Hydrogel beads for protecting oral peptide drugs and a method for preparing the same
By co-encapsulating Bowman-Birk inhibitors and therapeutic peptides in hydrogel beads, a pH-responsive core-shell structure was constructed, solving the degradation problem of peptide drugs in gastric acid and intestinal environment, achieving intestinal targeted delivery and intelligent release, and improving bioavailability and safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING TECH & BUSINESS UNIV
- Filing Date
- 2025-12-24
- Publication Date
- 2026-05-19
AI Technical Summary
Existing oral peptide drugs are easily degraded in the environment of gastric acid and intestinal proteases, resulting in low bioavailability. Furthermore, existing delivery systems have poor release kinetic control, leading to burst release effects and insufficient passive protection.
The Bowman-Birk inhibitor (BBI) and therapeutic peptides were co-encapsulated in pH-responsive hydrogel beads. A core-shell structure was constructed through ionic cross-linking and polyelectrolyte composites to achieve targeted release and active protection in the intestine. The protease-inhibiting activity of BBI was used to establish a low protease microenvironment in the intestinal region.
It significantly improves the oral bioavailability of peptide drugs, prolongs the active retention time, achieves gastrointestinal targeted delivery and intelligent release, enhances mechanical properties and drug loading, and reduces the risk of high systemic exposure.
Smart Images
Figure CN121714529B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a hydrogel bead for protecting oral peptide drugs and its preparation method. In particular, it utilizes a Bowman-Birk inhibitor (BBI) and a therapeutic peptide, such as a glucagon-like peptide-1 (GLP-1) analog such as smegglutide, co-encapsulated in pH-responsive hydrogel beads to achieve an intestinal-targeted and protective sustained-release delivery system, its preparation method, and its application. Background Technology
[0002] Oral peptide drugs have demonstrated significant efficacy in the treatment of type 2 diabetes and obesity. However, their oral delivery faces serious challenges: firstly, the drugs are prone to structural damage and inactivation in the highly acidic environment of the stomach; secondly, in the intestines, peptide drugs encounter abundant proteases (such as trypsin and chymotrypsin), leading to their rapid degradation, which is one of the core reasons for their extremely low oral bioavailability. Therefore, developing an oral delivery system that can effectively protect peptides and achieve precise intestinal-targeted release is crucial.
[0003] Hydrogel systems, as drug delivery carriers, have shown significant advantages in encapsulating and delivering peptide drugs, and have become a research hotspot in this field. Specifically, their advantages are reflected in the following aspects:
[0004] First, the three-dimensional network structure of hydrogels can effectively load peptide drugs through physical encapsulation or chemical bonding, providing them with a stable microenvironment. This structure acts as a physical barrier, isolating gastric acid and proteases to a certain extent, preventing peptide degradation and inactivation before reaching the target release site. Second, hydrogels possess excellent biocompatibility and biodegradability. Commonly used natural polymer materials, such as sodium alginate, chitosan, and gelatin, are widely available, highly safe, and biodegradable in vivo, avoiding the risk of long-term accumulation. Third, the release behavior of hydrogels is controllable and environmentally responsive. By selecting different polymer materials and controlling the crosslinking density, sustained-release control of peptide drugs can be achieved. More importantly, stimulus-responsive hydrogels can be designed and constructed. For example, pH-sensitive hydrogels maintain structural stability in the acidic environment of the stomach, while swelling or disintegrating under neutral or weakly alkaline conditions in the intestine, thereby achieving targeted intestinal release of drugs and greatly improving the accuracy of drug administration. Furthermore, as a multifunctional platform, hydrogels are easily functionalized, enabling more precise time-controlled release or synergistic delivery of other functional components.
[0005] However, existing technologies have major drawbacks:
[0006] (1) Poor control of drug release kinetics, with a significant "burst release effect".
