Magnetic microsphere as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedical engineering and drug delivery, in particular to a magnetic microsphere and a preparation method and application thereof. The preparation method of the magnetic microspheres comprises the following steps: dissolving curcumin in absolute ethyl alcohol to obtain a uniform solution; and injecting the uniform solution into water for self-assembly, and then freeze-drying to obtain the curcumin nanoparticles, the preparation method comprises the following steps: dissolving methacrylated gelatin, Fe3O4 coated SiO2 magnetic nanoparticles, curcumin nanoparticles and a photoinitiator in water to obtain an injection phase solution; forming uniform liquid drops from the injection phase solution through a micro-fluidic device, and then carrying out photocuring crosslinking to obtain the magnetic microspheres. The magnetic microspheres can be adsorbed to the esophagus in a targeted manner under the action of an external magnetic field, and curcumin is triggered by reflux gastric juice (gastric acid, pepsin and bile salt) to be released, so that the gastroesophageal reflux disease is treated.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and drug delivery, and in particular to a magnetic microsphere, its preparation method, and its application. Background Technology

[0002] Gastroesophageal reflux disease (GERD) is a common chronic digestive disorder characterized by frequent reflux of gastrointestinal contents into the esophageal lumen. Long-term accumulation can lead to esophageal mucosal inflammation, epithelial cell damage, and impaired barrier function. Current clinical treatment strategies primarily include proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs), both of which reduce reflux irritation by inhibiting gastric acid secretion. However, clinical studies have shown that approximately 40% of patients do not achieve complete symptom relief after treatment with these drugs, and long-term use may cause adverse reactions such as malabsorption, gut microbiota imbalance, and increased risk of infection. Therefore, existing therapies have limitations in both efficacy and safety, necessitating the development of novel and safer GERD treatment strategies.

[0003] Previous studies have shown that GERD-related esophageal epithelial barrier damage is not solely caused by direct corrosion from an acidic environment, but is closely related to the abnormal activation of inflammatory pathways. Chemokine-driven inflammatory responses are a crucial mechanism, and reactive oxygen species (ROS) are considered key mediators of the inflammatory cascade. In recent years, increasing evidence confirms that oxidative stress plays a central role in the pathogenesis of GERD. Specifically, excessive ROS can amplify local inflammatory responses by activating signaling pathways such as NF-κB, inducing the release of pro-inflammatory factors. Simultaneously, persistently elevated ROS and inflammatory factors further damage mitochondrial function, weakening the mitochondria's ability to scavenge free radicals. This interaction between ROS accumulation, inflammatory factor release, and mitochondrial dysfunction forms a vicious cycle, leading to escalating inflammation and ultimately severe damage to the structure and function of esophageal epithelial cells. Therefore, effectively regulating oxidative stress levels and blocking ROS-mediated inflammatory cascade responses has become a highly promising intervention strategy for the management and treatment of GERD.

[0004] Curcumin is a polyphenolic compound derived from natural plants, possessing significant anti-inflammatory and antioxidant activities. Its mechanism of action primarily includes reducing oxidative stress through indirect pathways such as direct free radical scavenging and regulation of the body's antioxidant enzyme system. However, curcumin itself suffers from poor water solubility and low bioavailability, limiting its widespread clinical application. Especially in the treatment of GERD, due to the unique anatomy and local environment of the esophagus, traditional administration methods struggle to achieve effective local drug delivery, resulting in limited efficacy.

[0005] Therefore, how to achieve precise delivery and sustained release of curcumin in the local environment of GERD, so as to effectively alleviate the inflammatory response and improve esophageal mucosal damage, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the problems of poor water solubility, low bioavailability, and unsatisfactory local treatment effects of curcumin in existing technologies, this invention provides a magnetic microsphere and its preparation method. This magnetic microsphere possesses dual functions of reactive oxygen species (ROS) scavenging and magnetic targeted adsorption, offering a new approach and technical pathway for the precise treatment of gastroesophageal reflux disease (GERD). First, this invention uses the Stöber method to synthesize SiO2-encapsulated Fe3O4 nanoparticles, obtaining core-shell magnetic nanomaterials with good biocompatibility and stable magnetism. Second, highly dispersed water-soluble curcumin nanoparticles are prepared via antisolvent precipitation to significantly improve the solubility and bioavailability of curcumin. The two types of nanoparticles are then introduced into a methacrylamide gelatin (GelMA) system, microfluidically shaped, and photocured under the action of a photoinitiator to obtain uniformly shaped, controllable-size nanohybrid hydrogel microspheres (i.e., magnetic microspheres). Under the influence of an external magnetic field, the nano-hybrid hydrogel microspheres can efficiently target and adhere to the esophagus, achieving targeted drug delivery and enrichment. At the same time, the curcumin nanoparticles loaded in the hydrogel can continuously scavenge ROS and inhibit the release of inflammatory factors, thereby effectively alleviating the chronic inflammatory response caused by GERD, promoting the repair of mitochondrial function in esophageal epithelial cells, and improving local tissue damage, showing excellent therapeutic potential and application prospects.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for preparing magnetic microspheres, comprising the following steps: Curcumin was dissolved in anhydrous ethanol to obtain a homogeneous solution; the homogeneous solution was injected into water to perform self-assembly, and then freeze-dried to obtain curcumin nanoparticles. Methacryloyl gelatin (GelMA), Fe3O4@SiO2 magnetic nanoparticles, curcumin nanoparticles and a photoinitiator were dissolved in water to obtain an injection phase solution. The injected phase solution is formed into uniform droplets using microfluidic technology, followed by photocuring and crosslinking to obtain the magnetic microspheres.

