Responsive sustained-release microspheres and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine production and relates to responsive sustained / controlled release microspheres and a preparation method and application thereof.The responsive sustained / controlled release microspheres comprise, by weight fraction, 160-200 parts of acetalized dextran, 40-80 parts of polylactic acid-glycolic acid copolymer, 10-30 parts of a drug, 3-7 parts of glucose oxidase and 0.5-1.5 parts of catalase; the mass ratio of the acetalized dextran and the polylactic acid-glycolic acid copolymer is 3-5:1.The microspheres can release insulin responsively according to blood glucose, once administration can stabilize blood glucose for 20 hours, and can ensure safety while effectively reducing blood glucose, and will not cause a series of life-threatening symptoms such as hypoglycemia due to excessive insulin injection.The blood glucose maintenance cycle can be adjusted according to the dose of the microspheres, and the purpose of long-term stable blood glucose is achieved.At the same time, the size and performance of the microspheres are uniform and stable.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical manufacturing technology, and relates to responsive sustained-release microspheres, their preparation methods, and applications. Background Technology

[0002] Insulin replacement therapy remains the most commonly used treatment for diabetes. However, inaccurate insulin dosage can lead to hypoglycemia, hyperglycemia, and diabetic ketoacidosis due to factors such as diet, intensity, and duration of physical activity. Furthermore, multiple daily insulin injections can result in poor patient compliance, injection site induration, abscesses, erythema, fat hypertrophy, or atrophy. Currently available long-acting insulins, such as insulin glargine, can be injected once daily. However, because long-acting insulins cannot guarantee rapid blood sugar reduction while ensuring safety, they initially require combination with other insulins, making their use complex and necessitating continuous dosage adjustments. To address this trade-off between safety and effectiveness, glucose-responsive insulin microspheres, which mimic the secretion pattern of pancreatic β-cells, can effectively control insulin release based on blood glucose levels.

[0003] Existing technology discloses a novel microsphere formulation containing small-diameter microspheres within an outer layer of microspheres using polylactic-co-glycolic acid copolymer (PLGA) as a carrier material. These small-diameter microspheres primarily utilize polyketides or their derivatives as a framework, with the model drug embedded within. Glucose oxidase and catalase triggers are dispersed on the exterior of the microspheres. However, this novel microsphere formulation exhibits high release rates at zero glucose content and in PBS (partially bismuth subunits), resulting in poor safety.

[0004] The prior art (He, C., Zeng, W., Su, Y., Sun, R., Xiao, Y., Zhang, B., … Chen, C. (2021). Microfluidic-based fabrication and characterization of drug-loaded PLGA magnetic microspheres with tunable shell thickness. Drug Delivery, 28(1), 692–699) discloses a method for fabricating a two-phase microfluidic chip. A schematic diagram of the two-phase microfluidic chip structure is shown below. Figure 1As shown in the figure; 1 is the external phase inlet; 2 is the internal phase inlet; 3 is the capillary inlet; 4 is the capillary outlet; 5 is the chip channel; 6 is the capillary; 7 is the PDMS chip; 8 is the PDMS substrate. The two-phase microfluidic chip consists of multiple channels. A certain proportion of internal and external phase solutions are injected into the feed channel. When these two liquid phases meet in the structure, they are separated into tiny droplets by surface tension, forming highly uniform droplets. After a series of microprocessing steps, they eventually form nearly spherical microspheres. Summary of the Invention

[0005] The purpose of this invention is to provide a more precise responsive sustained-release microsphere, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A responsive sustained-release microsphere, by weight, comprises 160-200 parts of acetalized dextran, 40-80 parts of polylactic acid-glycolic acid copolymer, 10-30 parts of drug, 3-8 parts of glucose oxidase and 0.5-1.5 parts of catalase;

[0008] The mass ratio of acetalized dextran to polylactic acid-glycolic acid copolymer is 3-5:1;

[0009] The structural formula of the acetalized dextran is:

[0010]

[0011] Where n is an integer between 120 and 450; the difference in molecular weight between polylactic acid-glycolic acid copolymer and acetalized dextran is less than 10,000.

[0012] In one preferred embodiment, the responsive sustained-release microspheres are composed of acetalized dextran and polylactic-co-glycolic acid copolymer as carriers, and drugs, glucose oxidase and catalase as contents.

[0013] In one preferred embodiment, the drug is insulin, exenatide, or glibenclamide.

[0014] In one preferred embodiment, the amount of the drug added is 12-22 parts.

[0015] Taking insulin as an example, in the composition of the microspheres, acetalized dextran (Ac-dex) and polylactic-co-glycolic acid copolymer (PLGA) together serve as drug carriers. Ac-dex is a moderately acid-degradable material; when blood glucose concentration is too high, glucose oxidase (GOx) converts glucose into gluconic acid, lowering the pH of the microenvironment and promoting the degradation of Ac-dex, thus releasing insulin. PLGA is a biocompatible material; controlling its mass ratio with Ac-dex within a suitable range can achieve optimal blood glucose response, combining efficacy and safety. Insulin is the main active drug, and GOx, as mentioned above, is a key component in blood glucose response. Catalase (CAT) effectively removes excess H2O2 from the body when GOx converts glucose into gluconic acid and H2O2, preventing inflammatory responses. Therefore, the responsive sustained-release microspheres prepared in this way are precisely controlled release microspheres; when the dextran content is zero, no insulin is released at all.

