High-internal-phase emulsion with nested structure, preparation method of high-internal-phase emulsion and porous material

Abstract

The invention discloses a high internal phase emulsion with a nested structure, a preparation method thereof and a porous material, the high internal phase emulsion has at least one layer of nested structure, the nested structure is an HIPE-1 / HIPE-2 structure, the HLB value of an emulsifier A used by the HIPE-1 is recorded as H1, the HLB value of an emulsifier B used by the HIPE-2 is recorded as H2, H1 and H2 meet the condition that H1 is greater than H2, and the difference between H1 and H2 is 8-12; according to the preparation method disclosed by the invention, the emulsifying agents in the HIPE-2 emulsion and the HIPE-1 emulsion are controlled to have a certain difference value, so that the interface structures of at least two different HIPE emulsions in the obtained high-internal-phase emulsion are stable and are immiscible with each other, and the finally obtained porous material has multistage pore diameters and provides a bionic carrier for tissue engineering, drug screening and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomimetic materials, and particularly relates to a high internal phase emulsion with a nested structure and a preparation method thereof and a porous material. BACKGROUND

[0002] The high internal phase emulsion (HIPE, the internal phase accounts for 74%) has high porosity (up to more than 90%) and good connectivity, and has become a new generation of carrier for 3D cell culture, but the prior art has significant defects: 1) single structure: the traditional HIPE is mostly a single-layer "water-in-oil" structure, and can only load a single drug or nutrient, and cannot simulate the real complex multilayer environment, so that the cells are difficult to differentiate into functional tissues during the culture process; 2) release disorder: the existing multilayer carrier is a layered coating, and only relies on the physical stacking of the coating adhesion, the interlayer bonding force is weak, and is easy to fall off, and the drug release cannot be accurately controlled, and cannot achieve the sustained gradient release required in the body; 3) difficult to prepare: when constructing the multilayer HIPE emulsion, the traditional shearing will cause the fusion of the interlayer interface due to the high viscosity of the HIPE, and the nested structure cannot be formed.

[0003] For biological composition engineering, the differentiation of cells requires different environments, nutrients and signals, and the traditional single-layer HIPE cannot meet this requirement, resulting in a low cell differentiation rate.

[0004] Therefore, developing a porous material capable of simulating the in-vivo environment is the key to breaking through the bottleneck of 3D cell culture. SUMMARY

[0005] In view of the defects of the prior art, the purpose of the present application is to provide a high internal phase emulsion with a nested structure and a preparation method thereof and a porous material.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a high internal phase emulsion with a nested structure, the high internal phase emulsion has at least one nested structure, the nested structure is a HIPE-1 / HIPE-2 structure, the HLB value of the emulsifier A used in the HIPE-1 is denoted as H1, the HLB value of the emulsifier B used in the HIPE-2 is denoted as H2, H1 and H2 satisfy: H1>H2, and the difference between them is 8-12, such as 8, 9, 10, 11, 12, etc.

[0008] The present application can make the interface structure of at least two different HIPE emulsions in the obtained high internal phase emulsion stable and not miscible by controlling the difference between the emulsifiers contained in the HIPE-2 emulsion and the HIPE-1 emulsion.

[0009] Different from the conventional W / O (water-in-oil) structure of emulsion, the HIPE-1 / HIPE-2 structure of the present application is a structure of HIPE-2 emulsion packing HIPE-1 emulsion, that is, in the high internal phase emulsion of the present application, a structure of HIPE-2 emulsion as a continuous phase and HIPE-1 emulsion as a dispersed phase.

[0010] Therefore, the porous material obtained from the high internal phase emulsion of the present application has at least two levels of pore sizes, specifically, a first level of pore size formed by the HIPE-1 emulsion and a second level of pore size formed by the HIPE-2 emulsion.

[0011] Preferably, the high internal phase emulsion has a two-layer nested structure, which is a HIPE-1 / HIPE-2 / HIPE-3 structure, the HLB value of the emulsifier C used in the HIPE-3 is denoted as H3, and H3 needs to satisfy: H2>H3, and the difference between H2 and H3 is 2-6, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.

[0012] The HIPE-1 / HIPE-2 / HIPE-3 structure of the present application is a structure of HIPE-3 emulsion packing HIPE-2 emulsion packing HIPE-1 emulsion, which can also be understood as a multi-layer nested structure of HIPE emulsion packing HIPE emulsion, or can be understood as a nested structure formed by three non-miscible and independent emulsions, wherein HIPE-3 is the outermost layer, constituting the continuous phase of the high internal phase emulsion, and HIPE-1 / HIPE-2 is the dispersed phase of the high internal phase emulsion, and at the same time, if a randomly selected certain dispersed phase cell is taken as a whole, it is a structure of HIPE-2 emulsion packing HIPE-1 emulsion, that is, in a randomly selected certain dispersed phase cell, HIPE-2 emulsion as a continuous phase and HIPE-1 as a dispersed phase; that is, the high internal phase emulsion with a two-layer nested structure of the present application, HIPE-3 as the outermost layer, HIPE-2 as the middle layer, and HIPE-1 as the innermost layer.

[0013] The present application realizes interfacial stability (high hydrophobicity in the outer layer, medium hydrophobicity in the middle layer, and low hydrophobicity in the inner layer) by using "gradient HLB value emulsifiers", and the interfacial tension between the layers is controlled at 10-15 mN / m, without fusion or shedding.

[0014] Preferably, H1 is 12-16, for example, 12, 13, 14, 15, 16, etc.

[0015] Preferably, the emulsifier A includes any one or a combination of at least two of polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, or polyglycerol ricinoleate.

