Tremella polysaccharide-based hydrogel microspheres as well as preparation method and application thereof

Abstract

The invention provides tremella polysaccharide-based hydrogel microspheres as well as a preparation method and application thereof, and belongs to the field of functional polysaccharide materials. The preparation method of the hydrogel microspheres comprises the following steps: (1) preparing a water phase containing tremella polysaccharide and a cationic polymer; (2) preparing an oil phase containing a surfactant; (3) preparing water-in-oil liquid drops through a capillary glass tube single-stage microfluidic device; and (4) introducing the oil phase containing the water-in-oil liquid drops into a receiving system containing acetic acid for crosslinking, thereby obtaining the product. The hydrogel microspheres can be prepared by adopting a simple device, the preparation process is mild and biologically friendly, and the obtained hydrogel microspheres are highly uniform in particle size and good in monodispersity. Meanwhile, the hydrogel microspheres prepared by the method have rich autofluorescence characteristics, can be used as a fluorescent tracing carrier or an imaging material and used for carrying various active substances, an exogenous dye loading step is omitted, the toxicity is reduced, the preparation process is simplified, and the hydrogel microspheres have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of functional polysaccharide materials, specifically relating to a Tremella fuciformis polysaccharide-based hydrogel microsphere, its preparation method, and its uses. Background Technology

[0002] Tremella fuciformis polysaccharides are a class of widely sourced natural fungal polysaccharides with excellent biocompatibility, immunomodulatory, and antioxidant activities. In recent years, they have attracted widespread attention for applications in functional foods, drug delivery, and tissue engineering. Therefore, developing monodisperse hydrogel microspheres based on Tremella fuciformis polysaccharides will help discover new properties of Tremella fuciformis polysaccharides and further expand their application scope.

[0003] Microfluidic droplet technology can precisely manipulate fluids at the micrometer scale to prepare highly monodisperse droplet templates, and is considered an ideal method for preparing uniform microspheres. Based on different gelation and solidification principles, physical crosslinking, chemical crosslinking, enzyme-induced crosslinking, and photocrosslinking have led to the development and preparation of various types of hydrogel microspheres using microfluidic technology. However, given the unique physicochemical properties of Tremella fuciformis polysaccharide, there is still no successful case of combining the precise molding capability of microfluidic emulsions with a suitable, mild, in-situ crosslinking mechanism for Tremella fuciformis polysaccharide. Specifically, how to design a reaction system that allows small molecules (such as acids) in the outer phase to diffuse through the oil phase intermediate layer, triggering a rapid crosslinking reaction between Tremella fuciformis polysaccharide in the inner aqueous phase and a specific crosslinking agent, thereby achieving uniform preparation, remains a pressing technical challenge in this field. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preparing and using Tremella fuciformis polysaccharide-based hydrogel microspheres.

[0005] This invention provides a method for preparing Tremella fuciformis polysaccharide-based hydrogel microspheres, comprising the following steps: (1) Prepare an aqueous phase containing Tremella polysaccharide and cationic polymer; (2) Prepare an oil phase containing surfactant; (3) Water-in-oil droplets were prepared by a single-stage microfluidic device using a capillary glass tube. (4) The oil phase containing water-in-oil droplets is introduced into the receiving system for cross-linking to obtain tremella polysaccharide-based hydrogel microspheres; the receiving system is an oil phase containing acetic acid.

[0006] Preferably, In step (1), the method for preparing the aqueous phase includes the following steps: (a) Prepare an aqueous solution of Tremella polysaccharide with a mass fraction of 0.05~1.5wt%; (b) Prepare an aqueous solution of cationic polymer with a mass fraction of 0.1 to 4.0 wt%; (c) Mix the aqueous solution of Tremella polysaccharide and the aqueous solution of cationic polymer at a volume ratio of (1~5):1, and adjust the pH to 5.7~6.4 to obtain the aqueous phase; And / or, in step (2), the method for preparing the oil phase includes the following steps: The surfactant is added to the continuous phase and mixed evenly to obtain an oil phase with a surfactant mass fraction of 0.5~10.0wt%; the continuous phase is selected from soybean oil, mineral oil, liquid paraffin, silicone oil or fluorinated oil; the surfactant is selected from polyglycerol polyricinoleate, Span80 or fluorinated oil-specific surfactant.

[0007] Preferably, In step (a), the mass fraction of the Tremella polysaccharide aqueous solution is 1.0 wt%. And / or, in step (b), the mass fraction of the cationic polymer aqueous solution is 2.0~4.0 wt%; And / or, in step (c), the volume ratio of the Tremella polysaccharide aqueous solution to the cationic polymer aqueous solution is 3:1; And / or, in step (c), the pH is adjusted to 5.9; And / or, the mass fraction of the surfactant in the oil phase is 5.0 wt%; And / or, the continuous phase is selected from soybean oil; And / or, the surfactant is selected from polyglycerol polyricinoleate.