[0008] Curcumin, derived from the rhizome of the ginger plant (Curcuma longa), possesses significant anti-inflammatory, antioxidant, antitumor, and neuroprotective biological activities. However, its clinical application is limited by its extremely low water solubility, bioavailability, and stability. Curcumin nanoparticles are drug delivery systems made by processing the natural polyphenol compound curcumin into nanoscale particles using nanotechnology. Nano-sizing significantly improves its dispersibility in aqueous phase, reduces rapid metabolism and clearance in vivo, thereby enhancing efficacy. This invention uses an antisolvent precipitation method to prepare curcumin nanoparticles. Furthermore, curcumin nanoparticles can achieve both sustained and controlled release, aiding in targeted delivery and increasing the cumulative concentration of curcumin in specific tissues or tumor sites. Curcumin nanoparticles show promising applications in cancer treatment, inflammatory diseases, cardiovascular diseases, and neurodegenerative diseases.

[0009] GelMA is a photosensitive crosslinked hydrogel material made from natural gelatin modified with methacrylic anhydride. It combines the biocompatibility and biodegradability of gelatin with excellent mechanical properties and structural stability after photocuring. GelMA offers several advantages in drug delivery. Its three-dimensional porous network structure allows for controllable pore size and mechanical strength by adjusting concentration, degree of methacrylization, and photocrosslinking conditions, thereby precisely regulating drug loading and release rates. GelMA can encapsulate various types of drugs, including small molecule compounds, proteins, nucleic acids, and nanoparticles, and provides excellent protection for bioactive molecules, reducing degradation and inactivation. GelMA can be fabricated into regular microspheres or fiber structures using microfluidic technology, enabling sustained release and even stimulus-responsive release in its drug delivery system. The magnetic microspheres in this invention can stably release encapsulated curcumin nanoparticles under the influence of refluxed gastric acid and bile salts. Furthermore, magnetic microspheres can encapsulate magnetic nanoparticles for targeted esophageal delivery therapy. GelMA has shown broad application prospects as a drug delivery tool, and is especially suitable for treatment strategies that require a balance between biocompatibility, biodegradability and precise release control.

[0010] The magnetic microspheres of this invention primarily rely on the stimulation of refluxed gastric juice (acid + pepsinogen) to degrade the hydrogel microspheres, thereby stably releasing the encapsulated curcumin. Commonly used methacryloyl chitosan (CSMA), methacryloyl hyaluronic acid (HAMA), and methacryloyl sodium alginate (AlgMA) are not suitable for this invention because AlgMA is more stable in the acidic environment of the stomach, while CSMA and HAMA are unaffected by pepsinogen. Low pH environments reduce the stability of ionic bonds, but their overall degradation efficiency is lower than that of gelma. Therefore, the methacryloyl gelatin of this invention is irreplaceable.

[0011] In a preferred embodiment of the present invention, the concentration of the homogenized solution is 1 mg / mL; the self-assembly temperature is 60~70°C and the time is 1 hour.

[0012] In a preferred embodiment of the present invention, the mass fraction of methacrylamide gelatin in the injected phase solution is 80 wt%, the mass fraction of Fe3O4@SiO2 magnetic nanoparticles is 8-10 wt%, the mass fraction of curcumin nanoparticles is 0.01 wt%, and the mass fraction of photoinitiator is 2-3 wt%; the photoinitiator is photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) or photoinitiator 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone).

[0013] In this invention, if the mass fraction of Fe3O4@SiO2 magnetic nanoparticles in the injected phase solution is higher than 10wt%, it will easily clog the microfluidic device channel; if the mass fraction of Fe3O4@SiO2 magnetic nanoparticles is lower than 8wt%, the microspheres will have insufficient magnetic force and will be difficult to be adsorbed by an external magnetic field.

[0014] In some embodiments of the present invention, when the mass fraction of curcumin nanoparticles is higher than 0.01 wt%, cytotoxicity increases; when the concentration is too low, the anti-inflammatory effect is insufficient.

[0015] In some embodiments of the present invention, the GelMA used is 90% substituted GelMA.