[0016] Preferably, the HIPE-1 has a viscosity of 2000-3000 cP, such as 2000 cP, 2200 cP, 2500 cP, 2600 cP, 2800 cP, 3000 cP, etc.

[0017] Preferably, H2 is 4-8, such as 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, etc.

[0018] Preferably, the emulsifier B comprises any one or a combination of at least two of sorbitan monostearate (Span 60), polyglyceryl stearate, sorbitan monopalmitate, or polyglyceryl isostearate.

[0019] Preferably, the HIPE-2 has a viscosity of 3000-4000 cP, such as 3000 cP, 3200 cP, 3500 cP, 3800 cP, 4000 cP, etc.

[0020] Preferably, the initiator used for the HIPE-2 comprises photoinitiator B, or a combination of photoinitiator B and thermal initiator B.

[0021] Preferably, H3 is 2-6, such as 2, 3, 4, 5, 6, etc.

[0022] Preferably, the emulsifier C comprises any one or a combination of at least two of sorbitan monooleate (Span 80), sorbitan trioleate, polyglyceryl oleate, or diglyceryl monooleate;

[0023] Preferably, the HIPE-3 has a viscosity of 4000-5000 cP, such as 4000 cP, 4200 cP, 4500 cP, 4800 cP, 5000 cP, etc.

[0024] Preferably, the initiator used for the HIPE-3 comprises photoinitiator C, or a combination of photoinitiator C and thermal initiator C.

[0025] Preferably, the HIPE-1 has a diameter of 70-90 μm, such as 70 μm, 72 μm, 75 μm, 78 μm, 80 μm, 82 μm, 85 μm, 88 μm, 90 μm, etc.

[0026] Preferably, the HIPE-2 has a layer thickness of 50-70 μm, such as 50 μm, 52 μm, 55 μm, 58 μm, 60 μm, 62 μm, 65 μm, 68 μm, 70 μm, etc.

[0027] Preferably, the layer thickness of the HIPE-3 is 50-70 μm, such as 50 μm, 52 μm, 55 μm, 58 μm, 60 μm, 62 μm, 65 μm, 68 μm, 70 μm, etc.

[0028] In the present application, the HIPE-1, HIPE-2, and HIPE-3 are all HIPE emulsions, which include an oil phase, an aqueous phase, an emulsifier, and an initiator, all of which are components conventionally used in HIPE emulsions, and the present application does not make excessive limitations, as long as the HLB value of the emulsifier in the HIPE-1, HIPE-2, and HIPE-3 meets the requirements defined in the present application. As for other components, the present application only exemplarily lists them as follows:

[0029] The HIPE-1 includes an oil phase A, an aqueous phase A, an emulsifier A, and an initiator A, the HIPE-2 includes an oil phase B, an aqueous phase B, an emulsifier B, and an initiator B, and the HIPE-3 includes an oil phase C, an aqueous phase C, an emulsifier C, and an initiator C.

[0030] Preferably, the oil phase A, oil phase B, and oil phase C all include a polymerizable monomer and a crosslinking monomer, and the polymerizable monomer and crosslinking monomer used in the HIPE-1, HIPE-2, and HIPE-3 of the present application can be the same or different, and the present application does not make excessive limitations, as long as any polymerizable monomer and crosslinking monomer that can meet the application requirements of the present application can be applied to the present application. The polymerizable monomer and crosslinking monomer that can be specifically selected in the present application are listed as follows:

[0031] Preferably, the polymerizable monomer accounts for 5-97% of the weight of the oil phase, such as 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 97%, etc., and further preferably 40-80%, and can include at least one water-insoluble monofunctional alkyl acrylate or alkyl methacrylate, a vinyl-based monomer, or a degradable polymerizable monomer.

[0032] Preferably, the monofunctional alkyl acrylate or alkyl methacrylate includes C4-C18 alkyl acrylate and / or C2-C18 alkyl methacrylate.

[0033] Preferably, the vinyl-based monomer includes styrene, chloroethylene, vinylidene chloride, isoprene, and chlorobutadiene, etc.

[0034] Preferably, the degradable polymerizable monomer includes PGA prepolymer (polyglycolic acid), PLA prepolymer (polylactic acid prepolymer), PCL prepolymer (polycaprolactone prepolymer), or PLGA prepolymer (poly(lactic-co-glycolic acid) prepolymer).

[0035] Preferably, the cross-linking agent is about 2-40% by weight of the oil phase, such as 2%, 5%, 10%, 20%, 30%, 40%, etc., further preferably 2-30%; this type of cross-linking monomer includes any one or a combination of at least two of the cross-linking agents containing two or more double bond-containing acrylate, methacrylate groups or divinylbenzene.

[0036] Preferably, the cross-linking agent is a multi-functional cross-linking monomer, including ethylene glycol dimethacrylate (EGDMA), hexanediol diacrylate (HDDA), polyethylene glycol diacrylate (PEGDA), trimethylolpropane trimethacrylate (TMPTA), divinylbenzene (DVB), etc.

[0037] Similarly, the present application does not limit the amount and specific type of emulsifier, as long as it can meet the limitation of the HLB value of the emulsifier of the present application.

[0038] Preferably, the amount of the emulsifier added is 1-20% of the mass of the oil phase, such as 1%, 2%, 3%, 5%, 8%, 10%, 12%, 15%, 18%, 20%.

[0039] Similarly, the present application does not limit the amount and specific type of initiator, any initiator that can meet the requirements of HIPE emulsion polymerization can be applied to the present application, the initiator can be an oil-soluble initiator, or a water-soluble initiator, the present application preferably is an oil-soluble initiator. The present application is exemplified as follows:

[0040] Preferably, the initiator is an oil-soluble initiator, the amount of the oil-soluble initiator added is 0.05-10% of the mass of the oil phase, such as 0.05%, 1%, 2%, 5%, 10%, etc. The oil-soluble initiator includes peroxide, such as benzoyl peroxide, or amine, azo, such as azobisisobutyronitrile.

