CN110755645A - Method for preparing monodisperse freeze-dried microbubbles based on microfluidics technology and application thereof - Google Patents
Method for preparing monodisperse freeze-dried microbubbles based on microfluidics technology and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
Abstract
The invention discloses a method for preparing monodisperse freeze-dried microbubbles based on a microfluidics technology and application thereof. The method obtains the size-controllable monodisperse emulsion by a microfluid rapid emulsification technology; freeze-drying the obtained monodisperse emulsion to obtain hollow microspheres which are the precursor of the microbubbles; and mixing the hollow microspheres with a buffer solution to obtain the monodisperse micro-bubble solution. The method utilizes the microfluid emulsification technology to prepare the hollow microspheres as the precursor of the microbubbles, the obtained microbubble freeze-dried powder is convenient to transport and store, is not limited by the requirements of instruments, fields, technical backgrounds of users and the like when in use, the size of the obtained microbubbles is uniform, and the size of the microbubbles and the type of gas in the core can be adjusted through the microfluid technology. In animal clinical experiments, the monodisperse freeze-dried microbubbles provided by the invention have stronger contrast effect than the traditional polydisperse microbubble contrast agents. The experiment of the invention has strong controllability, and the obtained microbubbles have wide application fields.
Description
Technical Field
The invention belongs to the technical field of biological material application, and particularly relates to a method for preparing monodisperse freeze-dried microbubbles based on a microfluid technology and application thereof.
Background
Microbubbles (1-10 m in diameter) of micron size have been widely used in the diagnosis of cardiovascular and superficial visceral diseases, tumors (Kanaka hetiarchchi et al Lab Chip,7, 463-468 (2007)). Compared with the traditional ultrasonic developer mainly prepared by chemical agents, the microbubbles are not only nontoxic and have no side effect, but also can provide clearer development contrast and higher signal-to-noise ratio. In some special cases, such as the diagnosis of ventricular fissures, conventional chemical ultrasound contrast agents have difficulty finding lesions, while microbubble ultrasound contrast agents provide significant contrast (Mehmet Kaya et al, Bubbe Sci Eng Technol.2(2), 33-40 (2010)).
Nowadays, microbubble ultrasonic developers replace traditional chemical developers and become mainstream ultrasonic developers in European and American medical markets. The main preparation form of the microbubble contrast agent commercialized at present is freeze-dried microcapsule, which can form a large amount of microbubbles after being stirred by adding water, thereby obtaining microbubble solution capable of blood injection. When microbubbles circulate through the ultrasound detection region with blood, we can clearly identify the distribution of microbubbles due to the large difference in the acoustic reflection properties of microbubbles (gas) and surrounding tissues (liquid and solid). Microbubbles provide extremely strong reflected signal contrast, particularly when the frequency of the ultrasound wave is close to the resonance frequency of the microbubble itself (Hangyu Lin, et al, Med Biol EngComput 54, 1317-. To date, the Global Market size for microbubble ultrasonography has exceeded $ 30 million and has maintained an annual average growth rate of 26% (Global Microbublles/Ultrasound controls Market Outlook: 2015-.
However, the commercial microbubble medical ultrasound contrast agents on the market still have many defects. First, microbubbles used in current microbubble medical contrast agents are all polydisperse microbubbles, i.e., the size of the bubbles varies from 1 micron to 10 microns in size distribution. The ultrasonic resonance frequency of the microbubbles is in inverse proportion to the size of the microbubbles (the resonance frequency is equal to the radius of the bubbles is equal to 3m/s), and the ultrasonic resonance frequencies of the microbubbles with different sizes are greatly different (Yoonjee Park, et al; Langmuir 2012,28, 3766-3772). In practical diagnosis, only a small fraction of the microbubbles in a polydispersed microbubble contrast agent are actually in the ultrasound resonance band, which not only reduces the intensity of signal reflections and reduces the signal-to-noise ratio, but also increases the risk of occlusion of the vessel by bubble collapse fusion, and makes the production and use of contrast agents inefficient and wasteful.
The prior art inventions also have various drawbacks. For example, application numbers 201610002404.3 and 201620004350.X both disclose a microfluidic chip patent technology for producing microbubbles. All of them can generate micro-bubbles in real time by adjusting the liquid flow rate and the gas pressure by injecting gas and liquid into the micro-channel. Compared with the patent, the limitations of the invention are that (1) the operation and maintenance of the microfluidic Chip require the user to have a certain professional knowledge, technical background and technical operation capability, and (2) the preparation efficiency of the micro-bubbles of the single-channel microfluidic Chip is low, and the production rate of the single-channel microfluidic Chip for preparing the micro-bubbles with the diameter of 5 microns is reported to be one million per second (Lab on a Chip 11(12),2023-9The solution of each microbubble needs 15-20 minutes and is difficult to respond to the instant requirement of clinical application; (3) the sizes and the concentrations of the microbubbles are difficult to independently adjust by using the traditional microfluidic method for producing the microbubbles, because the sizes of the microbubbles are in a negative correlation with the flow rate of the fluid, and a higher liquid flow rate is necessary for preparing smaller microbubbles, so that the lower bubble concentration is brought, and in practical ultrasonic contrast application, the lower bubble concentration is usually difficult to provide effective acoustic reflection; (4) in view of the low rate at which micro-bubbles are actually produced by microfluidics, the stability of micro-bubbles is often also affected during lengthy preparation, collection, transfer and transportation processes, further reducing the yield and use of micro-bubbles.