[0008] Preferably, In step (b), the cationic polymer is chitosan; And / or, in step (c), the pH adjustment is performed using a 1.0 wt% aqueous solution of sodium hydroxide.

[0009] Preferably, In step (3), when preparing water-in-oil droplets, the flow rate of the aqueous phase is 10~1500 μL / h; the flow rate of the oil phase is 200~10000 μL / h. And / or, in step (4), the volume fraction of acetic acid in the oil phase containing acetic acid is 0.05~0.5 v / v% And / or, in step (4), the crosslinking time is 10~120 min.

[0010] Preferably, in step (3), the flow rate of the aqueous phase is 150 μL / h and the flow rate of the oil phase is 1500 μL / h.

[0011] Preferably, in step (4), the volume fraction of the acetic acid is 0.1 v / v.

[0012] Preferably, the oil phase containing acetic acid is the oil phase prepared in step (2).

[0013] Further, the aqueous phase in step (1) may or may not contain genipin; when genipin is present, the mass fraction of genipin is 0.01~0.5wt%.

[0014] Preferably, the mass fraction of genipin is 0.1 wt%.

[0015] Furthermore, When the aqueous phase does not contain genie, in step (4), the crosslinking is performed in the oil phase containing acetic acid; preferably, the crosslinking time is 10~30 min. When the aqueous phase contains genipin, in step (4), the crosslinking is performed in the oil phase containing acetic acid; preferably, the crosslinking time is 30~120 min. When the aqueous phase contains genipin, in step (4), the crosslinking is first performed by standing in an oil phase that does not contain acetic acid, and then by crosslinking in an oil phase that contains acetic acid. Preferably, the standing time in the oil phase that does not contain acetic acid is 30-120 min; and the crosslinking time in the oil phase that contains acetic acid is 10-30 min.

[0016] Further, in step (4), after obtaining the tremella polysaccharide-based hydrogel microspheres, the excess oil phase is washed away with n-hexane; finally, the n-hexane is washed away with deionized water.

[0017] The present invention also provides a tremella polysaccharide-based hydrogel microsphere, which is prepared by the aforementioned method.

[0018] Preferably, the diameter of the hydrogel microspheres is 10~300 μm and the coefficient of variation (CV) is less than 5%.

[0019] The present invention also provides the use of the aforementioned Tremella fuciformis polysaccharide-based hydrogel microspheres in the fields of food engineering, drug delivery, cell labeling, bioimaging, or fluorescence sensing.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Uniform microsphere size: The droplet template generated by capillary glass tube microfluidics makes the size of tremella polysaccharide hydrogel microspheres controllable and has good monodispersity (CV < 3%).

[0021] (2) Structure and function can be designed: By adjusting the concentration and ratio of Tremella polysaccharide and cationic polymer (chitosan), introducing genipin or using ionic crosslinking, hydrogel microspheres with different crosslinking densities and mechanical properties can be obtained under mild conditions.

[0022] (3) Mild and bio-friendly process: The hydrogel microspheres prepared by this invention are suitable for encapsulating active substances (such as drugs, proteins, extracellular vesicles, etc.) under all-aqueous and mild acidic conditions without organic monomers or strong cross-linking agents.

[0023] (4) The device is simple and easy to expand: The device consists of common capillary glass tubes and injection pumps. It has a simple structure and low cost. It can be scaled up for production through multiple channels in parallel.

[0024] (5) Outstanding fluorescence properties of microspheres: The tremella polysaccharide-based hydrogel microspheres of the present invention have special and rich fluorescence luminescence properties, which can be used as fluorescent tracer carriers or imaging materials, eliminating the need for external dye loading steps, reducing toxicity and simplifying the preparation process.

[0025] (6) Edible materials: Both Tremella polysaccharide and chitosan are edible and biodegradable materials, and are expected to be used to improve the flavor of food, such as artificial caviar.

[0026] In summary, this invention provides a Tremella fuciformis polysaccharide-based hydrogel microsphere. This hydrogel microsphere can be fabricated using a simple apparatus, and the preparation process is mild and bio-friendly. The resulting hydrogel microspheres exhibit highly uniform particle size and good monodispersity. Furthermore, the Tremella fuciformis polysaccharide-based hydrogel microspheres prepared by this invention possess abundant autofluorescence properties and can be used as fluorescent tracer carriers or imaging materials for loading various active substances. This eliminates the need for exogenous dye loading steps, reduces toxicity, and simplifies the preparation process, demonstrating promising application prospects.

