CN110151986B - Biodegradable water-in-oil-in-water emulsion and preparation method thereof - Google Patents
Biodegradable water-in-oil-in-water emulsion and preparation method thereof Download PDFInfo
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
Abstract
The invention provides a biodegradable water-in-oil-in-water emulsion, which comprises an internal water phase, an internal oil phase and an external water phase which are separated by solid particles; the solid particles include a first type of solid particles and a second type of solid particles. Compared with the traditional water-in-oil-in-water emulsion system, the biodegradable water-in-oil-in-water emulsion provided by the invention can use no or less emulsifier, and the stability of the system is related to the degradation stability of solid particles by virtue of the effect of the solid particles with good biocompatibility on stabilizing the emulsion.
Description
Technical Field
The invention relates to the technical field of chemical engineering and biological preparations, in particular to a biodegradable water-in-oil-in-water emulsion and a preparation method thereof.
Background
The solid particle stable emulsion is a thermodynamically stable and kinetically unstable system which takes solid particles as an emulsifier to replace the traditional surfactant and is also called Pickering emulsion. The Pickering emulsion is mainly characterized in that part of solid particles can be adsorbed to the interface of an oil phase and a water phase to form one or more layers of isolating membranes in the preparation process. Research shows that when the size of the particles is large to a certain degree, the adsorption energy is several orders of magnitude larger than that of thermal motion, so that the adsorption process is irreversible, a stable emulsion system can be formed, and an isolation film formed by solid particles can well prevent merging of emulsion droplets and reduce precipitation of a dispersed phase. Recently, some researchers have applied Pickering emulsions to the field of biologicals with good results.
Vaccine immunologic adjuvants are a widely-demanded class of biological agents. Although vaccination is the most effective method for helping the body to prevent diseases today, most of general vaccines are quickly decomposed by the body after vaccination without special treatment, and the vaccination effect of a single vaccine is limited. As one of the methods for enhancing the vaccination effect, the vaccine adjuvant is a substance which can synergistically stimulate the immune response of the organism with an antigen, wherein a water-in-oil-in-water emulsion system is a more ideal vaccine adjuvant. Compared with an oil-in-water adjuvant, the water-in-oil-in-water adjuvant can wrap the vaccine in an internal water phase to realize long-acting slow release; compared with a water-in-oil adjuvant, the water-in-oil-in-water adjuvant can avoid the phenomenon of local inflammatory reaction overstimulation and has small side effect on organisms. However, the preparation process of the water-in-oil-in-water emulsion is complicated, the stability of the multiple emulsions is not ideal, and a large amount of emulsifiers and stabilizers are usually added to reduce the phenomenon of merging and demulsification among emulsion droplets when the water-in-oil-in-water emulsion is prepared, which greatly affects the practical application of the water-in-oil-in-water emulsion.
In view of the emulsion used in the field of biological preparation, there are many reports at present, and CN105688207A discloses a composite adjuvant for animal vaccine and its application, wherein the surface stability of the emulsion depends on polymer and emulsifier, and the emulsion does not contain solid particles and has no slow release effect, and has similar characteristics to the common oil-in-water emulsion. CN106511995A discloses an oil-in-water vaccine adjuvant, which uses a plurality of surfactants to stabilize the water-oil phase interface, the whole system has the effects of stimulating immunity and slow release, and the system does not contain solid particles. CN104013955A discloses an oil-in-water emulsion stabilized by solid particles, the use of the solid particles can effectively stimulate immunity, but the drug loading condition of the system depends on the automatic adsorption of drugs, the system cannot ensure good loading effect on various drugs, and the system also has the defect that the oil-in-water emulsion lacks a slow release function.
To date, no relevant report has been reported on the development of multiple Pickering emulsions for use in vaccine adjuvants. From the principle point of view, if the problem of combination of internal water phases in an oil phase of a water-in-oil-in-water emulsion can be well solved by utilizing the characteristics of the Pickering emulsion, the structure stability of the water-in-oil-in-water is maintained to the maximum extent, and the high-efficiency immune activation function is maintained, the application range of the water-in-oil-in-water emulsion adjuvant system can be widened. On the other hand, from the perspective of drug sustained release, the stability of the Pickering emulsion can be controlled by controlling the degradation rate of the solid particles, and then the sustained release rate of the drug can be controlled according to different requirements. In conclusion, the biodegradable water-in-oil-in-water Pickering emulsion has a wide application prospect in the field of vaccine adjuvants.
Disclosure of Invention
The invention provides a biodegradable water-in-oil-in-water emulsion and a preparation method thereof, which solve the problems of serious internal phase combination of the water-in-oil-in-water emulsion and dependence on excessive emulsifier in the prior art.
