Monodisperse core-shell microspheres and a method for preparing the same

Abstract

The application relates to the technical field of materials, in particular to a monodisperse core-shell microsphere and a preparation method thereof. The method comprises the following steps: S1, using a dispersion phase liquid containing a degradable polymer, forming monodisperse degradable polymer microspheres through a microfluidic process or an emulsification method; S2, combining horseradish peroxidase through one or more of the following methods: dopamine self-polymerization, electrostatic adsorption and chemical grafting on the surface of the degradable polymer microspheres, and under the catalysis of the horseradish peroxidase, crosslinking and coating the degradable polymer microspheres with a hydrogel material to obtain monodisperse core-shell microspheres with a hydrogel layer; the hydrogel material is a hydrogel system that can be crosslinked by enzyme initiation, such as hydroxyphenylpropionic acid hydrogel, silk fibroin or hyaluronic acid hydrogel. Through the method, highly monodisperse degradable core-shell microsphere materials can be obtained, the process operation is simple, and the method is beneficial to wide popularization and application.

Description

Technical Field

[0001] This invention relates to the field of materials technology, and in particular to a monodisperse core-shell microsphere and its preparation method. Background Technology

[0002] Core-shell microspheres are tiny particulate materials composed of a core and an outer shell. The core and shell structures are typically made of different materials, integrating the properties of both materials to achieve characteristics that a single core or shell material could not. The properties of core-shell microsphere composites make them ideal for pharmaceutical and biomedical applications, including cell encapsulation, cell research, targeted drug delivery, controlled drug release, cosmetic fillers, cell delivery, scaffold materials, the food industry, catalysis, and environmental monitoring. Currently, a wide range of technologies have been used to prepare core-shell microsphere materials, with common methods including coaxial nozzle extrusion, microfluidics, solvent evaporation, and self-assembly. However, existing technologies often struggle to produce monodisperse core-shell microspheres, and the presence of hydrogels in these microspheres enhances their cell adhesion, allowing cells to spread more effectively on their surface.

[0003] Microfluidics is a technique for controlling the mixing and flow of complex fluids at the microscale. It involves producing droplets through microchannels, using volumetric forces (e.g., syringe pumps) or pressure forces (e.g., pressure vessels) to propel immiscible continuous and dispersed phases through their respective microchannels. At the junctions of the microchannels, the continuous phase is subjected to compression or shearing forces against the dispersed phase, causing interfacial instability and breakage, thus forming dispersed droplets. These droplets are then transformed into core-shell microspheres through solidification, gelation, and cross-linking. The method for preparing core-shell microspheres using microfluidics combines microfluidics and particle preparation techniques, enabling the fabrication of microspheres with controllable particle size, shape, shell thickness, and core-shell structure. The preparation of core-shell microspheres using microfluidics represents an emerging field combining microfluidics and particle preparation technologies. On a microfluidic platform, precise fluid manipulation and chemical reactions allow for precise control over the shape, size, and core-shell structure of microspheres. This technology has broad application prospects, particularly in drug delivery, bioanalysis, and materials science, providing a higher level of controllability and customization for the research and application of microsphere materials.

[0004] However, existing microfluidic technologies for preparing core-shell microspheres mostly employ a dual-emulsion method, which is commonly used to prepare biocompatible microspheres such as PLLA. During the encapsulation process, unencapsulated or encapsulated cores are prone to occur. Existing dual-emulsion methods for preparing microsphere structures are complex and inefficient, typically requiring the use of three or more injection pumps to simultaneously control the flow rate, as well as the observation of microsphere formation through a microscope. This makes the operation complex and the process difficult to control. Summary of the Invention

[0005] In view of this, the present invention provides a monodisperse core-shell microsphere and its preparation method. The method of the present invention can obtain highly monodisperse biodegradable core-shell microsphere materials, and the process is simple and conducive to widespread application.

[0006] This invention provides a method for preparing monodisperse core-shell microspheres, comprising the following steps:

[0007] S1. Using a dispersed phase liquid containing biodegradable polymers, monodisperse biodegradable polymer microspheres are formed through microfluidic processes or emulsification.

[0008] S2. The surface of the biodegradable polymer microspheres is subjected to one or more of dopamine self-polymerization, electrostatic adsorption, and chemical grafting, combined with horseradish peroxidase. Under the catalysis of the horseradish peroxidase, the biodegradable polymer microspheres are cross-linked and coated with a hydrogel material to obtain monodisperse core-shell microspheres with a hydrogel layer. The hydrogel material is a hydrogel system that can be cross-linked by enzyme initiation, such as hydroxyphenylpropionic acid hydrogel, silk fibroin, or hyaluronic acid hydrogel.

[0009] In some embodiments of the present invention, the particle size of the biodegradable polymer microspheres is 10–1000 μm, preferably 30–250 μm, more preferably 40–200 μm, and even more preferably 50–180 μm.

[0010] In some embodiments of the present invention, the biodegradable polymer is at least one of PLA, PLGA, PCL, and PGA, preferably PLA.

[0011] In some embodiments of the present invention, the microfluidic process in step S1 includes:

[0012] An oily dispersed liquid containing a biodegradable polymer is injected into the inner phase channel of a coaxial microfluidic chip, and an aqueous continuous phase liquid is injected into the outer phase channel. Biodegradable polymer droplets are collected at the outlet end of the inner phase channel. The solvent of the oily dispersed liquid is removed, and preferably the mixture is washed with water and freeze-dried to obtain monodisperse biodegradable polymer microspheres.

[0013] The emulsification method in step S1 includes: stirring and emulsifying an oily dispersed phase liquid containing a biodegradable polymer and an aqueous continuous phase liquid to form a biodegradable polymer liquid, then removing the solvent from the oily dispersed phase liquid, preferably by washing with water and freeze-drying, to obtain monodisperse biodegradable polymer microspheres.

