Solid-liquid dual-phase embolic microspheres capable of multi-stage controlled release, their preparation methods, and applications

Abstract

This invention discloses solid-liquid biphase embolic microspheres capable of multi-stage controlled release, their preparation method, and applications. The microspheres comprise an oleophilic-hydrophobic liquid unit and hydrophilic Janus microspheres. The Janus microspheres include proportionally fused Janus ciliate microspheres with a ciliated structure and Janus porous microspheres with a porous structure, with the Janus microspheres coating the liquid unit. Different functional units with one or more active components can be loaded onto the interior of the liquid unit, the surface and / or interior of the Janus porous microspheres, and the Janus ciliate microspheres. The release rates of the functional units loaded on the interior of the liquid unit, the surface and / or interior of the Janus ciliate microspheres differ, forming at least two stages of multi-stage controlled release. This invention offers strong loading capacity, enables multi-drug synergy, allows for multi-stage sequential release, improves therapeutic stability, reduces burst release risk, and exhibits good versatility and delivery capability.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to solid-liquid dual-phase embolization microspheres capable of multi-stage controlled release, their preparation methods, and applications. Background Technology

[0002] Interventional therapies (such as transarterial embolization / chemoembolization) are widely used in the treatment of tumors, vascular malformations, and related diseases. Embolization is an interventional medical technique that involves injecting embolic material into a blood vessel to block blood flow to abnormal vessels or lesions, thereby achieving hemostasis, tumor shrinkage, or elimination of vascular malformations. Its core advantages lie in its minimally invasive and precise nature, and it is commonly used in the treatment of tumors, hemorrhagic diseases, and vascular malformations.

[0003] Currently, commonly used embolization materials in clinical practice include solid embolization microspheres and liquid embolization agents. These two types of embolization materials have been widely used in clinical practice, especially in tumor chemoembolization. However, the applicant has found that the above two types of embolization materials have the following defects: (1) The drug loading form is singular, making it difficult to simultaneously carry multiple types of drugs such as hydrophilic / hydrophobic, large and small molecules, and bioactive proteins / nucleic acids; (2) The release curve is difficult to be segmented and controlled according to the treatment window, often resulting in burst release or unstable release, leading to large fluctuations in local concentration, unstable efficacy, and the risk of toxic side effects; (3) Multimodal therapy (chemotherapy + immunotherapy + anti-angiogenesis + light / magnetic / imaging) requires the synergy of multiple carriers, which is complicated to operate, has poor component compatibility, and makes it difficult to achieve "single-platform integration". Therefore, this invention is derived from this. Summary of the Invention

[0004] In order to solve at least one of the above-mentioned technical problems, the present invention aims to provide solid-liquid dual-phase embolization microspheres capable of multi-stage controlled release, their preparation method and application.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0006] One objective of this invention is to provide a solid-liquid dual-phase embolization microsphere capable of multi-stage controlled release, comprising an oleophilic and hydrophobic liquid unit and hydrophilic Janus microspheres. The Janus microspheres include Janus ciliate microspheres with a ciliated structure and Janus porous microspheres with a porous structure. The Janus ciliate microspheres and Janus porous microspheres are fused together in a proportion to coat the liquid unit. Different functional units having one or more active components can be loaded inside the liquid unit, on the surface and / or inside the Janus porous microspheres and the Janus ciliate microspheres. Furthermore, the release rates of the functional units loaded inside the liquid unit, on the surface and / or within the porous channels of the Janus porous microspheres, and on the surface and / or inside the Janus ciliate microspheres differ to form at least two stages of multi-stage controlled release.

[0007] Furthermore, both the Janus ciliated microspheres and the Janus porous microspheres have asymmetric structures, with one side of the Janus ciliated microspheres having a ciliated structure and the other side having a smooth structure; one side of the Janus porous microspheres has a porous structure and the other side has a smooth structure; and the smooth structure in both the Janus porous microspheres and the Janus ciliated microspheres is composed of polymethyl methacrylate, while the ciliated structure and the porous structure are composed of block polymers with hydrophilic and hydrophobic segments of different molecular weights.

[0008] Furthermore, the preparation method of the Janus microspheres includes the following steps:

[0009] Preparation of the polymer oil phase: Take dichloromethane and dissolve polymethyl methacrylate and block polymer in a mass ratio of 7:1 to obtain an oil phase solution with a polymer mass fraction of approximately 4%.

[0010] Microfluidic emulsification into droplets: The above polymer oil phase solution and polyvinyl alcohol aqueous solution are emulsified in a microfluidic droplet forming channel, and the flow rate ratio of oil phase to water phase is controlled at 5:8, so that the polymer oil phase forms water-in-oil emulsion droplets in the polyvinyl alcohol aqueous phase.

[0011] Phase separation induces Janus structure formation: The above emulsion was left to stand at room temperature for 4 h to allow the polymethyl methacrylate phase and the block polymer phase to fully separate within the emulsion, forming an asymmetric structure prototype.

[0012] Curing and solvent evaporation: The emulsion with the asymmetric structure was heated and stirred at 40°C and 200 rpm for 2 hours to allow the dichloromethane to evaporate completely and the emulsion droplets to solidify into microspheres.

[0013] Centrifugation, washing and freeze-drying: The above emulsion droplets were solidified into a dispersion of microspheres by centrifugation at 6000 rpm, the supernatant was removed and the microspheres were resuspended in deionized water. The microspheres were washed three times to obtain Janus microspheres, which were then freeze-dried for storage.

[0014] In the preparation methods of Janus porous microspheres and Janus ciliated microspheres, the only difference is the molecular weight of the hydrophobic segments in the block polymers, and the molecular weight of the hydrophobic segments in the block polymers of the Janus porous microspheres is smaller than that of the block polymers of the Janus ciliated microspheres.

[0015] Furthermore, in the Janus microspheres, the ratio of Janus ciliated microspheres to Janus porous microspheres can be adjusted by changing the ratio of the Janus porous microspheres.

[0016] Furthermore, the functional units inside the liquid unit, on the surface and / or within the porous channels of the Janus porous microspheres, and on the surface and / or within the Janus ciliated microspheres are loaded through different loading methods to form a multi-level controlled release. The loading methods include physical adsorption / permeation, electrostatic / coordination / host-guest interaction, covalent coupling, or in-situ embedding.

[0017] Furthermore, the multi-stage controlled release includes: the release of functional units located on the surface or within the porous channels of the Janus porous microspheres in the first stage;

[0018] The release of functional units within the liquid unit in the second stage;

[0019] The third stage involves the release of functional units within the Janus microspheres.

[0020] Furthermore, the functional unit is loaded within the porous channels of the Janus porous microspheres via electrostatic adsorption. This electrostatic adsorption method includes treating the Janus porous microspheres with hydrochloric acid to hydrolyze the ester groups to carboxyl groups for loading positively charged drugs; or forming amide bonds with sodium aminoborane under nitrogen protection for loading negatively charged drugs; and / or

[0021] The functional unit is loaded inside the liquid unit by means of direct dissolution or dispersion within the liquid unit; and / or

[0022] The functional units are encapsulated inside the Janus microspheres during the synthesis process by being dissolved or dispersed in a dichloromethane solution.

[0023] Furthermore, the functional units include sustained-release drugs, biomacromolecules, nanocarriers, inorganic / hybrid nanostructures, and magnetic nanoparticles; when the functional unit is a magnetic nanoparticle, it can achieve controllable movement in various fluids.

[0024] Another object of the present invention is to provide a method for preparing solid-liquid dual-phase embolic microspheres as described in any of the above claims, comprising the following steps:

[0025] S1. Janus ciliated microspheres and Janus porous microspheres loaded with different functional units were prepared respectively.

