Glucose-responsive pickering emulsion and method of making the same

Abstract

The application discloses a glucose-responsive Pickering emulsion and a preparation method thereof, relates to the technical field of Pickering emulsion, and the emulsion comprises a water phase and an oil phase, the water phase contains a phenylboronic acid group alginate derivative and glucose, a benzene boronic acid group forms a dynamic B-O bond with a alginate molecular chain and a cis diol group on the glucose respectively, and the phenylboronic acid group alginate derivative forms a soft colloid particle and is distributed in the water phase and an oil-water interface. In the preparation, the phenylboronic acid group alginate derivative is prepared into an aqueous solution and the glucose is added, the pH is adjusted, the oil phase is added, and the Pickering emulsion is obtained through high-speed shearing emulsification. The emulsion can adjust the interface aggregation state of the soft colloid particle and the stable state of the emulsion according to the change of the glucose environment, so that the glucose response characteristic is given to the system, and the system can be used for preparing a glucose response release carrier.

Description

Technical Field

[0001] This invention relates to the field of Pickering emulsion technology, and more particularly to glucose-responsive Pickering emulsions and their preparation methods. Background Technology

[0002] Pickering emulsions are a class of emulsion systems that utilize the adsorption of solid or soft colloidal particles at the oil-water interface to form an interfacial layer, thereby inhibiting droplet coalescence. Among them, high internal phase Pickering emulsions have a high dispersed phase volume fraction and typically exhibit high viscosity or a gel state, showing potential applications in drug delivery and functional materials. Alginic acid and its derivatives possess good biocompatibility and biodegradability, and can be modified to achieve a certain degree of amphiphilicity through hydrophobic modification; therefore, they have been used to construct soft colloidal particles capable of adsorbing at the oil-water interface. Existing research indicates that alginate-based soft colloidal particles can not only form a particle adsorption layer at the oil-water interface but also form an interparticle network in the continuous phase, thereby maintaining the stability of the emulsion system.

[0003] To enable Pickering emulsions to respond to changes in the external environment, existing technologies further functionalize the emulsion particles using pH-responsive groups and phenylboronic acid groups. Among these, phenylboronic acid groups can form reversible BO bonds with compounds containing cis-diol structures, thereby modifying the emulsion properties by altering the crosslinking state, hydrophilicity / hydrophobicity, and interfacial behavior of the particles. Existing sugar-responsive Pickering emulsions typically utilize the hydrophilicity / hydrophobicity or conformational changes resulting from the binding of polymer particles or microgels containing phenylboronic acid groups to sugar molecules to regulate the adsorption behavior of the particles at the oil-water interface.

[0004] However, for alginate-based soft colloid particles, their molecular chains contain groups capable of dynamic bonding, leading to complex interactions within the particles, between particles, and at the oil-water interface. Current technologies lack sufficient research on the correlation between the aggregation state of soft colloid particles, the adsorption state at the oil-water interface, and the stability of high internal phase emulsions when the original dynamic bonding relationship between phenylboronic acid groups and alginate molecular chains changes after the addition of sugar molecules. Therefore, existing sugar-responsive Pickering emulsions, while utilizing changes in glucose concentration to regulate the interfacial structure and stability of high internal phase emulsions, still suffer from unclear relationships between the response mechanism and interfacial structure changes, and difficulty in effectively regulating the emulsion state based on the external glucose environment.

[0005] Furthermore, in applications where Pickering emulsions support hydrophobic substances, these substances are primarily distributed within the oil phase, and their release process is limited by the oil-water interface particle layer and the overall stability of the emulsion. Existing carriers typically rely on diffusion, carrier swelling, or natural emulsion instability to release the loaded material, resulting in limited responsiveness to changes in the external glucose environment. This makes it difficult to establish a direct correlation between the emulsion interface state and changes in glucose concentration, thus limiting the further application of Pickering emulsions in glucose-responsive release systems. Summary of the Invention

[0006] Based on the technical problems existing in the background technology, the present invention proposes a glucose-responsive Pickering emulsion and its preparation method. The emulsion can adjust the interfacial aggregation state of soft colloidal particles and the emulsion stability state according to changes in the glucose environment, thereby endowing the system with glucose-responsive characteristics, and can be used to prepare glucose-responsive release carriers.

[0007] The present invention proposes a glucose-responsive Pickering emulsion comprising an aqueous phase and an oil phase; The aqueous phase contains phenylboronic acid-based alginate derivatives and glucose; The phenylboronic acid-based alginate derivative includes an alginate molecular chain backbone and phenylboronic acid groups grafted onto the alginate molecular chain backbone. The concentration of the phenylboronic acid-based alginate derivative in the aqueous phase is 2-4 mg / mL, and the volume fraction of the oil phase is 80%-90%. The phenylboronic acid-based alginate derivative forms soft colloidal particles, which are distributed in the aqueous phase and at the oil-water interface between the aqueous and oil phases. At least a portion of the phenylboronic acid groups in the phenylboronic acid-based alginate derivative form a first dynamic BO bond with the cis-diol groups on the alginate molecular chain, and at least a portion of the phenylboronic acid groups form a second dynamic BO bond with the cis-diol groups on glucose.

[0008] Preferably, the preparation method of the phenylboronic acid-based alginate derivative is as follows: 3-Aminophenylborate salt was dissolved in water, formaldehyde was added, and the reaction was carried out at 20-30℃ for 2-4 hours; sodium alginate aqueous solution and cyclohexylisocyanate were added to the resulting solution, and the reaction was carried out at 20-30℃ in the dark for 12-24 hours; the product obtained from the reaction was dialyzed and freeze-dried to obtain the phenylborate-based alginate derivative. Preferably, the mass-to-volume ratio of sodium alginate, 3-aminophenylborate, formaldehyde, and cyclohexylisocyanate is 100 g: (4-8) g: (1-5) mL: (1-10) mL.