[0041] Preferably, the initiator is a water-soluble initiator, the amount of the initiator can be 1-20% of the molar mass of the polymerizable monomer of the oil phase, such as 1%, 2%, 5%, 10%, 15%, 20%, etc., or 0.001-10% of the mass of the aqueous phase, such as 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, 10%, etc.

[0042] Preferably, the initiator is an oil-soluble initiator, including any one or a combination of at least two of thermal initiators or photoinitiators.

[0043] The thermal initiator includes any one or a combination of at least two of benzoyl peroxide (BPO), azobisisobutyronitrile (AIBN), azobisisoheptyl nitrile (ABVN), di-tert-butyl peroxide (DTBP) or cumene hydroperoxide (CHP), and the addition amount is 0.1-10% of the mass of the oil phase, for example, 0.1%, 1%, 2%, 5%, 10% and the like, which is suitable for a 70-95℃ thermal curing process.

[0044] The photoinitiator includes any one or a combination of at least two of 2-hydroxy-2-methyl-1-phenyl-1-propanone (1173), 2,4,6-trimethylbenzoylphosphine oxide (TPO), isopropyl thioxanthone (ITX) or 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone (907), and the addition amount is 0.1-5% of the mass of the oil phase, for example, 0.1%, 1%, 2%, 3%, 4%, 5% and the like (the photoinitiator is more efficient, and the amount can be lower than the thermal initiator), which is suitable for a normal temperature UV curing process (wavelength of light source: 240-440 nm, for example, 240 nm, 250 nm, 300 nm, 350 nm, 400 nm, 440 nm and the like, curing time: 5-30 min, for example, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min).

[0045] In the present application, the water phase A, the water phase B and the water phase C are also conventionally selected in the art, and can be the same or different, and the present application does not make too many limitations, but only exemplary descriptions are made.

[0046] In the present application, the water phase includes deionized water, and can also include a water-soluble electrolyte. The introduction of the water-soluble electrolyte in the water phase can reduce the solubility of the oil phase components in water and maintain the structural stability of the water-in-oil system.

[0047] Preferably, the water-soluble electrolyte includes a chloride or sulfate of an alkaline earth metal (for example, calcium or magnesium) or a chloride or sulfate of an alkali metal (for example, sodium).

[0048] Preferably, the addition amount of the water-soluble electrolyte is 0.2-40% based on 100% of the total mass of the water phase, for example, 0.2%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% and the like.

[0049] Preferably, the water phase accounts for 75-97% of the total volume of the HIPE, for example, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 97% and the like.

[0050] Preferably, the oil-soluble solvent can be introduced into the HIPE-1, HIPE-2, and HIPE-3 as a diluent, preferably the oil-soluble solvent is ethyl acetate, toluene, xylene, etc., preferably the amount of addition is 2-10% of the mass of the oil phase, for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. The oil-soluble solvent of the present application can further reduce the viscosity of the HIPE.

[0051] In the present application, the HIPE-1, HIPE-2, and HIPE-3 can be prepared by the following preparation method:

[0052] (1) Mix the emulsifier, polymerizable monomer, crosslinking monomer, and initiator (oil-soluble initiator) to obtain an oil phase mixture;

[0053] (2) Mix deionized water and optionally electrolyte to obtain an aqueous phase, preferably the stirring rate of the mixing is 500-1000 rpm, for example 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, etc., preferably the mixing time is 5-20 min, for example 5 min, 8 min, 10 min, 15 min, 20 min, etc.

[0054] (3) Add the aqueous phase to the oil phase mixture dropwise, continue stirring after the addition is completed, to obtain the HIPE phase.

[0055] Preferably, in step (1), the stirring rate of the mixing is 500-1000 rpm, for example 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, etc., preferably the mixing time is 10-30 min, for example 10 min, 15 min, 20 min, 25 min, 30 min, etc., and preferably the mixing temperature is 25-35°C, for example 25°C, 28°C, 30°C, 32°C, 35°C, etc.

[0056] Preferably, in step (3), the rate of the dropwise addition is 2-10 mL / s, for example 2 mL / s, 3 mL / s, 4 mL / s, 5 mL / s, 6 mL / s, 7 mL / s, 8 mL / s, 9 mL / s, 10 mL / s, etc., and preferably the stirring rate is 800-1500 rpm, for example 800 rpm, 1000 rpm, 1200 rpm, 1500 rpm, etc., and preferably the temperature during the continued stirring is 40-50°C, for example 40°C, 42°C, 45°C, 48°C, 50°C, etc. Within this temperature range, the HIPE has a lower viscosity.

[0057] In a second aspect, the present application provides a method for preparing the high internal phase emulsion with nested structure according to the first aspect, the method comprising:

[0058] The HIPE-1 phase, the HIPE-2 phase and optionally the HIPE-3 phase are mixed by a microfluidic method to obtain the high internal phase emulsion with nested structure.

[0059] Preferably, the method comprises:

[0060] The HIPE-1 phase, the HIPE-2 phase and optionally the HIPE-3 phase are respectively input into different channels of a microfluidic device, and the mixing of the three phases is controlled in a manner of controlling the flow rate to obtain the high internal phase emulsion with nested structure.

[0061] The present application successfully constructs the high internal phase emulsion structure with nested structure by the microfluidic preparation method, and the porous material prepared from the high internal phase emulsion has secondary or tertiary pore size, can realize the simulation of complex environment, and achieves the gradient release of different drugs and nutrients.

