Preparation and application of ultrahigh insulin-loaded nanoparticles with glucose response

Abstract

The invention discloses a preparation method and application of glucose-responsive ultrahigh insulin-loaded nanoparticles. The preparation method comprises the following steps: step 1, preparing insulin-loaded cationic nanoparticles INS-PEI by adopting a sodium hydroxide alkalization-electrostatic adsorption method; and 2, jointly loading the Gox and INS-PEI nanoparticles onto carriers including but not limited to gel, seaweed microspheres and the like by adopting a vortex incubation method. According to the invention, INS-PEI is prepared by using a sodium hydroxide method under the electrostatic interaction, and the pH value of a microenvironment is reduced by GOx, so that insulin release is realized; the glucose-responsive ultrahigh insulin-loaded nanoparticle injection has the advantages of uniform particle size, high encapsulation efficiency and good slow release effect, effectively reduces the blood glucose level, stably controls the blood glucose, prolongs the drug action time, reduces the injection frequency, and improves the compliance of diabetic patients. And a thought is provided for treating the type I diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutics, and in particular relates to the preparation and application of glucose-responsive ultra-high insulin-loaded nanoparticles. Background Art

[0002] Diabetes is a metabolic disease characterized by hyperglycemia. Insulin injections are the preferred treatment for diabetes, requiring long-term, continuous administration to ensure efficacy, which inevitably places a heavy physical, mental, and financial burden on patients. Frequent intraperitoneal injections can cause allergic reactions, insulin neuritis, hypoglycemia, weight gain, lower limb edema, and subcutaneous nodules at the injection site.

[0003] Insulin (INS), a polypeptide drug, has an isoelectric point of 5.4. It exhibits negative charge at pH 7.4 and becomes positively charged at pH 5.4 and below. Polyethylenimine (PEI), a water-soluble cationic polymer, utilizes electrostatic interactions between insulin and cationic polymers to increase drug loading in carriers. In "closed-loop" insulin delivery systems, the sensitivity of the glucose-sensing element is crucial for achieving responsive insulin release. The sensor is typically integrated into a biomaterial matrix to respond to glucose concentration, driving physical or chemical changes in the matrix and thus releasing the encapsulated insulin. Glucose oxidase (GOx) is the most glucose-specific sensor, converting glucose into gluconic acid, which lowers the surrounding pH and triggers changes in the pH-sensitive carrier. When blood glucose concentrations are high, glucose reacts with GOx, significantly lowering the pH to the isoelectric point of insulin. This positively charges the insulin, causing intermolecular electrostatic repulsion to rupture the nanoparticles, thereby releasing the insulin and achieving long-lasting controlled release.

[0004] The present invention uses a cationic polymer as a carrier material, relying on changes in the pH of the microenvironment to achieve adsorption or desorption of insulin molecules by the polymer, thereby achieving loading and release. Through the electrostatic interaction between insulin and polymer molecules in the nanoparticles, insulin loading (electrostatic attraction) and glucose-responsive release (electrostatic repulsion) are simultaneously achieved, resulting in a formulation with a high insulin loading and a sensitive, long-lasting glucose response. This allows for better blood sugar control and regulatory effects, providing more options for the treatment of type 1 diabetes, improving drug administration safety and patient compliance, and possessing significant social and medical significance. Summary of the Invention

[0005] The present invention aims to provide the preparation and application of glucose-responsive ultra-high insulin-loaded nanoparticles. This discovery involves forming aggregates through electrostatic adsorption between insulin and cationic polymers, which are then loaded with GOx to form glucose-responsive insulin nanoparticles for subcutaneous injection to treat type 1 diabetes.

[0006] The present invention adopts an electrostatic adsorption-sodium hydroxide alkalization method to prepare insulin nanoparticles, which includes the following steps: In the first step, INS-PEI nanoparticles were prepared by electrostatic adsorption-sodium hydroxide alkalization method, specifically: 1-15 parts by weight of insulin are dissolved in 0.02-0.1 mM hydrochloric acid solvent, and 1-15 parts by weight of PEI are dissolved in pure water. The insulin mother liquor and the PEI mother liquor are mixed, and 0.1-0.3 parts by weight of sodium hydroxide are added to the INS-PEI mixture using a sodium hydroxide alkalization method to prepare INS-PEI nanoparticles.

[0007] In the second step, Gox and INS-PEI nanoparticles are co-loaded onto carriers including but not limited to gels, algae microspheres, etc. using a vortex incubation method.

[0008] Add 0.1-3 parts by weight of GOx to the nanoparticles from step 1 and vortex for 0.1-2 hours at 25°C-37°C. Centrifuge the resulting nanoparticles (5,000-10,000 g, 5-20 min) and remove the supernatant to obtain ultra-high insulin-loaded nanoparticles.

