Preparation method and application of porous core-shell hydrogel

Abstract

The invention discloses a preparation method and application of porous core-shell hydrogel, and belongs to the technical field of hydrogel microcarrier preparation and biomedical application. The invention provides a method for preparing porous core-shell hydrogel, which utilizes a coaxial microfluidic technology, accurately controls the size and shape of microspheres by combining a microfluidic chip with a gas shearing technology, and utilizes a porous shell formed by polyvinyl alcohol (PVA), sodium alginate (Alg) and polyethylene glycol (PEG) to prepare the porous core-shell hydrogel. A plurality of soft core materials (such as sodium carboxymethyl cellulose, collagen or matrigel) are wrapped, and core-shell hydrogel microspheres which are controllable in particle size, high in mechanical strength and porous in shell are rapidly prepared in one step in a full-water-phase system. The prepared core-shell microspheres are uniform in particle size and stable in structure, cell embedding protection and immune cell permeation can be achieved, and high-throughput and large-scale preparation can be achieved by adjusting parameters such as the flow rate.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel microcarrier preparation and biomedical application technology, and relates to a method for preparing porous core-shell hydrogels using microfluidic chips and gas shearing technology, as well as the resulting core-shell hydrogel microspheres. Specifically, it relates to a method for preparing porous core-shell hydrogels and their applications. Background Technology

[0002] Three-dimensional (3D) cell culture technology plays a central role in biomedical fields such as tissue engineering, drug screening, and immunotherapy evaluation. Successful 3D culture requires the construction of a highly biomimetic microenvironment that not only provides mechanical support but also ensures efficient nutrient exchange and intercellular signal transduction. However, traditional culture methods, such as the preparation of large hydrogel tissue blocks through patterning or spinning, generally suffer from limitations such as uncontrollable size, poor structural uniformity, and low capacity for internal nutrient and metabolic waste exchange.

[0003] To achieve precise control over the size and morphology of microcarriers, microfluidic technology utilizes the shearing action between immiscible fluids to prepare hydrogel microspheres with uniform particle size and good monodispersity. However, existing microfluidic technologies tend to produce solid microspheres, resulting in a homogeneous and uncontrollable growth environment for cells inside the microspheres, making it difficult to achieve regional culture of multiple cell types or substances.

[0004] To overcome this limitation, core-shell structures have been introduced into microcarrier design. The unique advantage of core-shell structures lies in enabling the partitioning and optimization of material functions: a robust outer shell (such as polyvinyl alcohol / sodium alginate) provides the necessary mechanical stability and protection for the entire microsphere, preventing premature degradation in in vitro or in vivo environments; while a soft core (such as collagen, matrix gel, or sodium carboxymethyl cellulose) can encapsulate various soft materials and active substances (such as cells and drugs) that are difficult to shape independently, thus providing a biomimetic microenvironment similar to the natural extracellular matrix (ECM). This design allows for the selection of different core materials to provide a more optimized and customized environment for three-dimensional cell culture.

[0005] Furthermore, the material transport efficiency of microspheres is crucial for ensuring long-term cell culture. Studies have shown that introducing macroporous structures or interconnected channels into hydrogel scaffolds can significantly improve scaffold permeability, accelerate nutrient transport, and facilitate the removal of metabolic waste. In addition, porosity allows larger substances, such as immune cells, to infiltrate and penetrate the outer shell into the inner core, which is particularly critical for constructing complex cell co-culture models and evaluating immunotherapies.

[0006] However, existing technologies face many challenges in fabricating microspheres that simultaneously possess a "core-shell structure" and "connected pores." For example, the fabrication of existing core-shell microspheres often employs cumbersome stepwise loading or surface coating methods. Furthermore, traditional microfluidic chip methods suffer from low flow rates, making it difficult to achieve high-throughput, large-scale fabrication to meet the needs of industrial or clinical applications.