[0027] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0028] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0029] Figure 1 The diagram shows the structure of the capillary glass tube microfluidic device described in this invention, as well as the diagram showing the preparation of W / O type monoemulsion droplets and the cross-linking molding of Tremella fuciformis polysaccharide-based hydrogel microspheres.

[0030] Figure 2 Optical microscope images of the water-in-oil droplets prepared in Example 1 of this invention and the Tremella fuciformis polysaccharide-based hydrogel microspheres obtained after cross-linking and cleaning: a is an optical microscope image of the water-in-oil droplets; b is an optical microscope image of the Tremella fuciformis polysaccharide-based hydrogel microspheres.

[0031] Figure 3 The image shows a fluorescence microscope image of the Tremella polysaccharide-based hydrogel microspheres prepared in Example 1 of this invention. From left to right, the images show the bright field, blue channel (B), green channel (G), and red channel (R) microscopy images.

[0032] Figure 4 Images of the Tremella polysaccharide-based hydrogel microspheres prepared in Example 1 of this invention under bright field (BF) and laser confocal microscopy at excitation wavelengths of 405 nm, 488 nm, 561 nm, and 640 nm.

[0033] Figure 5 Optical microscope images of the water-in-oil droplets prepared in Example 2 of the present invention, and the Tremella fuciformis polysaccharide-based hydrogel microspheres obtained after crosslinking and washing: a is an optical microscope image of the water-in-oil droplets; b is an optical microscope image of the crosslinked Tremella fuciformis polysaccharide-based hydrogel microspheres; c is an optical microscope image of the washed Tremella fuciformis polysaccharide-based hydrogel microspheres.

[0034] Figure 6 The image shows a fluorescence microscope image of the Tremella polysaccharide-based hydrogel microspheres prepared in Example 2 of this invention. From left to right, the images show the bright field, blue channel (B), green channel (G), and red channel (R) microscopy images.

[0035] Figure 7 The following images are of the water-in-oil droplets, genipin-crosslinked Tremella fuciformis polysaccharide-based hydrogel microspheres, acetic acid-crosslinked Tremella fuciformis polysaccharide-based hydrogel microspheres, and washed Tremella fuciformis polysaccharide-based hydrogel microspheres prepared in Example 3 of this invention: a) Optical microscope image of the water-in-oil droplets; b) Optical microscope image of the genipin-crosslinked Tremella fuciformis polysaccharide-based hydrogel microspheres; c) Optical microscope image of the acetic acid-crosslinked Tremella fuciformis polysaccharide-based hydrogel microspheres; d) Optical microscope image of the washed Tremella fuciformis polysaccharide-based hydrogel microspheres.

[0036] Figure 8 The image shows a fluorescence microscope image of the Tremella polysaccharide-based hydrogel microspheres prepared in Example 3 of this invention. From left to right, the images show the bright field, blue channel (B), green channel (G), and red channel (R) microscopy images.

[0037] Figure 9 The following are scanning electron microscope (SEM) images of the Tremella fuciformis polysaccharide-based hydrogel microspheres prepared in Examples 1, 2, and 3 of this invention: a) is an SEM image of the Tremella fuciformis polysaccharide-based hydrogel microspheres of Example 1; b) is an SEM image of the Tremella fuciformis polysaccharide-based hydrogel microspheres of Example 2; and c) is an SEM image of the Tremella fuciformis polysaccharide-based hydrogel microspheres of Example 3.

[0038] Figure 10Optical microscope images of the water-in-oil droplets prepared in Example 5 of the present invention and the Tremella fuciformis polysaccharide-based hydrogel microspheres obtained after cross-linking and cleaning: a is an optical microscope image of the water-in-oil droplets; b is an optical microscope image of the Tremella fuciformis polysaccharide-based hydrogel microspheres.

[0039] Figure 11 The image shows a fluorescence microscope image of the Tremella polysaccharide-based hydrogel microspheres prepared in Example 5 of this invention. From left to right, the images show the bright field, blue channel (B), green channel (G), and red channel (R) microscopy images. Detailed Implementation

[0040] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0041] I. Device Structure Used in This Invention A capillary glass tube single-stage microfluidic device, comprising: Injection capillary (inner tube): A glass capillary is used, the inlet end of which is connected to the liquid supply unit of the Tremella polysaccharide aqueous solution for introducing homogeneous solution (aqueous phase); the inner diameter of the capillary is preferably 20~150 μm.

[0042] The outer square tube and outlet capillary are made of glass. The inner cavity of the capillary envelops the outlet of the injection capillary to form a coaxial confluence structure. An oil phase containing a surfactant is introduced into the outer side to shear and disperse the aqueous phase flow to form water-in-oil (W / O) droplets. The inner diameter of the outer square tube is 1.0 mm; the inner diameter of the outlet capillary is preferably 100~500 μm.