[0062] Meanwhile, the preparation method provided by the present application solves the problems of microfluidic interfacial stabilization and precise wrapping of multilayer high-viscosity HIPE, ensures the uniformity and stability of the high internal phase emulsion with nested structure, and provides a biomimetic carrier for tissue engineering and drug screening.

[0063] Preferably, nitrogen is additionally introduced, and the introduction of nitrogen in the present application can maintain the pressure stability in the channel and avoid the blockage of the HIPE phase.

[0064] Preferably, the flow rate of the HIPE-1 phase is 45-55 μL / min, for example, 45 μL / min, 46 μL / min, 48 μL / min, 50 μL / min, 52 μL / min, 55 μL / min, etc.

[0065] Preferably, the flow rate of the HIPE-2 phase is 75-85 μL / min, for example, 75 μL / min, 76 μL / min, 78 μL / min, 80 μL / min, 82 μL / min, 85 μL / min, etc.

[0066] Preferably, the flow rate of the HIPE-3 phase is 95-105 μL / min, for example, 95 μL / min, 96 μL / min, 98 μL / min, 100 μL / min, 102 μL / min, 105 μL / min, etc.

[0067] Preferably, the flow rate of the nitrogen gas is 15-25 μL / min, such as 15 μL / min, 16 μL / min, 18 μL / min, 20 μL / min, 22 μL / min, 25 μL / min, etc.

[0068] Preferably, the HIPE-1 phase uses a channel diameter of 100-120 μm, such as 100 μm, 102 μm, 105 μm, 106 μm, 108 μm, 110 μm, 112 μm, 115 μm, 118 μm, 120 μm, etc.

[0069] Preferably, the HIPE-2 phase uses a channel diameter of 200-220 μm, such as 200 μm, 202 μm, 205 μm, 208 μm, 210 μm, 212 μm, 215 μm, 218 μm, 220 μm, etc.

[0070] Preferably, the HIPE-3 phase uses a channel diameter of 300-320 μm, such as 300 μm, 302 μm, 305 μm, 308 μm, 310 μm, 312 μm, 315 μm, 318 μm, 320 μm, etc.

[0071] Preferably, the nitrogen gas uses a central channel diameter of 50-60 μm, such as 50 μm, 52 μm, 55 μm, 56 μm, 58 μm, 60 μm, etc.

[0072] In a third aspect, the present application provides a porous material prepared from the high internal phase emulsion with nested structure of the first aspect.

[0073] The present application uses microfluidic to form a nested unit of "a layer of HIPE-2 emulsion packing HIPE-1 emulsion" or "a two-layer nested structure of HIPE-3 emulsion packing HIPE-2 emulsion packing HIPE-1 emulsion", etc., and after solidification, washing and drying, the tertiary pores of the HIPE-3 phase, the secondary pores of the HIPE-2 phase and the primary pores of the HIPE-1 phase are retained, thereby obtaining a high internal phase emulsion foam porous material with double or triple pore diameters.

[0074] The present application also provides a preparation method of the porous material as described, which comprises:

[0075] The high internal phase emulsion with nested structure of the first aspect is subjected to a solidification reaction and drying to obtain the hierarchical porous material.

[0076] Preferably, the curing reaction comprises a combination of photo-curing and thermal curing, preferably the light source wavelength of the photo-curing is 240-440 nm, such as 240 nm, 250 nm, 260 nm, 280 nm, 300 nm, 320 nm, 350 nm, 380 nm, 400 nm, 440 nm, etc., preferably the curing time of the photo-curing is 5-30 min, such as 5 min, 6 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0077] Preferably, the temperature of the thermal curing is 60-100℃, such as 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, 100℃, etc., and the time is 0.5-8 h, such as 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc.

[0078] The present application can ensure complete curing of the high internal phase emulsion by first performing photo-curing, and then performing thermal curing, so that the HIPE-3 phase of the outermost layer and the HIPE-2 phase of the middle layer of the high internal phase emulsion with a nested structure can be cured.

[0079] The present application realizes precise preparation and functional simulation of multi-layer HIPE through interface stabilization design, viscosity adaptation, and dynamic release control, and specifically:

[0080] (1) Multi-layer HIPE interface stabilization mechanism: The present application uses "gradient HLB value emulsifier" design, the outermost HIPE-3 uses high hydrophobic emulsifier, the middle HIPE-2 uses medium hydrophobic emulsifier, and the innermost HIPE-1 uses low hydrophobic emulsifier, which can avoid interface fusion, so that the high internal phase emulsion has at least one stable nested structure;

[0081] (2) Microfluidic multi-layer wrapping strategy: A four-channel coaxial microfluidic chip is used, the pore size of different channels is controlled, and the viscosity adaptation of "high viscosity of outer layer-low viscosity of inner layer" and the flow rate regulation of "slow flow rate of outer layer-fast flow rate of inner layer" are simultaneously used, to realize uniform wrapping of three-layer HIPE;

[0082] (3) Spatiotemporal gradient release design: The present application can also achieve the regulation of the degradation rate of each layer of material by introducing different polymerization monomers in the HIPE emulsion of different layers, for example, the outer layer HIPE-3 uses a PGA base, the degradation period is 7-10 days; the middle layer HIPE-2 selects a PLGA base, the degradation period is 14-21 days; the inner layer HIPE-1 selects a PCL base, the degradation period is 28-35 days, and can also be combined with the loading mode of drugs / nutrients to achieve the gradient mode of "outer layer drug release first-middle layer sustained release-inner layer slow release".

[0083] In a fourth aspect, the present application provides a use of the high internal phase emulsion with a nested structure of the first aspect or the porous material of the third aspect in 3D cell culture, tissue engineering scaffold, daily hygiene product, drug screening or drug loading.