The technical scheme of the invention is as follows:
a biodegradable water-in-oil-in-water emulsion comprising an internal aqueous phase, an internal oil phase, and an external aqueous phase separated by solid particles; the solid particles include a first type of solid particles and a second type of solid particles.
Preferably, the external water phase accounts for 60 to 90 percent by mass in the emulsion and comprises an aqueous solution and first solid particles, wherein the aqueous solution accounts for 92.5 to 99.5 percent by mass in the external water phase, and the first solid particles account for 0.5 to 5 percent by mass in the external water phase; the water solution is selected from one or more of purified water, normal saline and phosphate buffer solution.
Preferably, the external water phase further comprises a hydrophilic emulsifier, the mass percentage of the hydrophilic emulsifier in the external water phase is 0-2.5%, and the hydrophilic emulsifier is selected from emulsifiers with HLB (hydrophile-lipophile balance) values of more than 10, and comprises one or more of Tween-85, Tween-60, Tween-80, Tween-40 and Tween-20.
Preferably, the chemical composition of the first type of solid particles is biodegradable polymer and/or block copolymer of biodegradable polymer and water-soluble polymer, the biodegradable polymer is selected from one or more of poly (D, L-lactic acid-glycolic acid) copolymer, polycaprolactone and polyhydroxybutyrate, and the water-soluble polymer is selected from one or more of polyethylene glycol, polyethylene oxide, polypropylene oxide and ethylene oxide-propylene oxide block copolymer; wherein the molecular weight of the water-soluble polymer accounts for 1-50% of the molecular weight of the whole block polymer.
Preferably, the internal oil phase and the internal water phase are isolated and stabilized by the second type of solid particles and are water-in-oil emulsions, the particle size of the water-in-oil emulsions is between 50nm and 10 mu m, and the internal water phase accounts for 10 to 40 percent of the mass of the water-in-oil emulsions.
Preferably, the internal water phase is one or more of pure water, normal saline and buffered saline solution;
the internal oil phase comprises the following components by weight:
preferably, the second solid particles are selected from inorganic nanoparticles including one or more of fatty acid modified nano calcium carbonate, nano hydroxyapatite, nano montmorillonite and nano hydrotalcite; wherein the fatty acid is selected from one or more of caprylic acid, capric acid, lauric acid, arachidic acid, stearic acid, palmitic acid, oleic acid, linoleic acid and palmitic acid.
Preferably, the HLB value of the oleophilic emulsifier is between 3 and 6, and the oleophilic emulsifier is selected from Span series emulsifiers or compound emulsifiers of the Span series emulsifiers and Tween series emulsifiers.
Preferably, the particle size of the emulsion is between 1 and 50 microns; the average particle size of the first type of solid particles is between 10nm and 1000 nm; the average particle size of the second type of solid particles is between 10nm and 200 nm.
The other technical scheme of the invention is as follows:
a method for preparing biodegradable water-in-oil-in-water emulsion comprises the following steps:
(1) uniformly mixing medicinal mineral oil or vegetable oil, oleophilic emulsifier and fatty acid, adding the second solid particles, and performing ultrasonic dispersion to obtain an internal oil phase;
(2) adding the internal water phase into the internal oil phase, and shearing and uniformly dispersing to obtain a water-in-oil emulsion;
(3) ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles, or ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles after dissolving the hydrophilic emulsifier, so as to obtain an external aqueous phase;
(4) and (3) dropwise adding the water-in-oil emulsion into an external water phase, and shearing to uniformly disperse to obtain the water-in-oil-in-water emulsion.
Has the advantages that:
the biodegradable water-in-oil-in-water emulsion of the invention only uses solid particles or uses common surfactant and solid particles as phase interface stabilizing substances, compared with the traditional water-in-oil-in-water emulsion system, the biodegradable water-in-oil-in-water emulsion system can use no or less emulsifier, and the stability of the system is related to the degradation stability of the solid particles by virtue of the effect of the solid particles with good biocompatibility for stabilizing the emulsion. The adsorption of solid particles on the interface can also obviously increase the strength of a phase interface film, and overcome the problems of serious internal phase combination and dependence on excessive emulsifier of the common water-in-oil-in-water emulsion. Not only maintains the advantages of small side effect and sustained release of the traditional water-in-oil-in-water vaccine adjuvant, but also can regulate and control the rate and time of sustained release of the drug.