[0009] Preferably, the concentration of glucose in the aqueous phase is 2-50 mM, and the pH value of the aqueous phase is 7-8.

[0010] Preferably, the oil phase is squalene, and the volume fraction of the oil phase is 80%.

[0011] This invention proposes a method for preparing a glucose-responsive Pickering emulsion, as described above, and the method steps are as follows: S1. Dissolve 3-aminophenylborate salt in water, add formaldehyde, and react at 20-30℃ for 2-4 hours; add sodium alginate aqueous solution and cyclohexylisocyanate to the resulting solution, and react at 20-30℃ in the dark for 12-24 hours; dialyze and freeze-dry the product to obtain phenylborate-based alginate derivative. S2. Prepare an aqueous solution with a concentration of 2-4 mg / mL by the phenylboronic acid-based alginate derivative, add glucose to the aqueous solution, and adjust the pH of the resulting aqueous phase to 7-8. S3. Squalene, which serves as the oil phase, is added to the aqueous phase to make the volume fraction of the oil phase 80%-90%, and sheared at a speed of 15000-20000 rpm for 1-5 minutes to obtain a glucose-responsive Pickering emulsion. In this embodiment, at least a portion of the phenylboronic acid groups in the phenylboronic acid-based alginate derivative form a first dynamic BO bond with the cis-diol groups on the alginate molecular chain, and at least a portion of the phenylboronic acid groups form a second dynamic BO bond with the cis-diol groups on glucose.

[0012] Preferably, in step S1, the mass-to-volume ratio of sodium alginate, 3-aminophenylborate, formaldehyde, and cyclohexylisocyanate is 100g:(4-8)g:(1-5)mL:(1-10)mL.

[0013] Preferably, the sodium alginate aqueous solution has a mass concentration of 1-3 wt% and a pH of 3-4; the product obtained from the reaction is dialyzed using a dialysis membrane with a molecular weight cutoff of 3500 Da.

[0014] This invention proposes the application of a glucose-responsive Pickering emulsion in the preparation of a hydrophobic guest glucose-responsive release carrier, wherein the glucose-responsive Pickering emulsion is as described above.

[0015] Beneficial technical effects of the present invention: (1) This invention utilizes the dynamic BO bonds formed between the phenylboronic acid groups and the cis-diol groups on the alginate molecular chain in the phenylboronic acid-based alginate derivative, as well as the dynamic BO bonds formed between the phenylboronic acid groups and the cis-diol groups of glucose, to enable glucose to participate in the regulation of the dynamic cross-linking state within and between particles of the phenylboronic acid-based alginate derivative. As the glucose concentration increases, the aggregation degree of the soft colloidal particles decreases, and the wetting and adsorption state of the particles at the hydrophobic interface changes accordingly, thereby enabling the Pickering emulsion to produce a clear interfacial response to changes in the external glucose concentration.

[0016] (2) This invention introduces the above-mentioned dynamic BO bond regulation effect into the high internal phase Pickering emulsion system, so that changes in glucose concentration can be further transmitted to the particulate adsorption layer and emulsion droplet structure at the oil-water interface. Experimental results show that under the conditions of 3 mg / mL concentration of phenylboronic acid-based alginate derivative and 80% oil phase volume fraction, the average droplet diameter of the emulsion without glucose is 14.09 μm, and the average droplet diameter increases to 24.93 μm when the glucose concentration is 50 mM. This indicates that the glucose response regulation of the high internal phase Pickering emulsion state can be achieved by changing the glucose concentration to adjust the interfacial aggregation state and stability state of the emulsion.

[0017] (3) When the glucose-responsive Pickering emulsion of the present invention is used to load hydrophobic guests, it can convert changes in external glucose concentration into changes in the oil-water interface structure and further regulate the release behavior of hydrophobic guests. When Nile red is used as a model hydrophobic guest, the cumulative release in 24 hours is 20.87% in the absence of glucose, while in the presence of 50 mM glucose, the cumulative release in 6 hours reaches 62.86% and the cumulative release in 24 hours reaches 80.96%, indicating that the system can produce significantly different release behaviors according to the external glucose environment, providing a technical basis for constructing glucose-responsive hydrophobic substance release carriers. Attached Figure Description