[0026] S2. Preparation of solid-liquid dual-phase precursor: Janus ciliated microspheres and Janus porous microspheres loaded with different functional units are mixed and dispersed in deionized water according to a set mass ratio to obtain a mixed microsphere aqueous dispersion.

[0027] S3, Microsphere Mixed Dispersion: Add liquid units to the above mixed microsphere aqueous dispersion so that the liquid units are located near the oil-water interface;

[0028] S4. Emulsification and Pickering Coating: Place the disperser cutter head at the oil-water interface and emulsify for 10 min at 1000 rpm to form uniform droplets in the liquid unit. At the same time, Janus ciliated microspheres and Janus porous microspheres are adsorbed on the droplet surface to form a Pickering-like emulsion structure.

[0029] S5. Settling and washing to remove free emulsion: After emulsification, let stand for a preset time, shake to disperse, and then let stand for another preset time. Remove the supernatant, add deionized water to resuspend and gently shake to disperse. Repeat washing 3-5 times to remove unadsorbed microspheres and obtain clean solid-liquid biphase embolization microspheres.

[0030] Another object of the present invention is to provide the application of the solid-liquid biphasic embolization microspheres described in any of the above claims or the solid-liquid biphasic embolization microspheres obtained by the above preparation methods in the preparation of drugs for local drug delivery, interventional delivery, image-guided therapy or multimodal combination therapy.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] Janus microspheres include Janus ciliated microspheres and Janus porous microspheres. The ciliated and porous structures provide high specific surface area and multiple sites, enabling them to carry complex functional units with one or more active components and specific functions, as well as their drug-carrying structures, resulting in strong loading capacity. Multiple drugs and / or functional units can be integrated into functional superposition, reducing compatibility and operational complexity and achieving multi-drug synergy. Multi-stage sequential release can be achieved, improving efficacy stability and reducing burst release risk. Functional units can be flexibly replaced according to disease needs to enable applications in chemotherapy, immunotherapy, anti-angiogenesis, radiosensitization, imaging, and other fields, demonstrating good versatility. Furthermore, the particle size of the microspheres can be controlled to adapt to injection / catheter delivery, resulting in good deliverability. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 The solid phase microspheres in the solid-liquid dual-phase embolization microspheres of this invention are also known as Janus ciliated microspheres. Figure 1 (Figure a) and Janus porous microspheres ( Figure 1 SEM images of Figure b) and their respective specific surface areas (Figure b) Figure 1 (Figure C in the middle)

[0035] Figure 2 Fluorescent images of microspheres (including Janus ciliated microspheres, Janus porous microspheres, and Janus microspheres obtained by fusion).

[0036] Figure 3 Fluorescence images of Janus porous microspheres and Janus ciliated microspheres in different proportions, as well as Janus microspheres obtained after fusion.

[0037] Figure 4 The graph shows the relationship between the amount of Janus porous microspheres added in different proportions and the percentage of Janus porous microspheres on the surface of solid-liquid dual-phase embolization microspheres.

[0038] Figure 5 This document presents the drug release curves and cell regulation effects of solid-liquid biphasic embolization microspheres composed of doxorubicin electrostatically adsorbed in Janus porous microspheres, as described in Example 3 of the present invention. (Figure a shows a schematic diagram of the release of adsorbed loads from the porous channels and / or surface of the Janus porous microspheres; Figure b shows the appearance of doxorubicin electrostatically adsorbed on the Janus porous microspheres; Figure c shows the relationship between the release amount of doxorubicin and time; Figure d shows the relationship between the culture time of the solid-liquid biphasic embolization microspheres in Group A and the activity of tumor cells; Figure e shows the relationship between the amount of solid-liquid biphasic embolization microspheres used in Group A and the activity of tumor cells.)

[0039] Figure 6 This document presents the drug release curves and cell regulation effects of solid-liquid biphasic embolization microspheres composed of atezolizus embedded in Janus ciliated microspheres, as described in Example 3 of the present invention (Figure a shows a schematic diagram of the release of the load inside the iodized oil droplet; Figure b shows the appearance of atezolizus dispersed in the iodized oil droplet; Figure c shows the relationship between the release amount of the loaded atezolizus and time; Figure d shows the relationship between the culture time of the solid-liquid biphasic embolization microspheres in Group B and the activity of tumor cells; Figure e shows the relationship between the dosage of the solid-liquid biphasic embolization microspheres in Group B and the activity of immune cells).

[0040] Figure 7 This invention presents the drug release curve and cell regulation effect of solid-liquid biphasic embolization microspheres composed of bevacizumab dissolved in iodized oil droplets in Example 3 of the present invention (wherein figure a is a schematic diagram of the release of the embedded load inside Janus microspheres; figure b is an appearance view of bevacizumab embedded inside Janus ciliated microspheres; figure c is a graph showing the relationship between the release amount of the loaded bevacizumab and time; figure d is a graph showing the relationship between the culture time of solid-liquid biphasic embolization microspheres in group C and the activity of tumor cells; figure e is a graph showing the relationship between the amount of solid-liquid biphasic embolization microspheres used in group C and the activity of tumor cells).

[0041] Figure 8This invention relates to the motion control of solid-liquid biphase embolization microspheres composed of nanoparticles embedded in Janus ciliated microspheres and iodized oil droplets, as described in Example 4 of the present invention. (Figure a is a schematic diagram of the magnetic field control principle of solid-liquid biphase embolization microspheres loaded with magnetic nanoparticles; Figure b is an example diagram of the sample under magnetic field control and without magnetic field control; Figure c is a magnetization intensity-magnetic field curve; Figure d is a bright field motion sequence diagram in PBS channel and a fluorescence motion sequence diagram in simulated blood channel; Figure e is a comparison diagram of the motion velocity of solid-liquid biphase embolization microspheres loaded with magnetic nanoparticles in H2O, PBS, SPSS and Blood media.)

[0042] Figure 9 The figures show the drug release curves of the solid-liquid biphasic embolized microspheres in the three-drug loading system of Example 5 of the present invention (Figure a shows the drug release curves of the three drugs in PBS buffer (pH 7.4); Figure b shows the drug release curves in acidic buffer (pH 5.0).

[0043] Figure 10 The image shows the drug release curve of the solid-liquid biphase embolized microspheres in the three-drug delivery system after photothermal treatment in Example 5 of this invention. Detailed Implementation

[0044] In view of the shortcomings of the existing technology, the inventors of this case, through long-term research and extensive practice, have proposed a solid-liquid dual-phase embolization microsphere capable of multi-stage controlled release and its application.

[0045] The following description, in conjunction with preferred embodiments and accompanying drawings, further explains and illustrates the technical solution, its implementation process, and its principles. It should be particularly noted that the following embodiments are merely illustrative of the invention and should not be construed as limiting the scope of protection of the invention.

[0046] This invention provides a solid-liquid dual-phase embolic microsphere capable of multi-stage controlled release, such as... Figures 1 to 3 As shown, the device includes an oleophilic and hydrophobic liquid unit and hydrophilic Janus microspheres. The Janus microspheres include Janus ciliated microspheres with a ciliated structure and Janus porous microspheres with a porous structure. The Janus ciliated microspheres and Janus porous microspheres are fused together in proportion and coated on the outside of the liquid unit. Different functional units with one or more active components can be loaded inside the liquid unit, on the surface and / or in the porous channels of the Janus porous microspheres, and on the surface and / or inside the Janus ciliated microspheres. The release rates of the functional units loaded inside the liquid unit, on the surface and / or in the porous channels of the Janus porous microspheres, and on the surface and / or inside the Janus ciliated microspheres are different to form at least two stages of multi-stage controlled release.