[0016] In a preferred embodiment of the present invention, the step of forming uniform droplets from the injected phase solution using microfluidic technology refers to: adding a surfactant to an oily matrix to obtain a continuous phase solution; The injected phase solution and the continuous phase solution are respectively delivered to the input and output pipes of the microfluidic device to flow coaxially, so that the injected phase solution forms uniform droplets; The surfactant is at least one of Span 80, Tween 85, and Span 20; the oily matrix is ​​at least one of paraffin oil, castor oil, and silicone oil; the mass fraction of the surfactant in the continuous phase solution is 1-10 wt%, preferably 1-5 wt%, and more preferably 3-5 wt%.

[0017] In a preferred embodiment of the present invention, during coaxial flow, the flow rate of the injected phase solution is 0.1~1 mL / h (preferably 0.3~1 mL / h, more preferably 0.3~0.6 mL / h), the flow rate of the continuous phase solution is 1~10 mL / h (preferably 1~7 mL / h, more preferably 4~7 mL / h), and the system temperature is 35~50°C (preferably 35~45°C, more preferably 40~45°C).

[0018] When performing coaxial flow motion, the purpose of controlling the system temperature to 35~50°C is to prevent the methacrylamide gelatin from solidifying and clogging in the microfluidic chip.

[0019] In a preferred embodiment of the present invention, the photocuring crosslinking is performed using near-ultraviolet light with a wavelength of 405 nm, and crosslinking is carried out at room temperature for 3 to 5 minutes.

[0020] In this invention, the parameters for freeze drying are not particularly limited, and the freeze drying parameters (pressure, temperature, time) commonly used by those skilled in the art are adopted.

[0021] The Fe3O4@SiO2 magnetic nanoparticles used in this invention possess superparamagnetism and have a core-shell structure, comprising an internal Fe3O4 core and a SiO2 modification layer encapsulating the surface of the Fe3O4 core. The Fe3O4@SiO2 magnetic nanoparticles can be prepared by the Stöber method. The average particle size of the Fe3O4@SiO2 magnetic nanoparticles in this invention is 100–300 nm, preferably 100–200 nm, and more preferably 100–150 nm.

[0022] In this invention, the injected phase solution and the continuous phase solution are respectively delivered to the input and output pipes of the microfluidic device for coaxial flow. The injected phase solution flows coaxially through the capillary collection tube (located inside the output pipe) via the input pipe. The column of injected phase solution in the capillary collection tube is "broken" under the action of velocity difference and shear force, forming uniform droplets under the promotion of interfacial tension. The fluid shear force is generated by the high-speed relative flow of the inner phase in the capillary collection tube and the outer phase between the capillary collection tube and the output pipe in the channel of the coaxial flow microfluidic device and their interaction at the outlet.

[0023] The second technical solution of the present invention is a magnetic microsphere prepared by the above-mentioned preparation method.

[0024] The magnetic microspheres of this invention are obtained by encapsulating hydrophilic curcumin nanoparticles and highly biocompatible Fe3O4@SiO2 magnetic nanoparticles in methacrylamide gelatin, followed by photocuring and cross-linking. The magnetic microspheres of this invention are gel-state spheres with an average particle size of 75-100 μm, preferably 75-90 μm, and more preferably 80-85 μm; they exhibit long-term stability in aqueous solutions and can be attracted by an external magnetic field.

[0025] The magnetic microspheres of the present invention do not degrade after swelling and reaching equilibrium in water, saline or PBS (e.g., at room temperature or 4-25°C) and are left for one week (i.e., the size and morphology of the magnetic microspheres remain unchanged or essentially unchanged).

[0026] The third technical solution of the present invention is the application of the above-mentioned magnetic microspheres in the preparation of drugs for treating gastroesophageal reflux disease.

[0027] The magnetic microspheres of the present invention have a trigger release characteristic in a gastric acid + pepsin environment. After being placed in a mixture of simulated gastric acid and bile salts for 12 hours, the curcumin release rate reaches a predetermined level.

[0028] The magnetic microspheres of this invention can achieve targeted positioning in the esophagus under an external magnetic field, and the release of curcumin is triggered by refluxed gastric juice, thereby clearing ROS in the esophageal epithelium, inhibiting the secretion of inflammatory factors, achieving highly efficient anti-inflammatory and antioxidant effects, promoting the normal repair of chronic inflammation of the esophageal epithelium, and treating gastroesophageal reflux disease.

[0029] The curcumin nanoparticles prepared using the method of this invention have an average particle size of 200-500 nm, preferably 200-400 nm, and more preferably 200-300 nm. The curcumin nanoparticles of this invention can inhibit the production of IL-6, IL-8, TNF-α, and ROS in the esophageal mucosa, repair mitochondrial damage, and thereby improve chronic inflammatory damage of the esophageal mucosa.