[0018] Figure 1 The following are characterization diagrams of the effects of different glucose concentrations on the interfacial properties and aggregation behavior of SA-PBA proposed in this invention; (a) is the contact angle diagram of SA-PBA on a hydrophobic silicon wafer under different glucose concentrations, (b) is the dynamic interfacial tension variation diagram of SA-PBA under different glucose concentrations, and (c) is the fluorescence intensity diagram of SA-PBA under different glucose concentrations. Figure 2The following are characterization diagrams of the effect of glucose on the microstructure and structure of SA-PBA soft colloid particles proposed in this invention; (a) is the microstructure of SA-PBA without the addition of glucose, (b) is the microstructure of SA-PBA under 10mM glucose conditions, and (c) is a schematic diagram of glucose regulating the structure of SA-PBA soft colloid particles. Figure 3 The images show the appearance and gel properties of the SA-PBA stable Pickering emulsion at a concentration of 3 mg / ml under different oil volume fractions proposed in this invention. Figure 4 The images show the appearance, optical microscope images, and droplet size distribution of the SA-PBA stable Pickering emulsion at different glucose concentrations proposed in this invention. Figure 5 The image shows the appearance of the Pickering emulsion stabilized with SA-PBA at different glucose concentrations on the seventh day, along with optical microscope images and droplet size distribution. Figure 6 This is a graph showing the change in the stability index (TSI) of the SA-PBA-stabilized Pickering emulsion at different glucose concentrations proposed in this invention over time. Figure 7 The graph shows the apparent viscosity of the SA-PBA-stabilized Pickering emulsion at different glucose concentrations as a function of shear rate, as proposed in this invention. Figure 8 The rheological properties of the SA-PBA-stabilized Pickering emulsion at different glucose concentrations proposed in this invention are shown in the following figures: (a) is a strain scan test figure, and (b) is an angular frequency scan test figure. Figure 9 Scanning electron microscope images of the SA-PBA-stabilized Pickering emulsions with different glucose concentrations proposed in this invention after curing. Figure 10 Fluorescence microscopy images of the interface film of SA-PBA stable Pickering emulsion under different glucose concentrations proposed in this invention. Figure 11These are characterization diagrams of the interface structure, fluorescence distribution, and particle adsorption state of the SA-PBA stable emulsion droplets under different glucose concentrations proposed in this invention. Among them, (ad) are the interface structure morphology diagrams of the emulsion droplets under glucose concentrations of 0mM, 2mM, 10mM, and 50mM, respectively; (a1-d1), (a2-d2), and (a3-d3) are the fluorescence distribution diagrams of SA-PBA soft colloidal particles at the emulsion droplet interface under excitation channels of 405nm, 488nm, and 561nm, respectively; and (a4-d4) are schematic diagrams of the adsorption degree of SA-PBA soft colloidal particles at the emulsion droplet interface under different glucose concentrations. Figure 12 The QCM-D characterization diagrams of the adsorption and desorption behavior of SA-PBA at the oil-water interface proposed in this invention are shown below; (a) is a schematic diagram of SA-PBA adsorption at the oil-water interface, (b) is a diagram of the frequency change Δf over time during adsorption and desorption, (c) is a diagram of the dissipation change ΔD over time during adsorption and desorption, and (d) is a diagram of the ΔD-Δf relationship. Figure 13 The diagram shows the performance parameters of the adsorption layer formed by SA-PBA at the oil-water interface proposed in this invention. (a) shows the change of adsorption layer thickness over time, (b) shows the change of adsorption mass over time, (c) shows the change of adsorption layer modulus over time, and (d) shows the change of adsorption layer viscosity over time. Figure 14 The figures show the cell activity of SA-PBA and the drug release performance of SA-PBA-stable Pickering emulsion proposed in this invention. (a) is the cell activity evaluation figure of SA-PBA, (b) is the drug release standard curve, and (c) is the cumulative drug release of SA-PBA-stable Pickering emulsion in different glucose environments. Detailed Implementation

[0019] The present invention will be further explained below with reference to specific embodiments.

[0020] Example First, 6.00 g of sodium alginate (SA) was weighed and dissolved in 300 mL of deionized water to prepare a 2 wt% stock solution. Next, the pH of this sodium alginate solution was adjusted to 3.6 using 1 M hydrochloric acid solution. Simultaneously, 0.33 g of 3-aminophenylborate (3-APBA) was weighed and dissolved in 20 mL of deionized water. Then, 0.075 mL of formaldehyde was added to the 3-APBA solution, and the mixture was stirred at room temperature for three hours to ensure the Schiff base reaction was complete. Next, the formaldehyde-containing 3-APBA solution was added to the sodium alginate stock solution at pH 3.6, and the mixture was stirred vigorously for half an hour. Then, 0.33 mL of cyclohexylisocyanate was added to the reaction mixture, and the mixture was reacted at room temperature in the dark for 24 hours. Finally, the reaction product was purified by dialysis in deionized water using a 3500 Da dialysis bag for five days, with the dialysis fluid changed every eight hours. The final step is to freeze-dry the material to obtain the purified phenylboronic acid-based alginate derivative SA-PBA.

[0021] An aqueous solution of SA-PBA with a concentration of 3 mg / mL was prepared. Glucose was added to each SA-PBA aqueous solution to make glucose concentrations of 0 mM, 2 mM, 10 mM and 50 mM respectively. The pH of each aqueous phase was adjusted to 7.4 using NaOH solution.

[0022] Using squalene as the oil phase, squalene was added to the SA-PBA aqueous phases with different glucose concentrations to make the oil phase volume fraction 80%. Then, a high-speed shear dispersion emulsifier was used to shear emulsify at 18,000 rpm for 3 min to obtain high internal phase Pickering emulsions with glucose concentrations of 0 mM, 2 mM, 10 mM and 50 mM.

[0023] The contact angle of SA-PBA solution on a hydrophobic silicon substrate was measured using an ExperienceA-300 optical contact angle / surface tension meter. The dynamic interfacial tension (γ) of SA-PBA solutions with different glucose concentrations at the squalene / water interface was determined using a pendant drop method with a constant drop volume of 20 μL. The droplet shape was fitted to the Young-Laplace equation to calculate the γ value. Results are shown below. Figure 1 As shown.

[0024] Since phenylboronic acid is a molecule with a specific response to ortho-cis-diol, glucose can be a candidate for modulating the amphiphilicity of SA-PBA. The wetting performance of SA-PBA on a hydrophobic silicon substrate at different glucose concentrations was tested using an Experience a-300 optical contact angle / surface tension test. The contact angle is an indicator of the wetting performance and diffusion behavior of a droplet on a solid surface. When testing the contact angle on a hydrophobic solid surface, a larger contact angle indicates a poorer ability of the solution to wet the hydrophobic surface, meaning the solution is less hydrophobic and more hydrophilic. Figure 1 As shown in section (a), the interfacial contact angle between the droplet and the hydrophobic silica substrate was used to evaluate the wetting properties of SA-PBA at different glucose concentrations. The results showed that SA-PBA droplets exhibited the best wettability on the hydrophobic silica substrate with the smallest interfacial contact angle (62.1°) when no glucose was added to the solution. At a glucose concentration as high as 50 mM, the interfacial contact angle of the SA-PBA solution on the hydrophobic silica substrate reached as high as 82.9°, indicating extremely poor wettability. Therefore, different glucose concentrations in the solution lead to significant differences in the forces and intermolecular structures of the phenylboronic acid-alginic acid polymer.

