obtain calcium alginate beads. Immerse the calcium alginate beads in chitosan solution to form a film. After washing, freeze dry to obtain glucose-responsive microcapsules GR-NEMs.
[0039] In step (1), the concentration of sodium alginate in the sodium alginate solution is 0.005-0.02 g / mL, and the mass ratio of sodium alginate, EDC・HCl, NHS and L-cysteine hydrochloride is (8-12):(3-6):(2-4):(18-22).
[0040] In step (1), the pH is adjusted to 3.5-6.5, the activation time is 30-60 min, the stirring reaction temperature is 70-90℃, and the stirring reaction time is 4-6 h.
[0041] In steps (1) and (2), the molecular weight cutoff for dialysis is 3000-4000.
[0042] In step (2), the mass ratio of CYS-ALG, EDC・HCl, NHS and NI-NH2 is (18-22):(4-7):(2-4):(18-22), the concentration of CYS-ALG in the PBS solution is 0.01-0.05 g / mL, and the content of 2-nitroimidazolylamine (NI-NH2) in the N,N-dimethylformamide (DMF) solution is 0.01-0.05 g / mL.
[0043] The pH of the PBS solution is 7-8.
[0044] In step (2), the first stirring time in the dark is 20-40 min, and the second stirring time in the dark is 20-28 h.
[0045] In step (3), the mass ratio of NI-CYS-ALG, the loaded drug, and glucose oxidase is (6-10):(4-6):(0.5-1.5).
[0046] In step (3), the concentration of NI-CYS-ALG in the PBS solution of NI-CYS-ALG is 0.005-0.015 g / mL.
[0047] The drug in step (3) is one or more of small molecule drugs and protein polypeptide drugs.
[0048] In step (3), the ultrasonic self-assembly conditions are an ice bath, a power of 80-120 W, an ultrasonic interval of 2 seconds after 5 seconds, and a total ultrasonic time of 10-20 minutes.
[0049] In step (4), the voltage of the high-voltage electrostatic device is 8-12 kV, the addition rate is 0.5-2 mL / min, the pH of the chitosan solution is 3-5, and the film formation time is 10-30 min.
[0050] In step (4), the centrifugation speed is 10,000-15,000 rpm and the centrifugation time is 10-30 min.
[0051] In step (4), the volume ratio of the mixed solution to the calcium chloride solution is (3-5):(100-200), the concentration of GR-NPs in the mixed solution is 0.01-0.05 g / mL, and the mass fraction of calcium chloride in the calcium chloride solution is 1-2 wt%.
[0052] In step (4), the pH of the chitosan solution is 3-6 and the mass fraction is 0.1-0.3 wt%.
[0053] A glucose-responsive microcapsule prepared by the above method.
[0054] Application of a glucose-responsive microcapsule prepared by the above method.
[0055] Example 1
[0056] A glucose-responsive microcapsule is prepared by the following method:
[0057] (1) Weigh 0.2 g sodium alginate and dissolve it in 20 mL of ultrapure water. Stir at 37 °C until completely dissolved. Add 0.1 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC・HCl) and 0.06 g N-hydroxysuccinimide (NHS). Adjust the pH to 5.5. Activate in the dark for 45 min. Add 0.4 g L-cysteine hydrochloride and adjust the pH to 4.5. Stir the reaction for 5 h. Dialyze (molecular weight cutoff 3500) and freeze dry to obtain CYS-ALG.
[0058] (2) Dissolve 0.2 g CYS-ALG in 10 mL PBS (pH=7.4), add 0.05 g EDC・HCl and 0.03 g NHS, stir for 30 min in the dark for the first time, add 5 mL DMF solution containing 0.2 g 2-nitroimidazolylamine (NI-NH2), stir for 24 h in the dark for the second time, dialyze and freeze dry to obtain NI-CYS-ALG;
[0059] (3) Weigh 40 mg NI-CYS-ALG and dissolve it in 5 mL PBS (pH=7.4). Sonicate in an ice bath at 4°C for 15 min (100W, sonication for 5 s with a 2 s interval). Add 20 mg insulin (pre-dissolved in 3 mL of 0.01 mol / L hydrochloric acid) and 4 mg glucose oxidase. Sonicate in an ice bath at 4°C for 10 min (100W, sonication for 5 s with a 2 s interval). Centrifuge at 12000 rpm for 20 min. Wash the precipitate three times with ultrapure water and freeze-dry to obtain GR-NPs.
[0060] (4) Disperse GR-NPs in 5 mL of 2% sodium alginate solution to obtain a mixed solution. Stir for 20 min. Add 200 mL of 1.5 wt% calcium chloride solution to the mixed solution at a rate of 0.2 mL / min using a high-voltage electrostatic device (voltage 10 kV). Calcify for 15 min. After filtration, immerse the beads in 0.2 wt% chitosan solution (pH=4.6) for 30 min to form a film. Wash with ultrapure water 3 times and freeze-dry to obtain glucose-responsive microcapsules GR-NEMs.
[0061] Figure 1 The image shows a GR-NEM optical microscope image of the glucose-responsive microcapsules prepared in this embodiment. As can be seen from the image, the capsules are spherical with a smooth surface. The particle size of the microcapsules was measured to be 450 µm using ImageJ, and the drug loading was determined to be 7.61 ± 0.89% and the encapsulation efficiency to be 76.08 ± 8.91% by HPLC.