[0030] The fourth technical solution of the present invention is a microfluidic device for preparing the above-mentioned magnetic microspheres, comprising an input pipe, an output pipe, and a capillary collection tube; The outlet of the input pipe, the outlet of the output pipe, and the outlet of the capillary collection tube are coaxially aligned to form a composite flow outlet (i.e., the input pipe is coaxially nested inside the output pipe). The capillary collector is located inside the output pipe; the end of the input pipe extends into the center of the output pipe and into the capillary collector.

[0031] In some embodiments of the present invention, the microfluidic device further includes a connecting sleeve, a fixing bracket, an injection pump, an inner phase inlet, and an outer phase inlet; The input and output pipes are fixedly connected to the fixed bracket via connecting sleeves; The connecting bracket secures the position of the input and output pipes. The injected phase solution (internal phase fluid) is connected to the input pipeline through the internal phase inlet; The continuous phase solution (external phase fluid) is connected to the output pipe through the external phase inlet; The input pipe, output pipe, and capillary collection tube are cylindrical glass capillaries with an inner diameter in the micrometer range. The structure and dimensions of the connecting bracket, input pipe, output pipe, and capillary collection tube are designed in a "nested" relationship; The inner diameter of the inlet pipe is 30~80um, preferably 45~80um, and more preferably 45~60um; The microfluidic chip has a flat-tipped stainless steel injection needle at its inner phase inlet, which is connected to a syringe containing an aqueous phase via a PE tubing. The microfluidic chip also has a flat-tipped stainless steel injection needle at its outer phase inlet, which is connected to a syringe containing an oil phase via a PE tubing. The outlet of the capillary collection tube is located above the collector containing the oil phase and is slightly submerged in the collected phase solution.

[0032] In this invention, the diameter of the magnetic microspheres needs to be adjusted to 75~100um, more preferably 80~85um. However, conventional microfluidic devices in the prior art are difficult to prepare microspheres of this size. Therefore, a thinner capillary collection tube needs to be added to the output channel of conventional microfluidic devices in the prior art.

[0033] The syringe pump drives the inner phase fluid through the inlet pipe into the capillary collector, where it flows coaxially with the outer phase fluid driven by the syringe pump through the outlet pipe. The syringe pump is used to control the flow rates of the inner and outer phase fluids separately, thereby adjusting the droplet size and formation rate.

[0034] A magnetic GelMA aqueous solution in the injection phase solution is injected into a microfluidic device through a flat-mouthed stainless steel needle. The microfluidic device subjects the injection phase solution to fluid shear force within the continuous phase solution. The magnitude of this force is primarily determined by the properties and flow rates of the two-phase fluids (injection phase and continuous phase). This invention utilizes the immiscibility of oil and water, employing coaxial flow to break the injection phase solution into uniform droplets under fluid shear force in the oil phase collection liquid. Under defined conditions, the droplet size is determined by the flow rate and concentration of the injection phase solution, as well as the properties and flow rate of the continuous phase solution. Generally, within a controllable range of injection phase solution concentration, higher concentrations and flow rates of the injection phase solution and the lower flow rates of the continuous phase solution result in larger droplet sizes; conversely, lower concentrations and lower flow rates result in smaller droplet sizes.

[0035] The present invention has the following technical effects: (1) The magnetic microspheres of the present invention are simple to prepare, low in cost, have good biocompatibility, controllable size, and uniform size.

[0036] (2) Compared with traditional small molecule drugs, the present invention is derived from natural polyphenol curcumin, which has lower toxicity and side effects, and improves its water solubility and enhances its anti-inflammatory efficacy by making nanoparticles.

[0037] (3) This invention utilizes hydrogel microspheres to encapsulate magnetic nanoparticles, enabling targeted adsorption onto the esophagus under the influence of an external magnetic field. Simultaneously, it employs curcumin nanoparticles, a polyphenolic antioxidant, to effectively scavenge reactive oxygen species in the esophageal inflamed epithelium, inhibit the expression of inflammatory factors, and promote mitochondrial function repair by regulating the NF-κB signaling pathway, thus achieving more efficient anti-inflammatory and antioxidant properties.

[0038] (4) In this invention, curcumin nanoparticles are loaded into a hydrogel microsphere network to achieve sustained drug release under gastric acid environment. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This is a schematic diagram of the coaxial microfluidic device in this invention. Reference numerals: 1. Input pipe; 2. Output pipe; 3. Capillary collection tube; 4. Inner phase inlet; 5. Outer phase inlet; 6. Fixing bracket.

[0041] Figure 2 The images show a static image and a scanning electron microscope (SEM) image of the curcumin nanoparticle suspension prepared in Example 2 of this invention; wherein, a is a state image of the curcumin nanoparticle suspension after standing for 12 hours, and b is a scanning electron microscope image of the curcumin nanoparticles.

[0042] Figure 3 The images shown are optical microscope images and scanning electron microscope images of the magnetic microspheres prepared in Example 3 of the present invention; wherein, a is an optical microscope image and b is a scanning electron microscope image.