[0016] In one preferred embodiment, the method for preparing the acetalized dextran includes the following steps:

[0017] Under an inert gas atmosphere, dextran, pyridine 4-methylbenzenesulfonic acid and 2-ethoxypropylene were mixed evenly and reacted at 20-40℃ for 0.5-1.5h. Then, triethylamine was added to quench the reaction and the reaction was obtained. The reaction solution was washed, centrifuged and freeze-dried to obtain acetalized dextran.

[0018] In one preferred embodiment, the mass ratio of dextran, pyridine 4-methylbenzenesulfonate, and 2-ethoxypropylene is 900-1100:15.6:3100-3300.

[0019] In one preferred embodiment, the mass ratio of dextran to triethylamine is 1:0.7-0.8.

[0020] In one preferred embodiment, the molecular weight of the dextran is 20,000-70,000.

[0021] The molecular weight of dextran determines the molecular weight of acetalized dextran. If the molecular weight of acetalized dextran is too large or too small, the density difference between the two carrier materials will be too great, making it impossible to form microspheres.

[0022] In one preferred embodiment, the polylactic acid-glycolic acid copolymer has a molecular weight of 48,000-50,000.

[0023] In one preferred embodiment, the washing involves adding the reaction solution to water containing 0.1-0.2 wt% triethylamine and washing several times.

[0024] In one preferred embodiment, the centrifugation speed is 5000-7000 rpm and the centrifugation time is 10-20 min.

[0025] Based on the same inventive concept, this invention also claims protection for a method for preparing the responsive sustained-release microspheres, comprising the following steps:

[0026] S1. Prepare solutions of acetalized dextran, polylactic acid-glycolic acid copolymer, drugs, glucose oxidase, and catalase, mix them evenly to obtain a mixed emulsion; use the mixed emulsion as the chip internal phase injection solution.

[0027] S2. The inner phase injection solution and the outer phase solution are delivered into the two-phase microfluidic chip, and the responsive sustained-release microspheres are obtained through microfluidic preparation.

[0028] In one preferred embodiment, the external phase solution is a polyvinyl alcohol solution.

[0029] In one preferred embodiment, the mass concentration of the external phase solution is 1%-3%.

[0030] In one preferred embodiment, the receiving liquid is a mixture of polyvinyl alcohol and sodium chloride; wherein the ratio of polyvinyl alcohol to sodium chloride is 2:4-6.

[0031] In one preferred embodiment, the polyvinyl alcohol is a polyvinyl alcohol solution with a mass concentration of 1%-3%.

[0032] In one preferred embodiment, the sodium chloride is an aqueous solution of sodium chloride with a mass concentration of 2.5%-7.5%.

[0033] In one preferred embodiment, acetalized dextran and polylactic acid-glycolic acid copolymer are dissolved in an organic solvent to prepare a solution with a mass concentration of 0.5%-5%.

[0034] In one preferred embodiment, the acetalized dextran is prepared into a solution with a mass concentration of 3%-5%.

[0035] In one preferred embodiment, the polylactic acid-glycolic acid copolymer is prepared into a solution with a mass concentration of 0.5%-1%.

[0036] In one preferred embodiment, the organic solvent is one or both of dichloromethane or ethyl acetate.

[0037] In one preferred embodiment, the organic solvent is a mixed solution of dichloromethane and ethyl acetate, wherein the proportion of ethyl acetate in the mixed solution is 4 wt% to 9 wt%.

[0038] In one preferred embodiment, the drug, glucose oxidase, and catalase are dissolved in an acidic solution to prepare a solution with a mass concentration of 0.25%-7%.

[0039] In one preferred embodiment, the acid solution is a hydrochloric acid solution or an acetic acid solution.

[0040] In one preferred embodiment, the concentration of the acid solution is 0.1-0.5 mol / L.

[0041] In one preferred embodiment, the drug is prepared as a solution with a mass concentration of 5%-7%.

[0042] In one preferred embodiment, glucose oxidase is prepared as a solution with a mass concentration of 1%-1.5%.

[0043] In one preferred embodiment, catalase is prepared as a solution with a mass concentration of 0.25%-0.5%.

[0044] In one preferred embodiment, the microfluidic process parameters are: internal phase pressure of 0.15-0.25 MPa and external phase pressure of 0.20-0.50 MPa.

[0045] In one preferred embodiment, the responsive sustained-release microspheres have a particle size of 40-60 μm.

[0046] If the particle size is too small, the microspheres may enter vascular endothelial cells through capillary action and diffusion, leading to rapid absorption; however, they may also cause the drug to be released too quickly. Both of these situations will affect the sustained-release time and efficacy of the microspheres. If the particle size is too large, it may lead to uneven drug distribution and slow release, affecting the therapeutic effect. In addition, excessively large particle size may cause inflammatory responses, increase pain, and reduce patient compliance.