[0007] Existing delivery systems lack sufficient control over the release behavior of peptide drugs. Upon reaching the intestinal target site, the carrier often swells or disintegrates rapidly due to pH changes, resulting in a large release of the encapsulated drug within a short period, producing a significant "burst release effect." This burst release phenomenon not only may lead to excessively high local drug concentrations, posing potential safety hazards, but more seriously, it causes the vast majority of the peptide drug to be "concentratedly exposed" to the complex intestinal protease environment during the absorption window, greatly increasing the probability of degradation and thus making it difficult to effectively improve overall bioavailability.
[0008] (2) The protection model is passive and singular, lacking a proactive intervention mechanism.
[0009] The protection provided by existing technologies is essentially a passive barrier mode, primarily relying on the physical barrier effect of the carrier to delay the contact between the peptide and gastric acid and proteases. However, this passive protection is fragile and incomplete in the intestinal environment. Once the carrier disintegrates in the intestine, the drug loses its protection. Existing strategies generally lack the ability to actively intervene in and clear key intestinal degradation factors, failing to pre-alter the local microenvironment before drug release. This leaves the peptide drug directly exposed to high concentrations of proteases at the critical moment of release, constituting a major cause of its oral delivery failure.
[0010] Although BBI, a natural protease inhibitor extracted from soybeans, has long been considered an anti-nutritional factor, research has shown its potential applications in anti-cancer, anti-inflammatory, and blood sugar regulation. This invention aims to innovatively utilize the protease-inhibiting activity of BBI to transform it into a "natural protectant" for peptide drugs. Summary of the Invention
[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a hydrogel bead for protecting oral peptide drugs, its preparation method and application.
[0012] This system, through intelligent carrier design, enables the rapid release of Bowman-Birk inhibitors (BBI) and the slow release of therapeutic peptides, thereby actively establishing a protective low-protease microenvironment in the gut and significantly improving the oral bioavailability of the drug.
[0013] Specifically, to achieve the above objectives, the present invention first provides a hydrogel bead for protecting oral peptide drugs, characterized in that the hydrogel bead comprises a therapeutic peptide, a Bowman-Birk inhibitor (BBI), and a pH-responsive gel matrix formed by ionic crosslinking and polyelectrolyte complexation of sodium alginate, chitosan, and calcium ions; wherein the mass ratio of the therapeutic peptide to BBI is 1:1 to 1:9.
[0014] Preferably, the therapeutic polypeptide is a glucagon-like peptide-1 (GLP-1) analog. More preferably, the GLP-1 analog is smegglutinin.
[0015] The ratio of mannuronic acid to guluronic acid in the sodium alginate is 1:1 to 1:2.
[0016] The hydrogel beads have a particle size of 2.0-4.0 mm.
[0017] After the hydrogel beads were incubated in simulated gastric fluid at pH 1.2 for 2 hours, the cumulative release rate of the therapeutic peptide was less than 20%.
[0018] The Bowman-Birk inhibitor is prepared by a method comprising the following steps:
[0019] a) Isoelectric point precipitation: The pH of the soybean whey solution with a pH of 7.5 was adjusted to 4.0 with acid to carry out the first precipitation. The protein precipitate was collected after centrifugation.
[0020] b) Ammonium sulfate fractionation: Add ammonium sulfate to the supernatant obtained in step a) to make it saturated to 25%-65%, let it stand at 2-6℃ for 110-130 minutes, and then centrifuge to collect the second protein precipitate.
[0021] c) Gel filtration chromatography: After redissolving the precipitate obtained in step b), purification was performed using a Superdex 75 Increase 10 / 300 pre-packed gel filtration column, wherein the chromatography column was equilibrated with 0.1M sodium phosphate buffer solution.
[0022] The sample loading volume is 450-550 μL; the flow rate is controlled at 0.4-0.6 mL / min; the elution peak is detected at a wavelength of 280 nm and collected fractionally;
[0023] d) Collection and post-processing of active components: The trypsin inhibitory activity of each collected component was determined. Protein components with target inhibitory activity were collected, and after dialysis and freeze-drying, purified Bowman-Birk inhibitor products with a purity of over 90% were obtained.
