Beta-lactoglobulin nanofiber as adjuvant for oral delivery of therapeutic polypeptide drugs and application of beta-lactoglobulin nanofiber

Abstract

The invention discloses a beta-lactoglobulin nanofiber as an adjuvant for oral delivery of therapeutic polypeptide drugs and application of the beta-lactoglobulin nanofiber, and belongs to the technical field of drug delivery adjuvants, the nanofiber is formed by heat treatment of BLG under an acidic condition, and has good gastrointestinal tract stability and severe environment tolerance; in the oral administration process, the nanofibers can temporarily induce the activity of calcium-dependent calpain to be improved, so that the intestinal epithelial permeability is enhanced, and the absorption of polypeptide drugs is facilitated; the rapid recovery of the intestinal barrier function can also be promoted. Insulin is loaded in an enteric capsule coated with 3% of sodium alginate, and by utilizing the BLG nanofiber mediated intestinal barrier regulation effect, compared with subcutaneous injection, the oral administration of insulin shows that the area under a blood glucose curve (AUC) is obviously reduced in a mouse model, and the blood glucose AUC is reduced by about 20.8%. The nanofiber is high in safety and meets the clinical large-scale production requirement.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug delivery adjuvant, in particular to beta-lactoglobulin nanofiber as an adjuvant for oral delivery of therapeutic polypeptide drugs and its application. BACKGROUND

[0002] Currently, the global diabetes epidemic has affected 589 million adults, and epidemiological projections show that this number will rise to 853 million by 2050. This worrying growth trajectory highlights the urgent need for patient-friendly treatment options. Although subcutaneous insulin injection remains the standard clinical treatment, its associated needle phobia (affecting 20-30% of patients) and hypoglycemic risk (an average of 23 mild / moderate and severe events per person per year) can severely impair treatment effectiveness and patient quality of life.

[0003] Oral insulin administration has physiological benefits and can improve patient compliance, but the challenge is how to achieve sufficient absorption of insulin in the gastrointestinal tract during long-term administration while maintaining the integrity of the gastrointestinal barrier - which is particularly important for lifelong diabetes management. Existing strategies have some limitations. For example, chemical permeation enhancers (e.g., sodium saccharinate, sodium caprate) can increase the absorption rate of oral insulin to 5-8%, but often cause gastrointestinal irritation7,10, 12-14; ligand-functionalized nanocarriers (e.g., transferrin or cell-penetrating peptide-modified nanoparticles) can achieve cell-specific delivery, but face complex scale-up requirements11, 15-17; natural vesicles (e.g., bovine milk exosomes) exhibit biological efficacy, but are still economically unfeasible due to low abundance (5 mg / L of milk) and complex separation. These challenges require a new generation of delivery systems that can balance efficacy, safety, and scalability. SUMMARY

[0004] In view of the above technical problems, the present application provides beta-lactoglobulin nanofiber as an adjuvant for oral delivery of therapeutic polypeptide drugs and its application. The nanofiber is formed by heat-induced self-assembly of beta-lactoglobulin under acidic conditions, and has good gastrointestinal tolerance and food-grade biological safety; during oral administration, the nanofiber can temporarily induce an increase in endogenous calcium-dependent calpain activity, thereby reducing the integrity of the intestinal epithelial tight junction and enabling drug absorption across the intestinal mucosal wall; after entering the blood through the intestine, the nanofiber is rapidly degraded by gastrointestinal enzymes, and the intestinal barrier function is restored. The present application also provides a preparation method of the nanofiber, a drug delivery mode of the nanofiber and an enteric capsule encapsulating insulin, and evidence for the use of the nanofiber for oral insulin to reduce blood glucose.