[0084] Since the present application can control the degradation rate and loading of different nested layers, the porous material provided by the present application can simulate the "multi-region, multi-signal" microenvironment in the body, release nutrients / growth factors in a gradient, match the phased needs of cell differentiation, and further simulate the extracellular matrix (ECM) in the cell culture process.

[0085] Meanwhile, when the porous material provided by the present application is applied to tissue engineering, the outer layer simulates the subchondral bone microenvironment (loaded with osteogenic factors), the middle layer simulates the tide line region (loaded with cartilage-bone conversion factors), and the inner layer simulates the cartilage region (loaded with cartilage nutrition factors), achieving seamless regeneration at the interface.

[0086] Furthermore, the porous material provided by the present application can simulate the "tissue-stromal-vascular" multi-region microenvironment in the body, or simulate the tumor microenvironment: outer layer: simulating tumor stroma (loaded with stromal cells + inflammatory factors), middle layer: simulating tumor cell layer (loaded with tumor cells + chemotherapy drugs), inner layer: simulating the microenvironment around blood vessels (loaded with endothelial cells + nutrition factors), and therefore can be applied to drug screening to construct a 3D model closer to the in vivo environment.

[0087] And, the porous material can be applied to a drug carrier to constitute a precise drug delivery system, and has the advantages of local implantation type gradient drug release, specifically: the multi-layer degradation period difference of the porous material can realize a release mode of 'fast effect-sustained treatment-long-term maintenance', and is suitable for precise treatment of local diseases, for example, local drug delivery for fracture repair: outer layer (7-10 days of degradation): rapid release of anti-inflammatory drugs (such as ibuprofen) to control postoperative inflammation; middle layer (14-21 days of degradation): sustained release of bone repair factors (such as PDGF) to promote callus formation; inner layer (28-35 days of degradation): slow release of antibacterial drugs (such as vancomycin) to prevent implant-related infections; that is, the present application has the advantages of realizing multi-stage treatment with one implant, reducing the number of dressing changes, and reducing the risk of infection.

[0088] In particular, the porous material can be used in the field of sanitary napkins, dressings and other daily hygiene products, and one or more layers of the porous material with the same or different pore sizes are used as the core of sanitary napkins, dressings and other daily hygiene products, so that the porous material can absorb, guide, lock, prevent backflow or release liquids such as water, menstrual blood, blood, body fluid and nutrients, and the product has excellent properties such as lightness, dryness, long-acting and slow release.

[0089] Compared with the prior art, the present application has the following beneficial effects:

[0090] (1) Innovative structure: the present application creatively provides a high internal phase emulsion with a nested structure;

[0091] (2) Interfacial stability: the present application controls the interfacial tension between the layers to be 10-15 mN / m by designing the emulsifier with 'outer layer high hydrophobicity and inner layer low hydrophobicity', and the nested structure is stable;

[0092] (3) Innovative preparation method: the present application adopts a four-channel coaxial microfluidic and 'high viscosity-slow flow rate' adaptation strategy to overcome the delivery problem of multi-layer high viscosity HIPE;

[0093] (4) Innovative release control: the present application can realize the spatiotemporal gradient release of drugs / nutrients by 'degradation rate gradient and load mode adaptation'. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 A simple structure diagram of a four-channel microfluidic device used for the embodiment 1 of the present application;

[0095] Wherein, 1-center channel; 2-inner layer channel; 3-middle layer channel; 4-outer layer channel. DETAILED DESCRIPTION

[0096] The technical solutions of the present application are further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0097] Unless otherwise specified, the raw materials involved in the following specific embodiments of the present application are all conventional materials in the art and can be purchased through commercially available products. Some raw material information is as follows:

[0098] PLGA prepolymer (lactic acid: glycolic acid = 75:25): Sigma-Aldrich, model 75 / 25;

[0099] PCL prepolymer: NatureWorks, model 6800;

[0100] PGA prepolymer: Sigma-Aldrich, model P3884;

[0101] Span 60 (SPAN 60, HLB = 4.7), Span 80 (SPAN 80, HLB = 4.3): Croda UK;

[0102] Tween 80 (tween 80, HLB = 15), Tween 60 (tween 60, HLB = 14.9): Croda UK;

[0103] Polyglyceryl ricinoleate (HLB = 5.0): Shandong Binzhou Jinsheng New Material;

[0104] Polyglyceryl stearate (HLB = 5.2): purchased from Shandong Binzhou Jinsheng New Material.

[0105] Example 1

[0106] This embodiment provides a high internal phase emulsion with a nested structure and a porous material, wherein the high internal phase emulsion has a two-layer nested structure, which is a HIPE-1 / HIPE-2 / HIPE-3 structure, and the specific composition is as follows:

[0107] (1) The outermost layer, HIPE-3 layer (HIPE-3 phase, viscosity 4500 cP), comprising:

[0108] Oil phase C: PLGA prepolymer 45 parts, EGDMA 12 parts, ethyl acetate 6 parts;

[0109] Emulsifier C: SPAN80 8 parts;

[0110] Initiator C: BPO 0.5 parts, TPO 0.5 parts;

[0111] Water phase C: 4 wt% CaCl2deionized water solution, accounting for 78% of the total volume of HIPE-3 phase;

[0112] (2) middle layer, HIPE-2 (HIPE-2 phase, viscosity 3500 cP), comprising:

[0113] Oil phase B: PCL prepolymer 42 parts, TMPTA 10 parts, butyl acetate 8 parts;

[0114] Emulsifier B: SPAN60 6 parts;