[0024] On the other hand, the present invention also provides a method for preparing the aforementioned hydrogel beads, characterized by comprising the following steps:
[0025] (1) Preparation of gel precursor solution: Sodium alginate was dissolved in phosphate buffer solution with pH 6.5-7.0 and magnetically stirred until fully hydrated to form a homogeneous and transparent sodium alginate stock solution; then, the composite powder of therapeutic peptide and Bowman-Birk inhibitor was slowly and uniformly added to the sodium alginate stock solution under the condition of continuous ultrasonic dispersion and vortex oscillation to obtain a mixed gel precursor solution containing sodium alginate, therapeutic peptide and Bowman-Birk inhibitor;
[0026] (2) Preparation of heterogeneous hydrogel beads: The mixed gel precursor solution obtained in step (1) is vertically added to a calcium chloride solution and a chitosan solution under continuous and gentle stirring at a constant flow rate through a precision dropping device with a specific pore size. The distance between the outlet of the dropping device and the surface of the cross-linked solution is controlled at 8-15 cm to prepare hydrogel beads.
[0027] (3) Crosslinking treatment and stabilization: After washing the hydrogel beads obtained in step (2) with pre-cooled ultrapure water, they are immersed in a 0.5%-1.5% (w / v) sodium citrate solution for 1-5 minutes to quench unreacted calcium ions and optimize matrix stability. Then, the hydrogel beads are placed in a freeze dryer and cooled to below -40°C for 4-6 hours. Finally, they are freeze-dried for 48-60 hours under a vacuum of less than 10 Pa to obtain the dried co-encapsulated hydrogel beads.
[0028] In step (1), the concentration of the sodium alginate solution is 0.5%-2.5% (w / v). The initial mass ratio of the therapeutic peptide to the Bowman-Birk inhibitor is 1:1 to 1:9.
[0029] In step (2), the concentration of chitosan in the mixed crosslinking solution is 0.3%-0.8% (w / v). The concentration of calcium ions is 0.3-0.7M. The dropping process uses a syringe or peristaltic pump with a needle orifice diameter or tubing inner diameter of 0.5-1.0 mm. The distance between the outlet of the dropping device and the surface of the crosslinking solution is 6 cm to 18 cm. In step (2), the dropping rate is 10-90 mL / h.
[0030] The present invention also provides the application of the aforementioned hydrogel beads in the preparation of oral peptide drugs.
[0031] This invention constructs a pH-responsive hydrogel bead delivery system co-encapsulating a Bowman-Birk inhibitor and a therapeutic peptide, transforming the traditional anti-nutritional factor Bowman-Birk inhibitor into a highly effective protectant for oral peptide drugs, achieving time-sequential synergistic release and targeted delivery. Its key technical points are:
[0032] (1) Mechanism of gel sphere construction based on ionic crosslinking and polyelectrolyte composite
[0033] The construction of the hydrogel beads in this invention is a precise self-assembly process based on physicochemical crosslinking. Its core mechanism lies in utilizing the synergistic effect of ionic crosslinking and polyelectrolyte complexation to construct a "core-shell" structure network with pH-responsive characteristics. Specifically, it includes:
[0034] Primary “eggbox” network: guluronic acid (G) units on sodium alginate chains and Ca 2+ Specific coordination occurs, forming a rigid "egg box" structure, which constitutes the primary gel skeleton, achieving instantaneous shaping and initial drug encapsulation.
[0035] Secondary composite shell: Negatively charged SA and positively charged chitosan (CS) in the crosslinking solution undergo electrostatic interaction to form a polyelectrolyte complex, creating a tough secondary network. This network interpenetrates with the primary network and accumulates on the surface of the gel beads to form a dense "shell" layer, significantly enhancing the mechanical strength and barrier properties of the gel.