[0005] The present application provides an oral drug delivery adjuvant, which is composed of beta-lactoglobulin nanofibers prepared by the following steps:

[0006] (1) Dissolve beta-lactoglobulin in ultrapure water to 20 mg / mL, and adjust the pH of the solution to 2.0 by hydrochloric acid;

[0007] (2) Stir at 90 °C for 5 h to obtain precursor nanofibers rich in beta-sheet structure;

[0008] (3) Perform pulse tip sonication on the precursor nanofibers obtained in step (2); the pulse tip sonication is performed at 1 s on / 1 s off, 60 W for 15 min;

[0009] (4) Place the product of step (3) in a 100 kDa molecular weight cutoff dialysis bag, and dialyze against acidified ultrapure water to remove small molecular impurities;

[0010] (5) Neutralize the pH of the nanofiber solution after dialysis to physiological pH, and sterilize through a 0.22 μm filter membrane.

[0011] Further, the nanofibers can induce an increase in the level of endogenous calpain in the gastrointestinal environment, thereby enhancing the permeability of the intestinal epithelium, and are degraded by gastrointestinal enzymes to rapidly restore barrier integrity.

[0012] Further, the nanofibers are derived from food-grade beta-lactoglobulin, and no organic solvent or chemical crosslinking agent needs to be introduced after obtaining the precursor nanofibers.

[0013] The present application also provides an oral insulin delivery system, which comprises the oral drug delivery adjuvant and an enteric capsule coated with 3% sodium alginate and encapsulating insulin.

[0014] The present application also provides the use of the oral insulin delivery system in the preparation of a medicament for reducing blood glucose.

[0015] The medicament reduces blood glucose by the following routes:

[0016] (1) Simultaneously orally administer beta-lactoglobulin nanofibers and enteric capsules encapsulating insulin, and the capsules are released into the jejunum after staying in the stomach for 1-2 h;

[0017] (2) The beta-lactoglobulin nanofibers are pre-administered into the intestine, temporarily induce an increase in the expression of endogenous calpain in the local intestine, and enhance the permeability of the tight junction of the intestinal epithelium;

[0018] (3) Insulin is absorbed into blood circulation through the nanofiber-mediated channel, achieving blood glucose regulation; the nanofiber is then rapidly degraded by intestinal enzymes, and the intestinal barrier function is restored.

[0019] The beneficial technical effects of the present application include:

[0020] (1) Food-grade raw materials, simple process, low cost - the beta-lactoglobulin used is derived from dairy products, only acidification, heat treatment, ultrasonic and dialysis are required, without the need to add organic solvents or chemical cross-linking agents; the process conditions are mild and have good repeatability, and are suitable for large-scale production.

[0021] (2) Temporary regulation of intestinal barrier and improvement of absorption efficiency - the nanofiber can induce calcium-dependent calpain activity locally in the intestine, temporarily reducing the expression of tight junction proteins, allowing insulin to be absorbed through the intercellular space; the nanofiber is then degraded by intestinal enzymes, restoring the intestinal barrier function and reducing long-term safety risks.

[0022] (3) Good biocompatibility and safety - the beta-lactoglobulin itself is a food protein, and the nanofiber formed after acid and heat treatment is still a biodegradable material, with no obvious histopathological toxicity.

[0023] (4) Significant improvement in the bioavailability of oral insulin - oral administration experiments were conducted in healthy mice, mild and severe diabetic mouse models, and the results showed that, compared with the same dose of subcutaneous injection of insulin, oral insulin with nanofiber adjuvant can significantly reduce the cumulative AUC value of blood glucose by about 20.8%.

[0024] (5) Recycling of resources and sustainable production - about 45 g of nanofiber can be harvested per 1 kg of cheese produced, and dairy product processing waste can be converted into high-value pharmaceutical adjuvants, achieving resource recycling. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 : Schematic diagram of preparation and characterization results of beta-lactoglobulin nanofiber;

[0026] Figure 2 : In vitro biological safety evaluation of nanofiber;

[0027] Figure 3 : In vivo biological safety evaluation of nanofiber;

[0028] Figure 4 : Schematic diagram of reversible opening of intestinal barrier mediated by nanofiber;

[0029] Figure 5 : Intracellular calcium ion imaging and calpain inhibitor weakening of nanofiber-induced permeability;

[0030] Figure 6 : Comparison of blood glucose profile of nanofiber-mediated oral insulin and subcutaneous insulin injection. DETAILED DESCRIPTION

[0031] For a better understanding of the present application, it will now be described in further detail with reference to the accompanying drawings and specific examples, but the present application is not limited to the described examples.