[0047] During the microfluidic process, the internal and external phase solutions are transferred to the corresponding microfluidic system injection tubes, and the receiving solution is transferred to a 250 ml beaker. A pressure pump is used as the driving device to inject the solution into the two-phase microfluidic chip. After the chip outlet stabilizes and the droplets are sheared, the capillary is inserted below the liquid surface, and the microdroplets are collected while stirring. The stirring speed is set to 300-500 rpm. After the injection is completed, stirring is continued for 3-5 hours to allow the dichloromethane to evaporate completely, forming microspheres with a size of approximately 40-60 μm. These microspheres are then washed with ultrapure water and collected.

[0048] Based on the same inventive concept, the present invention also claims protection for the use of the responsive sustained-release microparticle in the preparation of reagents for treating diabetes.

[0049] The following is intended to further explain the present invention:

[0050] Microspheres, as a commonly used drug delivery system, face significant limitations in traditional manufacturing processes due to their high R&D costs, cycle time, material loss, microsphere particle size control, and production efficiency. These limitations include low encapsulation efficiency, inconsistent particle size, and high sterilization costs. Microfluidic technology, however, can produce microspheres with uniform size and controllable particle size, offering superior advantages in preparation efficiency, encapsulation efficiency, batch-to-batch repeatability, and stability, thus overcoming the difficulties in preparing microspheres for heat-sensitive and volatile drugs. The microspheres prepared in this invention exhibit more uniform biodegradation and drug release rates, preventing excessively rapid or slow drug release and improving therapeutic efficacy and safety. Furthermore, the microspheres prepared in this invention are less prone to aggregation, ensuring drug stability and long-lasting effects during storage, transportation, and in vivo release. Compared to existing technologies, the beneficial effects of this invention are:

[0051] This invention, through the selection of appropriate raw materials and the optimization of the preparation method of two-phase microfluidic chips, produces more uniform and stable blood glucose-responsive controlled-release microspheres, enabling uniform mass production, and the rapid blood glucose reduction and blood glucose maintenance effects produced by the microspheres are more stable.

[0052] This invention designs microspheres that can rapidly lower blood glucose and maintain stable blood glucose levels, combining effectiveness and safety. When blood glucose concentration is too high, GOx in the microspheres converts glucose into gluconic acid, lowering the pH of the microenvironment and promoting the degradation of Ac-dex, releasing insulin from the microspheres. When blood glucose returns to normal, Ac-dex degradation stops, and insulin release ceases, thus stabilizing and maintaining normal blood glucose levels, achieving a blood glucose-responsive insulin release effect. Compared with similar responsive formulations, these microspheres can stabilize blood glucose for up to 20 hours and, more importantly, ensure safety while effectively lowering blood glucose, avoiding life-threatening symptoms such as hypoglycemia caused by insulin overdose. The duration of maintaining normal blood glucose can be further adjusted by adjusting the dosage of the microspheres. Simultaneously, the size and performance of the microspheres are kept uniform and stable. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of a microfluidic chip structure in the prior art; 1-external phase inlet; 2-internal phase inlet; 3-capillary inlet; 4-capillary outlet; 5-chip channel; 6-capillary; 7-PDMS chip; 8-PDMS substrate;

[0054] Figure 2 These are the 1H NMR spectra of dextran and Ac-dex, where A is the 1H NMR spectrum of dextran and B is the 1H NMR spectrum of Ac-dex.

[0055] Figure 3 It is the insulin standard curve;

[0056] Figure 4The drug loading and encapsulation efficiency of microspheres prepared with different ratios of PLGA and Ac-dex are shown.

[0057] Figure 5 These are insulin release curves of microspheres prepared with different ratios of PLGA and Ac-dex;

[0058] Figure 6 These are the insulin pulse release curves of PLGA and Ac-dex microspheres with different ratios. The blue shaded area represents a 400 mg / dL glucose solution, and the white area represents a 100 mg / dL glucose solution.

[0059] Figure 7 These are the insulin release curves of microspheres (Ac-dex:PLGA = 3:1 and Ac-dex:PLGA = 5:1) over 2 weeks under physiological conditions;

[0060] Figure 8 The blood glucose-lowering curves of T1D mice injected with insulin (10 mg / kg), MPs (10 mg / kg), and MPs (20 mg / kg) are shown. One mouse is in each group. The red shaded area represents the feeding time.

[0061] Figure 9 The results simulated the blood glucose changes in T1D mice that eat three meals a day. Each group consisted of one mouse, which was injected with insulin (10 mg / kg) and MPs (20 mg / kg), respectively. The red shaded area represents the feeding time.

[0062] Figure 10 The hypoglycemic curves of T1D mice were obtained by subcutaneously injecting them with insulin (10 mg / kg) and MPs (20 mg / kg), respectively.

[0063] Figure 11 It is an intraperitoneal glucose tolerance test;

[0064] Figure 12 It is a hypoglycemic index experiment in healthy mice;

[0065] Figure 13 Images show microspheres prepared by microfluidic methods and microspheres prepared by conventional methods. Detailed Implementation

[0066] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the disclosure of this invention. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other. The polylactic acid-glycolic acid copolymer used in the embodiments of this invention has a molecular weight of 48,000.