[0043] Liquid supply unit: includes an aqueous phase injection pump and an oil phase injection pump, with an aqueous phase flow rate range of 10~1500 μL / h and an oil phase flow rate range of 200~10000 μL / h, used to precisely control the two-phase flow ratio.

[0044] Receiving unit: includes an oil phase container for collecting microdroplets, can be pre-filled with oil phase receiving liquid of different components (such as oil phase containing acetic acid or oil phase without acetic acid), and can be stirred or allowed to stand to achieve subsequent cross-linking and curing.

[0045] II. Preparation Method The method for preparing Tremella fuciformis polysaccharide-based hydrogel microspheres using the above-mentioned apparatus includes the following steps: 1. Preparation of homogeneous solution (aqueous phase) of Tremella fuciformis polysaccharide: (1) Dissolve a certain amount of Tremella polysaccharide in deionized water, preferably with a mass fraction of 0.05~1.5 wt%, to form a transparent solution; (2) Dissolve a certain amount of cationic polymer in deionized water, preferably with a mass fraction of 0.1 to 4.0 wt%, to form a transparent solution; the cationic polymer is preferably chitosan.

[0046] (3) Mix the Tremella polysaccharide solution and the cationic polymer solution in an appropriate volume ratio, and adjust the pH with an appropriate amount of dilute alkali (such as sodium hydroxide) to ensure complete dissolution. Then adjust the pH to a suitable range (e.g., 5.7~6.4) to obtain a stable and homogeneous Tremella polysaccharide-based aqueous solution. (4) If necessary, genipin (0.01~0.5 wt%) can be added to the aqueous phase as a covalent crosslinking agent, or it can be reserved for later addition.

[0047] 2. Preparation of the oil phase Soybean oil, mineral oil, liquid paraffin, silicone oil or fluorocarbon oil are selected as the continuous phase, and an appropriate amount of low HLB value surfactant (such as PGPR, Span80) or fluorocarbon oil-specific surfactant is added, preferably with a mass fraction of 0.5~10.0 wt%, and the mixture is thoroughly mixed to obtain a stable oil phase solution.

[0048] 3. Preparation of receiving liquid The receiving liquid is prepared by adding an appropriate amount of acetic acid to the continuous phase, or without adding acetic acid, as needed. (1) Ionic crosslinking scheme: A small amount of acetic acid is added to the oil phase, and the acetic acid diffuses from the oil phase inward to make the tremella polysaccharide and the cationic polymer undergo ionic crosslinking; (2) Genipin crosslinking scheme: The receiving liquid is a pure oil phase without acetic acid or the same oil phase as above. Genipin molecules can be covalently crosslinked with cationic polymers and Tremella polysaccharides respectively.

[0049] 4. Droplet formation and collection (1) Start the oil phase injection pump and set the flow rate Q. O (For example, 200~10000 μL / h) Introduce the oil phase into the outer square tube; (2) Start the aqueous phase injection pump and introduce the aqueous phase into the injection capillary at a set flow rate QW (e.g., 10~1500 μL / h); (3) Under the shearing action of the capillary outlet and the oil phase, the aqueous solution is dispersed into a water-in-oil emulsion with controllable size and uniform particle size. (4) The continuously flowing water-in-oil emulsion is collected by the receiving unit, which can be a petri dish, beaker or reaction flask containing a preset oil phase.

[0050] 5. Microsphere crosslinking and curing Option 1: Acetic acid diffusion-induced ionic crosslinking A certain amount of acetic acid is added to the receiving oil phase beforehand, and the mass fraction or volume fraction is set as needed (e.g., 0.1~0.5 v / v%). Acetic acid molecules diffuse in the oil phase and gradually enter the interior of the water-in-oil droplet, where they undergo protonation and electrostatic interactions with the amino groups on the cationic polymer (such as chitosan) and the carboxyl groups or other charged groups of Tremella fuciformis polysaccharide, inducing the formation of an ionic cross-linking network between the cationic polysaccharide and Tremella fuciformis polysaccharide. After an appropriate period of time (e.g., 10 min to 30 min), the aqueous phase inside the droplet transforms from a sol into a stable hydrogel, forming tremella polysaccharide-based hydrogel microspheres.

[0051] The prepared Tremella polysaccharide-based hydrogel microspheres have unique fluorescence luminescence properties and can be used for fluorescence microscopy imaging and tracing.

