Composite hydrogel microsphere delivery system and preparation method thereof

Abstract

The invention discloses a composite hydrogel microsphere delivery system and a preparation method thereof. The composite hydrogel microsphere delivery system comprises: (a) a plurality of plant-derived exosome-like nanovesicle (PDEV) cores; (b) a bioactive substance entrapped in the plant-derived exosome-like nanovesicle core or combined with the plant-derived exosome-like nanovesicle core; (c) a hydrogel matrix encapsulating the (a) and (b); plant fiber components are embedded in the hydrogel matrix. The system not only can utilize the macroscopic gastric acid protection and intestinal fixed-point release characteristics of the hydrogel microspheres, but also can provide a stable and friendly microenvironment for the PDEV nano-vesicles in the microspheres, so that an active carrier (PDEV) and an active endosome (ALA) thereof are completely and jointly delivered to an intestinal absorption part; and the dual problems of gastric acid degradation and poor intestinal absorption are solved synergistically.

Description

Technical Field

[0001] This application relates to a composite hydrogel microsphere delivery system and its preparation method, belonging to the fields of biomedicine, functional food and nanodelivery technology. Background Technology

[0002] Oral delivery is the ideal route of administration for active ingredients in pharmaceuticals and functional foods, but it faces a significant challenge from the gastrointestinal (GI) barrier. First, the highly acidic environment of the stomach (pH 1.0-3.0) and the presence of highly active digestive enzymes (such as pepsin) are extremely destructive to many bioactive substances and nanocarriers, which is the primary obstacle to achieving oral delivery.

[0003] To overcome the gastric acid barrier, biocompatible polymers (such as alginate) have been extensively studied for constructing pH-responsive hydrogels. Alginate can be prepared into hydrogel microspheres via mild ionogelation methods (e.g., crosslinking with calcium ions). These microspheres maintain structural stability in the acidic gastric environment but swell or disintegrate in the neutral intestinal environment, thus achieving "intestinal-targeted release" of active substances and avoiding their degradation in the stomach. However, traditional "macro" hydrogel encapsulation techniques only address the question of "where" the active substances are released, not "how" these substances (such as alpha-lipoic acid, curcumin, and other polyphenols) are efficiently absorbed after release into the intestine. These compounds generally suffer from inherent defects such as poor water solubility and low biomembrane permeability, resulting in low absorption efficiency and bioavailability even after targeted release.

[0004] On the other hand, nanodelivery technology, especially plant-derived exosome-like nanovesicles (PDEVs) (such as wheat germ vesicles), as a natural nanocarrier, has been shown to effectively encapsulate these hydrophobic substances and promote cellular uptake through their lipid membrane structure, and even exert synergistic therapeutic effects on their endogenous active substances (such as GABA and folic acid).

[0005] Therefore, this field faces an obvious technical challenge: if PDEVs, these sophisticated nanovesicles, are taken orally directly, they are highly susceptible to degradation in gastric acid; if they are simply encapsulated in traditional hydrogels, although they can resist gastric acid, the coarse network structure of the hydrogel and the preparation process may not provide sufficient microenvironmental stability for the PDEVs. Existing technologies either focus on complex nanoscale surface modifications of PDEVs (such as layer-by-layer encapsulation) to enhance their stability, which are relatively complex processes; or focus on preparing macroscopic dosage forms (such as tablets), which still release unprotected PDEVs after intestinal disintegration. Summary of the Invention

[0006] This invention aims to address the technical challenges of existing PDEV delivery systems, such as insufficient stability in the gastric acid environment, easy degradation, and premature leakage of internal active substances. This invention provides a novel "micron-nano" two-stage synergistic delivery system, comprising: (a) multiple plant-derived exosome-like nanovesicle cores; (b) a bioactive substance encapsulated within or bound to the plant-derived exosome-like nanovesicle cores; and (c) a hydrogel matrix encapsulating (a) and (b); wherein plant fiber components are embedded in the hydrogel matrix. This system not only utilizes the "macro" gastric acid protection and intestinal targeted release characteristics of hydrogel microspheres, but also provides a stable and friendly microenvironment for PDEV nanovesicles within the microspheres, thereby completely delivering the "active carrier (PDEV)" and its "active internally loaded substance (ALA)" to the intestinal absorption site, synergistically solving the dual problems of gastric acid degradation and poor intestinal absorption. Instead of nano-modifying the PDEV itself, this system embeds the PDEV nanovesicles loaded with bioactive substances (as the "core") entirely within a pH-responsive hydrogel microsphere matrix reinforced with homologous plant fibers (as the "shell").