[0047] Furthermore, such as Figure 1 As shown in Figures a and b, both Janus ciliated microspheres and Janus porous microspheres have asymmetric structures. One side of the Janus ciliated microsphere is a ciliated structure, and the other side is a smooth structure. One side of the Janus porous microsphere is a porous structure, and the other side is a smooth structure. The smooth structure is oleophilic and is preferably composed of polymethyl methacrylate (PMMA), but other alternatives such as polylactic acid, polylactide, polylactic acid / glycolic acid copolymer, or polystyrene can also be used. The porous structure has a large specific surface area and hydrophilicity, and the ciliated structure is also hydrophilic. The ciliated and porous structures are composed of block polymers with different molecular weights and consisting of hydrophilic and hydrophobic segments (in this embodiment, an example is a triblock polymer composed of polycaprolactone (PCL) and polyethylene glycol (PEG), i.e., PCL-b-PEG-b-PCL). Figure 2 As shown, Janus microspheres, formed by fusing Janus ciliated microspheres and Janus porous microspheres, form Pickering emulsion droplets on the surface of a liquid unit (iodized oil droplets in the prior art, as described in the examples below). Alternatively, the liquid unit may be one or more of existing polyacrylates, polyethylene glycols, and polyacrylonitriles.

[0048] Furthermore, the preparation method of Janus microspheres includes the following steps:

[0049] Preparation of the polymer oil phase: Take dichloromethane and dissolve polymethyl methacrylate and block polymer in a mass ratio of 7:1 to obtain an oil phase solution with a polymer mass fraction of approximately 4%.

[0050] Microfluidic emulsification into droplets: The above polymer oil phase solution and polyvinyl alcohol aqueous solution are emulsified in a microfluidic droplet forming channel, and the flow rate ratio of oil phase to water phase is controlled at 5:8, so that the polymer oil phase forms water-in-oil emulsion droplets in the polyvinyl alcohol aqueous phase.

[0051] Phase separation induces Janus structure formation: The above emulsion was left to stand at room temperature for 4 h to allow the polymethyl methacrylate phase and the block polymer phase to fully separate within the emulsion, forming an asymmetric structure prototype.

[0052] Curing and solvent evaporation: The emulsion with the asymmetric structure was heated and stirred at 40°C and 200 rpm for 2 hours to allow the dichloromethane to evaporate completely and the emulsion droplets to solidify into microspheres.

[0053] Centrifugation, washing and freeze-drying: The above emulsion droplets were solidified into a dispersion of microspheres by centrifugation at 6000 rpm, the supernatant was removed and the microspheres were resuspended in deionized water. The microspheres were washed three times to obtain Janus microspheres, which were then freeze-dried for storage.

[0054] The only difference between the preparation methods of Janus porous microspheres and Janus ciliated microspheres lies in the molecular weight of the hydrophobic segments in the block polymers. Furthermore, the molecular weight of the hydrophobic segments in the block polymers used in the Janus porous microsphere preparation method is smaller than that in the Janus ciliated microsphere preparation method. For example, in the Janus porous microsphere preparation method, the molecular weight of the hydrophobic segments (PCL segments) of the block polymer is 30 kDa, while in the Janus ciliated microsphere preparation method, the molecular weight of the PCL segments is 50 kDa.

[0055] In some preferred embodiments, the ratio of Janus microspheres to Janus porous microspheres is adjustable by adjusting the ratio of Janus microspheres to Janus ciliated microspheres.

[0056] In some preferred embodiments, functional units inside the liquid unit, on the surface and / or within the porous channels of Janus microspheres, and on the surface and / or within the Janus ciliated microspheres are loaded in different ways to form multi-level controlled release. The loading methods include physical adsorption / permeation, electrostatic / coordination / host-guest interaction, covalent coupling, or in-situ embedding.

[0057] In some preferred embodiments, the functional units include sustained-release drugs, biomolecules, nanocarriers, inorganic / hybrid nanostructures, and magnetic nanoparticles. It should be noted, however, that the functional units in the embodiments of the present invention refer to composite structures capable of carrying one or more active components and possessing specific functions. Examples include, but are not limited to:

[0058] (1) Traditional anticancer drugs: doxorubicin, cisplatin, atezolizumab, bevacizumab, etc.;

[0059] (2) Nanocarriers: liposomes, polymer micelles, polymer nanoparticles, nanogels, solid lipid nanoparticles;

[0060] (3) Inorganic / hybrid nanostructures: porous silicon, gold nanorods, carbon-based photothermal materials, metal oxides, MOF / COF, etc.;

[0061] (4) Biologically derived carriers: exosomes / cell membrane-encapsulated nanoparticles, protein nanocages.

[0062] It should be noted that when the functional unit is a magnetic nanoparticle (such as iron oxide nanoparticles), it can achieve controllable movement in various fluids through magnetic field control, thereby achieving targeted movement in blood vessels and longer-lasting embolization capability.

[0063] In some preferred embodiments, multi-stage controlled release includes the release of functional units located on the surface or within the porous channels of the Janus microspheres in the first stage, the release of functional units within the liquid unit in the second stage, and the release of functional units within the Janus microspheres in the third stage. Whether there are two or more stages depends on which parts are loaded with functional units, and this is not specifically limited here. Optionally, release can be further regulated by introducing cleavable linkages (acid-sensitive linkers (see Example 5 below), ROS-sensitive linkers, enzyme-sensitive linkers) or external trigger switches (photothermal (see Example 5 below), ultrasonic, magnetothermal / magnetomechanical).

[0064] In some preferred embodiments, to achieve the aforementioned multi-stage controlled release, the functional unit can be loaded into the porous channels of Janus porous microspheres via electrostatic adsorption. Electrostatic adsorption methods include hydrolyzing the ester groups of the Janus porous microspheres with hydrochloric acid to convert them into carboxyl groups for loading positively charged drugs, or forming amide bonds with sodium aminoborane under nitrogen protection to load negatively charged drugs. For example, direct treatment with 2M hydrochloric acid hydrolyzes the ester groups into carboxyl groups to carry positively charged drugs, such as DOX (doxorubicin); or forming amide bonds with sodium aminoborane under nitrogen protection to carry a positive charge for adsorbing negatively charged drugs. This loading method is mainly used for carrying drugs that are released immediately upon entering the body.

[0065] In some preferred embodiments, the functional units are loaded inside the liquid unit by directly dissolving or dispersing them within the liquid unit. For example, before preparing asymmetric microsphere-covered iodized oil droplets, the oil-soluble functional units are dissolved or dispersed in the iodized oil droplets for loading.

[0066] In some preferred embodiments, the functional units are encapsulated within the Janus microspheres during synthesis by dissolving or dispersing them in a dichloromethane solution. Examples include loading hydrophobic drugs by directly dissolving them in dichloromethane, loading hydrophilic drugs by first forming an oil-in-water solution and then emulsifying it, and loading oleic acid-modified nanoparticles by directly dispersing them in dichloromethane.

[0067] This invention also provides a method for preparing solid-liquid dual-phase embolic microspheres according to any one of the above claims, comprising the following steps:

[0068] S1. Janus ciliated microspheres and Janus porous microspheres loaded with different functional units were prepared according to the method described above.

[0069] S2. Preparation of solid-liquid dual-phase precursor: Janus ciliated microspheres and Janus porous microspheres loaded with different functional units are mixed and dispersed in deionized water according to a set mass ratio to obtain a mixed microsphere aqueous dispersion.

[0070] S3, Microsphere Mixed Dispersion: Add liquid units to the above mixed microsphere aqueous dispersion so that the liquid units are located near the oil-water interface;

[0071] S4. Emulsification and Pickering Coating: Place the disperser blade at the oil-water interface and emulsify for 10 minutes at 1000 rpm to form uniform droplets in the liquid unit. At the same time, Janus ciliated microspheres and Janus porous microspheres are adsorbed on the droplet surface to form a Pickering-like emulsion structure.