[0067] Example 1

[0068] Preparation and characterization of Ac-dex

[0069] 1. Preparation of Ac-dex

[0070] Weigh 1g of dextran and vacuum dry for 24h. Prepare a flame-dried round-bottom flask, purge with nitrogen, and place a clean, dry rotor at the bottom. Add 1g of dextran and 15.6mg of pyridine 4-methylbenzenesulfonic acid (PPTS) under nitrogen atmosphere, then add 10ml of ultra-dry DMSO and stir to dissolve the dextran. Subsequently, add 4.16ml of 2-ethoxypropene. Stir at room temperature for 1h (controlling the oil pan temperature at 30℃). After 1h, add 1ml of triethylamine to quench the reaction. Add this solution dropwise to 100ml of deionized water containing 0.1% triethylamine, wash three times, centrifuge (6000rpm, 15min), collect, and freeze-dry to obtain Ac-dex.

[0071] The prepared Ac-dex is as follows:

[0072]

[0073] Where n is an integer between 122 and 124.

[0074] 2. Characterization of Ac-dex

[0075] Approximately 10 mg of dextran and Ac-dex were dissolved in deuterated water and DMSO, respectively, and characterized using a 500 MHz nuclear magnetic resonance (NMR) spectra. The 1H NMR spectra of dextran and Ac-dex are shown below. Figure 2 As shown, A is the 1H NMR spectrum of dextran, and B is the 1H NMR spectrum of Ac-dex. The Ac-dex spectrum shows multiple signal peaks in the range of 1.0–1.4 ppm, all of which were identified as methyl hydrogen signal peaks. The dextran spectrum shows no signal peaks in this range and does not structurally contain methyl hydrogen. This indicates that the synthesis was successful. The product was identified as Ac-dex.

[0076] Example 2

[0077] Preparation of blood glucose-responsive controlled-release microspheres

[0078] The fabrication of the two-phase microfluidic chip was based on existing technology. He, C., Zeng, W., Su, Y., Sun, R., Xiao, Y., Zhang, B., Chen, C. (2021). Microfluidic-based fabrication and characterization of drug-loaded PLGA magnetic microspheres with tunable shell thickness. Drug Delivery, 28(1), 692–699.

[0079] Solution preparation

[0080] (1) 2% PVA aqueous solution: Weigh 10g PVA into a 1000ml beaker, add 500ml ultrapure water, seal with plastic wrap, poke holes in the surface, and mark the liquid level. Heat to 99℃ and set the speed to 300rpm, stirring until dissolved. After the solution returns to room temperature, add ultrapure water to the marked point, stir evenly, and filter with a 0.25μm filter membrane for later use.

[0081] (2) 2% PVA + 5% NaCl: Weigh 10g PVA into a 1000ml beaker, add 500ml ultrapure water, seal with plastic wrap, poke holes in the surface, and mark the liquid level. Heat to 99℃ and set the speed to 300rpm, stirring until dissolved. Weigh 25g NaCl and add it to the dissolved PVA solution, stirring until completely dissolved. After the solution returns to room temperature, add ultrapure water to the marked points, stir evenly, and filter through a 0.25μm filter membrane for later use.

[0082] (3) 0.1mol / L HCl: Take 250μL of concentrated hydrochloric acid (12mol / L), slowly add ultrapure water to 30ml, stir well and set aside.

[0083] (4) 0.5 mg / ml insulin standard solution: Take 1 mg of bovine insulin into a 2 ml volumetric flask, add 0.1 mol / L HCl solution until the concave meniscus reaches the mark, tighten the cap and shake well for later use.

[0084] 1. Emulsion Preparation

[0085] Weigh the raw materials according to the corresponding proportions. Ac-dex and PLGA are dissolved in dichloromethane (PLGA + Ac-dex: 8%, the specific concentrations of the two materials will be determined in subsequent experiments). Insulin, GOx, and CAT are dissolved in hydrochloric acid solution at pH 5 (Insulin: 5%, GOx: 1.25%, CAT: 0.25%). After all solutions are completely dissolved, mix them and homogenize them at 5000 rpm for 5 minutes to form an emulsion, which will be used as the in-chip injection solution. Add 3 ml of dichloromethane solution and 500 μl of hydrochloric acid solution.

[0086] 2. Preparation of microspheres

[0087] The internal and external phase solutions were transferred to the corresponding microfluidic system injection tubes, and the receiving solution was transferred to a 250 ml beaker. A pressure pump was used as the driving device to inject the solution into the two-phase microfluidic chip. The internal phase pressure was 0.15 MPa, and the external phase pressure was 0.20 MPa. After the chip outlet stabilized and the droplets were sheared, the capillary was inserted below the liquid surface, and the microdroplets were collected while stirring at a speed of 300 rpm. After injection, stirring continued for 4 hours to allow the dichloromethane to evaporate completely, forming microspheres approximately 50 μm in size. These microspheres were then washed with ultrapure water and collected.

[0088] 3. Determine the standard curve of insulin concentration using Coomassie Brilliant Blue G250 dye.