[0032] Example 1: Preparation, characterization, and safety evaluation of beta-lactoglobulin nanofiber

[0033] (1) Preparation of beta-lactoglobulin nanofiber: Beta-lactoglobulin solution (20 mg / mL, dissolved in ultrapure water) was acidified to pH 2.0 with hydrochloric acid and subjected to heat fibrillation at 90 °C for 5 hours. The obtained nanofiber was shortened in length by pulsed tip sonication (60 W, 1 second on / 1 second off for 15 minutes) and then purified by dialysis (100 kDa molecular weight cut-off (MWCO)) against acidified ultrapure water. For biological studies, the nanofiber solution was neutralized to physiological pH and sterilized by 0.22 μm filter.

[0034] (2) Characterization of beta-lactoglobulin nanofiber: 20 μL of 0.2 mg / mL solution was deposited on a freshly cleaved mica substrate. After 2 minutes of adsorption, the substrate was gently rinsed with ultrapure water and dried under a controlled nitrogen flow. Topography imaging was performed using a Bruker Multimode 8 AFM. The acquired images were flattened using NanoScope 9.1 software. Zeta potential measurement was performed using Zetasizer Nano-ZS90 (Malvern, UK). The results are shown in Figure 1

[0035] (3) Biological safety evaluation: The toxicity of nanofiber to Caco-2 cells was evaluated in vitro by measuring intracellular ATP levels and extracellular LDH release. Figure 2 The results showed that the cell survival rate was always > 95% in the concentration range of 0-120 μM, proving that it had no significant toxicity to intestinal epithelial cells. The excellent safety of nanofiber was confirmed by gavage to mice for 21 days (0.16 g / kg): (1) the inflammatory markers were not elevated; (2) the intestinal structure remained intact without tissue damage or macrophage infiltration; (3) the apoptosis rate did not change Figure 3

[0036] In summary, this example successfully prepared stable, uniform, and biocompatible beta-lactoglobulin nanofiber, which can be used as a safe and effective oral drug delivery adjuvant. ​​

[0037] Example 2: Study of β-lactoglobulin nanofiber-induced reversible opening of intestinal barrier

[0038] To verify the ability of nanofiber to reversibly induce opening of epithelial permeability in the intestine, in vivo experiments were carried out using a healthy mouse model (results shown in Figure 4

[0039] (1) Experimental materials and methods

[0040] Healthy C57BL / 6J male mice, body weight 25 g, were fasted overnight and randomly divided into 3 groups, 3 mice in each group, and were respectively given (i) PBS; (ii) 20 mg / mL monomer solution; or (iii) 20 mg / mL nanofiber solution, all with a volume of 200 μL, and jejunal tissue samples of the mice were collected at 0, 1, 2, 3, 4, 8, and 12 hours after gavage.

[0041] (2) FITC-dextran evaluation of intestinal permeability

[0042] After gavage of nanofiber solution, 4 kDa FITC-dextran (60 mg / kg) was given at 0, 1, 2, 3, 4, 8, and 12 hours after gavage, and the results showed that the peak concentration of nanofiber pre-administration was reached at 2 hours, which was 13.7 times higher than that of the control group (pre-administration of the same volume of PBS or protein monomer). In addition, mice were injected with FITC-dextran of different molecular weights (4, 10, 40, or 70 kDa) to evaluate the molecular weight-dependent permeability, which indicated that nanofiber preferentially promoted the absorption of 4-10 kDa compounds.

[0043] (3) Electron microscopy characterization of small intestinal epithelial tight junctions

[0044] Transmission electron microscopy of jejunal epithelium showed that after gavage of nanofiber, the tight junctions were remodeled over time: subtle openings appeared within 1 hour after gavage, and more obvious changes appeared within 2-4 hours, and importantly, the integrity of the tight junctions was completely restored within 8 hours after administration. No significant changes in the morphology of tight junctions were observed in the monomer treatment group, indicating that nanofiber has a specific permeability-enhancing effect.