[0014] In some embodiments of the present invention, in step S1, the coaxial microfluidic chip is a glass capillary chip or a PDMS chip; the inner phase channel outlet end is provided with a coaxial collection channel, and the straight-line distance between the inlet of the collection channel and the outlet of the inner phase channel is 30 to 1000 μm, preferably 50 to 500 μm.

[0015] In some embodiments of the present invention, in step S1, the aqueous continuous phase liquid is a PVA aqueous solution, and the flow rate can be adjusted by an injection pump to 1-100 ml / h, such as 10-50 ml / h, preferably 20-40 ml / h.

[0016] In some embodiments of the present invention, in step S1, the solvent of the oily dispersed phase liquid is at least one of dichloromethane, chloroform, ethyl acetate, acetone and tetrahydrofuran, preferably dichloromethane; the flow rate of the oily dispersed phase liquid is 0.1 to 50 ml / h, preferably 1 to 5 ml / h.

[0017] In some embodiments of the present invention, step S2 includes:

[0018] The biodegradable polymer microspheres were immersed in a dopamine solution, and then immersed in a horseradish peroxidase solution to allow the dopamine to self-polymerize and bind with the horseradish peroxidase, thus obtaining microspheres with horseradish peroxidase on their surface.

[0019] The microspheres with horseradish peroxidase on their surface were mixed with a hydrogel material and cross-linked to obtain monodisperse core-shell microspheres with a hydrogel layer.

[0020] In some embodiments of the present invention, in step S2, the concentration of the dopamine solution is 0.1-5 mg / mL, and the concentration of the horseradish peroxidase solution is 0.01-1 mg / mL, preferably 0.01-0.1 mg / mL;

[0021] The cross-linking reaction takes 0.1 to 5 minutes, and water is added to terminate the reaction and prevent excessive cross-linking.

[0022] This invention provides a monodisperse core-shell microsphere, prepared according to the method described above. The monodisperse core-shell microsphere has a smooth surface and can be used as a drug delivery, cell adhesion and cell delivery carrier, medical aesthetic filler, scaffold material, joint lubricant, and catalyst.

[0023] This invention primarily utilizes microfluidic technology combined with enzyme crosslinking to prepare biodegradable polymer microspheres with a core-shell structure. This results in core-shell microsphere samples exhibiting excellent consistency and stability in performance and function, while the process is simple to operate. Firstly, this invention employs microfluidic technology combined with enzyme crosslinking to achieve highly monodisperse core-shell microsphere samples. In this preparation process, by precisely controlling the fluid mixing and reaction processes within the microfluidic system, the size and shape of each microsphere are ensured to be very similar, thus achieving highly uniform core-shell microsphere samples. This monodispersity ensures a high degree of consistency in performance and function among the microspheres, enhancing their application stability. Secondly, the obtained highly monodisperse core-shell microsphere samples exhibit excellent stability and anti-aggregation properties. Because each microsphere is very similar in size and shape, the microspheres in the sample are not prone to sticking together, thus maintaining the independence and stability of the microspheres. This is crucial for some applications, especially where maintaining the dispersed state of the microspheres is required; highly monodisperse samples offer a significant advantage.

[0024] Furthermore, compared to the high cost and low efficiency of traditional dual-emulsion preparation methods, this invention offers a simpler approach by using microfluidic technology to prepare biodegradable polymer microspheres. In-situ enzymatic crosslinking then forms a hydrogel layer on the microsphere surface, thus constructing core-shell microspheres. This invention's embodiment, combining microfluidic technology with enzymatic crosslinking to prepare monodisperse core-shell microsphere samples, allows for more precise control. This microfluidic technology allows for precise control of experimental conditions and reaction parameters, ensuring a more reliable and reproducible microsphere preparation process. This precise control contributes to improved product quality, and highly monodisperse microsphere samples are preferred in applications requiring high consistency and accuracy. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The diagram shows the device design for fabricating capillary microfluidic chips according to the embodiments of this application; (a) is the chip design part, and (b) is the actual chip.

[0027] Figure 2 The following are schematic diagrams and physical images of the microfluidic system constructed in the embodiments of this application; (a) is a schematic diagram of the microfluidic system structure, and (b) is a physical image of the system.

[0028] Figure 3This is a schematic diagram of the microfluidic system in the preparation method of Example 1 of this application; (a) shows the main dimensions, and (b) shows the actual object and the spacing.

[0029] Figure 4 This is an optical microscope image of the PLLA microspheres obtained in Example 1 of this application;

[0030] Figure 5 This is an optical microscope image of the PLLA-GelHPA core-shell microspheres of Example 2 of this application;

[0031] Figure 6 The particle size of PLLA microspheres under different spacing conditions in Example 3 of this application;

[0032] Figure 7 The particle size of PLLA microspheres under different aqueous phase flow rates in Example 4 of this application;

[0033] Figure 8 The particle size of PLLA microspheres under different oil phase flow rates in Example 5 of this application;

[0034] Figure 9 This is a particle size distribution diagram of PLLA microspheres obtained in Example 1 of this application;

[0035] Figure 10 This is a particle size distribution diagram of PLLA-GelHPA core-shell microspheres in Example 2 of this application;

[0036] Figure 11 This is a SEM image of PLLA microspheres obtained in Example 1 of this application;

[0037] Figure 12 This is a SEM image of the PLLA-GelHPA core-shell microspheres from Example 2 of this application;

[0038] Figure 13 This is an elemental distribution diagram of the PLLA microspheres obtained in Example 1 of this application;

[0039] Figure 14 This is an elemental distribution diagram of the PLLA-GelHPA core-shell microspheres in Example 2 of this application;

[0040] Figure 15 Infrared spectra of PLLA microspheres from Example 1 and PLLA-GelHPA core-shell microspheres from Example 2;

[0041] Figure 16 Fluorescent images of the GelHPA hydrogel coating on the surface of the PLLA-GelHPA core-shell microspheres in Example 2;

[0042] Figure 17 The transfer of the PLLA microsphere suspension in Example 1 is shown;

[0043] Figure 18 The transfer of the PLLA-GelHPA core-shell microsphere suspension in Example 2 is shown.