[0089] Add 0.5 mg / ml insulin standard solution to 96-well plates at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μl, and then add 0.1 mol / L HCl solution to bring the total volume to 20 μl. Add 200 μl of Coomassie Brilliant Blue G250 dye reagent to each well and incubate at room temperature for 5 min. Measure the absorbance at 595 nm using a microplate reader. Plot a standard curve with absorbance on the ordinate and lg (lg) insulin concentration on the x-axis (e.g., ...). Figure 3 This serves as a reference for subsequent calculations of insulin concentration.

[0090] 4. Determination of drug loading and encapsulation efficiency of microspheres

[0091] Weigh approximately 10 mg of microspheres and dissolve PLGA and Ac-dex in 0.5 ml of dichloromethane. Sonicate for 10 min to ensure complete dissolution. Add 0.1 mol / L hydrochloric acid solution, vortex for 5 min, and centrifuge at 4000 rpm for 10 min. Add 20 μl of the supernatant to a 96-well plate, then add 200 μl of Coomassie Brilliant Blue G250 dye. React for 5 min, and measure the CD value of each well at 5995 nm using a microplate reader. Use a blank 0.1 mol / L hydrochloric acid solution as a control to calculate the total protein concentration. Perform three parallel tests. Calculate the drug loading and encapsulation efficiency of the microspheres using formulas to optimize the final microsphere formulation.

[0092] Drug loading = insulin mass / microsphere mass;

[0093] Encapsulation efficiency = Actual drug loading / Theoretical drug loading.

[0094] 5. Effect of the ratio of Ac-dex to PLGA on drug loading and encapsulation efficiency of microspheres

[0095] In the preparation of the above-mentioned glucose-responsive insulin controlled-release microspheres, different ratios of Ac-dex and PLGA were selected as carriers, including Ac-dex:PLGA = 1:1, Ac-dex:PLGA = 3:1, Ac-dex:PLGA = 5:1, and Ac-dex:PLGA = 7:1. The drug loading and encapsulation efficiency of each group were determined according to the above method. Figure 4 As shown.

[0096] The results show that among the four groups of microspheres with different proportions of carrier, the drug loading and encapsulation efficiency of the microspheres increased with the increase of the proportion of Ac-dex.

[0097] Example 3

[0098] In vitro release experiment of microspheres

[0099] Solution preparation

[0100] 0, 100, 200, 400 mg / dl glucose solutions: Weigh out 0, 30, 60, and 120 mg of glucose respectively, add 30 ml of ultrapure water, and label them for later use.

[0101] 1. Gradient Insulin Release Assay: Weigh four 10mg microspheres (using different ratios of Ac-dex and PLGA as carriers) into 1.5ml EP tubes, add 1ml of 0, 100, 200, and 400mg / dl glucose solution respectively, and place in a shaking incubator at 37℃ and 100rpm. Collect the supernatant from each tube every 2 hours. After sampling, transfer 20μl from each tube to a 96-well plate, add 200μl of Coomassie Brilliant Blue dye, react for 5min, and then measure the absorbance at 595nm using a microplate reader. Calculate the concentration and plot the curve.

[0102] The results are as follows Figure 5It was observed that the insulin release rate and amount increased with increasing concentration. At an Ac-dex:PLGA ratio of 1:1, the insulin release from the microspheres was minimal and unresponsive. At an Ac-dex:PLGA ratio of 7:1, insulin release was rapid in all concentration groups except for glucose concentration 0, with complete release within the first 2 hours. Furthermore, at a glucose concentration of 0, insulin release began after 6 hours. In contrast, Ac-dex:PLGA ratios of 3-5:1 showed better release and safety. However, at an Ac-dex:PLGA ratio of 5:1, release was lower at lower glucose concentrations, especially below 200 mg / dL, which is detrimental to glucose control. Compared to other groups, the Ac-dex:PLGA ratio of 3:1 microspheres exhibited better glycemic gradient responsiveness, maintaining an appropriate level of release, good responsiveness at each concentration, a more uniform release rate, and better safety.

[0103] Simultaneously, the gradient insulin release of the smart responsive sustained-release microspheres loaded with insulin, prepared in Example 1 of CN 111419825 A, was also tested. The outer microspheres used PLGA as the carrier material, the inner microspheres were mainly composed of polyacetal or its derivatives as the backbone, the model drug (insulin) was embedded within, and glucose oxidase and catalase triggers were dispersed on the outside of the small-diameter microspheres. The results showed that even at a glucose concentration of 0, the smart responsive sustained-release microspheres loaded with insulin began releasing insulin from the outset, and the release rate was relatively fast, even much greater than the release rate at the same concentration when Ac-dex:PLGA = 7:1. The release rate was even faster at other concentrations, which could lead to excessive insulin release, poor safety, and a high risk of hypoglycemia.

[0104] 2. Pulsating Insulin Release Assay: Weigh 10 mg of microspheres into a 1.5 ml EP tube, add 1 mL of 100 mg / dL glucose solution, place in a shaking incubator at 37℃ and 100 rpm, and collect the supernatant after 1 hour. Replace the medium with 400 mg / dL glucose solution and repeat the above steps. Perform three cycles, and measure the insulin release curves of the microspheres alternately in 100 mg / dL and 400 mg / dL glucose solutions (1 hour each). Results are as follows. Figure 6 As shown in the figure, insulin release exhibited a relatively rapid response in microspheres with three different carrier ratios (Ac-dex:PLGA = 7:1 / 5:1 / 3:1).