[0045] (4) Analysis of epithelial tight junction protein expression

[0046] The expression changes of tight junction proteins ZO-1, Occludin, and Claudin-1 in the jejunal tissue of mice were detected by Western blot, and the results were consistent with the electron microscopy results: ZO-1 expression decreased at 1-3 hours, while occludin and claudin-1 decreased at 2-4 hours, and the tight junction proteins underwent a transient but reversible regulation. ​

[0047] Figure 4 The results showed that β-lactoglobulin nanofibers could temporarily reduce tight junction protein expression, thereby enhancing intestinal permeability; subsequently, tight junction protein expression recovered, and intestinal barrier function was rapidly restored.

[0048] Example 3: Study on the mechanism of β-lactoglobulin nanofibers inducing intestinal barrier permeability

[0049] To verify the transient induction of calcium in the intestine by nanofibers 2+ Influx of calpain temporarily increases its activity and enhances epithelial permeability. This was validated in vitro using Caco-2 cells and an intestinal epithelial model based on Caco-2 cells (results are shown in...). Figure 5 (As shown).

[0050] (1) Intracellular calcium ion imaging

[0051] Intracellular calcium ion dynamics were monitored using the fluorescent probe Fluo-4 AM. Briefly, cells were washed with HEPES-buffered saline (HBSS) and then incubated with 5 μM Fluo-4 AM at 37 °C for 20 min. Afterward, cells were rinsed with HBSS and incubated for another 30 min to allow complete deesterification of AM esters. Images were acquired using a CLSM (excitation / emission: 485 / 515 nm), and fluorescence intensity was quantified using ImageJ.

[0052] (2) Establishment of Caco-2 cell monolayer

[0053] Caco-2 cells were cultured at 37 °C and humidified with 5% CO2 in DMEM medium (Thermo Scientific, Heracell 150i) supplemented with 10% FBS. To form a monolayer, the cells were cultured at a density of 1 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 1 / mL on 12 mm diameter Transwell® culture dishes (0.4 μm pore size polyester membrane; Corning 3460) and cultured for 21 days, with the medium changed every 2 days. Only monolayers of cells with an initial transepithelial resistivity (TEER) of at least 300 Ωcm² were used for subsequent experiments. TEER was measured using a Millicell ERS-2 voltmeter (Millipore).

[0054] (3) Calpain is the key medium that leads to increased permeability.

[0055] To investigate the molecular mechanism, monolayers were pretreated with specific inhibitors for 2 hours prior to nanofiber exposure: 500 nM MLCK inhibitor peptide 18 (MLCK inhibitor), 50 µM MDL-28170 (calpain inhibitor), 200 nM Bisindolymaleimide I (PKC inhibitor), 10 µM Y-27632 (ROCK inhibitor), 5 µM Adzmapimod (MAPK inhibitor). After 1 h of nanofiber treatment of monolayers, 4 kDa FITC-dextran (0.1 mM) was added to the apical chamber, and after 1 h of incubation, 50 μL of basolateral medium was taken and the fluorescence intensity (excitation / emission wavelength: 485 / 515 nm) was measured with a microplate reader to quantify 4 kDa FITC-dextran that had crossed the cell monolayer.

[0056] Example 4: In vivo efficacy evaluation of β-lactoglobulin nanofiber-mediated oral insulin delivery system

[0057] This example further verified the promotion effect of β-lactoglobulin nanofiber as a delivery adjuvant on the absorption of oral insulin and the hypoglycemic efficacy (results are shown in Figure 6

[0058] (1) Reagents and formulation

[0059] Insulin (human recombinant, content 100 IU / mg); β-lactoglobulin nanofiber: prepared according to the reference example 1; Sodium alginate-coated enteric capsule - the outer wall of the enteric capsule was coated with 3% sodium alginate solution, and the uniform mixture of insulin (mass ratio, insulin: aprotinin: inactive BSA protein = 3:1:1) was encapsulated. Each enteric capsule contains insulin (20 IU / kg).