[0052] Option 2: Genipin crosslinking In step 1, genipin was dissolved in the aqueous phase of tremella polysaccharide; Under suitable temperature (e.g., 20~37 ℃) and pH conditions, after an appropriate time (e.g., 30 min~120 min), genipin undergoes a covalent cross-linking reaction with the amino groups on the cationic polymer and some hydroxyl / amino sites in the tremella polysaccharide, gradually constructing a three-dimensional network structure inside the droplet. During the cross-linking process, genipin reacts with amino groups to produce a characteristic blue-green fluorescence, which further alters the fluorescence luminescence properties of the tremella polysaccharide-based hydrogel microspheres.

[0053] 6. Microsphere separation and washing After crosslinking, most of the oil phase is removed by centrifugation or static separation. After repeated washing with hexane and water, the residual oil phase and surfactant are removed, and finally, tremella polysaccharide-based hydrogel microspheres dispersed in the aqueous phase are obtained.

[0054] 7. Particle size and morphology control The size and distribution of microspheres can be controlled through the following methods: Adjusting the aqueous phase flow rate Q W Oil phase flow rate Q O ; Replace the injection capillary and outlet capillary with different inner diameters; Adjusting the viscosity of the aqueous phase (by changing the concentration of Tremella polysaccharide and cationic polymer); Under optimized conditions, monodisperse Tremella fuciformis polysaccharide hydrogel microspheres with diameters ranging from 10 to 300 μm and a particle size variation coefficient (CV) of less than 5% can be prepared.

[0055] The optimized preparation method of the present invention will be illustrated below through specific embodiments.

[0056] Example 1: Preparation of Tremella polysaccharide-based hydrogel microspheres using acetic acid-induced ionic crosslinking 1. Preparation of the aqueous phase Dissolve 0.02 g of Tremella fuciformis polysaccharide in 2 ml of deionized water, stir and heat to 40 °C until completely dissolved, to prepare a 1.0 wt% Tremella fuciformis polysaccharide solution; separately, dissolve 0.02 g of chitosan in 1 ml of deionized water to prepare a 2.0 wt% chitosan solution. Mix the two solutions at a volume ratio of 3:1 (Tremella fuciformis polysaccharide solution to chitosan solution), and adjust the pH to approximately 5.9 using a 1.0 wt% sodium hydroxide aqueous solution to obtain a homogeneous Tremella fuciformis polysaccharide-chitosan solution (aqueous phase).

[0057] 2. Preparation of the oil phase Polyglycerol polyricinoleate (PGPR) was added to edible soybean oil to a concentration of 5.0 wt%, and the mixture was stirred until homogeneous to obtain the oil phase.

[0058] 3. Preparation of receiving liquid Add 0.1% acetic acid by volume to the oil phase obtained in step 2, and mix well to obtain the receiving liquid.

[0059] 4. Preparation of hydrogel microspheres using microfluidic methods Hydrogel microspheres were prepared using a microfluidic method, with the aqueous phase flow rate Q set. w =150 μL / h, oil phase flow rate Q o =1500 μL / h. For example... Figure 1 As shown, a capillary microfluidic device can be used to form stable monodisperse water-in-oil droplets with a diameter of approximately 119.1 μm (CV value of 0.9%). Figure 2 (As shown in a). The oil phase containing water-in-oil droplets was introduced into a receiving liquid containing acetic acid. After standing at 25°C for 10 min, the acetic acid diffused into the aqueous phase, thus obtaining cross-linked and solidified Tremella fuciformis polysaccharide-based hydrogel microspheres within the droplets, with an average diameter of 71.0 μm (CV value of 4.7%). After washing with hexane and deionized water, the Tremella fuciformis polysaccharide-based hydrogel microspheres dispersed in deionized water had a particle size of 46.0 μm and a CV ≈ 2.5% (e.g., as shown in a). Figure 2 (as shown in b).

[0060] 5. Autofluorescence properties of hydrogel microspheres After washing and degreasing, fluorescent Tremella fuciformis polysaccharide-based hydrogel microspheres were obtained dispersed in water. Figure 3 As can be seen, monodisperse *Tremella fuciformis* polysaccharide-based hydrogel microspheres prepared using the method of this invention can be observed under a fluorescence microscope. These hydrogel microspheres exhibit significant autofluorescence. Multiple fluorescence patterns were observed under the fluorescence microscope, with an average fluorescence intensity of 25.8 in the blue channel (B), 12.22 in the green channel (G), and 12.63 in the red channel (R). Furthermore, the *Tremella fuciformis* polysaccharide-based hydrogel microspheres were observed using a laser confocal microscope, and the results are as follows... Figure 4 As shown, fluorescence can be observed in the hydrogel microspheres under different excitation wavelengths. These results demonstrate that the *Tremella fuciformis* polysaccharide-based hydrogel microspheres prepared in this invention possess abundant autofluorescence properties.