[0115] Initiator B: BPO 0.5 parts, TPO 0.5 parts;

[0116] Water phase B: 4 wt% CaCl2deionized water solution, accounting for 78% of the total volume of HIPE-3 phase;

[0117] (3) innermost layer, HIPE-1 layer (HIPE-1 phase, viscosity 2500 cP), comprising:

[0118] Oil phase A: PGA prepolymer 40 parts, DVB 11 parts, ethyl acetate 7 parts;

[0119] Emulsifier A: tween80 6 parts;

[0120] Initiator A: BPO 1 part;

[0121] Water phase A: 4 wt% CaCl2deionized water solution, accounting for 78% of the total volume of HIPE-3 phase;

[0122] The preparation methods of the three different emulsions are all carried out according to the following preparation method:

[0123] The oil phase components are mixed at 30°C and 500 rpm for 20 min, then the water phase is added dropwise to the oil phase at a dropwise rate of 5 mL / s, and after dropping, 800 rpm, 40°C continues to stir for 20 min, water bath heating can reduce the viscosity of the emulsion.

[0124] (4) Preparation method of high internal phase emulsion:

[0125] A four-channel coaxial microfluidic chip (structure see Figure 1 ): outer channel 4 (HIPE-3, inner diameter 300 μm), middle channel 3 (HIPE-2, inner diameter 200 μm), inner channel 2 (HIPE-1, inner diameter 100 μm), center channel 1 (nitrogen auxiliary, inner diameter 50 μm), driving system: "peristaltic pump + nitrogen pressure" composite driving for outer channel, peristaltic pump driving for middle / inner layer;

[0126] Flow rate setting: 100 μL / min for the outer layer, 80 μL / min for the middle layer, 50 μL / min for the inner layer, and 20 μL / min for the center nitrogen;

[0127] Wrap control: The nested morphology was observed in real time by microscope, and the flow rate was adjusted to stabilize the nested unit diameter at 280±20 μm, the inner layer HIPE diameter at 80±8 μm, the middle layer thickness at 60±6 μm, and the outer layer thickness at 60±6 μm;

[0128] Collection: The obtained high internal phase emulsion was collected.

[0129] (5) Preparation method of porous material

[0130] The high internal phase emulsion was injected into a polytetrafluoroethylene mold (5 cm×5 cm×0.5 cm), and then was irradiated with ultraviolet light (wavelength 365 nm) for 10 min (to solidify the outer layer and the middle layer), and then was heated in an oven at 90°C for 2 h (to deeply solidify the inner layer); after solidification, it was washed and dried to obtain the porous material.

[0131] Example 2

[0132] The present embodiment provides a high internal phase emulsion and a porous material with a nested structure.

[0133] The difference from Example 1 is that, in the present embodiment, a high internal phase emulsion and a porous material obtained therefrom with a one-layer nested structure (HIPE-1 / HIPE-2 structure) are not included.

[0134] Example 3

[0135] The present embodiment provides a high internal phase emulsion and a porous material with a nested structure.

[0136] The difference from Example 2 is that, in the present embodiment, the emulsifier B is SPAN80, and H2 is 4.3.

[0137] Example 4

[0138] The present embodiment provides a high internal phase emulsion and a porous material with a nested structure.

[0139] The difference from Example 1 is that, in the present embodiment, the emulsifier A is Tween60, the emulsifier B is polyglyceryl stearate, and the emulsifier C is Span85.

[0140] Example 5

[0141] The difference from Example 1 is that, in the present embodiment, the emulsifier A is polyglyceryl ricinoleate, the emulsifier B is Span40 (HLB=6.7), and the emulsifier C is Span80.

[0142] Example 6

[0143] This embodiment provides a high internal phase emulsion and porous material with a nested structure, wherein the high internal phase emulsion has a layer of nested structure, which is a HIPE-1 / HIPE-2 structure, and the specific composition is as follows:

[0144] (1) HIPE-2 (continuous phase, outer layer)

[0145] Oil phase B: PLA prepolymer (NatureWorks, type 4032) 30 parts, PCL prepolymer (NatureWorks, type 6800) 25 parts, crosslinking monomer (PEGDA, Mn=600, Macklin) 12 parts, oil-soluble solvent (ethyl acetate) 8 parts;

[0146] Emulsifier B: polyglycerol stearate (HLB=5.2, Shandong Binzhou Jinsheng New Material) 6 parts (H2=5.2);

[0147] Initiator B: benzoyl peroxide (BPO, Sinopharm) 0.5 parts, TPO 0.5 parts;

[0148] Water phase B: 3 wt% calcium chloride deionized water solution, accounting for 82% of the total volume of HIPE-2;

[0149] Preparation process: the oil phase components are stirred at 30°C and 700 rpm for 25 min until uniform, the water phase is added to the oil phase at a rate of 5 mL / min, and stirring is carried out at 42°C and 1100 rpm for 30 min to obtain a HIPE-2 phase with a viscosity of 3500 cP at 40°C.

