A method for preparing a two-chamber structure hydrogel microsphere
By controlling interfacial tension through coaxial microfluidic chips and electrostatic interactions, hydrogel microspheres with different two-chamber structures were prepared, overcoming the equipment limitations in existing technologies and achieving simple and efficient microsphere preparation and anti-tumor effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTHEAST UNIV
- Filing Date
- 2023-11-23
- Publication Date
- 2026-06-05
AI Technical Summary
Existing microfluidic devices are difficult to produce two-chamber hydrogel microspheres with different structures simultaneously, and require the use of organic solvents or surfactants to regulate the fluid interfacial tension, which limits the flexibility of the microsphere structure and the simplicity of the preparation method.
By constructing a coaxial microfluidic chip and utilizing electrostatic interaction and interfacial tension modulation of pure aqueous solution, hydrogel microspheres with different two-chamber structures, including Janus and core-shell structures, were prepared and loaded with iron source and ferroptosis inducer, respectively.
This technology enables the flexible fabrication of hydrogel microspheres with different two-compartment structures on a microfluidic chip, simplifying the operation, ensuring independent loading of iron sources and ferroptosis inducers, and promoting ferroptosis synergistically to enhance antitumor effects.
Smart Images
Figure CN117582902B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing two-chambered hydrogel microspheres. Background Technology
[0002] Ferroptosis is a form of programmed cell death, and in recent years, inducing ferroptosis in tumor cells has emerged as a novel cancer treatment strategy. Ferroptosis requires two conditions: first, providing the tumor with a certain amount of iron; and second, inducing lipid peroxidation in the tumor cell membrane. These two conditions need to work synergistically to achieve a highly effective anti-tumor effect. Therefore, it is necessary to develop a material that can simultaneously provide an iron source and induce lipid peroxidation.
[0003] Two-compartment hydrogel microspheres contain two independent chambers within a microcarrier, typically exemplified by bifacial Janus structures and hierarchical core-shell structures. Iron sources and ferroptosis inducers can be loaded separately into the two chambers of the Janus hydrogel microspheres, remaining independent within the microspheres. When injected into the tumor, they can be simultaneously released to synergistically induce ferroptosis in tumor cells.
[0004] Microfluidics is a microcarrier fabrication technique widely used to prepare hydrogel microspheres with layered structures. However, existing microfluidic devices can only produce hydrogel microspheres with fixed structures. For example, using an outlet-aligned coaxial capillary can only produce core-shell structured microspheres, while producing Janus microspheres requires the use of a theta-shaped capillary with side-by-side channels. In other words, existing technologies require different microfluidic devices to prepare two-chamber hydrogel microspheres with different structures, or require the use of organic solvents or surfactants to regulate the interfacial tension of the fluid to control the microsphere structure. Summary of the Invention
[0005] Purpose of the invention: The purpose of this invention is to provide a method for producing hydrogel microspheres with different two-compartment structures. This method can flexibly obtain hydrogel microspheres with different two-compartment structures by controlling the interfacial tension of pure aqueous solutions.
[0006] Technical solution: The preparation method of the two-chamber hydrogel microspheres of the present invention includes the following steps:
[0007] (1) Constructing a coaxial microfluidic chip: The coaxial microfluidic chip includes an inner phase capillary and an outer phase capillary arranged coaxially. The inner phase capillary is inserted into the outer phase capillary. Both the inner phase capillary and the outer phase capillary include capillary ends. The distance between the outlet of the capillary end of the inner phase capillary and the inlet of the capillary end of the outer phase capillary is 5 to 10 mm.
[0008] (2) Prepare internal phase solution and external phase solution:
[0009] An aqueous solution of sodium alginate doped with magnetic nanoparticles was used as the external phase solution, in which the mass fraction of sodium alginate was 1.8%; an aqueous solution of sodium alginate doped with ferrode activator was used as the internal phase solution, in which the mass fraction of sodium alginate was 0.9-2.4%.