[0043] Figure 4 The in vitro and in vivo therapeutic effects of the magnetic microspheres prepared in Example 4 of this invention are shown; where a represents the expression of inflammatory factors in the esophageal epithelial cell inflammation model, b represents the intracellular ROS content, and c represents the HE staining images of the esophageal mucosa of mice in each group after different treatments in the gastroesophageal reflux animal model.

[0044] Figure 5 This is a schematic diagram illustrating the working principle of the magnetic microspheres of this invention. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] This invention, through extensive research and screening, is the first to develop a magnetic microsphere constructed using microfluidic technology for the targeted delivery of curcumin nanoparticles to treat gastroesophageal reflux disease (GERD). Compared with existing technologies, the magnetic microspheres prepared in this invention possess both magnetic targeting and enrichment properties and gastric acid-bile salt-triggered release characteristics, enabling precise local drug delivery at GERD lesion sites. This effectively inhibits esophageal epithelial inflammation and oxidative stress, and repairs mitochondrial function and the mucosal barrier. The method provided by this invention offers advantages such as ease of operation, high throughput, controllable quality, low cost, and good biocompatibility, providing a new potential solution for the management and treatment of GERD.

[0051] Specifically, this invention utilizes microfluidic technology to co-load curcumin nanoparticles and Fe3O4@SiO2 magnetic nanoparticles onto a methacrylamide gelatin (GelMA) matrix, followed by photocuring and cross-linking under the action of a photoinitiator, thereby forming a uniformly structured and stable gel-like magnetic microsphere. These magnetic microspheres can achieve targeted localization in the esophagus under an applied magnetic field, and the release of curcumin nanoparticles is triggered by refluxed gastric juice. The released curcumin nanoparticles can efficiently scavenge local reactive oxygen species (ROS), inhibit excessive activation of the NF-κB signaling pathway, alleviate the release of inflammatory factors, repair mitochondrial function, and ultimately improve chronic inflammation and tissue damage of the esophageal epithelium. In vitro and in vivo experiments have verified that the curcumin-loaded magnetic hydrogel microspheres prepared in this invention exhibit significant advantages in both safety and efficacy, demonstrating their feasibility and application prospects as a novel drug delivery platform for the precise treatment of GERD.

[0052] Microfluidics, as an emerging material preparation method, can prepare microspheres with uniform particle size and controllable structure under continuous and stable operating conditions, providing an efficient platform for drug delivery and local treatment.

[0053] Fluid shear force, the "internal friction" generated by velocity differences within a fluid, is the core driving force for droplet generation in microfluidics. In a coaxial microfluidic device, the injected phase solution is injected into a capillary collection tube through an input channel, while the continuous phase solution flows coaxially along an output channel. When the continuous phase solution flows at high speed, its velocity gradient generates significant tangential and normal shear forces at the interface between the injected and continuous phase solutions. This shear force gradually thins the neck of the injected phase solution column and eventually cuts it off when the interfacial tension is insufficient to maintain a complete column, thus forming monodisperse droplets.

[0054] This invention provides a method for preparing curcumin-loaded magnetic microspheres by precisely controlling the two-phase flow rate ratio, fluid viscosity, and channel size to adjust the fluid shear force.

[0055] Coaxial microfluidic systems utilize concentrically arranged channels to precisely manufacture monodisperse droplets using strong and symmetrical shear forces between fluids. A typical coaxial system consists of two or more capillaries or microchannels with coincident central axes. The system comprises an inner injection capillary coaxially arranged with an outer channel, and a capillary collection tube nested within the outer channel. The dispersed phase fluid is pumped in through the injection capillary, while the continuous phase fluid flows in the outer channel, forming a uniformly sized microdroplet or microsphere perfectly enveloped by the continuous phase. Its 3D symmetrical shear is far more powerful and efficient than the 2D shear of planar T-junctions or flow focusing devices, thus enabling the stable "pulling apart" of the dispersed phase without extreme dependence on the viscosity of the collection phase, thereby producing uniform droplets. However, its manufacturing and assembly process is complex, especially the precise alignment of multiple capillaries, which is a significant technical challenge. Any slight misalignment can disrupt the symmetry of the shear force, affecting droplet quality. Secondly, the fine tips of the inner channels pose a high risk of clogging when using fluids that easily accumulate particulate matter (magnetic particles). Therefore, this invention provides a method for the controllable preparation of droplets ranging from micrometers to hundreds of micrometers in size by precisely adjusting the flow rate ratio of the injected phase solution to the continuous phase solution, the channel gap size, and the viscosity and interfacial tension of the two-phase fluids. Coaxial microfluidic systems have broad application potential in drug delivery, functional material synthesis, and bioanalysis.