[0009] The glucose-responsive ultra-high insulin-loaded nanoparticles prepared by the present invention can be injected subcutaneously to treat type I diabetes.

[0010] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention utilizes a nanostructured strategy to increase the drug loading capacity of the complex under the action of a cationic polymer, thereby achieving efficient insulin encapsulation. The method of the present invention is simple in process, easy to operate, high in encapsulation capacity, and good in stability.

[0011] (2) The nanoparticles prepared in the present invention have good drug loading, particle size distribution and stability. The insulin encapsulation efficiency is (99.66±0.05)%, the drug loading is (72.07±4.70)%, and the average particle size is (434.26±3.03) nm. The in vitro drug release results show that the release of insulin from the nanoparticles is slow, and the insulin in the nanoparticles is completely released after 48 h.

[0012] (3) Under high blood sugar stimulation, glucose oxidase is used for insulin sugar response release. Under normal blood sugar conditions, encapsulation is used to prevent insulin from being released in the form of nanoparticles to prevent hypoglycemia.

[0013] In summary, the preparation process of the present invention is simple and convenient, relatively stable and reliable, and can effectively control the release of insulin according to the blood glucose concentration after entering the body, and has great prospects in the treatment of type 1 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The particle size diagram of INS-PEI-GOx in the embodiment; Figure 2 Transmission electron microscopy image of INS-PEI-GOx; Figure 3 This is the in vitro release curve of INS-PEI-GOx.

[0015] Figure 4 This is the blood glucose change curve of INS-PEI-GOx in vivo experiment. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further described below in conjunction with specific implementation methods so that those skilled in the art can better understand the present invention and implement it.

[0017] Example The raw materials used in this embodiment include: Insulin API Tonghua Dongbao Pharmaceutical Co., Ltd. Polyethyleneimine Aladdin Reagent (Shanghai) Co., Ltd. Sodium hydroxide Chengdu Kelong Chemical Reagent Factory Glucose Chengdu Kelong Chemical Reagent Factory Glucose Oxidase Sangon Biotechnology (Shanghai) Co., Ltd. Hydrochloric acid Sichuan Xilong Scientific Co., Ltd. Citric acid Guangdong Guanghua Technology Co., Ltd. Trisodium Citrate Dihydrate Guangdong Guanghua Science and Technology Co., Ltd. Streptozotocin Solaibao Biotechnology Co., Ltd. The equipment used in this embodiment includes: Research Plus pipettes (Eppendorf, Germany), ZEN3600 nanoparticle size analyzer (Malvern Instruments Ltd., UK), CPA225B electronic balance (Sartorius Balance Co., Ltd.), KMH1 ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.), Ymnl-1000Y ultrasonic cell disruptor (Nanjing Emmanuel Instrument Equipment Co., Ltd.), CHA-S constant temperature oscillator (Changzhou Aohua Instrument Co., Ltd.).

[0018] The steps for glucose-responsive ultra-high insulin-loaded nanoparticles are as follows: The first step is to prepare INS-PEI nanoparticles using the sodium hydroxide alkalization method: Dissolve the prescribed amount of insulin (INS) in 0.012 M HCl and the prescribed amount of dopamine hydrochloride (PEI) in pure water. The INS and PEI stock solutions were then mixed uniformly in a specific ratio. After adding NaOH, the supernatant was removed by centrifugation, washed three times with pure water, and centrifuged again to obtain ultra-high insulin-loaded INS-PEI nanoparticles. The encapsulation efficiency, particle size, PDI, and potential were measured. The data are shown in the table.

[0019] (1) Determination of encapsulation efficiency and drug loading: INS-PEI nanoparticles were divided equally into two portions. One portion was then separated from the free components by high-speed centrifugation, and the free fraction was collected. The other nanoparticle sample was directly demulsified with dilute hydrochloric acid and diluted to the same multiple as the centrifuged insulin. The insulin content in the nanocarriers was determined by HPLC to calculate the INS-PEI encapsulation efficiency and drug loading. Encapsulation efficiency (%) = (1-Ca / Cb) × 100%, where Ca represents the free insulin concentration and Cb represents the insulin concentration in the unfractionated nanoparticles.

[0020] Drug loading (%) = Wa / Ww×100%, where Wa represents the mass of INS in the nanoparticle carrier, and Ww represents the mass of INS in the nanoparticle plus the mass of the carrier material.

[0021] (2) Determination of particle size and potential: The particle size and potential of the nanoparticles were measured using a ZEN3600 series nanolaser particle size analyzer. 0.1 mL of the solid lipid nanoparticle suspension was diluted to 1 mL. The nanoparticle size analyzer temperature was set to 25°C, the equilibration time was 2 minutes, and each sample was measured three times.