[0062] Example 2
[0063] Using diabetic mice as a model, the hypoglycemic effect of GR-NEMs was investigated. Two hours after gavage administration of GR-NEMs to diabetic mice, blood glucose levels decreased to 87.01 ± 5.16% of the initial value. By 10 hours, blood glucose concentration had decreased to the normal range, reaching 46.40 ± 1.52%, and remained stable for 24 hours. In the control group, after gavage administration of GR-NEMs, blood glucose only decreased slightly, but the blood glucose concentration remained within the normal range, without hypoglycemia. Subsequently, blood glucose concentration recovered, indicating that GR-NEMs can intelligently regulate blood glucose levels. H&E staining results showed that GR-NEMs did not cause significant damage to the heart, liver, spleen, lungs, kidneys, and small intestine of mice. Furthermore, blood biochemical tests showed that GR-NEMs had very low toxicity in mice, indicating that GR-NEMs have good biocompatibility.
[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing glucose-responsive microcapsules, characterized in that, Includes the following steps: (1) Sodium alginate was dissolved in water to obtain sodium alginate solution. EDC・HCl and NHS were added, and after adjusting the pH, it was activated in the dark. L-cysteine hydrochloride was added, and after adjusting the pH, the reaction was stirred. After dialysis and freeze drying, CYS-ALG was obtained. (2) Dissolve the CYS-ALG obtained in step (1) in PBS to obtain a CYS-ALG PBS solution, add EDC・HCl and NHS, stir for the first time in the dark, add DMF solution of NI-NH2, stir for the second time in the dark, dialyze and freeze dry to obtain NI-CYS-ALG. (3) The NI-CYS-ALG obtained in step (2) was dissolved in PBS by ultrasonic self-assembly and ultrasonically treated to obtain a PBS solution of NI-CYS-ALG. The loaded drug and glucose oxidase were added and ultrasonically treated again. After centrifugation, washing and drying, GR-NPs were obtained. (4) Disperse the GR-NPs obtained in step (3) in sodium alginate solution and stir to obtain a mixed solution. Add calcium chloride solution to the mixed solution through a high voltage electrostatic device to obtain calcium alginate beads. Immerse the calcium alginate beads in chitosan solution to form a film. After washing, freeze dry to obtain glucose-responsive microcapsules GR-NEMs.
2. The method for preparing glucose-responsive microcapsules as described in claim 1, characterized in that, In step (1), the concentration of sodium alginate in the sodium alginate solution is 0.005-0.02 g / mL, and the mass ratio of sodium alginate, EDC・HCl, NHS and L-cysteine hydrochloride is (8-12):(3-6):(2-4):(18-22).
3. The method for preparing glucose-responsive microcapsules as described in claim 1, characterized in that, In step (1), the pH is adjusted to 3.5-6.5, the activation time is 30-60 min, the stirring reaction temperature is 70-90℃, and the stirring reaction time is 4-6 h; in step (2), the first stirring time in the dark is 20-40 min, and the second stirring time in the dark is 20-28 h.
4. The method for preparing glucose-responsive microcapsules as described in claim 1, characterized in that, In step (2), the mass ratio of CYS-ALG, EDC・HCl, NHS and NI-NH2 is (18-22):(4-7):(2-4):(18-22), the concentration of CYS-ALG in the PBS solution is 0.01-0.05 g / mL, and the content of NI-NH2 in the DMF solution is 0.01-0.05 g / mL.
5. The method for preparing glucose-responsive microcapsules as described in claim 1, characterized in that, In step (3), the mass ratio of NI-CYS-ALG, the loaded drug and glucose oxidase is (6-10):(4-6):(0.5-1.5), and the concentration of NI-CYS-ALG in the PBS solution of NI-CYS-ALG is 0.005-0.015 g / mL.
6. The method for preparing glucose-responsive microcapsules as described in claim 1, characterized in that, The drug in step (3) is one or more of small molecule drugs and protein polypeptide drugs.
7. The method for preparing glucose-responsive microcapsules as described in claim 1, characterized in that, In step (3), the ultrasonic self-assembly conditions are an ice bath, a power of 80-120 W, 5 seconds of ultrasonication followed by a 2-second interval, and a total ultrasonic time of 10-20 min. In step (4), the voltage of the high-voltage electrostatic device is 8-12 kV, and the addition rate is 0.5-2 mL / min. The pH of the chitosan solution is 3-5, the film formation time is 10-30 min, the centrifugation speed is 10000-15000 rpm, and the centrifugation time is 10-30 min.
8. The method for preparing glucose-responsive microcapsules as described in claim 1, characterized in that, In step (4), the volume ratio of the mixed solution to the calcium chloride solution is (3-5):(100-200), the concentration of GR-NPs in the mixed solution is 0.01-0.05 g / mL, the mass fraction of calcium chloride in the calcium chloride solution is 1-2 wt%, the pH of the chitosan solution is 3-6, and the mass fraction is 0.1-0.3 wt%.
9. A glucose-responsive microcapsule prepared by the method of any one of claims 1-8.
10. An application of the glucose-responsive microcapsule as described in claim 9.