[0036] pH Smart Switch: This dual-network structure endows the gel with exceptional pH responsiveness. In gastric juice (pH 1.2), the SA carboxyl groups ionize, causing the network to contract and compact, closing pores and inhibiting release. In intestinal juice (pH 6.8), the SA carboxyl groups ionize, and electrostatic repulsion causes the network to relax and swell, opening pores and triggering release. This smart switch is the physicochemical basis for achieving targeted delivery into the gastrointestinal tract.
[0037] (2) Functional role transformation of anti-nutritional factors: The Bowman-Birk inhibitor (BBI) in soybean processing by-products is innovatively redefined from the traditional understanding of "anti-nutritional factors" as a "natural protectant" of oral peptide drugs. By co-encapsulating it with therapeutic peptides, its protease inhibitory activity is utilized to actively establish a low protease microenvironment in the intestinal tract, thus realizing a functional subversion from "digestive interference" to "drug protector".
[0038] (3) System design for synergistic release: Experiments have shown that, compared with gel beads without BBI, the present invention can significantly extend the active retention time of smegglutinin in a simulated intestinal environment from 2.5 hours to more than 4 hours.
[0039] (4) Excellent gastric protection and intelligent release: Utilizing sodium alginate and Ca 2+ The "egg box" cross-linking and the electrostatic interaction between sodium alginate and chitosan created a smart carrier that shrinks in the gastric acid environment and swells in the intestinal fluid, enabling targeted delivery of drugs to the gastrointestinal tract.
[0040] (5) Enhanced dense network and controlled release capability through multiple interactions: BBI and semaglutide form a stable complex through intermolecular hydrogen bonds and hydrophobic interactions. This complex not only directly and physically shields the cleavage sites of semaglutide, but also acts as an additional cross-linking point, interacting with the sodium alginate-chitosan-calcium ion carrier network, jointly enhancing the density, mechanical strength, and controlled release capability of the hydrogel, which is a key structural factor in achieving sustained release behavior.
[0041] (6) High-value utilization of natural by-products and system safety: This strategy transforms BBI in soybean whey wastewater into a high-value drug delivery functional component, realizing the green and high-value utilization of agricultural processing by-products. At the same time, through the sustained-release properties of hydrogels, BBI can establish an effective concentration locally rather than experiencing high systemic exposure, which is expected to minimize its potential impact on the normal digestion of dietary proteins and improve the biosafety of the system.
[0042] In summary, this invention has achieved excellent and positive results:
[0043] (1) Disruptive concept: For the first time, the traditional anti-nutritional factor BBI is creatively transformed into a "guardian" of peptide drugs, achieving "local inhibition and precise protection", providing a new paradigm for the high-value utilization of soybean processing by-products and oral delivery strategies.
[0044] (2) Significant intestinal protection and activity prolongation effect: Compared with gel beads that only encapsulate smegglutinin (1:0), after co-encapsulating BBI in this invention, the activity retention time of smegglutinin in the simulated intestinal environment was significantly prolonged from 2.5 hours to 4 hours (1.6 times that of the control group, P<0.05).
[0045] (3) Excellent gastric protection ability: After incubation in simulated gastric juice (pH 1.2) for 2 hours, the release of smegglutinin from all formulations of hydrogel beads was less than 15%, while free smegglutinin was completely degraded within 5 minutes. This indicates that the hydrogel beads can provide effective gastric protection for smegglutinin.
[0046] (4) Intelligent pH-responsive release: The hydrogel beads shrink in gastric juice to form a dense physical barrier; they swell rapidly in intestinal juice, opening pores and triggering drug release. This "protection in the stomach and release in the intestine" behavior perfectly meets the physiological requirements of oral drug administration.
[0047] (5) Enhanced mechanical properties and drug loading: The introduction of BBI enhances the density of the hydrogel network, improves the hardness and elasticity of the beads, and makes them more resistant to gastrointestinal peristalsis; the dense external and hollow internal gel structure exhibits high encapsulation efficiency and loading capacity for both smegglutinin and BBI. Attached Figure Description
[0048] Figure 1 The present invention provides a flowchart and schematic diagram of the preparation process of the co-encapsulated hydrogel beads.