[0061] Example 2: Preparation of Tremella polysaccharide-based hydrogel microspheres using genipin crosslinking and acetic acid-induced ionic crosslinking 1. Preparation of the aqueous phase Based on the aqueous phase prepared in Example 1, genipin with a concentration of 0.1 wt% was added, and the mixture was stirred and dissolved in the dark to obtain the aqueous phase.

[0062] 2. Preparation of the oil phase The preparation method of the oil phase is the same as that of the oil phase in Example 1.

[0063] 3. Preparation of receiving liquid The preparation method of the receiving liquid is the same as that of the receiving liquid in Example 1.

[0064] 4. Preparation of hydrogel microspheres using microfluidic methods Hydrogel microspheres were prepared using a microfluidic method, with the aqueous phase flow rate Q set. w =150 μL / h, oil phase flow rate Q o =1500 μL / h. Stable, monodisperse water-in-oil droplets can be formed using a capillary microfluidic device, with a droplet diameter of approximately 125.0 μm (CV value 0.8%). Figure 5 (As shown in a). The oil phase containing water-in-oil droplets was introduced into a receiving solution containing acetic acid and allowed to stand at 25°C for 60 min. Besides acetic acid-induced ionic crosslinking, genipin underwent covalent crosslinking with Tremella fuciformis polysaccharide and chitosan, thus obtaining Tremella fuciformis polysaccharide-based hydrogel microspheres that were crosslinked and solidified within the droplets, with an average diameter of 74.2 μm (CV value of 4.5%). Figure 5 (As shown in b). After washing with hexane and deionized water, the particle size of the Tremella fuciformis polysaccharide-based hydrogel microspheres dispersed in deionized water was 56.1 μm, and the CV was approximately 3.7% (as shown in b). Figure 5 (as shown in c).

[0065] 5. Autofluorescence properties of hydrogel microspheres After washing and degreasing, fluorescent Tremella fuciformis polysaccharide-based hydrogel microspheres were obtained dispersed in water. Figure 6 As shown, the hydrogel microspheres exhibited uniform size and uniform fluorescence distribution under a fluorescence microscope. The average fluorescence intensity of the blue channel (B) was 21.7; the average fluorescence intensity of the green channel (G) was 9.58; and the average fluorescence intensity of the red channel (R) was 38.42. These hydrogel microspheres demonstrated rich autofluorescence properties and exhibited a variety of fluorescence characteristics under a fluorescence microscope.

[0066] Example 3: Preparation of Tremella polysaccharide-based hydrogel microspheres by controlling the order of genipin crosslinking and acetic acid-induced ionic crosslinking. 1. Preparation of the aqueous phase The preparation method of the aqueous phase is the same as in Example 2.

[0067] 2. Preparation of the oil phase The preparation method of the oil phase is the same as in Example 2.

[0068] 3. Receive the oil phase The receiving oil phase is prepared in the same way as the oil phase, by adding polyglycerol polyricinoleate (PGPR) to edible soybean oil to a concentration of 5.0 wt%, and stirring until homogeneous. This receiving oil phase does not contain acetic acid.

[0069] 4. Preparation of receiving liquid The preparation method of the receiving liquid is the same as in Example 2.

[0070] 5. Preparation of hydrogel microspheres using microfluidic methods Hydrogel microspheres were prepared using a microfluidic method, with the aqueous phase flow rate Q set. w =150 μL / h, oil phase flow rate Q o =1500 μL / h. Stable, monodisperse water-in-oil droplets can be formed using a capillary microfluidic device, with a droplet diameter of approximately 123.2 μm (CV value 0.8%). Figure 7 (As shown in a). The oil phase containing water-in-oil droplets was introduced into a receiving oil phase without acetic acid. After standing at 25°C for 60 min, genipin covalently crosslinked with Tremella fuciformis polysaccharide and chitosan, thus obtaining Tremella fuciformis polysaccharide-based hydrogel microspheres that were crosslinked and solidified within the droplets. At this time, the morphology of the water-in-oil droplets did not change significantly (e.g., as shown in a). Figure 7 (As shown in b); when the receiving oil phase was replaced with a receiving liquid containing acetic acid and allowed to stand for 10 min, the morphology of the water-in-oil droplets changed, and the formation of hydrogel microspheres in the droplets could be clearly observed (e.g., Figure 7 (As shown in c). Under these conditions, the average diameter of the hydrogel microspheres was approximately 75.7 μm (CV value 4.3%); after washing with hexane and deionized water, the particle size of the Tremella fuciformis polysaccharide / chitosan hydrogel microspheres dispersed in deionized water was 52.3 μm, CV≈3.8% (as shown in c). Figure 7 (as shown in d).