[0010] (3) The inner phase capillary and the outer phase capillary are connected to the corresponding syringes through their respective spotting needles; the syringes pump the inner phase solution and the outer phase solution into the inner phase capillary and the outer phase capillary at the same flow rate through the injection pump; the positive terminal of the high voltage power supply is connected to the needle of the syringe for the outer phase solution or the inner phase solution, and the negative terminal of the high voltage power supply is connected to the receiving liquid; the injection pump is started and the high voltage power supply is turned on. Through electrostatic action, the solution at the capillary end outlet of the outer phase capillary is sprayed into the receiving liquid, and cross-linking reaction is carried out in the receiving liquid to form hydrogel microspheres with a two-chamber structure.
[0011] In step (1), the inner diameter of the capillary end in the inner phase capillary is 0.1 mm; the length of the capillary end is 1 / 3 to 1 / 2 of the total length of the inner phase capillary.
[0012] In step (1), the inner diameter of the capillary end in the external phase capillary is 0.15 mm; the length of the capillary end is 1 / 3 to 1 / 2 of the total length of the external phase capillary.
[0013] In step (2), the magnetic nanoparticles are ferric oxide nanoparticles or ferric oxide nanoparticles; in the external phase solution, the concentration of the magnetic nanoparticles is 0.5 to 5 mg / mL based on the mass of iron, preferably 1 mg / mL.
[0014] In step (2), the concentration of the ferrode death activator in the internal phase solution is 0.1 to 2 mg / mL, preferably 1 mg / mL.
[0015] In step (2), when the sodium alginate concentrations in the inner and outer phase solutions are the same, Janus microspheres with a two-sided structure are formed; when the sodium alginate concentration in the inner phase solution is greater than that in the outer phase solution, core-shell microspheres with the outer phase encapsulating the inner phase are formed; when the sodium alginate concentration in the inner phase solution is less than that in the outer phase solution, core-shell microspheres with the inner phase encapsulating the outer phase are formed. In the two-chamber hydrogel microspheres, one chamber is loaded with magnetic nanoparticles as an iron source, and the other chamber is loaded with ferroptosis inducers.
[0016] In step (3), the pumping flow rates of the inner and outer phase solutions are the same, ranging from 1 to 120 μL / min, with a preferred flow rate of 20 μL / min. The size of the hydrogel microspheres can be changed by adjusting the electric field strength and the flow rates of the inner and outer phases (keeping the inner and outer phase flow rates the same and adjusting them synchronously). As the electric field strength increases, the diameter of the microspheres decreases, and as the flow rate increases, the diameter of the microspheres increases.
[0017] In step (3), the voltage is 8 to 14 kV, preferably 12 kV.
[0018] In step (3), the receiving liquid is a calcium chloride aqueous solution with a mass fraction of 1-3%.
[0019] In step (3), the prepared hydrogel microspheres are left to stand in the receiving liquid for 2 hours, washed with pure water after standing, and finally stored in pure water.
[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The method of the present invention can flexibly obtain hydrogel microspheres with different two-compartment structures by controlling the interfacial tension of pure aqueous solutions, thereby realizing the preparation of hydrogel microspheres with different two-compartment structures through a microfluidic chip. The method is simple, easy to operate, reusable, and can precisely control the structural distribution of the two compartments of the microspheres. The two-compartment hydrogel microspheres prepared by the method of the present invention can simultaneously load iron sources and ferroptosis inducers. The two (iron sources and ferroptosis inducers) are independently loaded in the two compartments and work synergistically with each other, thereby achieving a good effect of promoting cell ferroptosis and anti-tumor. Attached Figure Description
[0021] Figure 1 This is a schematic diagram of the microfluidic chip used in this invention;
[0022] Figure 2 Microscopic images of hydrogel microspheres with different two-chamber structures prepared according to this invention are shown below. In these images, a represents a solution with sodium alginate concentrations of 1.8 wt% in the outer phase and 0.9 wt% in the inner phase; b represents a solution with sodium alginate concentrations of 1.8 wt% in the outer phase and 1.2 wt% in the inner phase; c represents a solution with sodium alginate concentrations of 1.8 wt% in the outer phase and 1.5 wt% in the inner phase; d represents a solution with sodium alginate concentrations of 1.8 wt% in both the outer and inner phases; e represents a solution with sodium alginate concentrations of 1.8 wt% in the outer phase and 2.1 wt% in the inner phase; and f represents a solution with sodium alginate concentrations of 1.8 wt% in the outer phase and 2.4 wt% in the inner phase. The outer phase is transparent, while the inner phase, containing iron oxide, is dark in color.