[0049] Figure 2 Macroscopic morphology of hydrogel beads prepared in different embodiments and comparative examples of the present invention.
[0050] Figure 3 : Test diagram of the textural properties of the hydrogel beads of the present invention.
[0051] Figure 4 : Scanning electron microscope (SEM) microstructure of the hydrogel beads of this invention.
[0052] Figure 5 : Statistical chart of the encapsulation efficiency of the hydrogel beads of this invention for smegglutinin and BBI.
[0053] Figure 6 : Schematic diagram of the swelling behavior of the hydrogel beads of the present invention under different pH conditions.
[0054] Figure 7 The in vitro cumulative release curves of smegglutinin and BBI in simulated gastrointestinal fluid using hydrogel beads of the present invention. Detailed Implementation
[0055] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0056] Example 1: Preparation of Smegglutinin / BBI co-encapsulated hydrogel beads (1:1)
[0057] 10 mg of smegglutide and 10 mg of BBI were dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 12 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain 1:1 co-encapsulated hydrogel beads.
[0058] The Bowman-Birk inhibitor (BBI) is prepared by a method comprising the following steps:
[0059] a) Isoelectric point precipitation: The pH of the soybean whey solution with a pH of 7.5 was adjusted to 4.0 with acid to carry out the first precipitation. The protein precipitate was collected after centrifugation.
[0060] b) Ammonium sulfate fractionation: Add ammonium sulfate to the supernatant obtained in step a) to make it saturated to 25%-65%, let it stand at 2-6℃ for 110-130 minutes, and then centrifuge to collect the second protein precipitate.
[0061] c) Gel filtration chromatography: After redissolving the precipitate obtained in step b), purification was performed using a Superdex 75 Increase 10 / 300 pre-packed gel filtration column, wherein the chromatography column was equilibrated with 0.1M sodium phosphate buffer solution.
[0062] The sample loading volume is 450-550 μL; the flow rate is controlled at 0.4-0.6 mL / min; the elution peak is detected at a wavelength of 280 nm and collected fractionally;
[0063] d) Collection and post-processing of active components: The trypsin inhibitory activity of each collected component was determined. Protein components with target inhibitory activity were collected, and after dialysis and freeze-drying, purified Bowman-Birk inhibitor products with a purity of over 90% were obtained.
[0064] Unless otherwise specified, the BBI used in the following examples and comparative examples was prepared according to the method described in Example 1.
[0065] Effects Description: This embodiment provides key evidence revealing the core mechanism of this invention: time-sequential synergistic release. The in vitro release curve clearly shows for the first time that after 2 hours of incubation in simulated gastric fluid (pH 1.2), the cumulative release rate of semaglutide was 12.28% (less than 20%). In simulated intestinal fluid, BBI rapidly released over 60% within 1 hour, while the release rate of semaglutide during the same period was only about 10%. This "protective agent first, therapeutic agent later" release kinetics experimentally verifies the feasibility of actively protecting peptide drugs by regulating the release sequence, providing a direct mechanistic basis for subsequent optimization of this invention.
[0066] Example 2: Preparation of Smegglutinin / BBI co-encapsulated hydrogel beads (1:3)
[0067] 10 mg of smegglutide and 30 mg of BBI were dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 12 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain 1:3 co-encapsulated hydrogel beads.
[0068] Effect Description: This embodiment also replicates the core mechanism of the present invention. When a higher amount of BBI is released, the release of semaglutide is nearly 10%. When the BBI ratio increases to 1:3, the active retention time and cumulative release rate of semaglutide begin to decrease slightly. Although its protective effect is still significantly better than Comparative Example 1, it is clearly inferior to Example 1. This phenomenon indicates that the enhancing effect of BBI on the gel network begins to appear, that is, the gradually increasing network density begins to hinder the later diffusion and release of semaglutide. This embodiment marks the critical point for performance optimization.