[0007] This invention also innovatively introduces plant fiber from PDEV extraction residue as a matrix enhancer, achieving full utilization of the original components while improving the mechanical strength of the microspheres. This system provides excellent gastric acid protection for PDEVs, allowing them to safely pass through the stomach and achieve targeted release in the intestinal environment, thereby significantly improving the delivery efficiency of active substances and synergistic therapeutic effects.

[0008] According to a first aspect of this application, a composite hydrogel microsphere delivery system is provided, comprising: (a) Multiple plant-derived exosome-like nanovesicle cores; (b) A bioactive substance contained within or bound to the core of the plant-derived exosome-like nanovesicles; (c) A hydrogel matrix encapsulating (a) and (b); The hydrogel matrix contains plant fiber components.

[0009] Optionally, the plant fiber component is derived from the plant-derived exosome-like nanovesicle plant material.

[0010] Optionally, the hydrogel matrix is ​​a pH-responsive hydrogel; The pH-responsive hydrogel is stable in acidic media with a pH of 1.0–3.0, and swells or disintegrates in neutral or alkaline media with a pH of 6.8–7.4.

[0011] Optionally, the hydrogel matrix is ​​selected from one or more of alginate, chitosan, gelatin, or pectin.

[0012] Optionally, the hydrogel matrix comprises alginate, cross-linked by multivalent cations; Preferably, the multivalent cation is a calcium ion.

[0013] Optionally, the average particle size of the composite hydrogel microspheres is from 100 µm to 2 mm.

[0014] Optionally, the plant-derived exosome-like nanovesicles are derived from plants of the Poaceae, Rutaceae, Zingiberaceae, or Rosaceae families; Preferably, the plant-derived exosome-like nanovesicles are selected from wheat sprouts, ginger, or grapefruit.

[0015] Optionally, the bioactive substance is selected from the group consisting of polyphenols, alkaloids, fat-soluble vitamins, or antioxidants; Preferably, the bioactive substance is selected from α-lipoic acid (ALA), curcumin, or resveratrol.

[0016] According to a second aspect of this application, a method for preparing the above-described composite hydrogel microsphere delivery system is provided, the method comprising: (1) Obtaining plant-derived exosome-like nanovesicle suspensions loaded with bioactive substances; (2) The plant-derived exosome-like nanovesicle suspension in step (1) is mixed with a solution containing a hydrogel precursor polymer, and plant fiber components are added to obtain a hybrid solution; (3) Disperse the hybrid solution into droplets, add a crosslinking agent solution, and solidify to obtain the composite hydrogel microsphere delivery system.

[0017] Optionally, the hydrogel precursor polymer is selected from sodium alginate; the crosslinking agent is selected from calcium chloride; Preferably, step (3) is achieved by a dropwise addition method or an emulsification-crosslinking method; Preferably, the hydrogel precursor polymer is present in a solution containing 1%-3% (w / v); where "w / v" refers to weight / volume percentage concentration. For example, "1%-3% (w / v)" means that each 100 mL of solution contains 1 g to 3 g of solute (i.e., hydrogel precursor polymer).

[0018] The crosslinking agent solution contains 1%-5% (w / v) of crosslinking agent.

[0019] Optionally, the mass ratio of the hydrogel precursor polymer to the plant fiber component is 3:1 to 4:1; Optionally, the volume ratio of the plant-derived exosome-like nanovesicle suspension loaded with bioactive substances to the solution containing the hydrogel precursor polymer is 1:2.

[0020] As one specific embodiment, the preparation method includes: (1) PDEV suspension is obtained from plant raw materials, and the plant residue remaining in the separation process is optionally processed into plant fiber filler. (2) The bioactive substance is loaded into the PDEV suspension to obtain a loaded PDEV suspension; (3) The loaded PDEV suspension is mixed with a hydrogel precursor solution containing alginate, and the plant fiber filler is added to the hybrid solution; (4) The hybrid solution is dispersed in a crosslinking agent solution by drop addition or emulsification, wherein the crosslinking agent solution contains polyvalent cations (preferably calcium chloride solution). (5) The hybrid solution droplets are instantaneously gelled in the crosslinking agent solution to form composite hydrogel microspheres loaded with PDEV; (6) Collect and wash the microspheres.