[0071] 6. Autofluorescence properties of hydrogel microspheres After washing and degreasing, fluorescent Tremella fuciformis polysaccharide-based hydrogel microspheres were obtained dispersed in water. Figure 8As shown, the hydrogel microspheres exhibited uniform size and uniform fluorescence distribution under a fluorescence microscope. The average fluorescence intensity of the blue channel (B) was 26.7; the average fluorescence intensity of the green channel (G) was 13.66; and the average fluorescence intensity of the red channel (R) was 58.66. These hydrogel microspheres demonstrated rich autofluorescence properties and exhibited a variety of fluorescence characteristics under the fluorescence microscope.

[0072] Example 4: Scanning electron microscopy observation The *Tremella fuciformis* polysaccharide-based hydrogel microspheres prepared in Examples 1, 2, and 3 were freeze-dried and then observed using scanning electron microscopy. Figure 9 As shown, the structures and morphologies of the Tremella polysaccharide hydrogel microspheres prepared under three different conditions are completely different. Among them, Figure 9 a represents the Tremella polysaccharide hydrogel microspheres prepared in Example 1. Figure 9 b represents the Tremella polysaccharide hydrogel microspheres prepared in Example 2. Figure 9 c represents the Tremella fuciformis polysaccharide hydrogel microspheres prepared in Example 3. The morphological structure diagram obtained by scanning electron microscopy illustrates that the gel network structure of the microspheres prepared in this invention can be adjusted, thereby obtaining hydrogel microspheres with different mechanical properties and abundant autofluorescence characteristics.

[0073] Example 5: Preparation of Tremella polysaccharide-based hydrogel microspheres using acetic acid-induced ionic crosslinking 1. Preparation of the aqueous phase Dissolve 0.02 g of Tremella fuciformis polysaccharide in 2 ml of deionized water, stir and heat to 40 °C until completely dissolved, to prepare a 1.0 wt% Tremella fuciformis polysaccharide solution; separately, dissolve 0.04 g of chitosan in 1 ml of deionized water to prepare a 4.0 wt% chitosan solution. Mix the two solutions at a volume ratio of 3:1 (Tremella fuciformis polysaccharide solution to chitosan solution), and adjust the pH to approximately 5.9 using a 1.0 wt% sodium hydroxide aqueous solution to obtain a homogeneous Tremella fuciformis polysaccharide-chitosan solution (aqueous phase).

[0074] 2. Preparation of the oil phase Polyglycerol polyricinoleate (PGPR) was added to edible soybean oil to a concentration of 5.0 wt%, and the mixture was stirred until homogeneous to obtain the oil phase.

[0075] 3. Preparation of receiving liquid Add 0.1% acetic acid by volume to the oil phase obtained in step 2, and mix well to obtain the receiving liquid.

[0076] 4. Preparation of hydrogel microspheres using microfluidic methods Hydrogel microspheres were prepared using a microfluidic method, with the aqueous phase flow rate Q set. w =150 μL / h, oil phase flow rate Qo =1500 μL / h. For example... Figure 10 As shown, a capillary microfluidic device can be used to form stable monodisperse water-in-oil droplets with a diameter of approximately 109.5 μm (CV value of 0.6%). Figure 10 (As shown in a). The oil phase containing water-in-oil droplets was introduced into a receiving liquid containing acetic acid. After standing at 25°C for 10 min, the acetic acid diffused into the aqueous phase, thus obtaining cross-linked and solidified Tremella fuciformis polysaccharide-based hydrogel microspheres within the droplets, with an average diameter of 66.1 μm (CV value of 4.2%). After washing with hexane and deionized water, the Tremella fuciformis polysaccharide-based hydrogel microspheres dispersed in deionized water had a particle size of 41.7 μm and a CV ≈ 3.5% (e.g., as shown in a). Figure 10 (as shown in b).

[0077] 5. Fluorescence and luminescence properties of hydrogel microspheres After washing and degreasing, fluorescent Tremella fuciformis polysaccharide-based hydrogel microspheres were obtained dispersed in water. Figure 11 As can be seen from the fluorescence microscope, monodisperse Tremella fuciformis polysaccharide-based hydrogel microspheres prepared using the method of this invention exhibit obvious autofluorescence characteristics. Multiple fluorescence patterns were observed under the fluorescence microscope, with the average fluorescence intensity of the blue channel (B) being 33.04; the average fluorescence intensity of the green channel (G) being 27.16; and the average fluorescence intensity of the red channel (R) being 22.42.