Drawings
FIG. 1 is a schematic diagram of the structure of a water-in-oil-in-water Pickering emulsion of the present invention;
FIG. 2a is a scanning electron micrograph of poly (D, L-lactic-co-glycolic acid) -polyethylene oxide (PLGA-PEO) block copolymer nanoparticles;
fig. 2b is a scanning electron microscope photograph of modified nano-hydroxyapatite particles;
FIG. 2c is a scanning electron microscope photograph of modified nano calcium carbonate particles;
FIG. 3a is an optical micrograph of the water-in-oil Pickering emulsion of example 1, with a scale bar of 25 μm;
FIG. 3b is an optical micrograph of the water-in-oil Pickering emulsion of example 2, with a scale bar of 25 μm;
FIG. 3c is an optical micrograph of the water-in-oil Pickering emulsion of example 3, with a scale bar of 25 μm;
FIG. 3d is an optical micrograph of the water-in-oil Pickering emulsion of example 4, with a scale bar of 25 μm;
FIG. 3e is an optical micrograph of the water-in-oil Pickering emulsion of example 5, with a scale bar of 25 μm;
FIG. 4 comparison of emulsion stability of comparative example 1 (left) and example 1 (right), with optical microscopy pictures at 25 μm scale;
FIG. 5 optical microscope comparison of emulsions after 14 days storage of comparative example 2 (left) and example 1 (right), with a scale bar of 25 microns.
In the figure: 1-an external aqueous phase; 2-solid particles of a first type; 3-a second type of solid particles; 4-an internal oil phase; 5-internal aqueous phase.
Detailed Description
In the following examples, the sources of all the raw materials are not particularly limited, and all the raw materials are commercially available.
Raw materials list
Instrument for measuring the position of a moving object
| Name (R) | Model number | Brand |
| Electronic balance | ME802E | METLER TOLEDO |
| Injection pump | LSP02-1B | Lange |
| Centrifugal machine | TGL-20M | Luxiang instrument |
| Freeze dryer | Alpha 1-2 LD plus | CHRIST |
| High-speed shearing dispersion machine | T25 digital ULTRA TURRAX | IKA |
| Ultrasonic cell crusher | BILON-650Y | Bilang (a Chinese character of 'Bilang') |
| Laser particle size analyzer | Nano-ZS90 | Malvern |
| Optical microscope | IX73 | OLYMPUS |
| Scanning electron microscope | Gemini SEM 300 | ZEISS |
The biodegradable water-in-oil-in-water emulsion of the present invention comprises an internal aqueous phase, an internal oil phase, and an external aqueous phase separated by solid particles; the solid particles comprise a first type of solid particles in the outer aqueous phase and a second type of solid particles in the inner aqueous phase.
Wherein the mass percentage of the external water phase in the emulsion is 60-90%, preferably 66-90%, more preferably 75-85%, and more preferably 80-85%; the external water phase comprises an aqueous solution and a first type of solid particles, wherein the mass percentage of the aqueous solution in the external water phase is 92.5-99.5%, preferably 95-99.5%, and further preferably 95-98.5%.
The mass percentage of the first solid particles in the external water phase is 0.5-5%, preferably 0.5-2.5%; the water solution is selected from one or more of purified water, normal saline and phosphate buffer solution; the first type of solid particles are selected from biodegradable polymers and/or block copolymers of the biodegradable polymers and water-soluble polymers, the biodegradable polymers are selected from one or more of poly (D, L-lactic acid-glycolic acid) copolymers, polycaprolactone and polyhydroxybutyrate, and the water-soluble polymers are selected from one or more of polyethylene glycol, polyethylene oxide, polypropylene oxide and block copolymers of ethylene oxide-propylene oxide; wherein the molecular weight of the water-soluble polymer accounts for 1-50% of the molecular weight of the whole block polymer.
Further, the external water phase also comprises a hydrophilic emulsifier, the mass percentage of the hydrophilic emulsifier in the external water phase is 0-2.5%, and the hydrophilic emulsifier is selected from emulsifiers with HLB (hydrophile-lipophile balance) values of more than 10, and comprises one or more of Tween-85, Tween-60, Tween-80, Tween-40 and Tween-20.
Wherein the internal oil phase and the internal water phase are isolated and stabilized by the second type of solid particles and are water-in-oil emulsion, the particle size of the water-in-oil emulsion is between 50nm and 10 mu m, and the mass proportion of the internal water phase in the water-in-oil emulsion is 10 to 40 percent.
The internal water phase is one or more of pure water, normal saline and buffer saline solution.
The internal oil phase comprises the following components by weight:
the second solid particles are selected from inorganic nano particles, including one or more of fatty acid modified nano calcium carbonate, nano hydroxyapatite, nano montmorillonite and nano hydrotalcite; wherein the fatty acid is selected from one or more of caprylic acid, capric acid, lauric acid, arachidic acid, stearic acid, palmitic acid, oleic acid, linoleic acid and palmitic acid.