[0056] This invention utilizes microfluidic technology to prepare methacrylamide gelatin (GelMA) microspheres (Cur-MNPs@GelMA microspheres, i.e., magnetic microspheres) loaded with curcumin nanoparticles (Cur) and magnetic nanoparticles (MNPs). These magnetic microspheres are generated into uniformly sized droplets using a microfluidic device and then photocrosslinked and cured to form a stable microsphere structure, exhibiting good biocompatibility and high adhesion. Simultaneously, the magnetic microspheres can achieve esophageal targeting with an external magnetic field, and the sustained release of curcumin is triggered by refluxed gastric contents (mainly gastric acid, pepsin, and bile salts). These microspheres can significantly inhibit the production of inflammatory factors (IL-6, IL-8, and TNF-α) and reactive oxygen species induced by gastric acid and bile salts, exerting anti-inflammatory and antioxidant effects by inhibiting the NF-κB signaling pathway and restoring mitochondrial function. In vitro and in vivo studies have shown that these magnetic microspheres can alleviate tissue damage, inhibit inflammation, and repair the epithelial barrier in a GERD mouse model. They have significant application potential in the treatment of GERD and other related fields. Furthermore, the preparation method provided by this invention has the advantages of high throughput, simple operation, and low cost, and is suitable for the local treatment and drug delivery of GERD.

[0057] The main advantages of this invention include: (1) This invention is the first to develop a magnetic microsphere with magnetic adsorption properties, adjustable size, high uniformity, and formed based on the encapsulation characteristics of methacrylamide gelatin and photocuring. A method is used to obtain uniformly sized and morphologically uniform magnetic droplets through fluid shear force in a coaxial microfluidic system, followed by photocuring and cross-linking to form curcumin-loaded magnetic microspheres. This method is controllable, low-cost, high-throughput, and requires no complex equipment.

[0058] (2) The present invention utilizes a microfluidic device assembled with multiple coaxial capillaries precisely aligned. Through fluid shear force, a magnetic aqueous solution of methacrylamide gelatin is used as the injection phase, and an oily solution mixed with surfactant is used as the continuous phase. The flow rate ratio between the injection phase and the continuous phase is precisely adjusted by an injection pump to control the fluid shear force to cut the injection phase into monodisperse droplets of the target size. After photocuring and crosslinking by light (405nm wavelength), highly biocompatible magnetic microspheres are finally obtained.

[0059] (3) The method of the present invention perfectly combines the characteristics of precise assembly of multiple capillaries, fine control of fluid shear force, and encapsulation of nano-curcumin and magnetic Fe3O4 nanoparticles, so that the magnetic microspheres loaded with curcumin nanoparticles have good biosafety, excellent size uniformity, high throughput, easy control of properties, and have potential application value in targeted local anti-inflammatory treatment.

[0060] (4) The components of the magnetic microspheres of the present invention are all biocompatible materials, and have excellent biocompatibility.

[0061] (5) The magnetic microspheres of the present invention can be targeted and adsorbed onto the esophagus under the action of an external magnetic field.

[0062] (6) The curcumin nanoparticles loaded with magnetic microspheres of the present invention can be stably released under the action of refluxed gastric acid and bile salts, thereby effectively clearing reactive oxygen species in the esophageal inflammatory epithelium, inhibiting the expression of inflammatory factors, and promoting mitochondrial function repair by regulating the NF-κB signaling pathway, thus achieving more efficient anti-inflammatory and antioxidant performance.

[0063] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0064] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0065] The microfluidic device used in the embodiments of the present invention is as follows: Figure 1 As shown: The diameter of the inlet at the end of the input pipe is 45~60µm, extending into the center of the output pipe. One end of the inner capillary collection tube extends slightly into the end of the input pipe through the output pipe, ensuring that the flow axes of the inner and outer fluids coincide to leave an annular gap for the outer phase to flow through. Injection needles are placed at the center of the inlet of both the input and output pipes, serving as inlets for the inner phase (injected phase solution) and the outer phase (continuous phase solution). The microfluidic device is sealed with epoxy resin. Throughout the assembly process, the pipes must be kept clean to avoid blockage or contamination of the microchannels, and to ensure that the final microfluidic chip has sufficient mechanical strength and sealing to prevent liquid leakage. The injected phase is connected to the injection needle on the input side of the microfluidic device via a syringe (placed in the injection pump) and a polyethylene tube.

[0066] Example 1: Preparation of magnetic nanoparticles First, 30 mL of anhydrous ethanol, 5 mL of deionized water, 0.1 g of Fe3O4 nanoparticles (particle size 10-20 nm), and 0.5 mL of ammonia (25%) were added to a reaction vessel and thoroughly mixed under magnetic stirring. The mixture was then ultrasonically agitated for 30 minutes to improve dispersion uniformity and promote stable suspension of the Fe3O4 nanoparticles in the solution. Under set water bath conditions (temperature controlled at 50°C, stirring speed at 800 rpm), the mixture was continuously stirred, with 50 μL of tetraethyl orthosilicate (TEOS) added dropwise every 30 minutes during stirring, four times in total, to ensure uniform deposition and controllable coverage of the silica shell. The entire reaction lasted for 2 hours. Particles were separated by adsorption using an external magnet, and the supernatant was discarded. The particles were washed four times alternately with ethanol and deionized water. Finally, the uniformly coated and highly biocompatible Fe3O4@SiO2 magnetic nanoparticles were redispersed in ethanol for storage.