[0044] Figure 19 The friction coefficient diagrams are for the PLLA microspheres of Example 1 and the PLLA-GelHPA core-shell microspheres of Example 2.

[0045] Figure 20 Image of the grinding disc of PLLA-GelHPA core-shell microspheres in Example 2;

[0046] Figure 21 Images showing the liveness and death of stem cells on PLLA microspheres in Example 1;

[0047] Figure 22 The images show the live and dead state of stem cells seeded on PLLA-GelHPA core-shell microspheres in Example 2.

[0048] Figure 23 The image shows a stained image of PLLA microsphere cell culture from Example 1.

[0049] Figure 24 This is a stained image of PLLA-GelHPA core-shell microsphere cell culture from Example 2. Detailed Implementation

[0050] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, the technical solution of the present invention will now be described in detail with reference to specific embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0051] This invention provides a method for preparing monodisperse core-shell microspheres, comprising the following steps:

[0052] S1. Using a dispersed phase liquid containing biodegradable polymers, monodisperse biodegradable polymer microspheres are formed through microfluidic processes or emulsification.

[0053] S2. The surface of the biodegradable polymer microspheres is subjected to one or more of dopamine self-polymerization, electrostatic adsorption, and chemical grafting, combined with horseradish peroxidase. Under the catalysis of the horseradish peroxidase, the biodegradable polymer microspheres are cross-linked and coated with a hydrogel material to obtain monodisperse core-shell microspheres with a hydrogel layer. The hydrogel material is a hydrogel system that can be cross-linked by enzyme initiation, such as hydroxyphenylpropionic acid hydrogel, silk fibroin, or hyaluronic acid hydrogel.

[0054] The method of this invention can obtain highly monodisperse biodegradable core-shell microspheres, and the process is simple to operate and easy to control precisely.

[0055] See Figure 1 This invention relates to a capillary microfluidic chip device. Firstly, a coaxial glass capillary microfluidic chip was designed using Solidworks. Alternatively, other materials with similar principles, such as polydimethylsiloxane (PDMS) chips, can also be used. In a specific embodiment, the main components of the capillary microfluidic chip device are a glass slide, multiple capillaries, and a dispensing needle. First, a circular capillary (1 mm outer diameter) is fixed to the glass slide using UV glue; the interior of this capillary is primarily an outer phase channel. Then, two cylindrical capillaries (inner / outer diameter: 0.5 mm / 0.8 mm) are gradually thinned using an igniter, and the tips are then sanded to the desired diameter. Under a microscope, two conical glass capillaries are inserted into the left and right sides of the outer circular glass tube, with their conical openings facing each other; the interiors of these two capillaries correspond to the inner phase channel and the collection channel, respectively. By observing and adjusting, ensure that the two capillaries are aligned coaxially, and gradually fine-tune the axial distance between them to 200 μm; it can also be adjusted between 30 and 1000 μm, preferably between 50 and 500 μm, for example, 50, 100, 300, 400, 500 μm, etc.

[0056] In this embodiment of the invention, two grooves are cut on both sides of the plastic end of a dispensing needle, allowing it to fit precisely into the contact area between the outer and incident circular tubes. The needle is then fixed at the connection between the left conical capillary and the outer circular capillary, and the remaining opening is sealed. A silicone tube is connected to the other end of the incident conical capillary, and a syringe is connected via the needle to inject the dispersed phase liquid. Alternatively, the silicone tube and needle can be connected at the dispensing needle to inject the continuous phase liquid. Furthermore, the connection between the tube and the capillary is glued together to secure it and prevent leakage. After the glue solidifies, a coaxial capillary microfluidic chip is obtained.

[0057] See Figure 2 , Figure 2 This diagram illustrates the structure and physical form of the microfluidic system according to an embodiment of the present invention. This microfluidic system primarily forms droplets and consists of two parts: a preparation device and a collection device. The preparation device includes a microfluidic chip and a syringe pump, while the collection device is mainly a beaker for holding the collected liquid. In this embodiment, the microfluidic chip can be fixed on a beaker, and the inlet end is connected to the syringe pump via a silicone tubing. By adjusting the relevant parameters of the syringe pump, the flow rates of the dispersed and continuous phases can be precisely controlled.

[0058] In some specific embodiments, the phase solution inside the microfluidic chip flows through a small-diameter circular capillary, which can be called the injection tube, with an aperture of 50–150 μm. Droplets form in another capillary, called the collection tube, which can also be connected to a beaker containing the collected liquid. The aperture of the collection tube can be 200–300 μm; the distance between the injection tube and the collection tube is as described above. In the above embodiments, the glass capillary is a hydrophilic material and does not require treatment when preparing oil-in-water droplets (such as PLLA droplets). The hydrophilicity of the capillary itself can be used to generate oil-in-water droplets; alternatively, further hydrophilic treatment can be performed to improve the hydrophilicity of the capillary and improve the formation of oil-in-water droplets.