[0007] Therefore, there is an urgent need to develop a method that can prepare novel hydrogel microspheres with porous shells, robust and stable structures, and customizable cores in one step using high-throughput microfluidic technology. Summary of the Invention

[0008] Therefore, the purpose of this invention is to provide a method for preparing porous core-shell hydrogels and its application. By using coaxial microfluidic technology to precisely control the size and morphology of microspheres, the prepared core-shell microspheres have uniform particle size and stable structure. Furthermore, high-throughput and large-scale preparation can be achieved by adjusting parameters such as flow rate.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing porous core-shell hydrogels, comprising the following steps: S1, Preparation of outer shell gel solution, core gel solution and microfluidic chip; The microfluidic chip includes an inlet I, an inlet II, a vent, and a core channel, a shell channel, and a gas channel arranged coaxially. The inlet I is connected to the inlet of the core channel, the inlet II is connected to the inlet of the shell channel, and the vent is connected to the inlet of the gas channel. The outlet of the core channel is located inside the shell channel, and the outlet of the shell channel is located inside the gas channel. S2, the outer shell gel solution is injected into the outer shell channel at a fixed flow rate to form an outer shell fluid; the inner shell gel solution is injected into the inner shell channel at a fixed flow rate to form an inner shell fluid; S3, inject gas into the gas channel at a fixed flow rate to shear the outer shell fluid and the core fluid, so that the core fluid is wrapped by the outer shell fluid and forms a core-shell droplet under the shearing action of the gas. S4, the core-shell droplets are collected in a curing solution containing a crosslinking agent, so that the shell forms a strong hydrogel layer; S5. Wash the solidified core-shell hydrogel with physiological saline or PBS buffer to obtain a porous core-shell hydrogel.

[0010] Among them, porous core-shell hydrogels mainly exist in the form of microspheres.

[0011] The shell gel solution is prepared by adding 0.5%~5% (w / v) polyvinyl alcohol, 0.5%~5% (w / v) sodium alginate and 1%~15% (w / v) polyethylene glycol to an aqueous solution and stirring until homogeneous; the polyethylene glycol has a molecular weight range of 2000~6000 Da.

[0012] Polyvinyl alcohol (PVA), sodium alginate (Alg), and polyethylene glycol (PEG) are used as pore-forming agents. After subsequent curing, they are eluted to form interconnected pores, thereby improving the permeability of the outer shell.

[0013] During cell culture, the solution outside the porous core-shell hydrogel is completely replaced with cell culture medium, and the cells are cultured as required.

[0014] Based on the above technical solution, the core gel solution is further prepared by adding a hydrogel material or matrix material for cell culture to the culture medium and mixing them. The hydrogel material is selected from one or more of sodium carboxymethyl cellulose, collagen, matrix gel, gelatin, hyaluronic acid, alginate, or their chemically modified derivatives. The sodium carboxymethyl cellulose has a mass-to-volume ratio of 0.2% to 1.0% (w / v), the collagen has a volume ratio of 10% to 90% (v / v), the matrix gel has a volume ratio of 10% to 90% (v / v), the gelatin has a mass-to-volume ratio of 2% to 10% (w / v), the hyaluronic acid has a mass-to-volume ratio of 0.1% to 1.0% (w / v), and the alginate has a mass-to-volume ratio of 0.2% to 3% (w / v).

[0015] In this process, when performing three-dimensional cell culture, the cells are uniformly suspended in an inner gel solution.

[0016] The matrix gel is Matrigel, and the other chemically modified derivatives are GelMA or HAMA.

[0017] Among them, the core gel solution containing suitable hydrogel materials or matrix materials for cell culture can encapsulate cells or active substances. Based on the above technical solution, the core gel solution further includes active substances, which are selected from one or more of animal or human cells, extracellular matrix proteins, growth factors, small molecule drugs, and immune cytokines.

[0018] Based on the above technical solution, further, the flow rate of the core gel solution in the core channel is 400~900 μL / min, the flow rate of the outer shell gel solution in the outer shell channel is 1.5~5 times that of the flow rate of the core gel solution in the core channel, and the flow rate of the gas in the gas channel is 1.5~3 L / min.

[0019] The gas flows at a rate of 1.5 to 3 L / min in the gas channel to provide the strong shear force required to shear high-viscosity fluids to form stable droplets, making it suitable for high-viscosity fluids and enabling high-throughput preparation.