[0067] Example 2: Preparation of curcumin nanoparticles Curcumin nanoparticles were prepared using an antisolvent precipitation method. First, curcumin was dissolved in anhydrous ethanol to prepare a homogeneous solution with a concentration of 1 mg / mL. Then, 1 mL of the curcumin-ethanol solution was drawn using a syringe and slowly added to 20 mL of deionized water at a constant rate of 1 mL / h using a syringe pump. During the reaction, the system temperature was maintained at 65°C, and the mixture was magnetically stirred at 300 rpm for 1 hour. This effectively promoted the rapid nucleation and self-assembly of curcumin molecules in the aqueous phase, forming a homogeneous and stable dispersion of curcumin nanoparticles. The resulting dispersion was freeze-dried to obtain solid curcumin nanoparticles with an average particle size of 275 nm.

[0068] The stability and morphology of the obtained product were characterized. For example... Figure 2 As shown in Figure a, the prepared curcumin nanoparticles and an equal mass of free curcumin were dissolved in the same volume of aqueous solution and allowed to stand for 12 hours. The results showed that the curcumin nanoparticle solution maintained good dispersibility, and no obvious precipitation was observed in the system. In contrast, the free curcumin solution exhibited low solubility and easy precipitation, verifying that nano-sizing significantly improved its water solubility and stability. Furthermore, the morphology of the nanoparticles was observed using scanning electron microscopy (SEM), such as... Figure 2 As shown in Figure b, the particles are clearly observed to be in a single dispersed state, further proving that the preparation method can obtain curcumin nanoparticles with good dispersion.

[0069] Example 3: Preparation of magnetic microspheres First, prepare the injection phase solution: Add 80 wt% methacrylamide gelatin, 10 wt% Fe3O4@SiO2 magnetic nanoparticles prepared in Example 1, 0.01 wt% curcumin nanoparticles prepared in Example 2, and 2 wt% photoinitiator LAP to 1 mL of deionized water. Place the mixture on a heating table and heat at 46°C in the dark until the methacrylamide gelatin is fully dissolved. Then, prepare the continuous phase solution: Add 3% surfactant Span 80 to the paraffin oil to enhance the stability of the oil phase and store at room temperature for later use.

[0070] Adopting such Figure 1 The microfluidic device shown connects the injected phase solution to the inner phase inlet needle of the coaxial microfluidic device via a syringe (placed in the injection pump) and a polyethylene tube; simultaneously, the continuous phase solution is connected to the outer phase inlet needle via another syringe (placed in the injection pump) and a polyethylene tube. The temperature of the entire system is maintained at 40-45°C to ensure the injected phase solution is in a suitable flow state. Under these conditions, the injected phase solution is subjected to fluid shear force in the continuous phase solution, thereby generating uniformly sized and regularly shaped magnetic droplets. When the flow rate of the continuous phase solution is fixed at 5 mL / h and the flow rate of the injected phase solution is 0.6 mL / h, the generated droplets have a concentrated particle size distribution of approximately 85 μm, exhibiting good uniformity. The droplets in the oil phase collection liquid are photocured and crosslinked at room temperature for 4 minutes (3-5 minutes is also acceptable) using near-ultraviolet light at a wavelength of 405 nm, and then washed three times sequentially with n-hexane and deionized water, followed by centrifugation to obtain magnetic microspheres. The results of optical microscopy observation are as follows. Figure 3 As shown in Figure a, the obtained microspheres have uniform particle size and complete and regular morphology; further detection by scanning electron microscopy reveals, for example... Figure 3 As shown in Figure b, the surface of the microspheres is smooth and exhibits a typical spherical structure.

[0071] Example 4: Application of magnetic microspheres in local targeted therapy for gastroesophageal reflux disease The magnetic microspheres prepared in Example 3 were lyophilized and placed in a simulated gastric acid and bile salt solution, and slowly stirred under constant temperature to simulate the drug release process in the gastrointestinal environment. Samples were taken every hour to detect changes in curcumin concentration. The results showed that at 1 hour, 6 hours, and 12 hours, the curcumin release rates were 19.9%, 76.8%, and 95.2%, respectively, indicating that the magnetic microspheres could continuously release the drug; demonstrating that the system possesses stable and controllable drug release performance. A gastric acid and bile salt stimulation model of esophageal epithelial cells was constructed. Esophageal epithelial cells were treated with a culture medium containing simulated gastric juice and bile salts for 10 minutes, three times a day, for a total of 5 days. Figure 4As shown in Figure a, the expression of inflammatory factors (IL-6, IL-8, and TNF-α) and the content of ROS in the treated esophageal epithelial cells were significantly increased. The cell model was treated with the magnetic microsphere extract prepared in Example 3 for 24 hours, as shown in Figure a. Figure 4 As shown in Figure b, it can significantly inhibit the production of inflammatory factors and reactive oxygen species induced by gastric acid and bile salts.