[0022] The preparation process of INS-PEI was optimized through experiments, including: (1a) Optimize the INS-PEI mass ratio: In the first step, the aforementioned formulation was maintained, but the ratio of INS to PEI was varied to prepare INS-PEI nanoparticles. The encapsulation efficiency and drug loading of the drug-loaded nanoparticles were investigated. The measured data are shown in Table 1. The experiments revealed that the encapsulation efficiency and drug loading were excellent at different mass ratios, with uniform particle size distribution and a stable size of approximately 420 nm. This indicates that the INS to PEI ratio has little effect on the particle size of the INS-PEI nanoparticles. A mass ratio of 2:1 achieved the best insulin encapsulation efficiency, so this was the ideal ratio.

[0023] Table 1 Effects of different mass ratios on INS-PEI particle size, PDI, encapsulation efficiency and drug loading

[0024] (1b) Optimize the quality of sodium hydroxide: In the first step, the mass ratio of INS to PEI was fixed at 2:1, the basic formulation remained unchanged, and the amount of sodium hydroxide was varied. The measured data are shown in Table 2. The results showed that as the proportion of sodium hydroxide increased, the insulin encapsulation efficiency and drug loading gradually decreased. The amount of sodium hydroxide did not affect the particle size or dispersibility of the INS-PEI nanoparticles. The final mass ratio of sodium hydroxide to PEI was determined to be 0.15:1, resulting in an encapsulation efficiency of 99.69 ± 0.05% and a drug loading of 74.57 ± 5.34%.

[0025] Table 2 Effect of the mass ratio of sodium hydroxide to PEI on INS-PEI particle size, PDI, encapsulation efficiency and drug loading

[0026] In the second step, GOx is incubated on a carrier loaded with INS-PEI nanoparticles, including but not limited to gels, microspheres, etc., by vortexing: Add a predetermined amount of GOx to a carrier containing the nanoparticles from the first step, including but not limited to gels and microspheres. Vortex at 25°C-37°C for 0.1-2 hours. Centrifuge the resulting nanoparticles (5,000-10,000 g, 5-20 min) and remove the supernatant to obtain the INS-PEI-GOx ultra-high insulin-loaded nanoparticles.

[0027] (1) Since GOx oxidizes glucose to produce gluconic acid to lower pH, which is the key to the release of insulin from INS-PEI-GOx nanoparticles, its content may have a significant impact on the release rate of insulin. The synthesis conditions of ultra-high insulin-loading nanoparticles were optimized by fixing the ratio of INS to PEI and the amount of sodium hydroxide and changing the ratio of GOx to PEI.

[0028] (2a) Optimizing the ratio of PEI to GOx In the first step, the mass ratio of INS to PEI was fixed at 2:1, and the mass ratio of sodium hydroxide to PEI was fixed at 0.15:1. Glucose oxidase at varying PEI to GOx mass ratios was then added to a carrier containing the nanoparticles from the first step, including but not limited to gels and microspheres, to produce ultra-high insulin-loaded nanoparticles. The particle size and PDI of the resulting INS-PEI-GOx nanoparticles were measured, and the insulin release time of the nanoparticles was determined by high-performance liquid chromatography. The data are shown in Table 3. The results show that increasing the mass of glucose oxidase shortened the release time. A 1:1 glucose oxidase to PEI mass ratio was selected to maximize sustained release.

[0029] Table 3 Insulin release time of INS-PEI-GOx nanoparticles with different PEI:GOx mass ratios

[0030] (2b) Optimize incubation time: At fixed mass ratios of INS to PEI and sodium hydroxide to PEI, GOx was incubated for varying times. The measured data are shown in Table 4. The results show no significant change in particle size with increasing incubation time, and the encapsulation efficiency and drug loading also remained similar. Therefore, considering the potential for sustained release of the nanoparticles later, a two-hour incubation time was selected.

[0031] Table 4 Effects of different incubation times on INS-PEI particle size, PDI, encapsulation efficiency and drug loading

[0032] Characterization of ultra-high insulin-loading nanoparticles (2b) The mass ratio of INS to PEI was fixed at 2:1, and the mass ratio of sodium hydroxide to PEI was fixed at 0.15:1. GOx was added at the same mass ratio as PEI to obtain ultra-high insulin-loaded nanoparticles. 0.1 mL of the ultra-high insulin-loaded nanoparticle suspension was diluted to 1 mL. The nanoparticle size analyzer temperature was set to 25 °C, and the equilibrium time was 2 min. The particle size and PDI were measured using a ZEN3600 nanoparticle size analyzer. The results are shown in Figure 2. Figure 1 The results showed that the particle sizes of ultra-high insulin-loaded nanoparticles were similar, which were 428.47±7.23 and PDI were 0.235±0.12.