[0059] This invention provides an oil-phase solution containing a biodegradable polymer, which is an oily dispersed liquid. The biodegradable polymer is primarily polylactic acid (PLA), including at least one of poly(L-lactide), poly(D-lactide), poly(PDLA), polylactic-co-glycolic acid copolymer (PLGA), poly(glycolic acid), and polycaprolactone (PCL), preferably polylactic acid, such as PLLA. The molecular weight of PLLA is further preferably 15901-16135. Furthermore, the solvent for the oily dispersed liquid can be at least one of dichloromethane, chloroform, ethyl acetate, acetone, and tetrahydrofuran, preferably dichloromethane. For example, biodegradable polymer powder such as PLLA is dissolved in dichloromethane (DCM) to prepare a 4-10% (w / v) oil-phase solution, preferably 5% (w / v).

[0060] Meanwhile, in this embodiment of the invention, an aqueous continuous phase liquid, also known as an aqueous solution, is prepared, primarily an aqueous solution of polyvinyl alcohol (PVA). The concentration of the aqueous phase (PVA) can be 1-3% (w / v), preferably 1% (w / v). Furthermore, the collecting liquid in the aforementioned collecting device is preferably a 0.5% (w / v) PVA aqueous solution. All polymers described in this embodiment of the invention are commercially available products, wherein the degree of PVA hydrolysis can be 87.0-89.0% (mol / mol).

[0061] In step S1 of this embodiment of the invention, monodisperse biodegradable polymer microspheres are prepared by injecting an oily dispersed liquid containing the biodegradable polymer into the inner phase channel of a coaxial microfluidic chip, and injecting an aqueous continuous phase liquid into the outer phase channel. At the outlet end of the inner phase channel, biodegradable polymer droplets are collected in a collection tube and can be collected in a beaker containing the collection liquid. Afterwards, the solvent in the oily dispersed liquid is removed, and the microspheres are washed with water and freeze-dried to obtain the final product.

[0062] Microfluidics is an advanced technology capable of controlling the mixing and flow rate of complex fluids at the microscopic scale. While core-shell microparticles prepared using microfluidics offer controllable morphology, the system structure is often complex, and the thickness of the shell is difficult to control. Droplet microfluidics, on the other hand, serves as a universal tool for manufacturing almost all types of microparticles. The convective flow patterns within the droplet facilitate mixing, while the components within the droplet are separated to avoid contamination and mutual interference. Therefore, since its emergence, microdroplet microfluidics has been extensively studied and applied both theoretically and practically. Droplet microfluidics can generate precisely calibrated droplets in a repeatable and designable manner. Furthermore, individual microdroplet reactors can operate independently, facilitating biochemical reactions and lab-on-a-chip applications.

[0063] In the process of generating biodegradable polymer droplets in this embodiment of the invention, the flow rate of the aqueous solution is adjusted by a syringe pump, which can be 1-100 ml / h, further 10-50 ml / h, preferably 20-40 ml / h, and even more preferably 30 ml / h. The flow rate of the oily dispersed liquid is also adjusted by another syringe pump, which can be 0.1-50 ml / h, specifically 1-5 ml / h, preferably 2-4 ml / h, and even more preferably 2 ml / h. The biodegradable polymer droplets collected in the beaker are preferably stirred at room temperature for 24 hours to volatilize dichloromethane. The obtained biodegradable polymer microspheres are washed with a large amount of water (generally deionized water in the laboratory) and freeze-dried for storage; the biodegradable polymer microspheres have uniform particle size, are monodisperse, and may have a porous structure; their particle size can be 30-250 μm, preferably 40-200 μm, more preferably 50-180 μm, and even more preferably 80-120 μm, and also have a certain degree of hydrophobicity.

[0064] After obtaining monodisperse biodegradable polymer microspheres, this embodiment of the invention directly immerses them in a dopamine solution of a certain concentration, enabling dopamine to adhere to the surface of the microspheres. Then, the microspheres with dopamine on their surface are immersed in a horseradish peroxidase (HRP) solution of a certain concentration for a certain period of time. Under weakly alkaline conditions (using a buffer solution or NaOH for adjustment, generally pH = 8.5), dopamine undergoes oxidative self-polymerization to form polydopamine. The o-benzoquinone groups on the polydopamine can covalently bind to the amino groups on the HRP enzyme, thereby immobilizing the HRP enzyme on the surface of the microspheres, achieving covalent binding between polydopamine and the enzyme, resulting in microspheres with horseradish peroxidase on their surface.

[0065] In this embodiment of the invention, the microspheres with horseradish peroxidase on their surface are then washed with water, and then hydrogen peroxide and a hydrogel material solution are added to carry out a cross-linking reaction. The hydrogel material is a hydrogel system that can be cross-linked by enzymes, such as hydroxyphenylpropionic acid gelatin, silk fibroin, or hyaluronic acid, and is further described as hydroxyphenylpropionic acid gelatin (GelHPA). After being activated by hydrogen peroxide (H2O2), horseradish peroxidase (HRP) can efficiently catalyze the free radical coupling of phenol and aniline derivatives. Taking GelHPA as an example, under the catalysis of horseradish peroxidase, the p-hydroxyphenylpropionic acid groups on GelHPA are activated. The presence of enzymes on the surface of polymer microspheres such as PLLA allows only the surface of the microspheres to cross-link and form a GelHPA hydrogel coating layer (shell). Similarly, silk fibroin itself contains groups that can be cross-linked by enzymes, or HPA can be grafted onto silk fibroin, hyaluronic acid hydrogel materials to achieve a better effect of enzyme cross-linking.