[0025] Figure 1 Part (b) illustrates the interfacial adsorption behavior of SA-PBA. In a fixed concentration (3 mg / ml) SA-PBA system, the IFT value increased from 59.57 to 80.96 mN / m with increasing glucose concentration. The results indicate that the adsorption capacity of SA-PBA at the oil-water interface is related to the glucose concentration. That is, the higher the glucose concentration in the solution, the weaker the adsorption capacity of SA-PBA at the oil-water interface, and the greater the change in interfacial tension. The trends of the dynamic interfacial tension test results and the contact angle measurement results are consistent, further demonstrating that glucose concentration can regulate the amphiphilicity of SA-PBA. Figure 1 As shown in section (c), the fluorescence intensity gradually decreases with increasing glucose concentration, indicating that the introduction of glucose disrupts the aggregation of SA-PBA soft particles in aqueous solution.

[0026] The microstructure of soft colloidal particles directly affects the physicochemical properties of high-internal-phase Pickering emulsions. TEM investigation of the effect of glucose on the self-assembly of SA-PBA to form soft colloidal microstructures is of significant guiding importance for understanding emulsion stability. In the absence of glucose in the environment, the particles formed by the self-assembly of SA-PBA shrink internally and decrease in size due to the formation of BO bonds between phenylboronic acid and SA chains. Simultaneously, the formation of covalent BO bonds between particles increases the degree of aggregation. Figure 2 (a) of the document. For example... Figure 2As shown in section (b), at 10 mM glucose, the degree of interparticle aggregation decreases, and the polymer chains in individual soft colloids can be significantly extended, with an increase in particle size. Therefore, the dynamic bond crosslinking ability and self-assembly behavior of SA-PBA are affected by glucose. The phenylboronic acid groups on SA-PBA soft colloid particles form covalent crosslinks with the cis-diol on the SA molecular chain through BO bonds. The formation of covalent crosslinks within the soft colloid particles is conducive to a more compact particle structure, and the BO bonds between particles promote aggregation and crosslinking. However, after the addition of glucose molecules, the dynamic covalent crosslinking between phenylboronic acid and SA chains is competitively replaced by glucose because the specific binding between the phenylboronic acid groups and the ortho-cis-diol on glucose is stronger than that between the cis-diol on the SA chain. This substitution disrupts the dynamic crosslinking network between soft colloid particles and makes the interior of the soft colloid particles loose. In addition, due to the excellent hydrophilicity of glucose, its binding with phenylboronic acid reduces the ability of SA-PBA solution to wet hydrophobic surfaces. Therefore, glucose can act as a hydrophobic switch for SA-PBA soft colloid particles. That is, the covalent cross-linking between phenylboronic acid and SA molecular chains on the soft colloid particles decreases with increasing glucose concentration, resulting in loosening of the soft colloid particles and thus reducing their wetting properties at the oil-water interface. Figure 2 Part (c) shows the self-assembled microstructure of SA-PBA.

[0027] Pickering emulsions with different dispersion volume fractions (Φ) were prepared using SA-PBA soft colloidal particles at a concentration of 3 mg / ml as the aqueous phase without the addition of glucose, such as... Figure 3 As shown in the diagram, observation of the emulsion appearance revealed that the emulsion layer of SA-PBA-stabilized Pickering emulsions increased with increasing dispersed volume fraction. When the dispersed volume fraction was <75%, the Pickering emulsion separated into a distinct aqueous phase (bottom) and emulsion layer (top) after standing for 12 hours. When the dispersed volume fraction was 75%, no aqueous layer precipitated after standing, but the emulsion layer flowed when inverted. However, when the Φ value was above 80%, the emulsion not only did not precipitate an aqueous layer after standing but also formed a gel-like state, and the emulsion layer did not flow when inverted. These results indicate that SA-PBA has good amphiphilicity and can stabilize high internal phase Pickering emulsions. Simultaneously, a small amount of soft colloidal particles can form a tight adsorption layer at the oil-water interface, completely covering the emulsion droplets and effectively preventing droplet aggregation. Since high internal phase Pickering emulsions (HIPPEs) typically have a minimum internal Φ value of 74%, this is not ideal. The above observations indicate that SA-PBA can form stable HIPPEs.

[0028] The microstructure of soft gel particles directly affects the physicochemical properties of hip-penetrating polyethylene (HIPPEs). SA-PBA backbones are grafted with phenylboronic acid groups that specifically bind to glucose. Therefore, the addition of glucose affects the microstructure of SA-PBA soft gel particles, thus impacting the stability of HIPPEs. Using 3 mg / ml SA-PBA as the polysaccharide emulsifier and 80% squalene as the oil phase, HIPPEs under different glucose conditions were prepared by adding different concentrations of glucose to the aqueous phase to study the effect of glucose concentration on the droplet size and stability of HIPPEs. The emulsion stability decreased with increasing droplet size. Figure 4 As shown, the appearance of the emulsion layer of each emulsion was highly similar at different glucose concentrations, indicating minimal influence from glucose concentration. Microscopic observation of the emulsion structure revealed that the HIPPEs emulsion droplets had the smallest droplet size at a glucose concentration of 0 mM, with an average diameter of 14.09 μm. The droplet size increased with increasing glucose concentration, reaching a maximum of 24.93 μm at a glucose concentration of 50 mM. Therefore, the addition of glucose led to larger droplets in SA-PBA-stabilized HIPPEs emulsions, resulting in decreased emulsion stability.