[0069] Example 3: Preparation of Smegglutinin / BBI co-encapsulated hydrogel beads (1:5)
[0070] 10 mg of smegglutide and 50 mg of BBI were dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 12 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain 1:5 co-encapsulated hydrogel beads.
[0071] Effects: This example further confirms the drawbacks of excessive BBI loading. At this ratio, the release of semaglutide was significantly inhibited, and its active retention time and bioavailability were significantly lower than in Example 1 (1:1). This clearly demonstrates that excessive BBI, due to its strong cross-linking effect, alters the release characteristics of the carrier, thus hindering the full realization of drug efficacy.
[0072] Example 4: Preparation of Smegglutinin / BBI co-encapsulated hydrogel beads (1:9)
[0073] 10 mg of smegglutide and 90 mg of BBI were dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 12 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain 1:9 co-encapsulated hydrogel beads.
[0074] Results: As an extreme ratio control, this study showed the worst release and protective effect of semaglutide. This result strongly demonstrates, from the opposite perspective, the unique advantage and necessity of the low-ratio (1:1 to 1:3) synergistic effect discovered in this invention, proving that the protective effect of BBI is not simply positively correlated with dosage.
[0075] Comparative Example 1: Smegglutinin hydrogel beads without BBI
[0076] 10 mg of smegglutide was dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 12 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain co-encapsulated hydrogel beads.
[0077] Results: This comparative example reveals the core deficiency of existing technologies (hydrogel encapsulation only). After incubation in simulated gastric fluid for 2 hours, the cumulative release rate of semaglutide reached 32.51%. However, in simulated intestinal fluid, due to the lack of protease inhibitors, the released semaglutide was rapidly degraded, with an activity retention time of only 2.5 hours. This result clearly demonstrates that a single physical barrier is insufficient to protect peptides, strongly proving the necessity and synergistic effect of co-encapsulating BBI with semaglutide, and providing a starting point for the proposed invention.
[0078] Comparative Example 2: Physically mixed semaglutide and BBI (without carrier encapsulation)
[0079] 10 mg of smegglutide and 10 mg of BBI were dissolved in a small amount of water and mixed together, without performing gelation steps such as dropping or cross-linking.
[0080] Results: When semaglutide enters gastric juice directly without encapsulation, it is completely degraded within five minutes. This demonstrates that without the protection of a pH-responsive hydrogel carrier, physically mixed BBI cannot provide effective protection for semaglutide in the environment of gastric acid and systemic dilution, highlighting the irreplaceable nature of the core concept of "co-encapsulation in a smart hydrogel" in this invention.
[0081] Comparative Example 3: Smegglutinin / BBI co-encapsulated hydrogel beads prepared by reducing the dropping distance
[0082] 10 mg of smegglutide and 10 mg of BBI were dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 6 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain co-encapsulated hydrogel beads.
[0083] Results: Gel beads prepared under these conditions exhibited poor sphericity, noticeable misshapenness, tailing, and adhesion, with a wide particle size distribution (1.5-4.0 mm) and poor product uniformity. Due to the irregular shape, their release behavior in simulated intestinal fluid was unstable and reproducible. This comparative example demonstrates that controlling the dropping distance is crucial for obtaining a uniform, high-quality product.
[0084] Comparative Example 4: Smegglutinin / BBI co-encapsulated hydrogel beads prepared by increasing the dropping distance
[0085] 10 mg of smegglutide and 10 mg of BBI were dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 18 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain co-encapsulated hydrogel beads.
[0086] Results: Under these conditions, the excessive kinetic energy of the droplets impacting the liquid surface led to a significant increase in the size of the formed gel beads (approximately 4.5 mm), resulting in a rough, wrinkled surface and potential damage to the internal structure. This resulted in a decrease in the encapsulation efficiency of semaglutide and an increase in burst release in gastric juice. This comparative example further demonstrates the importance of the optimal drop distance (6-18 cm, preferably 12 cm) for ensuring product performance.