[0020] Based on the above technical solution, further, the core channel, the outer shell channel, and the gas channel are all constructed of rigid materials, which are selected from one or more of stainless steel, capillary glass, ceramics, or polyetheretherketone; the inner diameter of the core channel is 50~800 μm, the inner diameter of the outer shell channel is 200~800 μm larger than the outer diameter of the core channel, and the inner diameter of the gas channel is 500~1500 μm larger than the outer diameter of the outer shell channel; the distance between the outlet end of the core channel and the outlet end of the outer shell channel is 0.2~2 mm.

[0021] Polyetheretherketone (PEEK) is abbreviated as PEEK.

[0022] Based on the above technical solution, the crosslinking agent is further described as a mixed solution containing 0.5%~5% (w / v) calcium chloride and 5mM~20mM sodium borate.

[0023] In this process, Alg and PVA in the outer shell gel solution are cured by a cross-linking agent, which cross-links Alg with calcium ions in the outer shell, and PVA forms a network through sodium borate, achieving double curing and forming a robust outer shell, thus forming a robust hydrogel layer.

[0024] The solidified core-shell hydrogel is washed with physiological saline or PBS buffer to remove polyethylene glycol and uncrosslinked components, thereby forming interconnected pores in the outer shell and obtaining a porous core-shell hydrogel.

[0025] Among them, rigid materials support medium-throughput and high-throughput fabrication.

[0026] Where mM refers to mmol / L.

[0027] Based on the above technical solution, the microfluidic chip is further constructed using soft lithography, glass or silicon etching, precision machining, or 3D printing.

[0028] Among them, soft lithography uses polydimethylsiloxane (PDMS); precision machining uses polymethyl methacrylate (PMMA) or polycarbonate.

[0029] Based on the above technical solution, the gas is further selected from one or more of air, nitrogen, and carbon dioxide.

[0030] Secondly, the present invention provides the application of a porous core-shell hydrogel prepared by the above method in three-dimensional cell culture, immunotherapy evaluation, or tissue engineering.

[0031] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a method for preparing porous core-shell hydrogels. Utilizing coaxial microfluidic technology, the size and morphology of microspheres are precisely controlled through a microfluidic chip combined with gas shearing technology. A porous shell formed from polyvinyl alcohol and sodium alginate (polyethylene glycol) encapsulates various soft core materials (such as sodium carboxymethyl cellulose, collagen, or matrix gel). This allows for the rapid one-step preparation of core-shell hydrogel microspheres with controllable particle size, high mechanical strength, and a porous outer shell in an all-aqueous system. The prepared core-shell microspheres exhibit uniform particle size and stable structure, enabling cell embedding and protection as well as immune cell penetration. Furthermore, high-throughput, large-scale preparation can be achieved by adjusting parameters such as flow rate.

[0032] 2. This invention introduces PEG as a pore-forming agent. After the outer shell is cross-linked and molded, it is eluted to form a through-pore structure on the outer shell of the microsphere. This significantly improves the permeation and transport efficiency of nutrients and the discharge efficiency of metabolic waste inside the microsphere, and allows larger cells such as immune cells to freely enter the core of the microsphere.

[0033] 3. The outer shell of this invention is made of a robust and stable hydrogel layer formed by double cross-linking of PVA and Alg, which provides the necessary mechanical strength and protection for the microspheres, preventing premature degradation during in vitro culture or in vivo transplantation. The soft inner core is composed of natural matrix materials, which can simulate the extracellular matrix environment, facilitating the encapsulation of various soft materials and active substances (such as living cells and drugs) that are difficult to shape independently. This provides a good three-dimensional scaffold and microenvironment for cell growth, and is beneficial for constructing complex cell co-culture systems and simulating in vivo tissue structures. 4. The method of this invention combines core-shell molding with porous outer shell construction, enabling simultaneous molding of core-shell microspheres and construction of a porous outer shell through a single microfluidic process. This eliminates the need for cumbersome step-by-step loading or subsequent surface treatment processes, resulting in a simple and efficient preparation process that is easy to scale up and industrialize. Therefore, the porous core-shell hydrogel microspheres prepared by this invention have broad application prospects in biomedical fields such as three-dimensional cell culture, immunotherapy model construction, immunotherapy evaluation, and tissue engineering. Attached Figure Description