[0021] Compared with existing technologies, the composite hydrogel microspheres loaded with plant-derived nanovesicles provided by this invention have the following significant beneficial effects: Excellent gastric acid protection (macroscopic protection): The hydrogel microspheres of this invention (such as calcium alginate gel) have a dense structure and do not swell in the gastric acid environment (pH 1.0-3.0), forming a robust physical barrier. This completely protects the internally encapsulated PDEV nanovesicles from destruction by gastric acid and pepsin, solving the fundamental problem of PDEV degradation in the stomach. pH-responsive release in the intestine: The hydrogel matrix is ​​pH sensitive. When the microspheres enter the neutral or weakly alkaline intestinal environment (pH 6.8-7.4), the alginate undergoes deprotonation and dissociates from calcium ions, leading to gel swelling and disintegration, thereby releasing the internal PDEV nanovesicles at specific points within the intestine. Waste utilization and mechanical enhancement (core advantage): This invention innovatively processes the plant residue (rich in cellulose) remaining after PDEV extraction and adds it back into the hydrogel matrix as a reinforcing filler. This not only significantly improves the mechanical strength and structural toughness of the microspheres, making them more resistant to the physical shear forces of gastrointestinal motility, but also achieves full utilization of plant raw materials, conforming to the green and natural concept of "using the original soup to cook the original food." Dual delivery and synergistic effect: This invention constructs a two-stage delivery system of "hydrogel protecting PDEV, and PDEV protecting active substances." Simple process and easy to scale up: The ion gelation method (drop addition method, emulsification method) used in this invention is simple, mild, and low-cost, making it extremely easy to achieve large-scale industrial production. Detailed implementation methods The present application is further illustrated below with reference to specific embodiments. The following descriptions are merely a few embodiments of the present application and are not intended to limit the present application in any way. Although the present application discloses preferred embodiments as follows, they are not intended to limit the present application. Any modifications or variations made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

[0022] Unless otherwise specified, the raw materials used in the embodiments of this application are all purchased commercially and used directly without any special treatment.

[0023] Example 1: Preparation of ALA@PDEV cellulose-reinforced composite hydrogel microspheres (dropping method) (1) Preparation of ALA@PDEV core: Fresh wheat sprouts were juiced and purified by differential centrifugation to obtain PDEV suspension (protein concentration 2000 µg / mL). The PDEV suspension was mixed with 800 µg / mL α-lipoic acid (ALA) solution at a volume ratio of 1:1, co-incubated, and then sonicated to prepare ALA-loaded PDEV suspension (i.e., ALA@PDEV). (1.5) Preparation of plant fiber filler: The precipitate remaining after differential centrifugation of PDEV in step (1) (i.e., plant residue) was taken. The residue was washed several times with deionized water to remove soluble impurities. Then it was freeze-dried and pulverized using a high-speed pulverizer and passed through a 100-mesh sieve to obtain wheat sprout plant fiber powder for later use. (2) Preparation of composite hydrogel microspheres: 2.0 g of sodium alginate and 0.5 g of plant fiber powder prepared in step (1.5) were accurately weighed and dispersed together in 97.5 mL of deionized water. The mixture was magnetically stirred overnight to obtain a homogeneous polymer precursor solution containing fiber-reinforced filler. The ALA@PDEV suspension prepared in step (1) was thoroughly mixed with the above polymer precursor solution at a volume ratio of 1:2 to obtain a hybrid solution to be gelled. A 2% (w / v) calcium chloride (CaCl2) aqueous solution was prepared as a crosslinking agent solution. Using a syringe with a 22G needle, the hybrid solution was added dropwise at a rate of 10 mL / h to the vigorously stirred calcium chloride crosslinking agent solution (200 mL). The droplets solidified instantly upon contact with the crosslinking agent to form white hydrogel microspheres. After continuous stirring and crosslinking for 30 minutes, the microspheres were collected with a sieve and washed three times with deionized water to remove residual calcium chloride on the surface. Characterization: Observation under an optical microscope showed that the obtained microspheres were regular in shape, uniformly spherical, and had an average particle size of about 1.3 mm.