[0066] In a specific embodiment of the present invention, the concentration of the dopamine solution can be 0.1–5 mg / mL, preferably 1–5 mg / mL, and further 2–3 mg / mL; during soaking, it is preferred to use an equal amount (50 mg) of microspheres to soak in a certain volume (3 ml) of dopamine solution. The concentration of the horseradish peroxidase solution can be 0.01–1 mg / mL, such as 0.01–0.1 mg / mL, preferably 0.02–0.05 mg / mL, and soaking for 24 hours [soaking for a shorter time can also be effective]. The gelHPA solution is usually a gelatin hydrogel of p-hydroxyphenylpropionic acid (HPA) with a concentration of 20% by mass / volume percentage. The temperature range of the cross-linking reaction is generally 25–30°C, and gelation is easier at lower temperatures; the time of the cross-linking reaction is preferably 0.1–5 minutes, further 0.5–2.5 minutes, such as 1 minute, 2 minutes, 3 minutes, etc., and excessive time will easily cause clumping. To avoid excessive cross-linking of the hydrogel layer and subsequent clumping, it is preferable to add 1 ml of water after 2 minutes of reaction to terminate the reaction, thus obtaining monodisperse core-shell microspheres with a uniform hydrogel layer on the surface. In the embodiments of the present invention, the hydrogel shell is mainly formed by in-situ enzymatic cross-linking. The degree of cross-linking of the hydrogel can be controlled by the concentration of hydrogen peroxide, enzyme concentration, and hydrogel concentration, thereby obtaining hydrogel shell layers of different thicknesses.

[0067] This invention provides a monodisperse core-shell microsphere, prepared according to the method described above. The monodisperse core-shell microsphere has a smooth surface and can be used as a drug delivery, cell adhesion and cell delivery carrier, medical aesthetic filler, scaffold material, joint lubricant, and catalyst preparation.

[0068] In a specific embodiment of the present invention, the monodisperse core-shell microspheres have a core-shell structure and are characterized by uniform particle size and monodispersity with single encapsulation. The core is composed of PLLA microspheres, and the shell covering the core is a hydrogel layer, which is further composed of a GelHPA hydrogel layer (which can be referred to as PLLA-GelHPA core-shell microspheres). The thickness of the shell layer is controllable (estimated to be between 0.1 and 100 micrometers).

[0069] PLLA, as a polymer, provides microspheres with a certain degree of stiffness and hardness, enabling them to provide support and prevent damage under heavy loads. PLLA microspheres offer mechanical support to articular cartilage and exhibit good biocompatibility and biodegradability, gradually degrading into lactic acid and being excreted in the body over time. While hydrogel microspheres are low in strength and brittle, the viscoelasticity of the hydrogel provides some wear resistance when applied to the microspheres. In osteoarthritis, smooth hydrogels effectively improve the lubrication of cartilage in affected areas, reducing friction and wear, extending joint lifespan, and thus improving the quality of life for osteoarthritis patients. However, due to the erosive effects of osteoarthritis, the lubrication of cartilage in affected areas typically decreases significantly. The presence of the hydrogel layer in the core-shell microspheres described in this embodiment provides a certain degree of lubrication. Therefore, the core-shell microspheres with the hydrogel layer provide both wear resistance and support due to the presence of the PLLA core.

[0070] Furthermore, PLLA is a hydrophobic material that easily adheres to the nozzle wall. GelHPA hydrogel is hydrophilic and contains RGD (Receptor Group Deposition) cell adhesion sites. The hydrogel shell of the core-shell microspheres described in this invention enhances the compatibility of the hydrophobic microspheres with the aqueous environment, preventing direct contact between the hydrophobic cores and between the hydrophobic cores and the needle or container wall. Microspheres without hydrogel coating have low suspension stability in water and may experience clogging during injection. The hydrogel layer on the surface of the PLLA-GelHPA core-shell microspheres contains sequences that promote cell adhesion, making cells more willing to adhere to the surface of the core-shell microspheres for spreading and proliferation, which is particularly beneficial for applications such as cell analysis research.

[0071] Furthermore, the shell and core materials of the monodisperse core-shell microspheres have a wide range of options. PLLA material can be replaced with PLGA or PLA, and GelHPA can be replaced with silk fibroin or hyaluronic acid hydrogel. Due to the controllability of microfluidics and other technologies, core-shell microspheres of different sizes can be prepared.

[0072] The embodiments of this invention mainly combine microfluidic technology with enzyme crosslinking, which can realize the preparation of monodisperse core-shell microspheres. The operation is simple and reduces the cost of the preparation equipment for core-shell microspheres. Moreover, core-shell microspheres such as PLLA-GelHPA have good monodispersity and also have better hydrophilicity and biocompatibility.

[0073] To better illustrate the present invention, further examples are provided below. In these examples, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art. PLLA has a molecular weight of 15901-16135 and an intrinsic viscosity of 0.47. PVA degree of alcoholysis: 87.0-89.0% (mol / mol).

[0074] Example 1

[0075] See Figure 3 , Figure 3 This is a schematic diagram of the microfluidic system in the preparation method of Example 1 of this application. The oil phase inlet of the injection tube (a small-diameter circular capillary through which the inner phase flows) has a diameter of 500 μm, and the water phase inlet of the outer phase channel has a diameter of 1000 μm; the droplet outlet of the collection tube (a coaxial capillary) has a diameter of 500 μm, and the droplet generation point is between the outlet of the injection tube and the inlet of the collection tube (diameter of 250 μm), and the distance between them is adjustable.

[0076] The preparation procedure for monodisperse biodegradable microspheres is as follows:

[0077] (a) Oil phase solution: PLLA powder was dissolved in dichloromethane (DCM) to prepare a 5% (w / v) solution;

[0078] (b) Aqueous solution: PVA aqueous solution (1%, w / v);

[0079] (c) Collection solution: 0.5% (w / v) aqueous solution of PVA.