[0072] S5. Settling and washing to remove free emulsion: After emulsification, let stand for a preset time, shake to disperse, and then let stand for another preset time. Remove the supernatant, add deionized water to resuspend and gently shake to disperse. Repeat washing 3-5 times to remove unadsorbed microspheres and obtain clean solid-liquid biphase embolization microspheres.

[0073] The present invention also provides the application of the solid-liquid biphasic embolization microspheres of any of the above-mentioned methods or the solid-liquid biphasic embolization microspheres prepared by the above-mentioned methods in the preparation of drugs for local drug delivery, interventional delivery, image-guided therapy or multimodal combined therapy.

[0074] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0075] Example 1: Preparation of Janus microspheres with different morphologies loaded with multiple functional units

[0076] (1) Preparation of Janus ciliated microspheres

[0077] Preparation of the polymer oil phase: Take 10 mL of dichloromethane and dissolve 350 mg of PMMA (molecular weight approximately 300 kDa) and 50 mg of PCL-b-PEG-b-PCL (triblock polymer, PCL segment with molecular weight approximately 50 kDa and PEG segment with molecular weight approximately 4 kDa) at a mass ratio of 7:1 to obtain an oil phase solution with a polymer mass fraction of approximately 4%.

[0078] Microfluidic emulsification droplet formation: The above polymer oil phase and PVA aqueous solution are emulsified in a microfluidic droplet formation channel, and the oil phase:water phase flow rate ratio is controlled at 5:8, so that the dichloromethane polymer oil droplets form water-in-oil (O / W) emulsion droplets in the PVA aqueous phase.

[0079] Phase separation induces Janus structure formation: The emulsion was left to stand at room temperature for 4 h to allow the PMMA phase and the PCL-b-PEG-b-PCL phase to fully separate inside the droplet, forming an asymmetric structure prototype.

[0080] Curing and solvent evaporation: The emulsion was heated and stirred at 40°C and 200 rpm for 2 hours to allow the dichloromethane to fully evaporate and the droplets to solidify into microspheres.

[0081] Centrifugation, washing, and lyophilization: The dispersion was centrifuged at 6000 rpm, the supernatant was removed, and the suspension was resuspended in deionized water. The washing was repeated three times to obtain Janus ciliated microspheres; then, they were lyophilized for storage.

[0082] Morphological characterization: SEM observation was performed, such as... Figure 1 As shown in the right side of Figure a, the prepared microspheres exhibit a Janus morphology that is "half smooth and half cilia" and have no obvious defects overall.

[0083] (2) Preparation of Janus porous microspheres

[0084] Preparation of the polymer oil phase: Take 10 mL of dichloromethane and dissolve 350 mg of PMMA (molecular weight approximately 300 kDa) and 50 mg of PCL-b-PEG-b-PCL (triblock polymer, PCL segment with molecular weight approximately 30 kDa and PEG segment with molecular weight approximately 4 kDa) at a mass ratio of 7:1 to obtain an oil phase solution with a polymer mass fraction of approximately 4%.

[0085] Microfluidic emulsification droplet formation: The above polymer oil phase and PVA aqueous solution are emulsified in a microfluidic droplet formation channel, and the oil phase:water phase flow rate ratio is controlled at 5:8, so that the dichloromethane polymer oil droplets form water-in-oil (O / W) emulsion droplets in the PVA aqueous phase.

[0086] Phase separation induces Janus structure formation: The emulsion was left to stand at room temperature for 4 h to allow the PMMA phase and the PCL-b-PEG-b-PCL phase to fully separate inside the droplet, forming an asymmetric structure prototype.

[0087] Curing and solvent evaporation: The emulsion was heated and stirred at 40°C and 200 rpm for 2 hours to allow the dichloromethane to fully evaporate and the droplets to solidify into microspheres.

[0088] Centrifugation, washing and lyophilization: The dispersion was centrifuged at 6000 rpm, the supernatant was removed and the mixture was resuspended in deionized water. The mixture was washed three times with water to obtain Janus porous microspheres, which were then lyophilized.

[0089] Morphological characterization: SEM observation was performed, such as... Figure 1 As shown in the right side of Figure b, the prepared microspheres exhibit a Janus morphology that is "half smooth and half porous" and have no obvious defects overall.

[0090] (3) Janus ciliated microspheres loaded with bevacizumab

[0091] Preparation of the polymer oil phase: Take 10 mL of dichloromethane and dissolve 350 mg of PMMA (molecular weight approximately 300 kDa) and 50 mg of PCL-b-PEG-b-PCL (triblock polymer, PCL segment with molecular weight approximately 50 kDa and PEG segment with molecular weight approximately 4 kDa) at a mass ratio of 7:1 to obtain an oil phase solution with a polymer mass fraction of approximately 4%.

[0092] Preparation of drug aqueous solution: Dissolve 10 mg of bevacizumab in 1 mL of deionized water to obtain drug aqueous solution.

[0093] To form a W1 / O emulsion: inject the drug aqueous solution into the polymer oil phase and emulsify it for 3 min using an ultrasonic emulsifier (100% power) to obtain a W1 / O emulsion.

[0094] Microfluidic W1 / O / W2 Biemulsion Droplet Formation: The above polymer oil phase and PVA aqueous solution are emulsified in a microfluidic droplet formation channel, and the oil phase:water phase flow rate ratio is controlled at 5:8, so that dichloromethane polymer oil droplets form water-in-oil W1 / O / W2 biemulsion droplets in the PVA aqueous phase.

[0095] Phase separation induces Janus structure formation: The emulsion was left to stand at room temperature for 4 h to allow the PMMA phase and the PCL-b-PEG-b-PCL phase to fully separate inside the droplet, forming an asymmetric structure prototype.

[0096] Curing and solvent evaporation: The emulsion was heated and stirred at 40°C and 200 rpm for 2 hours to allow the dichloromethane to fully evaporate and the droplets to solidify into microspheres.

[0097] Centrifugation, washing, and lyophilization: The dispersion was centrifuged at 6000 rpm, the supernatant was removed, and the suspension was resuspended in deionized water. The washing was repeated three times to obtain Janus ciliated microspheres; then, they were lyophilized for storage.

[0098] (4) Preparation of Janus porous microspheres loaded with doxorubicin by electrostatic adsorption

[0099] First, Janus porous microspheres were prepared according to the preparation steps of Janus porous microspheres in (2).

[0100] Surface charge regulation: 100 mg of Janus porous microspheres were weighed and dispersed in 5 mL of deionized water; an equal volume of 4M hydrochloric acid was also taken; the two were slowly mixed and reacted at room temperature for 6 h. At this time, an acid hydrolysis reaction of polycaprolactone (PCL) in the porous structure occurred, thereby generating exposed carboxyl molecules; after centrifugation and washing 3 times, the microspheres were dispersed in 10 mL of pH 9.0 buffer to obtain a charged Janus porous microsphere dispersion.

[0101] Doxorubicin adsorption loading: Dissolve 5 mg of doxorubicin hydrochloride in 1 mL of pH 9.0 buffer solution, add to 10 mL of the above microsphere dispersion, stir at 200 rpm in the dark for 6 h at room temperature; after centrifugation to remove the supernatant and washing with deionized water, Janus porous microspheres with doxorubicin electrostatic adsorption loading are obtained (prepare fresh for use).