[0150] (2) HIPE-1 (dispersed phase, inner layer)

[0151] Oil phase A: PCL prepolymer (NatureWorks, type 6800) 55 parts, crosslinking monomer (PEGDA, Mn=400, Macklin) 10 parts, oil-soluble solvent (butyl acetate) 6 parts;

[0152] Emulsifier A: Tween 80 (HLB=15.0, UK Redwood) 5 parts;

[0153] Initiator A: azobisisobutyronitrile (AIBN, Sinopharm) 0.8 parts;

[0154] Water phase A: 3 wt% calcium chloride deionized water solution, accounting for 82% of the total volume of HIPE-1;

[0155] Preparation process: same as HIPE-2, to obtain a HIPE-1 phase with a viscosity of 2300 cP at 40°C (HIPE-1 diameter 80±6 μm);

[0156] (3) Preparation method of high internal phase emulsion

[0157] A four-channel coaxial microfluidic chip is provided: an outer channel, a middle channel (HIPE-2, inner diameter 220 μm), an inner channel (HIPE-1, inner diameter 120 μm), and a center channel (nitrogen assisted, inner diameter 60 μm);

[0158] Driving system: driven by a peristaltic pump;

[0159] Flow rate setting: middle layer 75 μL / min, inner layer 45 μL / min, center nitrogen 15 μL / min;

[0160] Wrapping control: real-time observation of the nested structure by microscope, and adjustment of the flow rate to stabilize the diameter of the nested unit;

[0161] Collection: the obtained high internal phase emulsion is collected.

[0162] (4) Preparation method of porous material

[0163] The high internal phase emulsion is injected into a polytetrafluoroethylene mold (5 cm x 5 cm x 0.5 cm), first irradiated by ultraviolet light (wavelength 365 nm) for 5 min (solidifying the outer layer and the middle layer), and then heated in an 80°C oven for 5 h; after solidification, it is washed and dried to obtain the porous material.

[0164] Example 7

[0165] The present embodiment provides a high internal phase emulsion with a nested structure and a porous material, wherein the high internal phase emulsion has a one-layer nested structure, which is a HIPE-1 / HIPE-2 structure, and the specific composition is as follows:

[0166] (1) HIPE-2 (middle phase)

[0167] Oil phase B: vinyl toluene (Macklin, 98%) 55 parts, crosslinking monomer (TMPTA, Macklin) 10 parts, oil-soluble solvent (xylene) 6 parts;

[0168] Emulsifier B: polyglyceryl isostearate (HLB = 7.8) + Span 40 (HLB = 6.7) (mass ratio 2:1, composite HLB = 7.4) 8 parts;

[0169] Initiator B: BPO 0.5 parts, TPO 0.5 parts;

[0170] The remaining components and the preparation method are referred to Example 6, and the 40°C viscosity is 3800 cP.

[0171] (2) HIPE-1 (dispersion phase, inner layer)

[0172] Oil phase A: isoprene (Macrolin, 98%) 50 parts, styrene 5 parts, crosslinking monomer (DVB, Macrolin) 8 parts, oil-soluble solvent (ethyl acetate) 6 parts;

[0173] Emulsifier A: Tween 60 (HLB = 14.9, British ICI) 7 parts;

[0174] Initiator A: lauroyl peroxide (LPO, National Pharmaceutical Group) 0.9 parts;

[0175] Water phase A: 5 wt% sodium chloride deionized water solution, accounting for 78% of the total volume of HIPE-1;

[0176] Preparation process: same as HIPE-2, to obtain a HIPE-1 phase with a viscosity of 2500 cP at 40℃ (HIPE-1 diameter 75±5 μm);

[0177] (3) Preparation method of high internal phase emulsion:

[0178] A four-channel coaxial microfluidic chip is provided: an outer channel is empty, a middle channel (HIPE-2, inner diameter 210 μm), an inner channel (HIPE-1, inner diameter 110 μm), and a center channel (nitrogen auxiliary, inner diameter 55 μm);

[0179] Driving system: driven by a peristaltic pump;

[0180] Flow rate setting: middle layer 85 μL / min, inner layer 55 μL / min, center nitrogen 25 μL / min;

[0181] Encapsulation control: the nested morphology is observed in real time through a microscope, and the flow rate is adjusted to stabilize the diameter of the nested unit;

[0182] Collection: the obtained high internal phase emulsion is collected.

[0183] (4) Preparation method of porous material

[0184] The high internal phase emulsion is injected into a polytetrafluoroethylene mold (5 cm×5 cm×0.5 cm), first irradiated with ultraviolet light (wavelength 365 nm) for 20 min (solidifying the outer layer and the middle layer), and then heated in an 85℃ oven for 4 h; after solidification, it is washed and dried to obtain the porous material.

[0185] Comparative Example 1

[0186] This comparative example provides a high internal phase emulsion and a porous material with a nested structure.

[0187] The difference from Example 1 is that in this comparative example, the porous material is obtained by solidifying HIPE-2.

[0188] Comparative Example 2

[0189] This comparative example provides a high internal phase emulsion and porous material with nested structure.

[0190] The difference from Example 2 is that in this comparative example, the gradient of emulsifier HLB value is not controlled, specifically as follows:

[0191] HIPE-2 (outer layer) emulsifier: Tween 80 (HLB = 15.0) 6 parts is selected;

[0192] HIPE-1 (inner layer) emulsifier: Tween 60 (HLB = 14.9) 5 parts is selected.

[0193] Comparative Example 3 (high internal phase emulsion cannot be prepared)

[0194] This comparative example provides a high internal phase emulsion and porous material with nested structure.

[0195] The difference from Example 2 is that in the preparation method of the high internal phase emulsion provided in this comparative example, the positions of HIPE-2 and HIPE-1 are interchanged, i.e. a high internal phase emulsion with HIPE-2 / HIPE-1 structure and a porous material obtained therefrom are prepared.