[0080] (d) During droplet formation, the aqueous phase flow rate was 30 ml / h, and the oil phase flow rate was 2 ml / h (the flow rates were adjusted using syringe pumps). The distance between the outlet of the injection tube and the inlet of the collection tube was 200 μm, and PLLA droplets formed in the collection tube. The PLLA droplets collected in a beaker (containing the collection solution) were then stirred at room temperature for 24 hours to allow dichloromethane to evaporate. The resulting PLLA microspheres were washed with a large amount of deionized water and then lyophilized for storage.

[0081] See Figure 4 , Figure 4 This is an optical microscope image of PLLA microspheres fabricated using a capillary microfluidic chip. The image shows that the obtained PLLA microspheres exhibit uniform particle size and monodispersity.

[0082] Example 2

[0083] The GelHPA hydrogel shell was formed through in-situ enzymatic cross-linking, resulting in monodisperse core-shell microspheres. First, 50 mg of PLLA microspheres prepared in Example 1 were directly immersed in a 2 mg / ml dopamine solution (3 ml) at room temperature for 2 hours to achieve dopamine adhesion on the microsphere surface. Then, the dopamine-coated microspheres were immersed in a 0.04 mg / ml HRP enzyme solution for 24 hours to achieve polydopamine binding to the enzyme. Subsequently, the obtained microspheres were washed three times with deionized water, and 100 μl of H2O2 (10 mM) and 200 μl of 20% GelHPA solution were added. Cross-linking was carried out at 25–30 °C to form a GelHPA hydrogel layer. After two minutes of reaction, 1 ml of deionized water was added to terminate the reaction, resulting in monodisperse core-shell microspheres with a uniform hydrogel layer on the surface.

[0084] See Figure 5 , Figure 5 This is an optical microscope image of PLLA-GelHPA core-shell microspheres prepared by enzyme cross-linking. The image shows a transparent, uniform GelHPA hydrogel layer on the surface of the opaque PLLA microspheres.

[0085] Example 3

[0086] Compared to Example 1, the distance between the incident tube and the collecting tube in Example 1(d) was adjusted to 50, 400, and 1000 μm. See also Figure 6 , Figure 6 The particle size of PLLA microspheres is determined by the varying spacing between the injection tube and the collection tube.

[0087] It can be seen that, with other parameters remaining constant, the larger the distance between the inlet and the outlet, the larger the microsphere size. When the distances are 50, 200, 400, and 1000 μm, the resulting microsphere diameters are approximately 48, 100, 170, and 230 μm, respectively. Therefore, 100 μm PLLA microspheres were selected for subsequent gel layer preparation.

[0088] Example 4

[0089] Compared to Example 1, the aqueous phase flow rate in Example 1(d) was adjusted to 20, 40, and 50 ml / h. See also... Figure 7 , Figure 7 The figures show the particle size of PLLA microspheres under different aqueous flow rates. It can be seen that the higher the aqueous flow rate, the smaller the particle size of the microspheres, decreasing from approximately 140 μm to approximately 30 μm.

[0090] Example 5

[0091] Compared to Example 1, the oil phase flow rate in Example 1(d) was adjusted to 3 and 4 ml / h. See also Figure 8 , Figure 8 The figures show the particle size of PLLA microspheres at different oil phase flow rates. It can be seen that the higher the oil phase flow rate, the larger the microsphere size, reaching over 130 μm.

[0092] Furthermore, characterization tests were performed on Examples 1 and 2, and the results are as follows.

[0093] See Figure 9 , Figure 9 The PLLA microsphere particle size is shown in Example 1. It can be seen that the microsphere particle size is distributed at around 100 μm, and it has the characteristics of uniform particle size and monodispersity.

[0094] See Figure 10 , Figure 10 The particle size of the PLLA-GelHPA microspheres was statistically analyzed in Example 2. Due to the presence of a hydrogel layer on the surface, the PLLA-GelHPA core-shell microspheres have a slightly larger particle size than the PLLA microspheres, with the diameter increasing from approximately 102 μm to approximately 108 μm. They also exhibit a uniform size distribution.

[0095] See Figure 11 , Figure 11 The image shows the surface morphology of the freeze-dried PLLA microspheres as characterized by scanning electron microscopy (SEM) in Example 1. The surface morphology of the PLLA microspheres is porous, which is due to the surface pores formed when dichloromethane volatilizes during the curing process.

[0096] See Figure 12 , Figure 12 The image shows the surface morphology of the freeze-dried PLLA-GelHPA microspheres as characterized by SEM in Example 2. As can be seen from the image, the PLLA-GelHPA core-shell microspheres have a smooth surface due to the presence of the gel layer.

[0097] Porous structures can be used for drug encapsulation, thereby achieving drug loading and sustained release. By encapsulating hydrophobic drugs in a polymer core and then encapsulating them in a hydrogel layer, sustained release can be achieved. Therefore, core-shell microspheres with a hydrogel layer can achieve a greater degree of sustained release.

[0098] See Figure 13 , Figure 13 The image shows the elemental analysis of the freeze-dried PLLA microspheres characterized by X-ray energy dispersive spectroscopy (EDS) in Example 1. As can be seen from the figure, the PLLA microspheres are composed of carbon (C) and oxygen (O).

[0099] See Figure 14 , Figure 14 Elemental analysis of the freeze-dried PLLA-GelHPA microspheres characterized by EDS in Example 2 was performed. Nitrogen (N) was found in the PLLA-GelHPA, confirming the successful formation of the hydrogel layer on the surface of the PLLA-GelHPA microspheres.