[0105] 3. Insulin release curve under physiological conditions: Weigh 10 mg of microspheres into a 1.5 ml EP tube, add 1 ml of PBS solution (pH 7.4), and place in a shaking incubator at 37℃ and 100 rpm. After 24 hours, collect the supernatant from the tube, add fresh PBS solution (pH 7.4), and repeat the above steps. Measure the insulin release curves of the microspheres under alternating physiological conditions over 14 days. The results are as follows: Figure 7 As shown in the figure, insulin release exhibits good sustained-release properties in microspheres with two different carrier ratios (Ac-dex:PLGA = 5:1 / 3:1).

[0106] Example 4

[0107] In vivo mouse experiments with microspheres

[0108] Solution preparation

[0109] 1% streptozotocin (STZ) solution: Take several 1.5ml EP tubes, weigh 5mg STZ powder into each tube under light-protected conditions, add 0.5ml of pH=4.5 sodium citrate buffer to each tube before use, prepare on ice in the dark, use immediately after preparation, and inject within 5min after dissolution.

[0110] 15g / L glucose solution: Weigh 15g of glucose powder and dissolve it in 1L of ultrapure water.

[0111] 1. Establishment of type 1 diabetes (T1D) mouse model

[0112] Six- to eight-week-old male C57BL / 6N mice (approximately 23g) were used for acclimatization for one week. Before modeling, the mice were fasted for 12 hours (with water provided as usual). A single intraperitoneal injection of 150mg / kg of 1% STZ solution was administered. The buffer solution was filtered through a 0.22-micron membrane to remove impurities before use. Two hours later, the mice were given food and glucose solution (15g / L glucose). Blood glucose levels were measured after 72 hours and monitored. Three days later, blood glucose levels were measured in each group of mice. A random blood glucose level >16.7mmol / L and a fasting blood glucose level >11.1mmol / L were considered a successful model.

[0113] 2. T1D mouse hypoglycemic experiment (n=1)

[0114] First, T1D mice were divided into three groups: an insulin (10 mg / kg) group, a microspheres (MPs, 10 mg / kg) group, and an MPs (20 mg / kg) group, with five mice in each group. After subcutaneous injection of the corresponding dose, blood glucose levels were monitored using a glucometer. Blood glucose was measured via tail vein sampling. A 1.5 ml needle was used to puncture the tip of the mouse's tail, and blood was absorbed using a blood glucose meter strip before reading the value. After measurement, the puncture site was wiped clean with an alcohol swab. Excessive movement or loud talking was avoided, as stress can cause blood glucose levels to rise and fluctuate significantly in mice. The red shaded area indicates feeding times. Figure 8 In the insulin group, blood glucose levels rose rapidly after feeding and remained in a hyperglycemic state thereafter. The MPs (10 mg / kg) group, compared to the insulin (10 mg / kg) group, not only effectively lowered blood glucose but also exhibited glycemic responsiveness. However, during the response process, the lower insulin load led to significant fluctuations in blood glucose after feeding, although it eventually returned to a lower level after a period of time. Furthermore, the lower drug load may result in a shorter duration of efficacy. In contrast, the MPs (20 mg / kg) group, compared to the previous two groups, maintained a stable blood glucose level within the normal range for a longer period. Moreover, due to the glycemic responsiveness of this invention, this high-dose group still ensured the safety of the mice, preventing hypoglycemia due to increased dosage. Therefore, the period of stable blood glucose can be adjusted by regulating the dosage of the microspheres.

[0115] Based on the above experiments, 20 mg / kg was selected as the subsequent MPs dosage for T1D mice, with insulin as the control. T1D mice were divided into an insulin (10 mg / kg) group and an MPs (20 mg / kg) group, with one mouse in each group. They were fed three meals a day, simulating human meals, with each feeding lasting one hour. Figure 9 In the diagram, the red shaded areas indicate feeding times. Microspheres (10 mg / kg) and microspheres (20 mg / kg) were injected after the first feeding, followed by insulin (10 mg / kg) after each feeding. Figure 9 It can be seen that insulin can rapidly lower blood sugar after injection, but blood sugar rises to a hyperglycemic state immediately after eating, and blood sugar can only be lowered after another insulin injection. Overall, insulin requires frequent injections, and the fluctuation range of blood sugar in mice is very large. In contrast, the MPs (20mg / kg) group can maintain a stable blood sugar level for a whole day after a single injection after the first meal, with only a slight rise in blood sugar after each meal, and then a rapid return to normal.

[0116] 3. T1D mouse hypoglycemic experiment (n=5)

[0117] T1D mice were divided into an insulin (10 mg / kg) group and an MPs (20 mg / kg) group, with 5 T1D mice in each group. The mice had free access to food and water. The results are as follows: Figure 10 As shown, under free-feeding conditions, insulin could only control blood glucose for 4 hours, while the microspheres could stably control blood glucose for 20 hours, far exceeding the insulin group, demonstrating the long-lasting blood glucose-lowering ability of the microspheres. When the microspheres were administered at a dose of 10 mg / kg, they still exhibited blood glucose responsiveness and could lower blood glucose levels in mice to the normal range, but the effect was short-lived, lasting only 7 hours. Furthermore, the blood glucose levels in mice rose sharply after feeding, with large fluctuations, and only returned to a lower level after a period of fluctuation.