[0087] Comparative Example 5: Sodium alginate with M / G = 1:1
[0088] 10 mg of smegglutide and 10 mg of BBI were dissolved in 1 mL of deionized water and magnetically stirred to form a homogeneous drug solution. This drug solution was then mixed with 4 mL of 1.5% (w / v) sodium alginate solution and stirred continuously for 30 minutes to form a homogeneous precursor solution. Using a 5 mL syringe (0.7 mm orifice), the precursor solution was drawn up and the syringe needle tip was fixed at a height of 12 cm above the surface of the crosslinking solution. The solution was then vertically added dropwise at a constant flow rate of 40 mL / h to 50 mL of a mixed crosslinking solution containing 0.5% (w / v) chitosan and 0.5 M CaCl2. The mixture was allowed to crosslink at room temperature for 40 minutes. The gel beads were washed three times with deionized water to obtain co-encapsulated hydrogel beads.
[0089] Results: Due to the reduction in guluronic acid (G) units, the resulting "egg-box" structure was less stable and compact. The gel beads exhibited less shrinkage and lower mechanical strength in gastric juice, leading to an increased cumulative release rate of semaglutide in the stomach. In intestinal juice, the swelling rate was too rapid, resulting in faster drug release and a less effective sustained-release effect compared to Example 1 using sodium alginate with an M / G ratio of 1:2. This comparative example demonstrates that the M / G ratio of sodium alginate is a key parameter for regulating the gel's mechanical properties, barrier properties, and release behavior.
[0090] Table 1 Test results for each embodiment and comparative example
[0091]
[0092] Examples 1-4 systematically investigated the effects of different mass ratios of BBI to semaglutide (1:1 to 1:9) on the performance of gel beads and drug release behavior. As shown in Table 1, with the increase of the BBI ratio, the diameter of the gel beads increased from 3.20 mm (Example 1) to 3.80 mm (Examples 3 and 4). In contrast, Example 1 (dropping distance 12 cm) successfully prepared gel beads with a uniform diameter of approximately 3.20 mm. This size is significantly better than irregular particle size, more ergonomic, easier to swallow, and provides an ideal physical morphological basis for subsequent possible chewing administration. This demonstrates that controlling the dropping distance at 12 cm is key to obtaining high-quality, highly applicable products using this method.
[0093] Comparing the results of Example 1 with those of Comparative Examples 3 and 4 further validated the decisive influence of key process parameters on product properties. Comparative Example 3 (dropping distance 6 cm) exhibited poor gel bead formation and a wide particle size distribution due to insufficient droplet kinetic energy; while Comparative Example 4 (dropping distance 18 cm) resulted in significantly increased gel bead size, looser structure, and increased drug burst release due to excessive droplet impact energy on the liquid surface. In contrast, the gel beads prepared in Example 1 at a dropping distance of 12 cm showed high sphericity and uniform particle size (3.20 ± 0.16 mm), and demonstrated good release stability in a simulated gastrointestinal environment, fully demonstrating that controlling the dropping distance at 12 cm is a key process step for achieving high-quality product formation and controllable performance.
[0094] Comparative Example 5, using sodium alginate with an M / G ratio of 1:1, exhibited inferior roundness and elasticity of its gel beads compared to Example 1, which used sodium alginate with an M / G ratio of 1:2. This result confirms that the proportion of guluronic acid (G) units in sodium alginate directly affects the stability of the "egg-box" structure formed by its cross-linking with calcium ions. This proportion is the material basis for constructing a hydrogel system with ideal gastric protection and intestinal targeted release functions. Optimizing sodium alginate with an M / G ratio of 1:2 plays a crucial role in achieving time-sequential synergistic drug release.
[0095] In summary, this application has successfully created a novel oral peptide delivery system by encapsulating BBI and therapeutic peptides in a specific ratio (preferably 1:1 to 1:3) in hydrogel beads constructed using a specific process (dropping distance of 12 cm) and specific raw materials (M / G=1:2 sodium alginate).