[0060] (2) Establishment of diabetic mouse model: Severe and mild diabetic mouse models were established by intraperitoneal injection of streptozotocin (STZ). For the severe diabetic model, mice were injected intraperitoneally with freshly dissolved STZ (200 mg / kg) in 0.01 M citrate buffer (pH 4.5). The fasting blood glucose level was monitored daily, and mice with persistent hyperglycemia (>250 mg / dL) were included in the severe diabetic group.

[0061] ​(3) Animal grouping and dosing: C57BL / 6 mice were fasted and randomly divided into four experimental groups. Three groups of mice received 200 μL of the following reagents: (A) PBS, (B) 20 mg / mL monomer solution, or (C) 20 mg / mL nanofiber solution. Two hours after pretreatment, these groups were administered oral enteric-coated capsules containing 20 IU / kg insulin. Group D (drug control) was administered 2 IU / kg insulin by subcutaneous injection.

[0062] (4) Blood glucose change curve and serum insulin concentration determination

[0063] Group D (subcutaneous injection) reached the lowest blood glucose at 30 min, and then the blood glucose gradually rose within 4 h; Group B (oral insulin + monomer solution) showed no significant change in blood glucose, similar to Group A, indicating that oral insulin without adjuvant assistance was almost not absorbed; Group C (oral insulin + nanofiber solution) produced a slower but more sustained hypoglycemic effect within 6 h. Quantitative analysis showed that the blood glucose AUC value of the nanofiber group was significantly lower than that of PBS (42.7% reduction), monomer (44.1% reduction), and subcutaneous insulin (20.8% reduction). The serum insulin concentration had a good correlation with these blood glucose responses.

[0064] In summary, the present example demonstrates that the β-lactoglobulin nanofiber adjuvant can significantly improve the bioavailability of oral insulin and show good hypoglycemic effect and safety in vivo, and can provide a simple and feasible technical route for the delivery of oral polypeptide drugs (insulin).

[0065] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art should fall within the scope of protection determined by the claims of the present application.

Claims

1. An oral adjuvant for drug delivery, characterized in that, The oral adjuvant is composed of β-lactoglobulin nanofibers, which are prepared by the following steps: (1) Dissolve β-lactoglobulin in ultrapure water to 20 mg / mL, and adjust the pH of the solution to 2.0 with hydrochloric acid; (2) The precursor nanofibers rich in β-fold structure were obtained by stirring at 90 °C for 5 h. (3) The precursor nanofibers obtained in step (2) are subjected to pulse tip ultrasonic treatment; The conditions for the pulse tip ultrasonic treatment are: ultrasonic power 60 W, 1 s on / 1 s off, lasting 15 min; (4) Place the product from step (3) in a 100 kDa molecular weight cutoff dialysis bag and use acidified ultrapure water for dialysis to remove small molecule impurities; (5) Neutralize the pH of the dialysis nanofiber solution to physiological pH and sterilize it by passing it through a 0.22 μm filter membrane.

2. The oral adjuvant according to claim 1, characterized in that, The nanofibers can induce an increase in the level of endogenous calcium-dependent calpain in the gastrointestinal environment, thereby enhancing intestinal epithelial permeability and being degraded by gastrointestinal enzymes to rapidly restore barrier integrity.

3. The oral adjuvant according to any one of claims 1 to 2, characterized in that, The nanofibers are derived from food-grade β-lactoglobulin, and no organic solvents or chemical cross-linking agents need to be introduced after obtaining the precursor nanofibers.

4. An oral insulin delivery system, characterized in that, include: The oral adjuvant of claim 1 and the enteric-coated capsule coated with 3% sodium alginate and encapsulated with insulin.

5. Use of the oral insulin delivery system of claim 4 in the preparation of a medicament for lowering blood glucose.

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