[0023] Figure 3 Fluorescence images and corresponding capillary fluid structure diagrams of hydrogel microspheres with different two-chamber structures prepared in this invention.
[0024] Figure 4 This is a graph showing the relationship between the size of the two-chamber hydrogel microspheres prepared in this invention and voltage and flow rate;
[0025] Figure 5This is a diagram showing the effect of the two-chamber hydrogel prepared in this invention on promoting ferroptosis. Detailed Implementation
[0026] Example 1
[0027] The preparation method of the two-compartment hydrogel microspheres of the present invention includes the following steps:
[0028] (1) Fabrication of coaxial microfluidic chip:
[0029] like Figure 1 As shown, the coaxial microfluidic chip includes an inner-phase capillary and an outer-phase capillary arranged coaxially. The inner-phase capillary is inserted into the outer-phase capillary. A sampling needle is fixed at the joint between the inner-phase and outer-phase capillary to facilitate fluid introduction. All parts requiring fixation during assembly are finished with resin glue. The inner-phase capillary is made using a capillary with an inner diameter of 0.58 mm and an outer diameter of 1.14 mm. The front end of the capillary is drawn into a capillary tip with an inner diameter of 0.1 mm and a length of 10 mm using a flame torch (the total length of the inner-phase capillary is 30 mm, of which the capillary tip is 10 mm long). An outer phase capillary was fabricated using a capillary tube with an inner diameter of 0.58 mm and an outer diameter of 1.14 mm. The front end of the capillary was drawn into a capillary end with an inner diameter of 0.15 mm and a length of 10 mm using a flame torch (the total length of the outer phase capillary was 20 mm, of which the capillary end was 10 mm long). The front end of the capillary was ground flat using a needle grinder. Then, the inner phase capillary was coaxially inserted into the outer phase capillary. The distance between the outlet of the inner phase capillary and the inlet of the outer phase capillary was 5 mm. This length allows the inner and outer phase solutions to achieve a two-phase equilibrium within the outer phase capillary.
[0030] (2) Preparation of hydrogel microspheres by preparing internal and external phase solutions:
[0031] An aqueous solution of sodium alginate doped with ferric oxide nanoparticles was used as the external phase solution, in which the mass fraction of sodium alginate was 1.8% and the concentration of ferric oxide nanoparticles was 1 mg / mL based on the mass of iron. An aqueous solution of sodium alginate doped with ferrodesorption activator was used as the internal phase solution, in which the mass fraction of sodium alginate was 1.8% and the concentration of ferrodesorption activator was 1 mg / mL.
[0032] (3) Use two syringes to draw up the internal phase solution and the external phase solution respectively and connect them to the corresponding syringe pumps. Connect the corresponding syringe outlets (needles) to the spotting needles of the internal phase capillary and the external phase capillary with plastic tubing. Connect the positive terminal of the high voltage power supply to the needle (metal) of the external phase solution syringe and the negative terminal of the high voltage power supply to the receiving liquid. Start the syringe pump, and the flow rate of both the internal phase solution and the external phase solution is 20 μL / min. Turn on the high voltage power supply with a voltage of 12 kV. Through electrostatic action, the sodium alginate solution at the outlet of the external phase capillary is sprayed into the receiving liquid. A cross-linking reaction occurs in the receiving liquid to form hydrogel microspheres with a two-chamber structure. Let it stand for 2 hours, then wash it 3 times with pure water, and finally store it in pure water.