[0118] 4. Intraperitoneal glucose tolerance test (IPGTT)

[0119] T1D mice were divided into an insulin group and an MPs group, with 5 mice in each group. Five healthy mice were also selected as the Healthy group. Blood glucose levels were monitored in both groups of T1D mice after administration of the drugs until they returned to normal (around 100 mg / ml). Then, both groups of T1D mice were simultaneously injected intraperitoneally with glucose solution (1.5 g / kg), and blood glucose changes were monitored again.

[0120] like Figure 11 Healthy mice showed a brief increase in blood glucose after glucose injection, which then returned to normal. In the insulin group, blood glucose significantly increased after glucose injection, gradually rising to hyperglycemic levels over time. Mice in the MPs group showed a slight increase in blood glucose after glucose injection, which then rapidly returned to normal. This method of rapidly adjusting blood glucose concentration through glucose injection tested the effects of medication on healthy and hyperglycemic mice. The insulin group failed to control blood glucose, while the microsphere group showed even better blood glucose control than healthy mice. When the microspheres were administered at a dose of 10 mg / kg, although the blood glucose in mice responded normally after glucose injection and returned to normal within 2 hours, the time spent in the hyperglycemic range was significantly longer than with a dose of 20 mg / kg, failing to achieve immediate blood glucose reduction.

[0121] 5. Hypoglycemic index in healthy mice

[0122] Five healthy mice were divided into two groups: an insulin (10 mg / kg) group and a MPs (20 mg / kg) group. Blood glucose levels were monitored and recorded after each group was administered the medication. The hypoglycemic index was calculated as the concentration difference between the lowest and initial blood glucose levels divided by the time to reach the lowest blood glucose level.

[0123] The results are as follows Figure 12As shown, during the experiment, mice in the insulin group exhibited obvious symptoms of hypoglycemia, such as lethargy and hind limb weakness, while mice in the MPs group remained in a normal state, as indicated by the blood glucose curve ( Figure 11 It can also be seen that although the theoretical dosage of MPs was twice that of the Insulin group, it did not cause the mice's blood glucose to drop to hypoglycemia. When the theoretical dosage of MPs was the same as that of the Insulin group, i.e., 10 mg / kg, the mice had a higher minimum blood glucose level, a greater gap from the hypoglycemia threshold, higher safety, and relatively lower efficacy.

[0124] Meanwhile, the insulin-loaded smart responsive sustained-release microspheres prepared in Example 1 of the prior art CN 111419825 A were also tested, and mice also showed obvious hypoglycemic symptoms.

[0125] Comparative Example 1

[0126] In contrast, an insulin-loaded smart responsive sustained-release microsphere was prepared according to Example 1 of the prior art CN 111419825 A.

[0127] In vitro insulin release experiments using these microspheres showed that the blood glucose response was not significant, and the differences in insulin release across different glucose concentration gradients were minimal. In the PBS group (i.e., the glucose concentration was 0), insulin continued to be released, subsequently leading to excessive insulin release and causing hypoglycemia in the mice.

[0128] The microspheres of this invention release almost no insulin when the glucose concentration is 0, but as the glucose concentration gradually increases, the release of insulin exhibits a clear gradient and rapid response. Therefore, this invention has higher safety.

[0129] Comparative Example 2

[0130] Conventional methods for preparing glucose-responsive controlled-release microspheres

[0131] Solution preparation

[0132] (1) 2% PVA aqueous solution: Weigh 10g PVA into a 1000ml beaker, add 500ml ultrapure water, seal with plastic wrap, poke holes in the surface, and mark the liquid level. Heat to 99℃ and set the speed to 300rpm, stirring until dissolved. After the solution returns to room temperature, add ultrapure water to the marked point, stir evenly, and filter with a 0.25μm filter membrane for later use.

[0133] (2) 2% PVA + 5% NaCl: Weigh 10g PVA into a 1000ml beaker, add 500ml ultrapure water, seal with plastic wrap, poke holes in the surface, and mark the liquid level. Heat to 99℃ and set the speed to 300rpm, stirring until dissolved. Weigh 25g NaCl and add it to the dissolved PVA solution, stirring until completely dissolved. After the solution returns to room temperature, add ultrapure water to the marked points, stir evenly, and filter through a 0.25μm filter membrane for later use.

[0134] (3) 0.1mol / LHCl: Take 250μL of concentrated hydrochloric acid (12mol / L), slowly add ultrapure water to 30ml, stir well and set aside.

[0135] (4) 0.5 mg / ml insulin standard solution: Take 1 mg of bovine insulin into a 2 ml volumetric flask, add 0.1 mol / L HCl solution until the concave meniscus reaches the mark, tighten the cap and shake well for later use.