[0024] Mechanical strength: Compression tests were conducted on microspheres with added plant fiber powder and microspheres without it (control group). The microspheres with added plant fiber powder showed a compression modulus that was about 40% higher than the control group at 50% deformation, indicating a significant increase in mechanical strength and better resistance to physical shear.

[0025] Example 2: In vitro release and gastric acid protection study To verify the gastric acid protection and intestinal release characteristics of the present invention, in vitro release experiments were conducted on the cellulose-reinforced microspheres (loaded with ALA@PDEV) prepared in Example 1 and the unloaded ALA@PDEV nanoparticles (control group). (1) Simulated gastric juice (SGF, pH 2.0) release: Both groups of samples were placed in simulated gastric juice (containing pepsin) and shaken at 37°C. The results showed that after 2 hours, the encapsulation rate of ALA in the control group (without ALA@PDEV) dropped sharply to less than 40%, and severe aggregation and precipitation of PDEV were detected. In contrast, the hydrogel microspheres of this invention released less than 5% of ALA into the medium, and the microspheres maintained their intact morphology (thanks to fiber reinforcement), demonstrating their excellent gastric acid protection ability. (2) Release from simulated intestinal fluid (SIF, pH 7.4): The sample treated in SGF for 2 hours was transferred to simulated intestinal fluid (containing trypsin) and shaken at 37°C. Results showed that the control group had largely degraded with no further release. Upon entering the SIF, the hydrogel microspheres of this invention began to swell significantly and gradually disintegrate, with the internally loaded ALA@PDEV nanovesicles being slowly released into the medium. At 4 hours, the cumulative ALA release rate reached approximately 85%, achieving programmed release in the intestinal environment. Conclusion: The hydrogel microsphere system of this invention successfully protects the PDEV core from gastric acid degradation and achieves targeted release in a simulated intestinal environment, overcoming a major obstacle to oral delivery of PDEVs.

[0026] Example 3: Preparation of curcumin@ginger PDEV composite hydrogel microspheres (emulsification-crosslinking method) To demonstrate the versatility of the preparation method of this invention, this embodiment uses an emulsification-crosslinking method to prepare microspheres of different particle sizes, and changes the PDEV source and active material.

[0027] (1) Preparation of curcumin@ginger-PDEV core: Fresh ginger was juiced and purified by differential centrifugation to obtain ginger-derived PDEV suspension (protein concentration adjusted to 2000 µg / mL). Curcumin was dissolved in a small amount of ethanol and diluted with PBS, then mixed with PDEV suspension at a ratio of 1:1 (final curcumin concentration 800 µg / mL), co-incubated, and then sonicated to prepare curcumin-loaded PDEV suspension (i.e., Curcumin@Ginger-PDEV).

[0028] (2) Preparation of composite hydrogel microspheres (emulsification method): Prepare a 1.5% (w / v) sodium alginate aqueous solution and add 0.5% (w / v) ginger plant fiber micropowder (preparation method is the same as in Example 1.5) to obtain a polymer precursor solution. Mix the Curcumin@Ginger-PDEV suspension prepared in step (1) with the polymer precursor solution at a volume ratio of 1:2 to obtain a hybrid solution (aqueous phase). Preparation of oil phase: Take 100 mL of liquid paraffin and add 1% (v / v) Span 80 as an emulsifier. Slowly add the aqueous phase to the vigorously stirred (800 rpm) oil phase and continue emulsifying for 15 minutes to form a stable W / O emulsion. Under continuous stirring, slowly add 20 mL of a crosslinking solution containing 5% (w / v) calcium chloride and 0.5% (v / v) Tween 80 to the emulsion. Continue stirring and crosslinking for 1 hour to solidify the sodium alginate microdroplets. Stop stirring and allow the mixture to separate into layers. Discard the upper oil phase and collect the lower microspheres. Wash the microspheres alternately with isopropanol and deionized water to remove the oil phase and unreacted substances, and finally freeze-dry.

[0029] Characterization: The obtained microspheres were found to have a uniform particle size distribution, with an average particle size of approximately 180 µm, as determined by a laser particle size analyzer.