The HLB value of the oleophilic emulsifier in the internal oil phase is between 3 and 6, and the oleophilic emulsifier is selected from Span series emulsifiers or compound emulsifiers of the Span series emulsifiers and Tween series emulsifiers.
The particle size of the emulsion is between 1 and 50 mu m; the average particle size of the first type of solid particles is between 10nm and 1000 nm; the average particle size of the second type of solid particles is between 10nm and 200 nm.
The preparation method of the biodegradable water-in-oil-in-water emulsion comprises the following steps:
(1) uniformly mixing medicinal mineral oil or vegetable oil, oleophilic emulsifier and fatty acid, adding the second solid particles, and performing ultrasonic dispersion to obtain an internal oil phase;
(2) adding the internal water phase into the internal oil phase, and shearing and uniformly dispersing to obtain a water-in-oil emulsion;
(3) ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles, or ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles after dissolving the hydrophilic emulsifier, so as to obtain an external aqueous phase;
(4) and (3) dropwise adding the water-in-oil emulsion into an external water phase, and shearing to uniformly disperse to obtain the water-in-oil-in-water emulsion.
Wherein in the step (1), the weight percentages of the mineral oil or the vegetable oil, the oleophilic emulsifier and the fatty acid are as follows:
in the step (1), the second solid particles are selected from inorganic nano particles, including one or more of fatty acid modified nano calcium carbonate, nano hydroxyapatite, nano montmorillonite and nano hydrotalcite; wherein the fatty acid is selected from one or more of caprylic acid, capric acid, lauric acid, arachidic acid, stearic acid, palmitic acid, oleic acid, linoleic acid and palmitic acid.
In the step (1), the HLB value of the oleophilic emulsifier is between 3 and 6, and the oleophilic emulsifier is selected from Span series emulsifiers or compound emulsifiers of Span series emulsifiers and Tween series emulsifiers.
In the step (2), the internal water phase is one or more of pure water, normal saline and buffer saline solution.
In the step (3), the first type of solid particles are selected from biodegradable polymers and/or block copolymers of biodegradable polymers and water-soluble polymers, the biodegradable polymers are selected from one or more of poly (D, L-lactic acid-glycolic acid) copolymers, polycaprolactone and polyhydroxybutyrate, and the water-soluble polymers are selected from one or more of polyethylene glycol, polyethylene oxide, polypropylene oxide and ethylene oxide-propylene oxide block copolymers; wherein the molecular weight of the water-soluble polymer accounts for 1-50% of the molecular weight of the whole block polymer.
In the step (3), the mass percentage of the hydrophilic emulsifier in the external water phase is 0-2.5%, and the hydrophilic emulsifier is selected from emulsifiers with HLB (hydrophile-lipophile balance) values larger than 10, and comprises one or more of Tween-85, Tween-60, Tween-80, Tween-40 and Tween-20.
In the step (3), the aqueous phase solution is selected from one or more of purified water, normal saline and phosphate buffer.
The grain diameter of the emulsion prepared in the step (4) is between 1 and 50 mu m; the average particle size of the first type of solid particles is between 10nm and 1000 nm; the average particle size of the second type of solid particles is between 10nm and 200 nm.
Example 1
This example provides a water-in-oil-in-water emulsion prepared from poly (D, L-lactic-co-glycolic acid) (PLGA) and nano calcium carbonate, and referring to fig. 1, the present invention is shown in a schematic structural diagram of the water-in-oil-in-water Pickering emulsion, and the preparation process includes the following steps:
1. preparation of PLGA nanoparticles:
accurately, 0.8g of PLGA was dissolved in 35mL of acetone, dropped into a 0.25% aqueous PVA solution using a syringe pump, and evaporated by stirring overnight while being open. Freezing the solution dispersed with the PLGA nano-particles at-22 ℃, and then freeze-drying under the condition of 0.12kPa to obtain the PLGA nano-particles. The resulting particles had an average particle diameter of 168nm and a zeta potential of-3.09 mV, determined by Dynamic Light Scattering (DLS).
2. Hydrophobic modification of nano calcium carbonate:
0.1g of stearic acid is accurately dissolved in 100mL of ethanol, 1g of nano calcium carbonate is added into the ethanol, ultrasonic dispersion is carried out, and dispersion is carried out for 1h at 80 ℃. The precipitate was collected by centrifugation at 8000rpm for 15min using a centrifuge. Washing with ethanol for three times, drying at 80 deg.C for 12h, grinding, and collecting the product. As shown in fig. 2c, is a scanning electron microscope photograph of the modified nano calcium phosphate particles.