[0102] (5) Janus ciliated microspheres loaded with magnetic nanoparticles

[0103] Preparation of the polymer oil phase: Take 10 mL of dichloromethane and dissolve 350 mg of PMMA (molecular weight approximately 300 kDa), 50 mg of PCL-b-PEG-b-PCL (a triblock polymer, with the PCL segment having a molecular weight of approximately 50 kDa and the PEG segment approximately 4 kDa) and 10 mg of magnetite nanoparticles at a mass ratio of 7:1 and disperse them thoroughly to obtain an oil phase solution with a polymer mass fraction of approximately 4%.

[0104] Microfluidic emulsification droplet formation: The above polymer oil phase and PVA aqueous solution are emulsified in a microfluidic droplet formation channel, and the oil phase:water phase flow rate ratio is controlled at 5:8, so that the dichloromethane polymer oil droplets form water-in-oil (O / W) emulsion droplets in the PVA aqueous phase.

[0105] Phase separation induces Janus structure formation: The emulsion was left to stand at room temperature for 4 h to allow the PMMA phase and the PCL-b-PEG-b-PCL phase to fully separate inside the droplet, forming an asymmetric structure prototype.

[0106] Curing and solvent evaporation: The emulsion was heated and stirred at 40°C and 200 rpm for 2 hours to allow the dichloromethane to fully evaporate and the droplets to solidify into microspheres.

[0107] Centrifugation, washing, and lyophilization: The dispersion was centrifuged at 6000 rpm, the supernatant was removed, and the suspension was resuspended in deionized water. The washing was repeated three times to obtain Janus ciliated microspheres; then, they were lyophilized for storage.

[0108] (6) Comparison of specific surface area of ​​Janus microsphere nanostructures

[0109] Samples: S1: Symmetrical PMMA microspheres (unstructured) S2: Blank Janus ciliated microspheres (lyophilized) S3: Blank Janus porous microspheres (lyophilized).

[0110] Weigh 100 mg of microspheres and place them in BET sample tubes to a filling height of approximately 10 mm. Degas under a nitrogen flow at 40 °C for 12 h. Then, use a specific surface area and pore size analyzer to collect adsorption-desorption isotherms at liquid nitrogen temperature (77 K); each sample should be tested at least three times. Specific surface area is calculated using linear fitting within the P / P0 = 0.05–0.30 interval; Figure 1 As shown in Figure c, compared to Janus ciliated microspheres, Janus porous microspheres exhibit a higher BET specific surface area, thus providing more sites for electrostatic adsorption. Furthermore, the specific surface area of ​​Janus porous microspheres is close to 10 m² / g. 2 / g, the specific surface area of ​​Janus ciliated microspheres is close to 5 m². 2 / g, while the specific surface area of ​​the smooth structure of Janus microspheres is less than 1 m². 2 / g, which shows that both Janus porous microspheres and Janus ciliated microspheres have high specific surface area and can provide multiple sites.

[0111] Example 2: Preparation and Surface Morphology Control of Solid-Liquid Dual-Phase Iodized Oil Droplets

[0112] Fluorescent labeling: When preparing Janus ciliated microspheres and Janus porous microspheres, 5 μg of Brønsted red fluorescent dye and coumarin 6 fluorescent dye were added to the oil solution respectively to label them with red fluorescence and green fluorescence respectively.

[0113] Microsphere mixed dispersion: 200 mg of Janus ciliated microspheres and Janus porous microspheres loaded with different functional units were weighed and dispersed in 40 mL of deionized water to obtain a mixed microsphere aqueous dispersion.

[0114] Adding iodized oil to form a solid-liquid biphase precursor: Slowly add 500 μL of iodized oil to the above mixed microsphere dispersion so that the iodized oil is located near the oil-water interface.

[0115] Emulsification and Pickering Coating: The disperser blade was placed at the oil-water interface and emulsified for 10 min at approximately 1000 rpm to form uniform droplets of iodized oil. Simultaneously, Janus ciliated microspheres and Janus porous microspheres adsorbed onto the droplet surface, forming a Pickering-like emulsion structure (see...). Figure 2 (Fused image). Janus ciliated microspheres exhibit red fluorescence, while Janus porous microspheres exhibit green fluorescence.

[0116] (4) Standing and washing to remove free microspheres: After emulsification, let stand for 30 min; after gently shaking to disperse, let stand for 1 min, carefully aspirate the supernatant; add deionized water to resuspend and gently shake to disperse; repeat washing 3–5 times to remove excess unadsorbed microspheres and obtain clean solid-liquid biphase embolization microspheres.

[0117] (5) Surface distribution characterization: see as follows Figure 3 As shown, observations using fluorescence microscopy / confocal microscopy revealed that the droplet surface was co-coated with red and green microspheres. Figure 3 and Figure 4It can be seen that the ratio of Janus porous microspheres to Janus ciliated microspheres on the surface of the solid-liquid dual-phase embolization microspheres can be adjusted. For example... Figure 4 As shown, by adjusting the proportion of Janus porous microspheres in the microsphere mixed dispersion, the proportion of Janus porous microspheres on the surface of the solid-liquid two-phase embolization microspheres can be effectively adjusted.

[0118] Example 3: Multi-stage controlled drug release and cell regulation effects

[0119] This example is used to verify the "fast / medium / slow" three-stage release:

[0120] Phase 1: Release of adsorbed loads from the porous channels and / or surface of Janus porous microspheres (e.g.) Figure 5 (As shown in Figure a); Second stage: Diffusion and release of internal load in iodized oil (as shown in Figure a) Figure 6 (As shown in Figure a); Third stage: The internal embedding load of Janus microspheres is released with delayed release as the material degrades (e.g., Figure 7 (As shown in Figure a).

[0121] 3.1 Solid-liquid dual-phase embolization microspheres were prepared according to the Janus porous microspheres and Janus ciliated microspheres ratio of 8:2 as in Example 2, and the following three types of example samples were constructed respectively:

[0122] Group A: Doxorubicin was electrostatically adsorbed onto Janus porous microspheres, blank Janus ciliated microspheres, and blank iodized oil droplets (e.g., ... Figure 5 (As shown in Figure b).

[0123] Group B: Atezolizus were dispersed in iodized oil droplets and blank Janus ciliated microspheres and blank Janus porous microspheres (e.g. Figure 6 (As shown in Figure b).

[0124] Group C: Bevacizumab embedded inside Janus ciliated microspheres, along with blank Janus porous microspheres and blank iodized oil droplets (e.g., ... Figure 7 (As shown in Figure b).

[0125] 3.2 Drug release curves and cell-regulating effects of solid-liquid biphasic embolization microspheres composed of doxorubicin drug electrostatically adsorbed in Janus porous microspheres:

[0126] Take 10 mg of group A solid-liquid biphasic embolization microspheres, put them into a dialysis bag and seal it; place the dialysis bag in a centrifuge tube containing 50 mL PBS (pH=5.0 and 7.4) and shake at 37℃ and 100 rpm.

[0127] Sampling and Replenishment (Time Point Settings): Take 2 mL of external fluid at time points of 2 h, 4 h, 6 h, 12 h, 24 h, 36 h, and 48 h, and replenish it with an equal volume of fresh PBS of the same pH value.

[0128] Drug quantification and cumulative release calculation: The DOX concentration of the sample solution was obtained by UV quantification, converted into release amount, and a cumulative release curve was plotted. The release results showed an early, distinct release phase, followed by a decrease and plateauing of the release rate (e.g., ...). Figure 5 As shown in Figure C, the drug release reaches 80% of the loading capacity within 48 hours.