[0033] (2c) INS-PEI-GOx was diluted with pure water to a suitable concentration and dropped onto the copper grid. It was allowed to dry naturally and a transmission electron microscope photo of the morphology was taken. The results are shown in Figure 2 The results showed that the nanoparticles were regular, complete, and consistent in morphology, and their particle sizes were consistent with the data from the particle size analyzer.

[0034] Step 3: In vitro release experiment of INS-PEI-GOx: In vitro drug release studies were conducted using a direct dissolution method. The constant temperature oscillator was set at 37°C and the oscillation frequency was constant at 100 r / min. Three replicates were set up for each group. INS-PEI-GOX was placed in 50 mL of normal saline solution (pH = 7.4), 50 mL of 400 mg / dL glucose solution (pH = 7.4), and 50 mL of 100 mg / dL glucose solution (pH = 7.4) for release. 1 mL of sample was taken at predetermined time points within 0-48 hours. The sample was rotated at 8000 r·min. -1 Centrifuge for 10 min under the same conditions, add the sample into the chromatographic column for analysis, and calculate the release rate. The results are shown in Figure 3 .

[0035] Step 4: In vivo efficacy test: (1) Experimental animals: SD male rats, weighing 220 g ± 2 g, were fed for one week before the experiment.

[0036] (2) Experimental animal models: A rat model of type 1 diabetes was established by high-dose streptozotocin injections to destroy pancreatic B cells. Male Sprague-Dawley rats were fasted for 4-6 hours before administration. For three consecutive days, 1% streptozotocin (w / v) in 0.1M citric acid-trisodium citrate buffer was injected intraperitoneally at a dose of 50 mg / kg. One week after modeling, success was considered when fasting blood glucose levels remained above 13.9 mmol.

[0037] (3) Experimental groups: Group 1: healthy group (0.9% saline subcutaneous injection); Group 2: Control group: (subcutaneous injection of insulin); Group 3: Drug administration group: (INS-PEI-GOX subcutaneous injection) There were 3 groups in total, with 5 subjects in each group.

[0038] (4) Results of efficacy experiments: The level of blood sugar directly indicates the effectiveness of treatment. Figure 4 The results showed that the drug-treated group was more effective than the control group and could control and stabilize blood sugar for a longer period of time.

[0039] The technical solutions provided by the present invention are not limited to the above-mentioned embodiments. Any technical solutions formed by transformation and substitution of the structure and method of the present invention are within the protection scope of the present invention.

Claims

1. A method for preparing ultra-high insulin-loaded nanoparticles, characterized by: Including steps: In the first step, INS-PEI nanoparticles were prepared by electrostatic adsorption-sodium hydroxide alkalization method, specifically: 1-15 parts by weight of insulin are dissolved in 0.02-0.1 mM hydrochloric acid solvent, and 1-15 parts by weight of PEI are dissolved in pure water. The insulin mother solution and the PEI mother solution are mixed, and 0.15-4.5 parts by weight of sodium hydroxide are added to the INS-PEI mixture by a sodium hydroxide alkalization method to prepare INS-PEI nanoparticles; In the second step, Gox and INS-PEI nanoparticles were co-loaded onto carriers including but not limited to gels and algae microspheres using a vortex incubation method. Add 0.1-3 parts by mass of GOx to the nanoparticles in the first step, vortex at 25°C-37°C for 0.1-2 h, centrifuge the obtained nanoparticles (5000-10000 centrifugal force, 5-20 min), and remove the supernatant to obtain ultra-high insulin-loaded nanoparticles.

2. The method for preparing ultra-high insulin-loading nanoparticles according to claim 1, wherein: In the mixed solvent of insulin and polyethyleneimine, the volume ratio of insulin to polyethyleneimine is (1~15): (1~15).

3. The method for preparing ultra-high insulin-loading nanoparticles according to claim 1, wherein: In the mixed solvent of insulin and PEI, the mass ratio of PEI to sodium hydroxide solution is 1:(0.1~0.3).

4. The method for preparing ultra-high insulin-loading nanoparticles according to claim 1, wherein: The usage ratio of the glucose oxidase and the polyethyleneimine is (0.1-3):

1.

5. The ultra-high insulin loading nanoparticles according to claim 1, characterized in that: For preparing a pharmaceutical composition for treating type 1 diabetes by subcutaneous injection, The pharmaceutical composition can significantly improve the bioavailability of insulin in the body, prolong the duration of action of insulin, and significantly reduce the frequency of administration.

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