[0072] Further in vivo validation experiments were conducted. Eight-week-old C57BL / 6 mice were selected, and a gastroesophageal reflux disease model was established using esophagoduodenal side-to-side anastomosis. Figure 4 As shown in Figure c, HE staining revealed elongation of the lamina propria papillae and epithelial erosion in the esophageal mucosa of the model group mice. After surgery, mice were observed routinely until day 14 and randomly divided into four groups (n=10 per group): a simulated surgery group, a gastroesophageal reflux control group, a curcumin-only treatment group, and a magnetic microsphere treatment group. The simulated surgery group and the gastroesophageal reflux control group received a normal diet; the curcumin-only treatment group received 100 mg / kg curcumin nanoparticles via gavage twice daily; in the magnetic microsphere treatment group, a magnet was fixed externally to the chest of the mice to achieve targeted enrichment of magnetic microspheres in the esophagus, followed by gavage administration of magnetic microspheres (containing 100 mg / kg curcumin nanoparticles) twice daily. On day 14 of treatment, esophageal tissue samples were taken from the mice for pathological and molecular-level analysis. The results are as follows: Figure 4 As shown in Figure c, HE staining under a microscope revealed that magnetic microspheres can effectively alleviate the thickening of the basal cell layer of the esophageal epithelium and the elongation of the papillae of the lamina propria caused by gastroesophageal reflux, confirming that magnetic microspheres can effectively improve chronic esophageal epithelial inflammation caused by gastroesophageal reflux.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing magnetic microspheres, characterized in that, Includes the following steps: Curcumin was dissolved in anhydrous ethanol to obtain a homogeneous solution; the homogeneous solution was injected into deionized water for self-assembly, and then freeze-dried to obtain curcumin nanoparticles. Methacrylamide gelatin, Fe3O4@SiO2 magnetic nanoparticles, curcumin nanoparticles and photoinitiator were dissolved in deionized water to obtain the injection phase solution; The injected phase solution is formed into uniform droplets using microfluidic technology, followed by photocuring and crosslinking to obtain the magnetic microspheres.

2. The preparation method according to claim 1, characterized in that, The concentration of the homogeneous solution is 1 mg / mL; the self-assembly temperature is 60~70°C and the time is 1 hour.

3. The preparation method according to claim 1, characterized in that, The injected phase solution contains 80 wt% methacrylamide gelatin, 8-10 wt% Fe3O4@SiO2 magnetic nanoparticles, 0.01 wt% curcumin nanoparticles, and 2-3 wt% photoinitiator; the photoinitiator is photoinitiator LAP or photoinitiator 2959.

4. The preparation method according to claim 1, characterized in that, The process of forming uniform droplets from the injected phase solution using microfluidic technology refers to: adding a surfactant to an oily matrix to obtain a continuous phase solution; and delivering the injected phase solution and the continuous phase solution to the input and output pipes of the microfluidic device respectively for coaxial flow, thereby forming uniform droplets from the injected phase solution. The surfactant is at least one of Span 80, Tween 85, and Span 20; the oily matrix is ​​at least one of paraffin oil, castor oil, and silicone oil; and the mass fraction of the surfactant in the continuous phase solution is 1-10 wt%.

5. The preparation method according to claim 4, characterized in that, When coaxial flow is performed, the flow rate of the injected phase solution is 0.1~1 mL / h, the flow rate of the continuous phase solution is 1~10 mL / h, and the system temperature is 35~50°C.

6. The preparation method according to claim 1, characterized in that, Photocuring crosslinking is performed using near-ultraviolet light with a wavelength of 405nm at room temperature for 3-5 minutes.

7. A magnetic microsphere prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the magnetic microspheres as described in claim 7 in the preparation of a medicament for treating gastroesophageal reflux disease.

9. A microfluidic device for preparing the magnetic microspheres of claim 7, characterized in that, Includes input pipes, output pipes, and capillary collection pipes; The outlets of the input pipe, the output pipe, and the capillary collection tube are coaxially aligned to form a composite flow outlet. The capillary collection tube is located inside the output pipe; the end of the input pipe extends into the center of the output pipe and into the capillary collection tube.

10. The microfluidic device according to claim 9, characterized in that, It also includes a connecting sleeve, a fixing bracket, an injection pump, an internal phase inlet, and an external phase inlet; The input and output pipes are fixedly connected to the fixed bracket via connecting sleeves; The internal phase inlet is used to introduce the injection phase solution into the input pipeline; The external phase inlet is used to introduce a continuous phase solution into the output pipeline.

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