[0100] See Figure 15 , Figure 15 Infrared spectra of PLLA microspheres and PLLA-GelHPA microspheres. The comparison shows that PLLA exhibits higher infrared intensity at 1740 cm⁻¹. -1 The characteristic peak at 3200 cm⁻¹ is due to the effect of the ester group. -1 The broad peaks on the left and right represent the OH stretching vibration, at 1604 cm⁻¹. -1 The characteristic peaks at these locations are due to the asymmetric stretching vibrations of the COO layer in the hydrogel. These characteristics qualitatively indicate the presence of a GelHPA hydrogel shell on the PLLA microspheres. Therefore, comparison with the infrared results confirms the successful formation of the GelHPA hydrogel layer.

[0101] See Figure 16 , Figure 16 This image shows the fluorescence of the GelHPA hydrogel coating on the surface of PLLA-GelHPA core-shell microspheres. Fluorescently labeled GelHPA is visible using laser confocal microscopy (grafting and imaging are common techniques). The carboxyl groups of GelHPA can bind to Rhodamine 123, forming Rhodamine 123-grafted GelHPA. Rhodamine 123 emits green light under confocal laser illumination. Observing the prepared PLLA-GelHPA microspheres under the green light channel using a confocal microscope reveals a green outline on the microsphere surface. The PLLA microspheres are surrounded by a fluorescent layer, which is the GelHPA gel coating.

[0102] Example 6

[0103] See Figure 17 A 1ml pipette was used to aspirate a suspension of PLLA microspheres, which were then ejected from the tip of the pipette. The aggregation of the microspheres on the pipette tip wall was observed. It can be seen that the hydrophobic PLLA microspheres are prone to self-aggregation. Microspheres not coated with hydrogel are ejected unevenly from the pipette tip, and some microspheres aggregate and adhere to the inner wall of the pipette tip.

[0104] See Figure 18PLLA-GelHPA core-shell microspheres were aspirated into a 1ml pipette tip. Under injection force, the hydrogel-encapsulated polylactic acid microspheres were smoothly expelled from the tip without any residue. The hydrogel-coated core-shell microspheres exhibit high suspension stability and maintain good injectability even at high volume fractions, which is beneficial for implanting large numbers of microspheres into small tissues.

[0105] Example 7

[0106] See Figure 19 The lubrication performance of the microspheres was measured in linear reciprocating mode using a tribological testing machine (UMT-5, Bruker Nano Inc, Germany) (tested by a third-party testing platform). The physiological state of a joint was simulated by sliding the upper sample along the lower sample surface. All samples were prepared in PBS suspension and added to the contact surface as a lubricant between the upper and lower samples. It was observed that the coefficient of friction (COF) of the core-shell microspheres was lower than that of the PLLA microspheres, which have a higher coefficient of friction, indicating that the hydrogel layer of the core-shell microspheres provides a lubricating effect in addition to acting as a ball bearing.

[0107] Core-shell microspheres, based on a hydrogel shell and a polymer core, offer certain advantages in the treatment of osteoarthritis. The hydrogel shell effectively reduces surface roughness, endowing the microspheres with the ability to reduce friction and wear. The polymer core loads drugs, while the thickness of the hydrogel shell can be adjusted to control the drug release cycle. The core and shell of the microspheres can be modulated, allowing for fine-tuning of their biochemical and biomechanical properties to meet the needs of various tissues.

[0108] See Figure 20 The depth and width of the wear marks on the steel disc were observed (from left to right: PBS group, PLLA microsphere group, and PLLA-GelHPA core-shell microsphere group). Based on the surface morphology of the worn surface of the steel disc, it can be seen that the wear marks of the PLLA-GelHPA core-shell microspheres are lighter in color, indicating that the presence of the hydrogel coating reduces the wear effect of the microspheres on the substrate.

[0109] Example 8

[0110] See Figure 21 (Green represents live cells, red represents dead cells). Stem cells were cultured on PLLA microspheres, and live / dead cells were stained on the third day of culture to observe cell growth on the microspheres (culture and staining are familiar to industry professionals). The image shows that the cells on the surface of the PLLA microspheres are mostly viable.

[0111] See Figure 22Stem cells were cultured on PLLA-GelHPA core-shell microspheres, and the cell growth on the microspheres was observed. The core-shell microsphere group showed more cells on its surface, which is attributed to the better biocompatibility of GelHPA hydrogel as a natural polymer material.

[0112] See Figure 23 On day 3 of cell culture, the cytoskeleton and nuclei were stained to observe the cell spreading on the surface of the PLLA microspheres. Cells showed some adhesion and spreading on the PLLA microsphere surface, which is attributed to the hydrophobicity of the PLLA microspheres.

[0113] See Figure 24 Nuclear skeleton staining was performed on PLLA-GelHPA core-shell microspheres cultured for three days. Cells were observed to almost completely encapsulate the microspheres, resulting in cell clusters. This is due to the presence of cell adhesion sites (RGDs) in the GelHPA hydrogel, which allow for greater cell adhesion and spreading on the surface of the core-shell microspheres.

[0114] Example 9

[0115] Monodisperse biodegradable microspheres were prepared according to the steps in Example 1;

[0116] Furthermore, monodisperse core-shell microspheres were obtained following the steps of Example 2. The difference was that the reaction time was changed to five minutes. In the resulting monodisperse core-shell microspheres, the thickness of the hydrogel shell layer increased to 5 μm.