[0136] 1. Emulsion Preparation

[0137] Weigh the raw materials according to the corresponding proportions. Ac-dex and PLGA are dissolved in dichloromethane (PLGA + Ac-dex: 8%, the specific concentrations of the two materials will be determined in subsequent experiments), and insulin, GOx, and CAT are dissolved in hydrochloric acid solution at pH 5 (Insulin: 5%, GOx: 1.25%, CAT: 0.25%). After all solutions are completely dissolved, mix them together and use a homogenizer set to 5000 rpm for 5 minutes to stir the two phases into an emulsion.

[0138] 2. Preparation of microspheres

[0139] The emulsion was injected directly into the receiving liquid while stirring at 300 rpm using a syringe. Stirring continued for 4 hours to allow the dichloromethane to evaporate completely, forming microspheres of approximately 200 μm in size. These microspheres were then washed with ultrapure water and collected.

[0140] The microspheres prepared using microfluidic control in Example 2 and the microspheres prepared using conventional injection methods in Comparative Example 2 are as follows: Figure 13 As shown in the image, it is clear that the microspheres prepared by the microfluidic method are more uniform and have a reasonable size of around 50 μm. In contrast, the microspheres prepared by conventional methods are very uneven in size and excessively large, reaching 200 μm.

[0141] Since the biodegradation rate of microspheres is closely related to their specific surface area, uniform size can ensure that the biodegradation rate of each microsphere tends to be consistent. The uniformity of the biodegradation rate and drug diffusion rate of microspheres has a significant impact on drug release. Experiments have shown that microspheres prepared by conventional methods exhibit larger fluctuations in blood drug concentration and unstable blood glucose control due to the excessively rapid or slow release of some microspheres. Under the same dosage, the responsiveness and gradient properties of microspheres prepared by conventional methods are significantly worse than those of the microspheres prepared in Example 1.

[0142] In terms of safety, microspheres with uniform size and controllable particle size not only enable more stable drug release, improving therapeutic efficacy and safety, but also better adapt to the in vivo environment, reducing immune system rejection and minimizing inflammation and other adverse reactions. Furthermore, they are more stable during in vivo release, storage, and transportation, less prone to aggregation or degradation, ensuring drug stability and long-lasting effects. Experiments have shown that, at the same dosage, the stability and long-lasting effect of microspheres prepared by conventional methods are significantly inferior to those prepared in Example 1.

[0143] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A responsive sustained-release microsphere, characterized in that, By weight, it includes 160-200 parts acetalized dextran, 40-80 parts polylactic acid-glycolic acid copolymer, 10-30 parts drug, 3-7 parts glucose oxidase and 0.5-1.5 parts catalase; The mass ratio of acetalized dextran to polylactic acid-glycolic acid copolymer is 3-5:1; The structural formula of the acetalized dextran is: Where n is an integer between 120 and 450; the difference in molecular weight between polylactic acid-glycolic acid copolymer and acetalized dextran is less than 10,000.

2. The responsive sustained-release microspheres according to claim 1, characterized in that, The responsive controlled-release microorganism The spheres use acetalized dextran and polylactic-co-glycolic acid copolymer as carriers, containing drugs, glucose oxidase, and... The contents consist of catalase.

3. The responsive sustained-release microspheres according to claim 1, characterized in that, The drug is insulin. Exenatide or glibenclamide.

4. The responsive sustained-release microspheres according to any one of claims 1-3, characterized in that, The acetalization The preparation method of dextran includes the following steps: Under an inert gas atmosphere, dextran, pyridine 4-methylbenzenesulfonic acid, and 2-ethoxypropene were mixed thoroughly and reacted at 20-40°C for 0.5-1.5 h. Triethylamine was then added to quench the reaction, yielding the reaction solution. The solution was then washed, centrifuged, and cooled. Freeze-drying yields acetalized dextran.

5. The responsive sustained-release microspheres according to claim 4, characterized in that, Dextran, 4-methylbenzenesulfonate The mass ratio of acid pyridine to 2-ethoxypropylene is 900-1100:15.6:3100-3300; the mass ratio of dextran to triethylamine is 1:0.7-0.

8.

6. The responsive sustained-release microspheres according to claim 4, characterized in that, The molecular weight of the dextran The molecular weight is 20,000-70,000; the molecular weight of the polylactic acid-glycolic acid copolymer is 48,000-50,000.

7. The method for preparing responsive sustained-release microspheres according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Prepare solutions of acetalized dextran, polylactic acid-glycolic acid copolymer, drugs, glucose oxidase, and catalase, mix them evenly to obtain a mixed emulsion; use the mixed emulsion as the chip internal phase injection solution. S2. The inner phase injection solution and the outer phase solution are delivered into the two-phase microfluidic chip, and the responsive sustained-release microspheres are obtained through microfluidic preparation.

8. The preparation method according to claim 7, characterized in that, The external phase solution is a polyvinyl alcohol solution; The mass concentration of the external phase solution is 1%-3%.

9. The preparation method according to claim 7, characterized in that, The microfluidic process parameters are: internal phase pressure of 0.15-0.25 MPa and external phase pressure of 0.20-0.50 MPa; the particle size of the responsive controlled-release microspheres is 40-60 μm.

10. The use of the responsive sustained-release microspheres according to any one of claims 1-6 in the preparation of reagents for treating diabetes.

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