[0078] As demonstrated in the above examples, using Tremella fuciformis polysaccharide and cationic polymers (especially chitosan as the cationic polymer) as the aqueous phase components, and crosslinking with acetic acid and / or genipin, Tremella fuciformis polysaccharide-based hydrogel microspheres with abundant autofluorescence can be successfully prepared. Previously, the inventors used sodium alginate and chitosan as the aqueous phase components, crosslinking with acetic acid. While this yielded hydrogel microspheres, they only exhibited significant autofluorescence at excitation wavelengths of 405 nm and 488 nm. This application selects Tremella fuciformis polysaccharide as the matrix, and prepares a mixed aqueous solution with chitosan as the cationic polymer. This allows for the preparation of hydrogel microspheres with even richer autofluorescence properties, endowing them with new functions and expanding their application range. These hydrogel microspheres can be directly used as long-term cell / drug delivery markers, microfluidic-encoded microspheres, or in vivo / in vitro imaging probes, eliminating the need for exogenous dye loading, reducing toxicity, and simplifying the preparation process, demonstrating promising application prospects.

[0079] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing Tremella fuciformis polysaccharide-based hydrogel microspheres, characterized in that, Includes the following steps: (1) Prepare an aqueous phase containing Tremella polysaccharide and cationic polymer; (2) Prepare an oil phase containing surfactant; (3) Water-in-oil droplets were prepared by a single-stage microfluidic device using a capillary glass tube. (4) The oil phase containing water-in-oil droplets is introduced into the receiving system for cross-linking to obtain tremella polysaccharide-based hydrogel microspheres; the receiving system is an oil phase containing acetic acid.

2. The method according to claim 1, characterized in that: In step (1), the method for preparing the aqueous phase includes the following steps: (a) Prepare an aqueous solution of Tremella polysaccharide with a mass fraction of 0.05~1.5wt%; (b) Prepare an aqueous solution of cationic polymer with a mass fraction of 0.1 to 4.0 wt%; (c) Mix the aqueous solution of Tremella polysaccharide and the aqueous solution of cationic polymer at a volume ratio of (1~5):1, and adjust the pH to 5.7~6.4 to obtain the aqueous phase; And / or, in step (2), the method for preparing the oil phase includes the following steps: The surfactant is added to the continuous phase and mixed evenly to obtain an oil phase with a surfactant mass fraction of 0.5~10.0 wt%; the continuous phase is selected from soybean oil, mineral oil, liquid paraffin, silicone oil or fluorinated oil; the surfactant is selected from polyglycerol polyricinoleate, Span80 or fluorinated oil-specific surfactant.

3. The method according to claim 2, characterized in that: In step (a), the mass fraction of the Tremella polysaccharide aqueous solution is 1.0 wt%. And / or, in step (b), the mass fraction of the cationic polymer aqueous solution is 2.0~4.0 wt%; And / or, in step (c), the volume ratio of the Tremella polysaccharide aqueous solution to the cationic polymer aqueous solution is 3:1; And / or, in step (c), the pH is adjusted to 5.9; And / or, the mass fraction of the surfactant in the oil phase is 5.0 wt%; And / or, the continuous phase is selected from soybean oil; And / or, the surfactant is selected from polyglycerol polyricinoleate.

4. The method according to claim 3, characterized in that: In step (b), the cationic polymer is chitosan; And / or, in step (c), the pH adjustment is performed using a 1.0 wt% aqueous solution of sodium hydroxide.

5. The method according to claim 1, characterized in that: In step (3), when preparing water-in-oil droplets, the flow rate of the aqueous phase is 10~1500 μL / h; the flow rate of the oil phase is 200~10000 μL / h. And / or, in step (4), the volume fraction of acetic acid in the oil phase containing acetic acid is 0.05~0.5 v / v% And / or, in step (4), the crosslinking time is 10~120 min.

6. The method according to claim 1, characterized in that: The aqueous phase in step (1) may or may not contain genipin; when genipin is present, the mass fraction of genipin is 0.01~0.5wt%.

7. The method according to claim 6, characterized in that: When the aqueous phase does not contain genie, in step (4), the crosslinking is performed in the oil phase containing acetic acid; When the aqueous phase contains genipin, in step (4), the crosslinking is performed in the oil phase containing acetic acid; When the aqueous phase contains genie, in step (4), the crosslinking is first allowed to stand in an oil phase that does not contain acetic acid; then crosslinking is performed in an oil phase that contains acetic acid.

8. The method according to claim 1, characterized in that: In step (4), after obtaining the tremella polysaccharide-based hydrogel microspheres, the excess oil phase is washed away with n-hexane; finally, the n-hexane is washed away with deionized water.

9. A type of Tremella fuciformis polysaccharide-based hydrogel microsphere, characterized in that: It is prepared by the method described in any one of claims 1 to 8.

10. Use of the Tremella fuciformis polysaccharide-based hydrogel microspheres according to claim 9 in the fields of food engineering, drug delivery, cell labeling, bioimaging or fluorescence sensing.

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