3. Preparation of water-in-oil emulsion:
accurately and uniformly mixing 0.2g of stearic acid, 12.6g of liquid paraffin, 1g of span 85 and 0.2g of modified nano calcium carbonate, performing ultrasonic dispersion to obtain an oil phase, and uniformly mixing 5.8g of 0.9% NaCl solution and 0.2g of tween80 to obtain a water phase. The two phases were mixed and dispersed for 10min at 12000rpm to give 20g of water-in-oil emulsion. The prepared emulsion has stable property, and no water phase is obviously separated after 14 days.
4. Preparation of water-in-oil-in-water emulsion:
accurately and uniformly mixing 0.2g of PLGA nano particles, 0.1g of tween80 and 19.7g of 0.9 percent NaCl solution, performing ultrasonic dispersion to obtain an external water phase, mixing 4g of water-in-oil emulsion with the external water phase, and performing dispersion for 2min at the rotating speed of 8000rpm to obtain 24g of water-in-oil-in-water emulsion. The resulting emulsion was observed under a microscope to have a distinct multiple emulsion structure as shown in FIG. 3 a. After being stored for 14 days at room temperature, the coating is layered, and no obvious liquid drop coalescence phenomenon exists.
Example 2
The embodiment provides a water-in-oil-in-water emulsion prepared from PLGA and nano-hydroxyapatite, which comprises the following steps:
1. preparation of PLGA nanoparticles:
accurately, 0.8g of PLGA was dissolved in 35mL of acetone, dropped into a 0.25% aqueous PVA solution using a syringe pump, and evaporated by stirring overnight while being open. Freezing the solution dispersed with the PLGA nano-particles at-22 ℃, and then freeze-drying under the condition of 0.12kPa to obtain the PLGA nano-particles. The resulting particles had an average particle diameter of 168nm and a zeta potential of-3.09 mV, as determined by DLS.
2. Hydrophobic modification of nano hydroxyapatite:
0.1g of stearic acid is accurately dissolved in 100mL of ethanol, 1g of nano-hydroxyapatite is added into the ethanol and is subjected to ultrasonic dispersion, and the mixture is reacted for 4 hours at 80 ℃. The precipitate was collected by centrifugation at 8000rpm for 15min using a centrifuge. Washing with ethanol for three times, drying at 80 deg.C for 12h, grinding, and collecting the product. As shown in fig. 2b, is a scanning electron microscope photograph of the modified nano-hydroxyapatite particles.
3. Preparation of water-in-oil emulsion:
accurately taking 0.2g of stearic acid, 12.6g of liquid paraffin, 1g of span 85 and 0.2g of modified nano-hydroxyapatite, uniformly mixing and ultrasonically dispersing to form an oil phase, and taking 5.8g of 0.9% NaCl solution and 0.2gtween 80, uniformly mixing to form a water phase. The two phases were mixed and dispersed for 10min at 12000rpm to give 20g of water-in-oil emulsion. The prepared emulsion has stable property, and no water phase is obviously separated after 14 days.
4. Preparation of water-in-oil-in-water emulsion:
accurately and uniformly mixing 0.2g of PLGA nano particles, 0.1g of tween80 and 19.7g of 0.9 percent NaCl solution, performing ultrasonic dispersion to obtain an external water phase, mixing 4g of water-in-oil emulsion with the external water phase, and performing dispersion for 2min at the rotating speed of 8000rpm to obtain 24g of water-in-oil-in-water emulsion. The resulting emulsion was observed under a microscope to have a distinct multiple emulsion structure as shown in FIG. 3 b. After being stored for 14 days at room temperature, the coating is layered, and no obvious liquid drop coalescence phenomenon exists.
Example 3
The embodiment provides a method for preparing a water-in-oil-in-water Pickering emulsion by adopting Polycaprolactone (PCL) and nano calcium carbonate, which comprises the following steps:
1. preparing PCL nano particles:
0.8g of PCL is accurately dissolved in 35mL of dichloromethane, dropped into 0.25% of PVA aqueous solution by using a syringe pump, ultrasonically dispersed for 10min by 195W to form emulsion, and stirred and volatilized overnight when the solution is opened. Freezing the solution dispersed with PCL nano particles at-22 ℃, and freeze-drying under the condition of 0.12kPa to obtain the PCL nano particles. The resulting particles had an average particle diameter of 311nm and a zeta potential of-2.97 mV, as determined by DLS.
2. Hydrophobic modification of nano calcium carbonate:
0.1g of stearic acid is accurately dissolved in 100mL of ethanol, 1g of nano calcium carbonate is added into the ethanol and is subjected to ultrasonic dispersion, and the reaction is carried out for 1h at the temperature of 80 ℃. The precipitate was collected by centrifugation at 8000rpm for 15min using a centrifuge. Washing with ethanol for three times, drying at 80 deg.C for 12h, grinding, and collecting the product. As shown in fig. 2c, is a scanning electron microscope photograph of the modified nano calcium phosphate particles.