Claims
1. A method for preparing hydrogel beads for protecting oral semaglutide, characterized in that, The preparation method includes the following steps: (1) Preparation of gel precursor solution: Sodium alginate was dissolved in phosphate buffer solution at pH 6.5-7.0 and magnetically stirred until completely hydrated to form a homogeneous and transparent sodium alginate stock solution; subsequently, the composite powder of smegglutide and Bowman-Birk inhibitor was slowly and uniformly added to the sodium alginate stock solution under the condition of continuous ultrasonic dispersion and alternating vortex oscillation to obtain a mixed gel precursor solution containing sodium alginate, smegglutide and Bowman-Birk inhibitor; the mass ratio of smegglutide to Bowman-Birk inhibitor was 1:1 to 1:3; the ratio of mannulic acid to guluronic acid in sodium alginate was 1:2; the concentration of sodium alginate stock solution was 0.5%-2.5%; (2) Preparation of heterogeneous hydrogel beads: The mixed gel precursor solution obtained in step (1) is vertically added to a calcium chloride solution and a chitosan solution under continuous and gentle stirring at a constant flow rate through a precision dropping device with a specific pore size. The distance between the outlet of the dropping device and the surface of the crosslinked solution is controlled at 12 cm to prepare hydrogel beads. The concentration of the chitosan solution is 0.3%-0.8%; the concentration of calcium chloride is 0.3-0.7 M; the dropping device is a syringe or a peristaltic pump with a needle orifice diameter or tubing inner diameter of 0.5-1.0 mm; the dropping rate is 10-90 mL / h. (3) Crosslinking treatment and stabilization: After washing the hydrogel beads obtained in step (2) with pre-cooled ultrapure water, they are immersed in 0.5%-1.5% sodium citrate solution for 1-5 minutes to quench unreacted calcium ions and optimize matrix stability. Then, the hydrogel beads are placed in a freeze dryer and cooled to below -40°C for 4-6 hours. Finally, they are freeze-dried for 48-60 hours under vacuum conditions below 10Pa to obtain the dried co-encapsulated hydrogel beads.
2. The preparation method according to claim 1, characterized in that, The finished hydrogel beads have a particle size of 2.0-4.0 mm.
3. The preparation method according to claim 1, characterized in that, After the hydrogel beads were incubated in simulated gastric fluid at pH 1.2 for 2 hours, the cumulative release rate of the smegglutinin was less than 20%.
4. The preparation method according to claim 1, characterized in that, The Bowman-Birk inhibitor was prepared by the following steps: a) Isoelectric point precipitation: The pH of the soybean whey solution with a pH of 7.5 was adjusted to 4.0 with acid to carry out the first precipitation. The protein precipitate was collected after centrifugation. b) Ammonium sulfate fractionation: Add ammonium sulfate to the supernatant obtained in step a) to make it saturated to 25%-65%, let it stand at 2-6℃ for 110-130 minutes, and then centrifuge to collect the second protein precipitate. c) Gel filtration chromatography: After redissolving the precipitate obtained in step b), purification was performed using a Superdex 75 Increase 10 / 300 gel filtration pre-packed chromatography column, wherein the chromatography column was equilibrated with 0.1M sodium phosphate buffer solution. The sample loading volume is 450-550 μL; the flow rate is controlled at 0.4-0.6 mL / min; the elution peak is detected at a wavelength of 280 nm and collected stepwise; d) Collection and post-processing of active components: The trypsin inhibitory activity of each collected component was determined. Protein components with target inhibitory activity were collected, and after dialysis and freeze-drying, purified Bowman-Birk inhibitor products with a purity of over 90% were obtained.
5. Hydrogel beads for protecting oral semaglutide prepared according to any one of claims 1-4.
6. The use of the hydrogel beads for protecting the oral smegglutide drug as described in claim 5 in the preparation of the oral smegglutide drug.