[0129] Tumor cell viability (CCK-8) evaluation: HepG2 tumor cells were digested, counted, and seeded into 96-well plates at a density of 5000 cells per well. They were incubated overnight at 37°C with 100 μL of complete culture medium in 5% CO2. Group A solid-liquid biphasic embolizing agent was added to the cells at a concentration of 64 μg / mL. After culturing for 48 h, 36 h, 24 h, 12 h, and 6 h, the cells were washed three times with 100 μL PBS, and then incubated with 100 μL of CCK-8 reagent at 37°C for 2 h. OD was measured using a microplate reader. 450 Cell viability was calculated, with three replicates per group. Figure 5 As shown in Figure d, with increasing culture time, more DOX was released and cell activity decreased, demonstrating an effective cell-killing function.

[0130] HepG2 tumor cells were treated with group A solid-liquid biphasic embolization agent at concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL. After culturing for 48 hours, the cells were washed three times with 100 μL PBS, and then incubated with 100 μL CCK-8 reagent at 37°C for 2 hours. The OD was measured using a microplate reader. 450 Cell viability was calculated, with three replicates per group. Figure 5 As shown in Figure e, with the increase of the embolic agent dosage, more DOX was released and the cell activity was worse, demonstrating an effective cell-killing function.

[0131] 3.3 Drug release curves and cellular regulatory effects of solid-liquid biphasic embolic microspheres composed of atezolizumab dissolved in iodized oil droplets:

[0132] Take 10 mg of group B solid-liquid biphasic embolization microspheres, put them into a dialysis bag and seal it; place the dialysis bag in a centrifuge tube containing 50 mL PBS (pH=5.0 and 7.4) and shake at 37℃ and 100 rpm.

[0133] Sampling and Replenishment (Time Point Settings): Take 2 mL of external fluid at time points of 2 h, 4 h, 6 h, 12 h, 24 h, 36 h, and 48 h, and replenish it with an equal volume of fresh PBS of the same pH value.

[0134] Drug quantification and cumulative release calculation: The concentration of atezolizus in the sample solution was obtained by ELISA, converted into release rate, and a cumulative release curve was plotted. The release results showed an early, distinct release phase, followed by a decrease and plateauing of the release rate (e.g., ...). Figure 6 As shown in Figure c), 60% of the drug loading is released within 48 hours.

[0135] Tumor cell viability (CCK-8) evaluation: Atezolizumab is a PD-L1 antibody that promotes immune cell proliferation and enhances immune response by blocking the PD-1 / PD-L1 immunosuppressive pathway, thereby relieving the suppressed state of immune cells. Therefore, its effect should be reflected in the activity of immune cells, rather than a decrease in tumor cell activity. In 96-well plates, 100 μL of anti-CD3 coating solution (e.g., 10 μg / mL, prepared in PBS) and PD-L1-Fc coating solution (e.g., 10 μg / mL, prepared in PBS) were added to each well. After incubation at 37°C for 2 h, the coating solution was discarded, and the cells were washed twice with PBS. After Jurkat cell isolation and counting, the cells were seeded into 96-well plates at a density of 5000 cells per well, incubated overnight at 37°C with 100 μL of complete medium containing 100 U / mL IL-2, and cultured at 5% CO2. The solid-liquid biphasic embolizing agent (Group B) was added to cells at a concentration of 64 μg / mL. After culturing for 48 h, 36 h, 24 h, 12 h, and 6 h, 100 μL of CCK-8 reagent was added, and the cells were incubated at 37℃ for 2 h. The OD was measured using a microplate reader. 450 Cell viability was calculated, with three replicates per group. Figure 6 As shown in Figure d, with increasing culture time, more atezolizumab was released, and the cell activity was higher, demonstrating effective immune cell proliferation function.

[0136] HepG2 tumor cells were treated with group B solid-liquid biphasic embolization agent at concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL. After culturing for 48 hours, the cells were washed three times with 100 μL PBS, and then incubated with 100 μL CCK-8 reagent at 37°C for 2 hours. The OD was measured using a microplate reader. 450 Cell viability was calculated, with three replicates per group. Figure 6 As shown in Figure e, with the increase of the embolic agent dosage, more atezolizumab was released, and the cell activity was higher, demonstrating an effective immune cell proliferation function.

[0137] 3.4 Drug release curves and cellular regulatory effects of solid-liquid biphasic embolization microspheres composed of bevacizumab encapsulated in Janus ciliated microspheres:

[0138] Take 10 mg of group C solid-liquid biphasic embolization microspheres, put them into a dialysis bag and seal it; place the dialysis bag in a centrifuge tube containing 50 mL PBS (pH=5.0 and 7.4) and shake at 37℃ and 100 rpm.

[0139] Sampling and Replenishment (Time Point Settings): Take 2 mL of external fluid at time points of 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, and replenish with an equal volume of fresh PBS of the same pH value.

[0140] Drug quantification and cumulative release calculation: The concentration of bevacizumab in the sample solution was quantified using ELISA, converted into release rate, and a cumulative release curve was plotted. The release results showed an early, distinct release phase, followed by a decrease and plateauing of the release rate (e.g., ...). Figure 7 As shown in Figure c), 60% of the drug loading is released within 120 hours.

[0141] Tumor cell viability (CCK-8) evaluation: Bevacizumab inhibits vascular endothelial cell proliferation and migration by neutralizing VEGF-A and blocking the binding of VEGF to its receptors (VEGFR1 / 2). HUVEC vascular endothelial cells were digested, counted, and seeded into 96-well plates at a density of 5000 cells per well. They were cultured overnight at 37°C with 100 μL of complete medium containing 100 U / mL VEGF-A and allowed to adhere. A solid-liquid biphasic embolic agent (Group C) was added to the cells at a concentration of 64 μg / mL. After culturing for 120 h, 96 h, 72 h, 48 h, and 24 h, the cells were washed three times with 100 μL of PBS, and then incubated with 100 μL of CCK-8 reagent at 37°C for 2 h. OD was measured using a microplate reader. 450 Cell viability was calculated, with three replicates per group. Figure 7 As shown in Figure d, with increasing culture time, more cells are released and cell activity decreases, demonstrating an effective function in inhibiting the proliferation of vascular wall cells.

[0142] HUVEC vascular endothelial cells were treated with group C solid-liquid biphasic embolization agent at concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL, respectively. After culturing for 120 hours, the cells were washed three times with 100 μL PBS, incubated with 100 μL CCK-8 reagent at 37°C for 2 hours, and the OD was measured using a microplate reader. 450 Cell viability was calculated, with three replicates per group. Figure 7 As shown in Figure e, with the increase of the embolic agent dosage, more bevacizumab was released and the cell activity was worse, demonstrating an effective function in inhibiting the proliferation of vascular wall cells.

[0143] Example 4: Motion Control Triggered by External Magnetic Field

[0144] Solid-liquid dual-phase embolization microspheres were prepared according to the Janus porous microspheres and Janus ciliated microspheres in an 8:2 ratio as described in Example 2. Janus ciliated microspheres loaded with iron tetroxide nanoparticles, blank Janus porous microspheres, and blank iodized oil droplets were also constructed.

[0145] A certain volume of the solid-liquid dual-phase embolic agent dispersion was placed in a transparent glass bottle and allowed to stand to allow it to disperse evenly. A neodymium iron boron magnet was placed close to the outer wall of the bottle, and the enrichment behavior of the sample under the action of the magnetic field gradient was recorded. Figure 8 The left side of Figure b shows the sample in a dispersed state inside the bottle; the right side shows a distinct black enrichment area near the magnet, indicating that the sample can be rapidly attracted and enriched by an external magnetic field, thereby achieving positioning, aggregation, or recovery.