3. Preparation of water-in-oil emulsion:
accurately and uniformly mixing 0.2g of stearic acid, 12.6g of liquid paraffin, 1g of span 85 and 0.2g of modified nano calcium carbonate, performing ultrasonic dispersion to obtain an oil phase, and uniformly mixing 5.8g of 0.9% NaCl solution and 0.2g of tween80 to obtain a water phase. The two phases were mixed and dispersed for 10min at 12000rpm to give 20g of water-in-oil emulsion. The prepared emulsion has stable property, and no water phase is obviously separated after 14 days.
4. Preparation of water-in-oil-in-water emulsion:
accurately taking 0.2g of PCL nano particles, 0.1g of tween80 and 19.7g of 0.9 percent NaCl solution, uniformly mixing and ultrasonically dispersing into an external water phase, taking 4g of water-in-oil emulsion, mixing with the external water phase, and dispersing for 2min at the rotating speed of 8000rpm to obtain 24g of water-in-oil-in-water emulsion. The resulting emulsion was observed under a microscope to have a distinct multiple emulsion structure as shown in FIG. 3 c. After being stored for 14 days at room temperature, the coating is layered, and no obvious liquid drop coalescence phenomenon exists.
Example 4
This example provides a method for preparing water-in-oil-in-water emulsion using PLGA-PEO-PLGA and nano calcium carbonate, comprising the following steps:
1. preparation of PLGA-PEO-PLGA nanoparticles:
accurately taking 0.8g of PLGA-PEO-PLGA, dissolving in 35mL of acetone, dripping the acetone into 0.25% PVA water solution by using a syringe pump, and stirring and volatilizing the mixture overnight after opening. Freezing the solution dispersed with the PLGA-PEO-PLGA nano particles at-22 ℃, and then freeze-drying under the condition of 0.12kPa to obtain the PLGA-PEO-PLGA nano particles. The resulting particles had an average particle diameter of 166nm and a zeta potential of-16.0 mV, as determined by DLS. As shown in fig. 2a, scanning electron micrograph of PLGA-PEO block nanoparticles.
2. Hydrophobic modification of nano calcium carbonate:
0.1g of stearic acid is accurately dissolved in 100mL of ethanol, 1g of nano calcium carbonate is added into the ethanol and is subjected to ultrasonic dispersion, and the reaction is carried out for 1h at the temperature of 80 ℃. The precipitate was collected by centrifugation at 8000rpm for 15min using a centrifuge. Washing with ethanol for three times, drying at 80 deg.C for 12h, grinding, and collecting the product. As shown in fig. 2c, is a scanning electron microscope photograph of the modified nano calcium phosphate particles.
3. Preparation of water-in-oil emulsion:
accurately and uniformly mixing 0.2g of stearic acid, 12.6g of liquid paraffin, 1g of span 85 and 0.2g of modified nano calcium carbonate, performing ultrasonic dispersion to obtain an oil phase, and uniformly mixing 5.8g of 0.9% NaCl solution and 0.2g of tween80 to obtain a water phase. The two phases were mixed and dispersed for 10min at 12000rpm to give 20g of water-in-oil emulsion. The prepared emulsion has stable property, and no water phase is obviously separated after 14 days.
4. Preparation of water-in-oil-in-water emulsion:
accurately and uniformly mixing 0.2g of PLGA-PEO-PLGA nano particles, 0.1g of tween80 and 19.7g of 0.9 percent NaCl solution, performing ultrasonic dispersion to obtain an external water phase, mixing 4g of water-in-oil emulsion with the external water phase, and performing dispersion for 2min at the rotating speed of 8000rpm to obtain 24g of water-in-oil-in-water emulsion. The resulting emulsion was observed under a microscope to have a distinct multiple emulsion structure as shown in fig. 3 d. After being stored for 14 days at room temperature, the coating is layered, and no obvious liquid drop coalescence phenomenon exists.
Example 5
This example provides a method for preparing water-in-oil-in-water Pickering emulsion from copolymerized hydroxybutyric acid (P (3-HB-co-4-HB)) and nano calcium carbonate, comprising the following steps:
1. preparing the copolymerized hydroxybutyrate nano particles:
accurately taking 0.8g of copolymerized hydroxybutyric acid to dissolve in 35mL of dichloromethane, dripping the copolymerized hydroxybutyric acid into 0.25% PVA aqueous solution by using a syringe pump, carrying out ultrasonic dispersion for 10min by 195W to form emulsion, and stirring and volatilizing the emulsion overnight while opening. Freezing the solution dispersed with the copolymerized hydroxybutyric acid nano particles at-22 ℃, and freeze-drying under the condition of 0.12kPa to obtain the copolymerized hydroxybutyric acid nano particles. The resulting particles had an average particle diameter of 248nm and a zeta potential of-3.82 mV, determined by DLS.