[0030] Example 4: Universality of different active substances with PDEV sources To further demonstrate the platform versatility of the technical solution of this invention, similar steps to those in Example 1 (droplet addition method) or Example 3 (emulsification method) were used, with the following combinations and substitutions: Combination A: Grapefruit-derived PDEV, loaded with resveratrol, was prepared using the droplet addition method of Example 1, with the addition of grapefruit pomace fiber. Combination B: Wheat sprout PDEV, loaded with coenzyme Q10 (CoQ10), was prepared using the emulsification method of Example 3 (adding the corresponding wheat sprout fiber to the sodium alginate solution).

[0031] Results: Both combinations successfully prepared well-morphologically sound composite hydrogel microspheres encapsulating PDEV cores. In vitro release experiments showed similar gastric acid protection properties and intestinal pH-responsive release behavior as in Example 2. This demonstrates the broad applicability of the present invention, serving as a general platform technology for delivering various PDEVs and different bioactive substances.

[0032] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A composite hydrogel microsphere delivery system, characterized in that, include: (a) Multiple plant-derived exosome-like nanovesicle cores; (b) A bioactive substance contained within or bound to the core of the plant-derived exosome-like nanovesicles; (c) A hydrogel matrix encapsulating (a) and (b); The hydrogel matrix contains plant fiber components.

2. The composite hydrogel microsphere delivery system according to claim 1, characterized in that, The plant fiber component is derived from the plant-derived exosome-like nanovesicle plant material.

3. The composite hydrogel microsphere delivery system according to claim 1, characterized in that, The hydrogel matrix is ​​a pH-responsive hydrogel; The pH-responsive hydrogel is stable in acidic media with a pH of 1.0–3.0, and swells or disintegrates in neutral or alkaline media with a pH of 6.8–7.

4.

4. The composite hydrogel microsphere delivery system according to claim 1, characterized in that, The hydrogel matrix is ​​selected from one or more of alginate, chitosan, gelatin or pectin.

5. The composite hydrogel microsphere delivery system according to claim 4, characterized in that, The hydrogel matrix contains alginate, which is cross-linked by multivalent cations; Preferably, the multivalent cation is a calcium ion.

6. The composite hydrogel microsphere delivery system according to claim 1, characterized in that, The average particle size of the composite hydrogel microspheres is 100 µm to 2 mm.

7. The composite hydrogel microsphere delivery system according to claim 1, characterized in that, The plant-derived exosome-like nanovesicles are derived from plants of the Poaceae, Rutaceae, Zingiberaceae, or Rosaceae families; Preferably, the plant-derived exosome-like nanovesicles are selected from wheat sprouts, ginger, or grapefruit.

8. The composite hydrogel microsphere delivery system according to claim 1, characterized in that, The bioactive substances are selected from the group consisting of polyphenols, alkaloids, fat-soluble vitamins, or antioxidants; Preferably, the bioactive substance is selected from α-lipoic acid, curcumin, or resveratrol.

9. A method for preparing the composite hydrogel microsphere delivery system according to any one of claims 1 to 8, characterized in that, The preparation method includes: (1) Obtaining plant-derived exosome-like nanovesicle suspensions loaded with bioactive substances; (2) The plant-derived exosome-like nanovesicle suspension in step (1) is mixed with a solution containing a hydrogel precursor polymer, and plant fiber components are added to obtain a hybrid solution; (3) Disperse the hybrid solution into droplets, add a crosslinking agent solution, and solidify to obtain the composite hydrogel microsphere delivery system.

10. The preparation method according to claim 9, characterized in that, The hydrogel precursor polymer is selected from sodium alginate; the crosslinking agent is selected from calcium chloride. Preferably, step (3) is performed by a dropwise addition method or an emulsification-crosslinking method; Preferably, in the solution containing the hydrogel precursor polymer, the content of the hydrogel precursor polymer is 1%-3% (w / v); The crosslinking agent solution contains 1%-5% (w / v) of the crosslinking agent. Preferably, the mass ratio of the hydrogel precursor polymer to the plant fiber component is 3:1 to 4:1; Preferably, the volume ratio of the plant-derived exosome-like nanovesicle suspension loaded with bioactive substances to the solution containing the hydrogel precursor polymer is 1:2.