[0034] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0035] Figure 1 The diagram below shows the structure of the porous core-shell microsphere construction device of the present invention: A is a partial enlarged view of the outlet end of the core channel, the outer shell channel and the gas channel; Figure 2 Bright-field diagram of porous core-shell microspheres prepared with different core gel solutions in Example 2 of this invention; Figure 3Statistical diagram of the size distribution of porous core-shell microspheres prepared with different core gel solutions in Example 2 of the present invention; Figure 4 This is a confocal fluorescence image used to verify the pore permeability of the microspheres in Example 3 of the present invention; Figure 5 This is a confocal fluorescence image of the microspheres used for cell encapsulation and NK cell infiltration in Example 4 of the present invention; In the figure: 1. Foam box; 2. Metal block embedded in the foam box; 3. Chip clip; 4. Microfluidic chip; 5. Inlet I; 6. Inlet II; 7. Vent; 801. Gas channel; 802. Outer shell channel; 803. Core channel; 9. Droplet. Detailed Implementation

[0036] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0037] Example 1 The fabrication process of the microfluidic chip: A PDMS chip containing inlet I, inlet II, a vent, and various channels is fabricated by molding. A glass slide (76×26mm) and the PDMS chip are cleaned using oxygen plasma, and the glass slide is bonded to the surface of the PDMS chip. A stainless steel capillary with an inner diameter of 0.3mm is inserted into inlet I of the bonded chip, a stainless steel capillary with an inner diameter of 0.9mm is inserted into inlet II and fitted over it, and a stainless steel capillary with an inner diameter of 2mm is inserted into the vent and fitted over it. All three are kept coaxial and their positions are fixed using AB glue.

[0038] Example 2 Fabrication process of porous core-shell microspheres: vertically fixing the chip Figure 1 The chips were pre-cooled in a foam box with crushed ice. A mixed gel solution of 1.5% Alg + 3% PVA + 12.5% ​​PEG3500 was injected into injection port II at a rate of 1500 μL / min; 0.3% CMC solution, 40% collagen 1 (CO1) solution, and 30% Matrigel solution were injected into the core channel at a rate of 600 μL / min; nitrogen gas was introduced into the vent at a rate of 2 L / min; droplets were formed by gas shearing, and the droplets were collected and solidified in a cell culture dish containing 2% calcium chloride and 10 mM sodium borate aqueous solution. The solidified microspheres were collected and washed three times with PBS buffer to obtain porous core-shell microspheres. The obtained microspheres were photographed and their dimensions were counted using a phase contrast microscope to obtain the results. Figure 2 and Figure 3 .

[0039] Example 3 Verification of pore permeability: Porous core-shell microspheres with a core of 0.3% CMC prepared in Example 2 were added to an aqueous solution containing 200 nm diameter green fluorescent particles (EGFP). The mixture was gently shaken and incubated at room temperature for a certain period. Appropriate amounts of microspheres were taken at 3 h, 12 h, and 24 h, and the distribution of the fluorescent particles was observed under a laser confocal fluorescence microscope. Figure 4 Bright green fluorescent spots appeared on the inner side of the microsphere shell and near the core region, indicating that fluorescent particles with a diameter of about 200 nm could penetrate into the interior of the microsphere through the shell pores.