[0146] Hysteresis loop test: Magnetic Janus ciliated microspheres (“microspheres”) are used; a solid-liquid dual-phase embolic agent (“microsphere-coated iodized oil”) is formed by coating the magnetic microspheres. A vibrating sample magnetometer (VSM) or equivalent magnetic field testing equipment is used to scan within the applied magnetic field strength range (e.g., -2 kOe to +2 kOe) to obtain the magnetization-magnetic field curve. Figure 8 The two S-shaped curves shown in Figure c indicate that the sample exhibits typical paramagnetic / ferromagnetic response characteristics: the magnetization intensity rises rapidly with increasing magnetic field and tends to saturate, while the magnetization intensity can fall back after the field is removed. Furthermore, the solid-liquid dual-phase particles (microspheres coated with iodine oil) show a considerable overall magnetic response, indicating that "microsphere-coated droplets" can effectively transfer magnetism to the macroscopic particle level, making the droplets maneuverable under a magnetic field. The difference between the two curves can be used to reflect the difference in the content of magnetic components per unit mass of sample, as well as the change in equivalent magnetic moment caused by the "coating structure" (e.g., the increase in total magnetic moment due to the integration of multiple microspheres on the droplet surface or the difference caused by dispersion and dilution).

[0147] Microfluidic straight channels (e.g., PDMS-glass slide bonding channels) were fabricated, with channel width / height set to the order of 1000 μm. The channels were sequentially perfused with different media: a PBS liquid channel and a simulated blood liquid channel (fresh sheep blood). To facilitate tracking, fluorescent labels (e.g., red fluorescence) could be introduced into the solid-liquid biphase particles during fabrication, allowing for tracking of their motion in fluorescence mode. Permanent magnets were placed on the sides of the channels to create a transverse magnetic field gradient. The movement of the particles within the channels was recorded in real-time using a microscope. By changing the position of the magnets (e.g., switching from the left to the right side of the channel), it was observed whether the direction of particle motion could reversibly switch with changes in the magnetic field direction. Figure 8As shown in Figure d, the left column shows the bright-field motion sequence within the PBS channel, and the right column shows the fluorescence motion sequence within the simulated blood channel. White arrows indicate the direction of particle motion under the influence of the magnetic field. It can be seen that the particles can continuously move along the magnetic field gradient direction within the channel, and their direction of motion changes accordingly after the magnet's position is changed, indicating that the system possesses reversible magnetic field-driven motion and navigation capabilities.

[0148] For example Figure 8 The video recorded in the middle d-axis was tracked frame by frame to obtain particle centroid displacement-time data; velocity was calculated, and the average velocity and standard deviation of the particle trajectory were statistically analyzed. The media included H2O (deionized water), PBS, SPSS (physiological saline), and Blood (anticoagulated sheep blood).

[0149] like Figure 8 The figure shows that the particle velocity was similar in H2O, PBS, and SPSS media, but significantly decreased in the Blood group. This result indicates that the system can maintain stable magnetically driven motion in low- and medium-viscosity media; in whole blood, due to higher viscosity, additional resistance from cellular and protein components, and interfacial interactions, the velocity decreased, but it still exhibited a tendency to respond to magnetic traction, thus providing evidence for magnetically controlled localization in complex in vivo environments.

[0150] Example 5: Multi-stage controllable drug release process

[0151] 5.1 A solid-liquid biphasic embolization microsphere system allows three drugs to be located in different structural domains and exhibit rapid, intermediate, and slow sequential release:

[0152] Phase 1 (rapid release): Doxorubicin is electrostatically adsorbed onto the pore surface of Janus porous microspheres; Phase 2 (intermediate release): Atezolizumab is located inside the iodized oil droplets and undergoes diffusion release; Phase 3 (slow release): Bevacizumab is embedded inside the Janus ciliated microspheres and achieves long-term release as the polymer degrades.

[0153] 5.2 The above embodiments were constructed as follows: 1. Janus porous microspheres adsorbing doxorubicin, 2. Janus ciliated microspheres encapsulating bevacizumab, 3. Iodized oil droplets dissolving atezolizumab.

[0154] Janus porous microspheres adsorbed with doxorubicin and Janus ciliated microspheres encapsulated with bevacizumab were mixed at a mass ratio of 2:8, with a total weight of 200 mg, and dispersed in 40 mL of deionized water. 500 μL of iodized oil dissolved in atezolizumab was slowly added to the oil-water interface. The disperser blade was placed at the oil-water interface, and emulsification was carried out at 1000 rpm for 10 min to form uniform droplets covered by the two types of microspheres, thus constructing a three-drug loaded solid-liquid biphasic embolic microsphere. After standing for 30 min, the supernatant was aspirated; the microspheres were resuspended in water and gently shaken; the washing was repeated 3–5 times to remove free microspheres, yielding the final sample, denoted as the three-drug loaded solid-liquid biphasic embolic microsphere.

[0155] 10 mg of three-drug-loaded solid-liquid biphasic embolic microspheres were placed in a dialysis bag and dialyzed in PBS buffer (pH 7.4) and acidic buffer (pH 5.0), with a dialysate volume of 50 mL. Dialysis was performed at the following sampling time points: 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, and 240 h, with 3 mL of dialysate collected immediately and immediately replenished with an equal volume of fresh dialysate at the same pH. DOX was quantified by UV spectroscopy, while bevacizumab and atezolizumab were quantified using an ELISA kit, as shown below. Figure 9 The drug release curves shown indicate that the three drugs are released sequentially, with the release occurring in a neutral environment (e.g., under certain conditions). Figure 9 As shown in Figure a), the drug curves of all three drugs are relatively flat, while the release rate increases under acidic conditions (e.g., Figure 9 As shown in Figure b), this facilitates rapid drug release upon contact with the tumor, while allowing for slower drug release as the tumor microenvironment improves or the drug is removed from the tumor tissue, thus achieving controlled release. It should be noted that the drug release rate is calculated as (released amount / carried amount) * 100%, determined by... Figure 9 It was found that at pH 5.0, doxorubicin was the first drug to reach 100% release over time, followed by atezolizumab. Bevacizumab did not reach 100% release within the monitoring period. The overall release rate of the three drugs was higher at pH 5.0 than at pH 7.4. The release curve was flatter at pH 7.4, reflecting the sustained-release characteristics under neutral conditions. Therefore, the controllable release of the three drugs can be effectively regulated by pH value.

[0156] 5.3 Photothermal acceleration of drug release enables an effective and controllable multi-stage drug release process. Similarly, three-drug loaded solid-liquid biphasic embolization microspheres are constructed.

[0157] 10 mg of three-drug-loaded solid-liquid biphasic embolic microspheres were placed in a dialysis bag and dialyzed in PBS buffer (pH 7.4) to a volume of 50 mL. The microspheres were irradiated with a 740 nm laser for 2 min. At sampling time points of 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, and 240 h, 3 mL of dialysate was collected. An equal volume of fresh dialysate at the same pH was immediately added, followed by irradiation with a 740 nm laser for 2 min. DOX was quantified by UV spectroscopy, while bevacizumab and atezolizumab were quantified using an ELISA kit. Figure 10 The drug release curves shown indicate that, with the addition of photothermal stimulation, the release rates of doxorubicin and atezolizumab, which are released via diffusion, are faster, while the release curve of bevacizumab, which is released via degradation, shows little change (compared to...). Figure 9 As shown in Figure a), this allows for effective and controllable multi-stage drug release. Therefore, it can be seen that... Figure 10 As shown, compared with the drug release system at pH 7.4, after applying photothermal treatment, the release rates of doxorubicin and atezolizumab increased while the release rate of bevacizumab remained unchanged. Thus, the controllable release of the three drugs can be effectively regulated through photothermal treatment.