[0196] Performance test

[0197] The samples provided in the examples and comparative examples are subjected to performance tests, and the method is as follows:

[0198] (1) Structural stability: observe the nested units under a microscope, count the fusion rate, and the calculation formula is as follows:

[0199] Fusion rate = number of fused units / total number of units x 100%;

[0200] (2) Pore size characteristics: observed by scanning electron microscope (SEM, TESCAN MIRA3), randomly measured 50 primary pores, 100 secondary pores, and 100 tertiary pores (if any), and the average value was taken;

[0201] (3) Porosity: liquid displacement method (anhydrous ethanol), the sample was vacuum dried and weighed (m1), and after the ethanol was degassed, it was weighed (m2), the drainage method was used to measure the volume V, and the following calculation was performed:

[0202] Porosity = (m2-m1) / (ρethanol x V) x 100%;

[0203] (4) Cell activity: BMSCs (1 x 10 5 were inoculated, and after 7 days of culture, the CCK-8 method was used for testing, and the survival rate = (test group OD - blank group OD) / (control group OD - blank group OD) x 100%;

[0204] (5) Cell differentiation: BMSCs were inoculated, and after 21 days of culture, immunofluorescence staining (alkaline phosphatase ALP) was performed, and the differentiation rate (ALP positive cell number / total cell number x 100%) was calculated;

[0205] (6) Mechanical properties: Instron 5967, sample size 10mm x 10mm x 5mm, compression rate 1mm / min, stress at 8% strain;

[0206] The test results are as follows:

[0207] Table 1

[0208]

[0209] Note: Comparative Example 3 cannot form a nested structure, and its pore size presents a random distribution in the range of 50-300 μm.

[0210] From the examples and performance tests, it can be seen that the nested structure of the high internal phase emulsion provided by the present application is stable, the fusion rate is ≤3%, and the porous material obtained presents a hierarchical pore size distribution. When it is used for cell culture, the cell survival rate is ≥91%, and the differentiation rate is ≥75%, which is significantly better than single-stage HIPE porous material or non-gradient / physically stacked material.

[0211] The technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above process steps, that is, it does not mean that the present application must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement on the present application, equivalent replacement of the materials selected by the present application, addition of auxiliary ingredients, selection of specific methods, etc. all fall within the protection scope and disclosure scope of the present application.

Claims

1. A high internal phase emulsion with a nested structure, characterized in that, The high internal phase emulsion has at least one nested structure, which is a HIPE-1 / HIPE-2 structure. The HLB value of emulsifier A used in HIPE-1 is denoted as H1, and the HLB value of emulsifier B used in HIPE-2 is denoted as H2. H1 and H2 satisfy: H1 > H2, and the difference between them is 8-12.

2. The nested internal phase emulsion according to claim 1, characterized in that, The high internal phase emulsion has a two-layer nested structure, namely HIPE-1 / HIPE-2 / HIPE-3 structure. The HLB value of the emulsifier C used in HIPE-3 is denoted as H3. H3 needs to satisfy: H2 > H3, and the difference between H2 and H3 is 2-6.

3. The nested internal phase emulsion according to claim 1 or 2, characterized in that, H1 is 12-16; And / or, the emulsifier A comprises any one or a combination of at least two of polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, or polyglycerol ricinoleate. And / or, the viscosity of the HIPE-1 is 2000-3000 cP; And / or, the H2 is 4-8; And / or, the emulsifier B comprises any one or a combination of at least two of sorbitan monostearate (Span 60), polyglycerol stearate, sorbitan monopalmitate, or polyglycerol isostearate; And / or, the viscosity of the HIPE-2 is 3000-4000 cP; And / or, the initiator used in the HIPE-2 includes photoinitiator B, or a combination of photoinitiator B and thermal initiator B; And / or, the H3 is 2-6; And / or, the emulsifier C comprises any one or a combination of at least two of the following: sorbitan monooleate (Span 80), sorbitan trioleate, polyglycerol oleate, or dipolyglycerol monooleate; And / or, the viscosity of the HIPE-3 is 4000-5000 cP; And / or, the initiator used in the HIPE-3 includes photoinitiator C, or a combination of photoinitiator C and thermal initiator C.

4. The high internal phase emulsion with a nested structure according to any one of claims 1-3, characterized in that, The diameter of HIPE-1 is 70-90 μm; Preferably, the thickness of the HIPE-2 layer is 50-70 μm; Preferably, the thickness of the HIPE-3 layer is 50-70 μm.

5. A method for preparing a high internal phase emulsion with a nested structure as described in any one of claims 1-4, characterized in that, The preparation method includes: The HIPE-1 phase, HIPE-2 phase, and optionally HIPE-3 phase are mixed by a microfluidic method to obtain the high internal phase emulsion with nested structure.

6. The preparation method according to claim 5, characterized in that, The preparation method includes: HIPE-1 phase, HIPE-2 phase and optionally HIPE-3 phase are respectively input into different channels of the microfluidic device, and the three-phase mixing is controlled by controlling the flow rate to obtain the high internal phase emulsion with nested structure. Preferably, the process also includes introducing nitrogen gas for assistance.

7. The preparation method according to claim 6, characterized in that, The flow rate of the HIPE-1 phase is 45-55 μL / min; Preferably, the flow rate of the HIPE-2 phase is 75-85 μL / min; Preferably, the flow rate of the HIPE-3 phase is 95-105 μL / min; Preferably, the flow rate of the nitrogen gas is 15-25 μL / min.

8. The preparation method according to claim 6 or 7, characterized in that, The channel diameter used in the HIPE-1 phase is 100-120 μm; Preferably, the channel diameter used in the HIPE-2 phase is 200-220 μm; Preferably, the channel diameter used in the HIPE-3 phase is 300-320 μm; Preferably, the diameter of the central channel used for the nitrogen gas is 50-60 μm.

9. A porous material, characterized in that, It is prepared from the high internal phase emulsion with a nested structure according to any one of claims 1-4.

10. The use of the high internal phase emulsion with a nested structure according to any one of claims 1-4 or the porous material according to claim 9 in 3D cell culture, tissue engineering scaffolds, daily chemical hygiene products, drug screening or drug loading.

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