[0033] The preparation method of Example 1 was used, keeping the mass fraction of sodium alginate in the external phase solution constant at 1.8%. The only difference was adjusting the concentration of sodium alginate in the internal phase solution to 0.9 wt%, 1.2 wt%, 1.5 wt%, 2.1 wt%, and 2.4 wt%, respectively, to prepare hydrogel microspheres. The flow rates of both the internal and external phase solutions were 20 μL / min, and the voltage was 12 kV. Microscopic images of the hydrogel microspheres prepared based on different concentrations of sodium alginate in the internal phase solution are shown below. Figure 2 As shown, through Figure 2 It can be seen that when the sodium alginate concentration in both the inner and outer phase solutions is 1.8 wt%, Janus microspheres with symmetrical two sides are formed. The outer phase containing iron oxide nanoparticles in the two chambers of the Janus microspheres is an opaque chamber, while the inner phase is a transparent chamber. When the sodium alginate concentration in the outer phase solution remains constant, and the sodium alginate concentration in the inner phase solution becomes 2.1 wt%, two-chambered hydrogel microspheres are formed with the outer phase partially enveloping the inner phase in a crescent shape. When the sodium alginate concentration in the outer phase solution remains constant, and the sodium alginate concentration in the inner phase solution continues to increase to 2.4 wt%, core-shell structured hydrogel microspheres are formed, with the inner phase acting as the core and the outer phase enveloping the inner phase as the outer shell, which is also the outer chamber. When the sodium alginate concentration in the outer phase solution remains constant, and the sodium alginate concentration in the inner phase solution decreases to 1.5 wt%, two-chambered hydrogel microspheres are formed where the inner phase partially encapsulates the outer phase in a crescent shape. When the inner phase concentration decreases to 1.2 wt%, the degree to which the inner phase encapsulates the outer phase increases. When the inner phase concentration decreases to 0.9 wt%, the inner phase completely encapsulates the outer phase, forming core-shell structured microspheres. In this case, the outer phase has a core as the inner chamber, and the inner phase has a shell as the outer chamber. The hydrogel microspheres with different two-chambered structures obtained above all have regular shapes and uniform particle sizes. Figure 3 As shown, taking typical Janus, core-shell (inner phase is the core) and core-shell (outer phase is the core) structures as examples, the outer phase contains iron oxide and is yellow, while the inner phase contains a green fluorescent dye and is transparent under a light microscope. In the outer capillary, the inner and outer phases can form a stable flow pattern with clear boundaries, and the fluorescence pattern confirms the formation of hydrogel microspheres with corresponding flow structures. Figure 4As shown, taking Janus microspheres prepared when the concentration of sodium alginate in both the inner and outer phases is 1.8 wt% as an example, the particle size of the microspheres decreases with increasing voltage; the particle size of the microspheres also decreases with decreasing flow rates in both phases. This invention can prepare various hydrogel microspheres with different two-chamber structures using a coaxial microfluidic chip, and the interfacial tension can be changed by adjusting the concentration of the hydrogel precursor aqueous solution, without the need for organic solutions and surfactants.
[0034] The two-chambered hydrogel microspheres prepared in Example 1 were used for antitumor therapy:
[0035] Janus two-compartment microspheres were prepared using a precursor solution with a sodium alginate concentration of 1.8 wt% in both the inner and outer phases. The resulting microspheres had a particle size of 200 μm. The outer phase contained 1 mg / mL (based on elemental iron) of iron oxide nanoparticles, and the inner phase contained 1 mg / mL of the ferroptosis activator erastin. As a control, three other types of two-compartment hydrogel microspheres with the same particle size were prepared: microspheres with only iron oxide nanoparticles loaded in the outer phase and no drug loaded in the inner phase; microspheres with only the ferroptosis activator loaded in the inner phase and no drug loaded in the outer phase; and microspheres with no drug loaded in either the inner or outer phase. U87 cells (human glioblastoma cells) were then cultured at a concentration of 1 × 10⁻⁶. 4 The microspheres were seeded at the specified density in 96-well plates and cultured for 24 hours. The culture medium was then removed, and fresh culture medium containing the four types of microspheres described above was added to each well, with 200 microspheres per well. The microspheres were then co-incubated with U87 cells for one day, and cell proliferation activity was assessed using the CCK8 assay kit. Results are as follows: Figure 5 As shown, the microspheres without drugs and those loaded with only iron oxide showed no significant cytotoxicity; cell proliferation was unaffected, and cell survival rates were both greater than 95%. Cell activity was inhibited in the microsphere group loaded with only erastin, with a survival rate of approximately 70%. However, the microsphere groups loaded with iron oxide and erastin in their respective compartments showed significantly reduced cell proliferation, with a survival rate of approximately 40%, demonstrating a more significant tumor cell killing ability compared to the microsphere group loaded with only ferroptosis activator. This is because iron oxide nanoparticles alone, as an iron source, release low concentrations of iron ions, which do not affect cell activity. Microspheres loaded with erastin can release erastin to induce ferroptosis, activating lipid peroxidation of the cell membrane, damaging the cell membrane and its function, leading to inhibition of cell proliferation and metabolism. This induced oxidative death is iron-dependent; iron ion deficiency cannot fully trigger ferroptosis. However, microspheres loaded with iron oxide and erastin in their respective compartments can simultaneously trigger iron ion release, enhancing erastin-induced ferroptosis and achieving a highly efficient anti-tumor effect.