[0029] In addition, the state of the emulsion layer and the droplet size distribution of the emulsion were observed after the emulsion was left to stand for seven days. Figure 5 As shown, without the addition of glucose, the emulsion maintained a stable emulsion layer height, and the droplet size did not change significantly. At glucose concentrations of 2 and 10 mM, a very distinct oil phase precipitated on the upper layer of the emulsion, and the droplet size increased significantly. At a glucose concentration of 50 mM, the emulsion layer essentially broke down, and even optical microscopy could not produce a complete and uniform image of the emulsion droplets. This phenomenon indicates that glucose reduces the amphiphilicity of SA-PBA soft colloid particles, causing them to lose their ability to maintain a stable emulsion. Simultaneously, this result also indicates that the glucose response of SA-PBA is persistent, meaning that the emulsion stimulation response becomes more significant over time.

[0030] The TSI value can be used as an indicator of emulsion stability; that is, the lower the TSI value, the higher the emulsion stability. Multiple light scattering (PSS) technology utilizes the intensity of the backscattered light spot formed after near-infrared light passes through an opaque emulsion sample to study the stability changes of the emulsion system at different times and locations. For example... Figure 6As shown, the change curves of TSI values over time within 6 hours for HIPPEs emulsions stabilized by SA-PBA at different glucose concentrations were tested to further clarify the effect of different glucose concentrations on the physical stability of the emulsions. The results show that the TSI values of all emulsions increase with the extension of time, indicating that the emulsion stability decreases over time. In addition, as the glucose concentration increases, the TSI value of the emulsion rises rapidly, indicating that the addition of glucose is not conducive to the adsorption of SA-PBA at the oil-water interface, making the emulsion more unstable. The specific dynamic binding between the hydrophobic group phenylboronic acid and glucose weakens the amphiphilicity of SA-PBA soft colloidal particles, and the glucose binding also causes changes in the microstructure of the SA-PBA soft colloid. These results further confirm that the emulsion has glucose sensitivity, and the glucose content in the environment can control the stability of the SA-PBA emulsion.

[0031] The rheological property of an emulsion is an important indicator for evaluating emulsion stability, and rheological tests are used to analyze the relationship between the micro-interfacial viscoelasticity and the macroscopic emulsion stability. The shear viscosity of the emulsion was tested to further clarify the effect of glucose concentration on the HIPPE emulsion stabilized by SA-PBA soft particles, as shown in Figure 7 . The results show that the viscosity of all emulsions decreases with the increase of shear rate, exhibiting typical shear-thinning behavior. At a fixed shear rate, the viscosity of the emulsion stabilized by SA-PBA without added glucose is the highest, and the viscosity of the emulsion formed with added glucose gradually decreases. The change in apparent viscosity is related to the formation of the dense layer and particle network structure in the continuous interfacial phase. Therefore, the formation of dynamic B-O covalent bonds between glucose and phenylboronic acid destroys the microstructure of SA-PBA soft colloidal particles and reduces the interfacial wettability of the alginate derivative. The variation trend of viscosity is consistent with the changes in the droplet distribution size and TSI value of the emulsion.

[0032] The effect of the particle microstructure on the rheological behavior of the emulsion was further investigated by oscillation scanning. The magnitudes of the storage modulus (G') and loss modulus (G") of the emulsion can be used to judge the viscous or liquid-like behavior (G'<G") and the elastic or gel behavior (G'>G") of the sample. As shown in Figure 8 part (a), the linear viscoelastic region (LVR) was determined by strain scanning test, and the LVR was determined to be 1%. Figure 8Part (b) presents the functional relationships between the storage modulus and dissipation modulus of SA-PBA-stabilized HIPPE emulsions with different glucose concentrations as a function of angular frequency. Experimental results show that, in strain scanning experiments conducted at a strain amplitude of 1%, the storage modulus of the emulsion is consistently greater than its dissipation modulus, indicating that the emulsion primarily exhibits elastic or gel-like behavior and can effectively reduce deformation under external stress. With increasing glucose concentration, the G' and G" of the emulsion show a significant decreasing trend. When the glucose concentration is 50 mM, the G' of the emulsion drops to its minimum value (17 Pa), indicating that the addition of glucose alters the microstructure of the soft colloidal particles, leading to a decrease in emulsion stability. This suggests that when the glucose concentration in the environment is too high, the embedding of hydrophilic small molecules disrupts the covalent cross-linked network structure formed between the amphiphilic alginate polymer chains, resulting in a significant change in the particle structure at the interface and a marked weakening of the emulsion's rheological properties.

[0033] The surface morphology of SA-PBA soft colloidal particles-stabilized emulsion droplets was observed using SEM. A high-internal-phase Pickering emulsion was prepared using styrene (containing 2 mol% azobisisobutyronitrile) as the oil phase. After emulsion curing, the interfacial microstructure was observed using SEM. Figure 9 As shown, amphiphilic alginate-based polymer soft colloid particles are unevenly dispersed and adhered to the droplet surface. The distribution of SA-PBA soft colloid particles on the droplet surface varies significantly under different glucose concentrations. When only SA-PBA soft colloid particles stabilize the HIPPEs emulsion, many particles aggregate on the droplet surface and between large droplets, forming tighter bridging and network structures between droplets. The bridging structure between droplets indicates the formation of a gel network, increasing the physical stability of the emulsion. At lower glucose concentrations, less aggregation occurs on the droplet surface, but bridging structures composed of small particles still appear between droplets. However, at higher glucose concentrations, the particle distribution on the droplet surface becomes sparser. Only a few soft colloid particles exist on the emulsion microspheres prepared at a glucose concentration of 50 mM, indicating a significant reduction in the number of colloidal particles adsorbed at the oil-water interface. These results indicate that the addition of glucose disrupts the dynamic cross-linking network between SA-PBA chains, as the covalent cross-linking between phenylboronic acid and SA chains dissociates and preferentially forms more stable BO bonds with glucose monomers. Meanwhile, due to the good hydrophilicity of glucose, the hydrophobicity of amphiphilic SA-PBA soft colloid particles is reduced, which induces the soft colloid particles to desorb from the oil-water interface and migrate into the aqueous phase, resulting in glucose-responsive demulsification of the emulsion.