2. Hydrophobic modification of nano calcium carbonate:
0.1g of stearic acid is accurately dissolved in 100mL of ethanol, 1g of nano calcium carbonate is added into the ethanol and is subjected to ultrasonic dispersion, and the reaction is carried out for 1h at the temperature of 80 ℃. The precipitate was collected by centrifugation at 8000rpm for 15min using a centrifuge. Washing with ethanol for three times, drying at 80 deg.C for 12h, grinding, and collecting the product. As shown in fig. 2c, is a scanning electron microscope photograph of the modified nano calcium phosphate particles.
3. Preparation of water-in-oil emulsion:
accurately and uniformly mixing 0.2g of stearic acid, 12.6g of liquid paraffin, 1g of span 85 and 0.2g of modified nano calcium carbonate, performing ultrasonic dispersion to obtain an oil phase, and uniformly mixing 5.8g of 0.9% NaCl solution and 0.2g of tween80 to obtain a water phase. The two phases were mixed and dispersed for 10min at 12000rpm to give 20g of water-in-oil emulsion. The prepared emulsion has stable property, and no water phase is obviously separated after 14 days.
4. Preparation of water-in-oil-in-water emulsion:
accurately and uniformly mixing 0.2g of copolymerized hydroxybutyric acid nano particles, 0.1g of tween80 and 19.7g of 0.9 percent NaCl solution, performing ultrasonic dispersion to obtain an external water phase, mixing 4g of water-in-oil emulsion with the external water phase, and performing dispersion for 2min at the rotating speed of 8000rpm to obtain 24g of water-in-oil-in-water emulsion. The resulting emulsion was observed under a microscope to have a distinct multiple emulsion structure as shown in figure 3 e. After being stored for 14 days at room temperature, the coating is layered, and no obvious liquid drop coalescence phenomenon exists.
Comparative example 1 preparation of water-in-oil emulsion without solid particles in the internal phase
Accurately taking 0.2g of stearic acid, 13.6g of liquid paraffin and 1g of span 85, uniformly mixing and ultrasonically dispersing to form an oil phase, and taking 5.8g of 0.9% NaCl solution and 0.2g of tween80, uniformly mixing to form a water phase. The two phases were mixed and dispersed for 10min at 12000rpm to give 20g of water-in-oil emulsion. FIG. 4 is a graph comparing the water-in-oil emulsion prepared in comparative example 1 (left) and example 1 (right), and from the results, it can be seen that the emulsion prepared in example 1 showed a clear separation of the aqueous phase at the bottom after one day and almost complete separation of the aqueous phase after 21 days.
Comparative example 2 preparation of Water-in-oil-in-Water emulsion without solid particles in the internal phase
1. Preparation of water-in-oil emulsion:
accurately taking 0.2g of stearic acid, 13.6g of liquid paraffin and 1g of span 85, uniformly mixing and ultrasonically dispersing to form an oil phase, and taking 5.8g of 0.9% NaCl solution and 0.2g of tween80, uniformly mixing to form a water phase. The two phases were mixed and dispersed for 10min at 12000rpm to give 20g of water-in-oil emulsion. The emulsion prepared had a clear aqueous phase which separated out at the bottom after one day and almost completely separated out after 21 days.
2. Preparation of water-in-oil-in-water emulsion:
accurately and uniformly mixing 0.2g of PLGA, 0.1g of tween80 and 19.7g of 0.9 percent NaCl solution, performing ultrasonic dispersion to obtain an external water phase, mixing 4g of water-in-oil emulsion with the external water phase, and performing dispersion for 2min at the rotating speed of 8000rpm to obtain 24g of water-in-oil-in-water emulsion. FIG. 5 shows a comparison between comparative example 2 (left) and example 1 (right) after storage for 14 days, and it can be seen that in comparative example 2, when no solid particles are used as an emulsifier, the water-in-oil emulsion is unstable and the resulting water-in-oil-in-water structure is unstable.
It will be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not limited thereto. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and substance of the invention, and these modifications and improvements are also considered to be within the scope of the invention.