[0158] In summary, the solid-liquid biphasic embolization microspheres of this invention, comprising Janus microspheres (including Janus ciliated microspheres and Janus porous microspheres), provide high specific surface area and multiple sites, enabling them to carry complex structural functional units with one or more active components and specific functions, as well as their drug-carrying components, exhibiting strong loading capacity. They allow for the integration of multiple drugs and / or functional units into functional superposition, reducing compatibility and operational complexity and achieving multi-drug synergy. They enable multi-stage sequential release, improving therapeutic stability and reducing burst release risk. Functional units can be flexibly replaced according to disease needs to achieve applications in chemotherapy, immunotherapy, anti-angiogenesis, radiosensitization, imaging, and other fields, demonstrating good versatility. Furthermore, the particle size of the microspheres can be controlled to adapt to injection / catheter delivery, resulting in good deliverability.

[0159] All aspects, embodiments, features, and examples of this invention are to be regarded as illustrative in all respects and are not intended to limit the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0160] Throughout this invention, wherever a composition is described as having, containing, or including specific components, or wherever a process is described as having, containing, or including specific process steps, it is contemplated that the compositions taught in this invention are also substantially composed of or comprised of the described components, and that the processes taught in this invention are also substantially composed of or comprised of the described process steps.

[0161] Unless otherwise specifically stated, the use of the terms “include, include, including” or “have, has, or having” should generally be understood as open-ended and non-restrictive.

[0162] It should be understood that the order of the steps or the order in which specific actions are performed is not particularly important, as long as the teachings of this invention remain operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0163] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A solid-liquid dual-phase embolic microsphere capable of multi-stage controlled release, characterized in that, The system comprises an oleophilic and hydrophobic liquid unit and hydrophilic Janus microspheres. The Janus microspheres include Janus ciliated microspheres with a ciliated structure and Janus porous microspheres with a porous structure. The Janus ciliated microspheres and Janus porous microspheres are fused together in a specific ratio to coat the liquid unit. Different functional units with one or more active components can be loaded inside the liquid unit, on the surface and / or inside the Janus porous microspheres and Janus ciliated microspheres. Furthermore, the release rates of the functional units loaded inside the liquid unit, on the surface and / or inside the Janus porous microspheres and Janus ciliated microspheres differ to form at least two stages of multi-stage controlled release.

2. The solid-liquid dual-phase embolization microspheres according to claim 1, characterized in that, Both the Janus ciliated microspheres and the Janus porous microspheres have asymmetric structures. One side of the Janus ciliated microspheres is a ciliated structure, and the other side is a smooth structure. One side of the Janus porous microspheres is a porous structure, and the other side is a smooth structure. The smooth structure in both the Janus porous microspheres and the Janus ciliated microspheres is composed of polymethyl methacrylate, while the ciliated structure and the porous structure are composed of block polymers with hydrophilic and hydrophobic segments of different molecular weights.

3. The solid-liquid dual-phase embolization microspheres according to claim 2, characterized in that, The preparation method of the Janus microspheres includes the following steps: Preparation of the polymer oil phase: Take dichloromethane and dissolve polymethyl methacrylate and block polymer in a mass ratio of 7:1 to obtain an oil phase solution with a polymer mass fraction of 4%. Microfluidic emulsification into droplets: The above polymer oil phase solution and polyvinyl alcohol aqueous solution are emulsified in a microfluidic droplet forming channel, and the flow rate ratio of oil phase to water phase is controlled at 5:8, so that the polymer oil phase forms water-in-oil emulsion droplets in the polyvinyl alcohol aqueous phase. Phase separation induces Janus structure formation: The above emulsion was left to stand at room temperature for 4 h to allow the polymethyl methacrylate phase and the block polymer phase to fully separate within the emulsion, forming an asymmetric structure prototype. Curing and solvent evaporation: The emulsion with the asymmetric structure was heated and stirred at 40°C and 200 rpm for 2 hours to allow the dichloromethane to evaporate completely and the emulsion droplets to solidify into microspheres. Centrifugation, washing and freeze-drying: The above emulsion droplets were solidified into a dispersion of microspheres by centrifugation at 6000 rpm, the supernatant was removed and the microspheres were resuspended in deionized water. The microspheres were washed three times to obtain Janus microspheres, which were then freeze-dried for storage. In the preparation methods of Janus porous microspheres and Janus ciliated microspheres, the only difference is the molecular weight of the hydrophobic segments in the block polymers, and the molecular weight of the hydrophobic segments in the block polymers of the Janus porous microspheres is smaller than that of the block polymers of the Janus ciliated microspheres.

4. The solid-liquid dual-phase embolic microspheres according to claim 3, characterized in that, In the Janus microspheres, the ratio of Janus ciliated microspheres to Janus porous microspheres can be adjusted by changing the ratio of the Janus porous microspheres.

5. The solid-liquid dual-phase embolic microspheres according to claim 1, characterized in that, The functional units inside the liquid unit, on the surface and / or inside the Janus porous microspheres and the Janus ciliated microspheres are loaded through different loading methods to form a multi-level controlled release. The loading methods include physical adsorption / permeation, electrostatic / coordination / host-guest interaction, covalent coupling or in-situ embedding.

6. The solid-liquid dual-phase embolic microspheres according to claim 5, characterized in that, The multi-stage controlled release includes: the release of functional units located on the surface or within the porous channels of the Janus porous microspheres in the first stage; The release of functional units within the liquid unit in the second stage; The third stage involves the release of functional units within the Janus microspheres.

7. The solid-liquid dual-phase embolization microspheres according to claim 6, characterized in that, The functional units are loaded within the porous channels of the Janus porous microspheres via electrostatic adsorption. This electrostatic adsorption includes treating the Janus porous microspheres with hydrochloric acid to hydrolyze the ester groups to carboxyl groups for loading positively charged drugs; or forming amide bonds with sodium aminoborane under nitrogen protection for loading negatively charged drugs; and / or The functional unit is loaded inside the liquid unit by means of direct dissolution or dispersion within the liquid unit; and / or The functional units are encapsulated inside the Janus microspheres during the synthesis process by being dissolved or dispersed in a dichloromethane solution.

8. The solid-liquid dual-phase embolization microspheres according to claim 1, characterized in that, The functional units include sustained-release drugs, biomacromolecules, nanocarriers, inorganic / hybrid nanostructures, and magnetic nanoparticles; when the functional unit is a magnetic nanoparticle, it can achieve controllable movement in various fluids.

9. The method for preparing solid-liquid dual-phase embolic microspheres according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Janus ciliated microspheres and Janus porous microspheres loaded with different functional units were prepared respectively. S2. Preparation of solid-liquid dual-phase precursor: Janus ciliated microspheres and Janus porous microspheres loaded with different functional units are mixed and dispersed in deionized water at a set mass ratio to obtain a mixed microsphere aqueous dispersion. S3, Microsphere Mixed Dispersion: Add liquid units to the above mixed microsphere aqueous dispersion so that the liquid units are located near the oil-water interface; S4. Emulsification and Pickering Coating: Place the disperser blade at the oil-water interface and emulsify for 10 minutes at 1000 rpm to form uniform droplets in the liquid unit. At the same time, Janus ciliated microspheres and Janus porous microspheres are adsorbed on the droplet surface to form a Pickering-like emulsion structure. S5. Settling and washing to remove free emulsion: After emulsification, let stand for a preset time, shake to disperse, and then let stand for another preset time. Remove the supernatant, add deionized water to resuspend and gently shake to disperse. Repeat washing 3-5 times to remove unadsorbed microspheres and obtain clean solid-liquid biphase embolization microspheres.

10. The use of the solid-liquid biphasic embolization microspheres according to any one of claims 1-8 or the solid-liquid biphasic embolization microspheres obtained by the preparation method according to claim 9 in the preparation of drugs for local drug delivery, interventional delivery, image-guided therapy or multimodal combined therapy.

Images