[0034] The microstructure of SA-PBA soft gel particles aggregates at the oil-water interface is crucial for understanding the stability of HIPPEs. The intrinsic luminescence properties of SA-PBA soft gel particles can be used to pinpoint their adsorption locations at the oil-water interface. Figure 10The analysis was conducted using the selected excitation channel (488 nm). A green fluorescent layer was observed around the HIIPEs droplets, confirming the adsorption of SA-PBA soft colloidal particles at the oil-water interface. The adsorption and dynamic assembly of SA-PBA soft colloidal particles at the oil-water interface formed a dense polymer layer, which created a stable aggregation barrier for the emulsion droplets, thus improving emulsion stability. Because the microstructure of the SA-PBA soft colloidal particles changes with increasing glucose concentration, the thickness of the dense layer adsorbed at the droplet interface varies. With increasing glucose concentration, the emulsion interface film thickness decreases, and the fluorescence intensity weakens. These results indicate that in-situ study of the emulsion interface film through interface visualization provides a more intuitive and clear understanding of the emulsion stabilization mechanism.

[0035] The high surface area of ​​luminescent microspheres provides an excellent matrix for biomolecule attachment, and their luminescent properties can be detected with high sensitivity. The particle-particle interactions at the interface were explored by adsorbing polysaccharide soft colloidal particles onto polystyrene microspheres via emulsion curing. CLSM images showed that the adsorption of polysaccharide soft colloidal particles on the surface of emulsion droplets was correlated with glucose concentration. Figure 11 Images of polystyrene microspheres in three fluorescence channels at a glucose concentration of 0 mM show that the polysaccharide soft colloidal particles completely cover the surface of the microspheres, and all three channels exhibit strong fluorescence emission. With increasing glucose concentration, the number of polysaccharide soft colloidal particles adsorbed on the surface of the emulsion microspheres decreases, and their distribution becomes uneven, leading to a decrease in fluorescence intensity. Particularly at a glucose concentration of 50 mM, only a few polysaccharide soft colloidal particles adhere to the surface of the emulsion microspheres, and their fluorescence emission intensity is extremely weak. To further elucidate the mechanism by which SA-PBA soft colloidal particles stabilize HIPPE emulsions, a schematic diagram illustrates the adsorption behavior of SA-PBA at the oil-water interface, such as... Figure 11 The (a4-d4) section shows the effect of glucose regulation on the microstructure of polysaccharide soft gel particles, which controls the amount of these particles adhering to the surface of polystyrene microspheres. These results indicate that the stability and droplet size of HIPPEs depend on the structure of the soft gel particles. Glucose-driven glucose regulation weakens the interparticle interactions in SA-PBA soft gel particles.

[0036] Dissipative quartz crystallography-diparticle (QCM-D) is a real-time interface multidimensional tracking technology that enables online, label-free analysis of various interface types. Therefore, QCM-D can be used to analyze the stability mechanism of amphiphilic polysaccharide emulsifiers in stabilizing HIPPE emulsions at the microscale, elucidating the influence of glucose presence on the adsorption kinetics and interfacial properties of SA-PBA at the oil-water interface. Figure 12Part (a) describes the interfacial adsorption behavior of SA-PBA at the oil-water interface. Here, Δf and ΔD represent the changes in frequency and dissipation, respectively. Δf is the phenomenon caused by the adsorption and desorption of soft colloidal particles at the oil film interface, while ΔD reflects the rigid conformation of the interfacial film. Figure 12 As shown in sections (b) and (c), a sustained negative Δf and an increase in positive ΔD were observed during the adsorption process, indicating the deposition of a large number of polysaccharide soft colloidal particles on the sensor. The sharp decrease in Δf value indicates that the soft colloidal particles were rapidly adsorbed onto the sensor. The viscosity and density of the polysaccharide soft colloidal solution are higher than those of the aqueous solution, leading to an initial decrease. The polysaccharide soft colloidal solution containing glucose interacted weakly with the oil film and reached adsorption equilibrium at the end of the 23-hour adsorption period, while the pure polysaccharide soft colloidal solution maintained a slower linear deposition and did not reach adsorption equilibrium even at the end of the 23-hour adsorption period. A sharp increase in Δf value was observed during desorption, due to the rapid desorption of unbound or loosely bound polysaccharide soft colloidal particles, while the sharp decrease in ΔD value indicates that the remaining polysaccharide soft colloidal particles adsorbed on the sensor surface formed a more compact film structure.

[0037] Furthermore, the change in the slope value of K(ΔD / Δf) can be used to further qualitatively analyze the conformational changes and adsorption kinetics of the adsorption film, allowing for direct comparison of the film's properties. A higher K value indicates greater energy dissipation per unit frequency and a larger water-soluble film. If the K value does not change significantly, it means that no conformational change occurred during adsorption or desorption. A non-constant or discontinuous K value indicates that conformational changes occur within the oil film. For adsorption kinetics, the scattering curve represents rapid kinetics, while the continuous curve represents slow kinetics. The ΔD-Δf of SA-PBA adsorption is shown below. Figure 12As shown in section (d), the Df curves for all cases exhibit a kinetic slope during adsorption. That is, all samples initially show a scattered curve (rapid kinetics), indicating a fast adsorption rate of the polysaccharide soft particles in the initial stage. Subsequently, it transitions to a continuous curve (slow kinetics), indicating a slow transition from monolayer adsorption at the oil-water interface to multilayer adsorption. Furthermore, changes in film structure are accompanied by conformational changes and reduced hydration. Before kinking, the steeper slope of the pure polysaccharide soft colloidal solution indicates a more flexible film formed by the adsorbed SA-PBA molecules. After kinking, the lower K2 value indicates that the polysaccharide soft colloidal particles form a rigid film on the sensor surface. Notably, the polysaccharide soft colloidal solution without added glucose shows a significant inflection point K3 during adsorption, and the K3 value is greater than the K2 value. This may be because the polysaccharide soft colloidal particles form covalent crosslinks through BO ester bonds formed by phenylboronic acid and diols on the polysaccharide chain, resulting in a thicker multilayer film. During desorption, the Df curves for all samples show a significant change in slope. In the initial stage of desorption, the ΔD-Δf plot is a scatter plot with a very high K4 value, mainly due to the decrease in viscosity and adsorption density of the polysaccharide soft colloidal solution on the adsorption layer after water washing. Subsequently, the ΔD-Δf plots of all samples turn into continuous curves, and K5 decreases, indicating that the film formed by the remaining polysaccharide soft colloidal particles becomes more rigid due to the desorption of unbound or loosely bound polysaccharide soft colloidal particles.