Claims
1. A method for preparing two-chambered hydrogel microspheres, characterized in that, Includes the following steps: (1) Constructing a coaxial microfluidic chip: The coaxial microfluidic chip includes an inner phase capillary and an outer phase capillary arranged coaxially. The inner phase capillary is inserted into the outer phase capillary. Both the inner phase capillary and the outer phase capillary include capillary ends. The distance between the outlet of the capillary end of the inner phase capillary and the inlet of the capillary end of the outer phase capillary is 5 to 10 mm. (2) Preparation of internal and external phase solutions: Sodium alginate aqueous solution doped with magnetic nanoparticles was used as the external phase solution; Sodium alginate aqueous solution doped with ferrode death activator was used as the inner phase solution; In the external phase solution, the mass fraction of sodium alginate is 1.8%; In the inner phase solution, the mass fraction of sodium alginate is 0.9–2.4%. When the sodium alginate concentrations in the inner and outer phase solutions are the same, Janus microspheres with a bifacial structure are formed. When the sodium alginate concentration in the inner phase solution is greater than that in the outer phase solution, core-shell microspheres with the outer phase encapsulating the inner phase are formed. When the sodium alginate concentration in the inner phase solution is less than that in the outer phase solution, core-shell microspheres with the inner phase encapsulating the outer phase are formed. (3) The inner phase capillary and the outer phase capillary are connected to the corresponding syringes through their respective spotting needles; the syringes pump the inner phase solution and the outer phase solution into the inner phase capillary and the outer phase capillary at the same flow rate through the injection pump; the positive terminal of the high voltage power supply is connected to the needle of the syringe for the outer phase solution or the inner phase solution, and the negative terminal of the high voltage power supply is connected to the receiving liquid; the injection pump is started and the high voltage power supply is turned on. Through electrostatic action, the solution at the capillary end outlet of the outer phase capillary is sprayed into the receiving liquid, and cross-linking reaction is carried out in the receiving liquid to form hydrogel microspheres with a two-chamber structure.
2. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (1), the inner diameter of the capillary end in the inner phase capillary is 0.1 mm; the length of the capillary end is 1 / 3 to 1 / 2 of the total length of the inner phase capillary.
3. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (1), the inner diameter of the capillary end in the external phase capillary is 0.15 mm; the length of the capillary end is 1 / 3 to 1 / 2 of the total length of the external phase capillary.
4. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (2), the magnetic nanoparticles are ferric oxide nanoparticles or ferric oxide nanoparticles; in the external phase solution, the concentration of the magnetic nanoparticles is 0.5 to 5 mg / mL based on the mass of iron.
5. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (2), the concentration of the ferrodegeneration activator in the internal phase solution is 0.1–2 mg / mL.
6. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (3), the pumping flow rates of the internal phase solution and the external phase solution are the same, ranging from 1 to 120 μL / min.
7. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (3), the voltage is 8~14kV.
8. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (3), the receiving liquid is a calcium chloride aqueous solution with a mass fraction of 1-3%.
9. The method for preparing two-chambered hydrogel microspheres according to claim 1, characterized in that: In step (3), the prepared hydrogel microspheres are placed in the receiving liquid and then washed with pure water. Finally, they are stored in pure water.