[0117] As can be seen from the above embodiments, the core-shell microspheres of the present invention have the characteristics of uniform particle size and monodispersity. Due to the presence of hydrogel, the surface is smooth, exhibiting high suspension stability and maintaining good injectability even at high volume fractions. They also have a lubricating effect, reducing wear on the substrate and demonstrating good biocompatibility. In particular, the GelHPA hydrogel contains cell adhesion sites (RGD), which is beneficial for cell survival. The hydrogel shell in the embodiments of the present invention is mainly formed through in-situ enzymatic cross-linking. The degree of cross-linking can be controlled by the reaction time, hydrogen peroxide concentration, enzyme concentration, and hydrogel concentration to obtain hydrogel shells of different thicknesses. It can be used for drug delivery, cell adhesion and cell delivery carriers, medical aesthetic fillers, scaffold materials, joint lubrication, and catalyst preparation, showing promising application prospects.

[0118] The above examples are only used to illustrate the technical features and implementation process of the present invention, and are not intended to limit the technical solutions of the present invention. It should be noted that those skilled in the art can still make modifications or equivalent substitutions to the present invention without departing from the principle of the present invention, and all such modifications or substitutions are covered by the protection of the present invention.

Claims

1. A method for preparing monodisperse core-shell microspheres, characterized by, The method comprises the following steps: S1, using a liquid containing degradable polymer in the dispersed phase, forming monodisperse degradable polymer microspheres by microfluidic process or emulsification method; S2, the surface of the degradable polymer microspheres is combined with horseradish peroxidase by one or more of dopamine self-polymerization, electrostatic adsorption and chemical grafting, and under the catalysis of the horseradish peroxidase, the degradable polymer microspheres are cross-linked and coated with hydrogel material to obtain monodisperse core-shell microspheres with a hydrogel layer; the hydrogel material is a hydrogel system that can be cross-linked by enzyme initiation.

2. The production method according to claim 1, characterized by, The hydrogel material is hydroxyphenylpropionic acid hydrogel, silk fibroin or hyaluronic acid hydrogel.

3. The preparation method according to claim 1, characterized in that, The particle size of the degradable polymer microspheres is 10-1000 μm.

4. The production method according to claim 3, characterized by, The particle size of the degradable polymer microspheres is 30-250 μm.

5. The production method according to claim 4, characterized by, The particle size of the degradable polymer microspheres is 40-200 μm.

6. The production method according to claim 5, wherein The particle size of the degradable polymer microspheres is 50-180 μm.

7. The preparation method according to claim 1, characterized in that, The degradable polymer is at least one of PLA, PLGA, PCL and PGA.

8. The production method according to claim 7, characterized by, The degradable polymer is PLA.

9. The method of any one of claims 1-8, wherein, The microfluidic process in step S1 comprises: Injecting an oily dispersed phase liquid containing degradable polymer into the inner phase channel of a coaxial microfluidic chip, injecting an aqueous continuous phase liquid into the outer phase channel, collecting degradable polymer droplets at the outlet end of the inner phase channel, removing the solvent of the oily dispersed phase liquid, and then washing with water and freeze-drying to obtain monodisperse degradable polymer microspheres; The emulsification method in step S1 comprises: stirring and emulsifying the oily dispersed phase liquid containing degradable polymer and the aqueous continuous phase liquid to form degradable polymer liquid, then removing the solvent of the oily dispersed phase liquid, and then washing with water and freeze-drying to obtain monodisperse degradable polymer microspheres.

10. The method of claim 9, wherein, In step S1, the coaxial microfluidic chip is a glass capillary chip or a PDMS chip; the outlet end of the inner phase channel is provided with a coaxial collection channel, and the straight-line distance between the inlet of the collection channel and the outlet of the inner phase channel is 30-1000 μm.

11. The method of claim 10, wherein, In step S1, the straight-line distance between the inlet of the collection channel and the outlet of the inner phase channel is 50-500 μm.

12. The method of claim 10, wherein, In step S1, the aqueous continuous phase liquid is a PVA aqueous solution, and the flow rate is adjusted by a syringe pump to be 1-100 mL / h.

13. The method of claim 12, wherein, In step S1, the flow rate of the aqueous continuous phase liquid is adjusted by a syringe pump to be 10-50 mL / h.

14. The method of claim 13, wherein, In step S1, the flow rate of the aqueous continuous phase liquid is adjusted by a syringe pump to be 20-40 mL / h.

15. The preparation method according to claim 12, characterized in that, In step S1, the solvent of the oily dispersed phase liquid is at least one of dichloromethane, chloroform, ethyl acetate, acetone and tetrahydrofuran; and the flow rate of the oily dispersed phase liquid is 0.1-50 mL / h.

16. The method of claim 15, wherein, In step S1, the solvent of the oily dispersed phase liquid is dichloromethane; and the flow rate of the oily dispersed phase liquid is 1-5 mL / h.

17. The method of making according to any one of claims 1-8, wherein, The step S2 comprises: The degradable polymer microspheres are immersed in a dopamine solution and then in a horseradish peroxidase solution, so that the dopamine is self-polymerized and combined with the horseradish peroxidase, to obtain microspheres with horseradish peroxidase on the surface; The microspheres with horseradish peroxidase on the surface are mixed with a hydrogel material to perform a cross-linking reaction, to obtain monodisperse core-shell microspheres with a hydrogel layer.

18. The method of claim 17, wherein, In the step S2, the concentration of the dopamine solution is 0.1-5 mg / mL, and the concentration of the horseradish peroxidase solution is 0.01-1 mg / mL. The cross-linking reaction is performed for 0.1-5 minutes, and water is added to terminate the reaction to prevent over-cross-linking.

19. The method of claim 18, wherein, In the step S2, the concentration of the horseradish peroxidase solution is 0.01-0.1 mg / mL.

20. A monodisperse core-shell microsphere characterized by, The monodisperse core-shell microspheres obtained by the preparation method according to any one of claims 1-19 have smooth surfaces and are used for drug delivery, cell adhesion and cell delivery carriers, medical and beauty filling materials, scaffold materials, bone and joint lubrication and catalyst preparation.

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