[0038] The thickness and quality of the thin film can be quantitatively analyzed using the Sauerbrey model. Figure 13 Parts (a) and (b) show the thickness and mass of the adsorbed membrane during the adsorption and desorption processes of SA-PBA. During adsorption, polysaccharide soft colloidal particles rapidly form a loose film in the initial stage of adsorption. Then, with the continuous adsorption of the emulsifier, more soft colloidal particles aggregate through intermolecular interactions to form corresponding microstructures, and this aggregation of polysaccharide soft colloidal particles contributes to the rigidity of the film. This results in hf and mf increasing over time, with the pure polysaccharide soft colloidal solution exhibiting the highest adsorbed membrane thickness and mass, mainly attributed to the formation of dense multilayer adsorption by the polysaccharide soft colloidal particles. Furthermore, the viscosity ηf and elastic modulus μf of the adsorbed membrane were quantitatively analyzed using the Dfind-Voigt model. Figure 13Sections (c) and (d) show the trends of shear modulus μf and shear viscosity ηf over time during adsorption and desorption. The results indicate that pure polysaccharide soft colloidal particles exhibit higher μf and ηf during adsorption. This may be due to the formation of multilayer adsorption after dynamic cross-linking between soft colloidal particles via BO bonds. The presence of glucose suggests that the SA-PBA soft colloidal particles are induced to desorb from the interface, leading to a reduction in the thickness and mass of the adsorbed film. Furthermore, the reduced stability of the HIPPE emulsion stabilized by SA-PBA soft particles may be due to the lower viscosity and elastic structure exhibited after the addition of glucose.

[0039] SA-PBA's good water dispersibility, low CMC, and responsiveness to external stimuli make it a potential candidate for applications in the biomedical field. The biocompatibility of SA-PBA was assessed using the CCK-8 assay, and L929 cells were used for cell viability evaluation. Figure 14 As shown in section (a), after incubating L929 cells with different concentrations of SA-PBA for 24 hours, it was found that SA-PBA promoted cell growth, especially after incubation at a concentration of 4 mg / ml for 24 hours, the cell growth rate reached 160%. These results provide direct evidence for the good biocompatibility of SA-PBA. Therefore, the SA-PBA-stable HIPPEs system can be used as a drug delivery system for hydrophobic drugs. Glucose can be used to regulate the aggregation behavior of SA-PBA at the oil-water interface, and an in vitro Nile Red release model was constructed under different glucose conditions. The oil phase contained squalene with 1 mg / ml Nile Red, and the aqueous phase contained 3 mg / ml SA-PBA. 3500 Da dialysis bags containing the drug-loaded emulsion were placed in 100 ml of dialysate (50% methanol) containing different glucose concentrations, and in vitro drug release experiments were conducted in a constant-temperature shaker. Figure 14 Part (b) shows the standard curve of Nile Red release in aqueous solution, with the actual release concentration calculated using UV absorbance. The cumulative drug release of SA-PBA soft colloidal particles-stabilized drug-loaded HIPPEs under different glucose concentrations is shown in the figure. Figure 14As shown in section (c), Nile Red is released rapidly in the initial stage, followed by a slower release. This is likely because unencapsulated Nile Red is released rapidly from the dialysis bag, while encapsulated Nile Red is released slowly, resulting in a slower release rate and longer release time. Furthermore, when glucose is absent in the external environment, the release rate of Nile Red tends to plateau, with a final cumulative release of 20.87% after 24 hours. When the glucose concentration in the external environment is low, the final cumulative release of Nile Red is 53.51% and 60.33%, respectively. When the glucose concentration in the external environment reaches 50 mM, the cumulative release of Nile Red reaches 62.86% within 6 hours, then the release rate plateaus, with a final cumulative release as high as 80.96% after 24 hours. The experimental results indicate that the release rate and cumulative release of Nile Red are related to the stability of the emulsion under different glucose concentrations in the environment. In the absence of glucose in the environment, SA-PBA soft gel particles form a tight covalently cross-linked network at the oil-water interface, making it difficult to release the hydrophobic drug encapsulated by the softener. As the glucose content in the environment increases, after phenylboronic acid on SA-PBA forms a covalent cross-link with glucose, the soft gel particles desorb from the interfacial film, the emulsion becomes unstable, and the hydrophobic drug is rapidly released. Therefore, HIPPE emulsions stabilized by SA-PBA soft gel particles exhibit the potential for sustained-release drugs under glucose stimulation.