[0040] Example 4 Cell encapsulation and NK cell infiltration assays: CACO2 cells were uniformly dispersed at a density of 5 x 10^7 cells / ml in 40% CO1 solution and kept on ice. Microspheres were prepared according to the method in Example 2. The obtained microspheres were cultured in DMEM high-glucose medium containing 20% ​​FBS at 37°C and 5% CO2. An appropriate amount of NK-92 cells was centrifuged at 1200 rpm for 3 min, resuspended in 1 ml of DMEM serum-free medium, and 1 μl of Celltraker (Green) was added and gently mixed. The mixture was incubated at 37°C and 5% CO2 for 1 h. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml of NK-92-specific medium. 1 ml of the above CACO2 microspheres was placed in a 24-well plate, fluorescently labeled NK-92 cells were added, and after incubation for 3 days, the CACO2 microspheres were observed under a laser confocal microscope to observe the NK cell infiltration. Figure 5 Green fluorescent bright spots are visible within the microspheres and cell clusters.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing porous core-shell hydrogels, characterized in that, Includes the following steps: S1, Preparation of outer shell gel solution, core gel solution and microfluidic chip; The microfluidic chip includes an inlet I, an inlet II, a vent, and a core channel, a shell channel, and a gas channel arranged coaxially. The inlet I is connected to the inlet of the core channel, the inlet II is connected to the inlet of the shell channel, and the vent is connected to the inlet of the gas channel. The outlet of the core channel is located inside the shell channel, and the outlet of the shell channel is located inside the gas channel. S2, the outer shell gel solution is injected into the outer shell channel at a fixed flow rate to form an outer shell fluid; The core gel solution is injected into the core channel at a fixed flow rate to form an inner shell fluid. S3, inject gas into the gas channel at a fixed flow rate to shear the outer shell fluid and the core fluid, so that the core fluid is wrapped by the outer shell fluid and forms a core-shell droplet under the shearing action of the gas. S4, the core-shell droplets are collected in a curing solution containing a crosslinking agent, so that the shell forms a strong hydrogel layer; S5. Wash the solidified core-shell hydrogel with physiological saline or PBS buffer to obtain a porous core-shell hydrogel.

2. The method for preparing a porous core-shell hydrogel according to claim 1, characterized in that, The shell gel solution is prepared by adding 0.5%~5% (w / v) polyvinyl alcohol, 0.5%~5% (w / v) sodium alginate and 1%~15% (w / v) polyethylene glycol to an aqueous solution and stirring until homogeneous; the polyethylene glycol has a molecular weight range of 2000~6000 Da.

3. The method for preparing a porous core-shell hydrogel according to claim 1, characterized in that, The core gel solution is prepared by adding a hydrogel material or matrix material for cell culture to a culture medium and mixing them. The hydrogel material is selected from one or more of sodium carboxymethyl cellulose, collagen, matrix gel, gelatin, hyaluronic acid, alginate, or their chemically modified derivatives. The sodium carboxymethyl cellulose content is 0.2%~1.0% (w / v), the collagen content is 10%~90% (v / v), the matrix gel content is 10%~90% (v / v), the gelatin content is 2%~10% (w / v), the hyaluronic acid content is 0.1%~1.0% (w / v), and the alginate content is 0.2%~3% (w / v).

4. The method for preparing a porous core-shell hydrogel according to claim 3, characterized in that, The core gel solution also contains active substances, which are selected from one or more of animal or human cells, extracellular matrix proteins, growth factors, small molecule drugs, and immune cytokines.

5. The method for preparing a porous core-shell hydrogel according to claim 1, characterized in that, The core gel solution flows at a rate of 400-900 μL / min in the core channel, the outer shell gel solution flows at a rate of 1.5-5 times that of the core gel solution in the core channel, and the gas flows at a rate of 1.5-3 L / min in the gas channel.

6. The method for preparing a porous core-shell hydrogel according to claim 1, characterized in that, The core channel, the outer shell channel, and the gas channel are all constructed of a rigid material selected from one or more of stainless steel, capillary glass, ceramic, or polyetheretherketone. The inner diameter of the core channel is 50-800 μm, the inner diameter of the outer shell channel is 200-800 μm larger than the outer diameter of the core channel, and the inner diameter of the gas channel is 500-1500 μm larger than the outer diameter of the outer shell channel. The distance between the outlet end of the core channel and the outlet end of the outer shell channel is 0.2-2 mm.

7. The method for preparing a porous core-shell hydrogel according to claim 1, characterized in that, The crosslinking agent is a mixed solution containing 0.5%~5% (w / v) calcium chloride and 5mM~20mM sodium borate.

8. The method for preparing a porous core-shell hydrogel according to claim 1, characterized in that, The microfluidic chip is constructed using soft lithography, glass or silicon etching, precision machining, or 3D printing.

9. The method for preparing a porous core-shell hydrogel according to claim 1, characterized in that, The gas is selected from one or more of air, nitrogen, and carbon dioxide.

10. The application of a porous core-shell hydrogel prepared by the method according to any one of claims 1 to 9 in three-dimensional cell culture, immunotherapy evaluation or tissue engineering.

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