Claims (9)
1. A biodegradable water-in-oil-in-water emulsion comprising an internal aqueous phase, an internal oil phase, and an external aqueous phase separated by solid particles; the solid particles comprise a first type of solid particles and a second type of solid particles;
the external water phase comprises an aqueous solution and a first solid particle, the chemical composition of the first solid particle is a biodegradable polymer and/or a block copolymer of the biodegradable polymer and a water-soluble polymer, the biodegradable polymer is selected from one or more of poly (D, L-lactic acid-glycolic acid) copolymer, polycaprolactone and polyhydroxybutyrate, and the water-soluble polymer is selected from one or more of polyethylene glycol, polyethylene oxide, polypropylene oxide and a block copolymer of ethylene oxide-propylene oxide;
the internal oil phase and the internal water phase are isolated and stabilized by the second type of solid particles and are water-in-oil emulsion; the internal oil phase comprises the following components by weight: 80-95% of medicinal mineral oil or vegetable oil, 1-5% of fatty acid, 0.5-5% of second solid particles and 0-10% of oleophilic emulsifier; the second solid particles comprise one or more of fatty acid modified nano calcium carbonate, nano hydroxyapatite, nano montmorillonite and nano hydrotalcite; wherein the fatty acid is selected from one or more of caprylic acid, capric acid, lauric acid, arachidic acid, stearic acid, palmitic acid, oleic acid, linoleic acid and palmitic acid;
the preparation method of the biodegradable water-in-oil-in-water emulsion comprises the following steps:
(1) uniformly mixing medicinal mineral oil or vegetable oil, oleophilic emulsifier and fatty acid, adding the second solid particles, and performing ultrasonic dispersion to obtain an internal oil phase;
(2) adding the internal water phase into the internal oil phase, and shearing and uniformly dispersing to obtain a water-in-oil emulsion;
(3) ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles, or ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles after dissolving the hydrophilic emulsifier, so as to obtain an external aqueous phase;
(4) and (3) dropwise adding the water-in-oil emulsion into an external water phase, and shearing to uniformly disperse to obtain the water-in-oil-in-water emulsion.
2. The biodegradable water-in-oil-in-water emulsion according to claim 1, wherein the external water phase is present in the emulsion at a mass percentage of 60% to 90%, wherein the aqueous solution is present in the external water phase at a mass percentage of 92.5% to 99.5%, and the first type of solid particles is present in the external water phase at a mass percentage of 0.5% to 5%; the water solution is selected from one or more of purified water, normal saline and phosphate buffer solution.
3. The biodegradable water-in-oil-in-water emulsion according to claim 2, wherein the external water phase further comprises a hydrophilic emulsifier, the mass percentage of the hydrophilic emulsifier in the external water phase is 0-2.5%, the hydrophilic emulsifier is an emulsifier with HLB value more than 10, and the hydrophilic emulsifier is one or more selected from Tween-85, Tween-60, Tween-80, Tween-40 and Tween-20.
4. The biodegradable water-in-oil-in-water emulsion according to claim 2, wherein the molecular weight of the water-soluble polymer is 1 to 50% of the molecular weight of the whole block polymer.
5. The biodegradable water-in-oil-in-water emulsion according to claim 1, wherein the particle size of the water-in-oil emulsion is between 50nm and 10 μm, and the internal aqueous phase is 10 to 40% by weight of the water-in-oil emulsion.
6. The biodegradable water-in-oil-in-water emulsion according to claim 5, wherein said internal aqueous phase is one or more of pure water, physiological saline, and buffered saline.
7. The biodegradable water-in-oil-in-water emulsion according to claim 6, wherein the lipophilic emulsifier has HLB value between 3 and 6 and is selected from Span series emulsifiers or compound emulsifiers of the Span series emulsifiers and Tween series emulsifiers.
8. The biodegradable water-in-oil-in-water emulsion according to any one of claims 1 to 7, wherein the particle size of said emulsion is between 1 μm and 50 μm; the average particle size of the first type of solid particles is between 10nm and 1000 nm; the average particle size of the second type of solid particles is between 10nm and 200 nm.
9. The method for preparing a biodegradable water-in-oil-in-water emulsion according to claims 1 to 8, comprising the steps of:
(1) uniformly mixing medicinal mineral oil or vegetable oil, oleophilic emulsifier and fatty acid, adding the second solid particles, and performing ultrasonic dispersion to obtain an internal oil phase;
(2) adding the internal water phase into the internal oil phase, and shearing and uniformly dispersing to obtain a water-in-oil emulsion;
(3) ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles, or ultrasonically and uniformly mixing the aqueous phase and the first type of solid particles after dissolving the hydrophilic emulsifier, so as to obtain an external aqueous phase;
(4) and (3) dropwise adding the water-in-oil emulsion into an external water phase, and shearing to uniformly disperse to obtain the water-in-oil-in-water emulsion.
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