[0040] In summary, this application utilizes the dynamic bonding characteristics between phenylboronic acid groups and cis-diol groups to enable SA-PBA to dynamically assemble into soft colloidal particles with different microstructures under different glucose environments, thereby establishing the relationship between interfacial microstructure and emulsion properties. The phenylboronic acid groups in the SA-PBA soft colloidal particles form dynamic BO bonds with the ortho-cis-diol on the SA chain, creating a dynamic cross-linked structure within and between the soft colloidal particles. This allows the soft colloidal particles to adsorb and form a thicker interfacial film at the oil-water interface, reducing emulsion droplet aggregation and droplet size. Simultaneously, the soft colloidal particles in the continuous phase form a cross-linked network structure through dynamic BO bonds, further enhancing emulsion stability. As glucose concentration increases, the binding between phenylboronic acid groups and the ortho-cis-diol on glucose competes with the dynamic binding between phenylboronic acid groups and the ortho-cis-diol on the SA chain. Consequently, some phenylboronic acid groups bind to glucose instead, reducing the original dynamic BO bonds between phenylboronic acid groups and the SA chain. This weakens the dynamic cross-linking network within and between the soft colloid particles, making the internal structure of the soft colloid particles looser. Simultaneously, the wetting and adsorption capacity of the soft colloid particles at the oil-water interface decreases, resulting in reduced interfacial adsorption and increased emulsion droplet size and decreased emulsion stability. Therefore, the microstructure of SA-PBA soft colloid particles and their adsorption state at the oil-water interface can be adjusted by changing the external glucose concentration, thereby achieving glucose-responsive regulation of the Pickering emulsion's stable state. Furthermore, an in vitro emulsion release system was constructed using this glucose-responsive characteristic. The results showed that the release rate and cumulative release amount of the model hydrophobic guest are correlated with the glucose concentration in the external environment. Therefore, by regulating the assembly and aggregation state of polysaccharide soft colloidal particles at the interface through external glucose stimulation, the emulsion interface structure, emulsion stability, and hydrophobic guest release behavior can be synergistically regulated, providing a technical basis for constructing glucose-responsive alginate-based Pickering emulsions and hydrophobic guest release carriers.

Claims

1. A glucose-responsive Pickering emulsion, characterized in that, Includes aqueous phase and oil phase; The aqueous phase contains phenylboronic acid-based alginate derivatives and glucose; The phenylboronic acid-based alginate derivative includes an alginate molecular chain backbone and phenylboronic acid groups grafted onto the alginate molecular chain backbone. The concentration of the phenylboronic acid-based alginate derivative in the aqueous phase is 2-4 mg / mL, and the volume fraction of the oil phase is 80%-90%. The phenylboronic acid-based alginate derivative forms soft colloidal particles, which are distributed in the aqueous phase and at the oil-water interface between the aqueous and oil phases. At least a portion of the phenylboronic acid groups in the phenylboronic acid-based alginate derivative form a first dynamic BO bond with the cis-diol groups on the alginate molecular chain, and at least a portion of the phenylboronic acid groups form a second dynamic BO bond with the cis-diol groups on glucose.

2. The glucose-responsive Pickering emulsion according to claim 1, characterized in that, The preparation method of the phenylboronic acid-based alginate derivative is as follows: Dissolve 3-aminophenylborate salt in water, add formaldehyde, and react at 20-30℃ for 2-4 hours; add sodium alginate aqueous solution and cyclohexylisocyanate to the resulting solution, and react at 20-30℃ in the dark for 12-24 hours; dialyze and freeze-dry the product to obtain the phenylborate-based alginate derivative.

3. The glucose-responsive Pickering emulsion according to claim 2, characterized in that, The mass-to-volume ratio of sodium alginate, 3-aminophenylborate, formaldehyde, and cyclohexylisocyanate is 100 g: (4-8) g: (1-5) mL: (1-10) mL.

4. The glucose-responsive Pickering emulsion according to claim 1, characterized in that, The concentration of glucose in the aqueous phase is 2-50 mM, and the pH value of the aqueous phase is 7-8.

5. The glucose-responsive Pickering emulsion according to claim 1, characterized in that, The oil phase is squalene, and the volume fraction of the oil phase is 80%.

6. A method for preparing a glucose-responsive Pickering emulsion, wherein the glucose-responsive Pickering emulsion is as described in any one of claims 1-5, characterized in that, The steps are as follows: S1. Dissolve 3-aminophenylborate salt in water, add formaldehyde, and react at 20-30℃ for 2-4 hours; add sodium alginate aqueous solution and cyclohexylisocyanate to the resulting solution, and react at 20-30℃ in the dark for 12-24 hours; dialyze and freeze-dry the product to obtain phenylborate-based alginate derivative. S2. Prepare an aqueous solution with a concentration of 2-4 mg / mL by the phenylboronic acid-based alginate derivative, add glucose to the aqueous solution, and adjust the pH of the resulting aqueous phase to 7-8. S3. Squalene, which serves as the oil phase, is added to the aqueous phase to make the volume fraction of the oil phase 80%-90%, and sheared at a speed of 15000-20000 rpm for 1-5 minutes to obtain a glucose-responsive Pickering emulsion. In this embodiment, at least a portion of the phenylboronic acid groups in the phenylboronic acid-based alginate derivative form a first dynamic BO bond with the cis-diol groups on the alginate molecular chain, and at least a portion of the phenylboronic acid groups form a second dynamic BO bond with the cis-diol groups on glucose.

7. The preparation method according to claim 6, characterized in that, In step S1, the mass-to-volume ratio of sodium alginate, 3-aminophenylborate, formaldehyde, and cyclohexylisocyanate is 100 g: (4-8) g: (1-5) mL: (1-10) mL.

8. The preparation method according to claim 6, characterized in that, The sodium alginate aqueous solution has a mass concentration of 1-3 wt% and a pH of 3-4; the product obtained from the reaction is dialyzed using a dialysis membrane with a molecular weight cutoff of 3500 Da.

9. The application of a glucose-responsive Pickering emulsion in the preparation of a hydrophobic guest glucose-responsive release carrier, characterized in that, The glucose-responsive Pickering emulsion is as described in any one of claims 1-5.

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