Disclosure of Invention
Aiming at various defects in the prior art, the invention aims to provide a method for preparing monodisperse freeze-dried microbubbles based on a microfluidics technology and application thereof. Compared with other commercialized microbubble developers on the market, the method has the main advantages that (1) the prepared microbubbles have high size uniformity, and the enhancement effect of ultrasonic development is better by adjusting the size of bubbles through a microfluid technology and (2). Compared with other patent inventions for directly preparing microbubbles by a microfluidics technology, the method has the main advantages that (1) no technical background requirement is required for users, (2) microbubbles of freeze-dried powder can be immediately dissolved and used without waiting for the production of the microbubbles in clinical use, (3) the concentration and the size of the microbubbles can be independently regulated and controlled, (4) the freeze-dried microbubble powder has good stability and can be immediately used after long-term storage, and (5) the type of gas in the microbubble core can be a certain gas or a mixture of a plurality of gases, and the selection range is wide.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a method for preparing monodisperse freeze-dried microbubbles based on a microfluidics technology comprises the following steps:
Assembling a droplet generation device assembled by a microfluidic chip or a glass capillary;
Injecting an aqueous solution containing a surfactant and a trichloromethane solution dissolved with liposome into the microfluidic chip from a water phase inlet and an oil phase inlet respectively by using a mechanical pump or a pneumatic pump as a drive, wherein the aqueous solution contains Pluronic-F68 surfactant with the volume fraction of 2.5%, the trichloromethane solution contains liposome with the mass fraction of 0.1% -1%, the flow rate of the aqueous phase is 1-50ml/hr, the flow rate of the oil phase is 0.1-10ml/hr, the ratio of the flow rate of the aqueous phase to the flow rate of the oil phase is 1/5-1/20, and the flow rate ratio of the flow rate of the aqueous phase to the flow rate of the oil phase is adjusted to prepare the oil/water emulsion with the diameter of 5-50 mu m and uniform distribution; wherein, the liposome is distributed at the interface of the oil/water emulsion due to the self amphipathy;
And (3) collecting and standing the oil/water emulsion obtained in the step (2) for separation, skimming an upper aqueous solution, transferring the rest emulsion into a freeze dryer, and carrying out ultra-low temperature drying at-80 ℃ to obtain hollow microspheres wrapped by the liposome, namely monodisperse freeze-dried microbubbles.
The improved method is characterized in that the microfluidic chip is a PDMS microfluidic chip, and the preparation method of the PDMS microfluidic chip comprises the following steps:
designing a micro-fluid pipeline with the depth of 50 microns and the width of 50 microns by using engineering drawing software AutoCAD, and printing the micro-fluid pipeline as a photoetching template on a transparent plastic sheet, wherein the micro-fluid pipeline is provided with a water phase inlet, an oil phase inlet and a product collecting outlet, and the calibers of the water phase inlet and the oil phase inlet are 1 mm;
secondly, printing a photoetching template on a silicon wafer substrate by using a soft photoetching technology, wherein the projection size ratio of patterns on the photoetching template on the silicon wafer substrate is 1: 1;
thirdly, uncovering the mould
Uniformly coating PDMS silica gel on a silicon wafer substrate, wherein the thickness of the layer is 5mm, removing residual bubbles in the silica gel by using a vacuum pump, moving the silica gel into a 70 ℃ oven to heat for 1 hour, and removing the mold to obtain a pipeline, wherein the silicon wafer substrate can be repeatedly used for removing the mold;
the fourth step, bonding
And placing the microfluid pipeline and the glass substrate into a plasma reaction box, irradiating the surfaces with ultraviolet light for 1 minute to ensure that the surfaces of the material A and the glass substrate are fully hydroxylated, and quickly taking out the microfluid pipeline and the glass substrate after the reaction is finished to ensure that the surfaces of the microfluid pipeline and the glass substrate are lightly contacted to complete bonding, thereby obtaining the microfluid chip.
As a modification, the specific components of the liposome in the step 2 and the step 3 are as follows: the liposome contains 60mg of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1, 2-hexacosanyl-sn-glycero-3-phosphocholine) per 100mg of liposome; 10mg of 1, 2-dipalmitoyl-sn-glycerol-3-phosphate, sodium salt; 20mg of 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanomine (1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine); and 10mg of 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ carboxy (polyethylene glycol) -2000 ].
As an improvement, the mass fraction of the liposome in the chloroform solution is 0.5%, and the liposome emulsion has higher stability and utilization at the ratio.
The improvement is that the microfluidic chip is a droplet generation device assembled by a glass capillary, and the preparation method of the droplet generation device comprises the following steps:
step i, pipe making
Taking a round capillary as an inner tube, and stretching one end of the round capillary by using a capillary stretcher to form a streamline contraction port, wherein the size of the port is 0.1 mm; taking another square glass capillary as an outer tube; the outer diameter of the inner pipe is not larger than the inner diameter of the outer pipe, the difference is not larger than 0.5 mm, and the inner pipe and the outer pipe are fully cleaned and dried before use;
step ii, assembling
Carefully inserting the inner pipe obtained in the step i into the outer pipe by about 5mm, respectively connecting the far end of the inner pipe, the joint of the inner pipe and the outer pipe and the far end of the outer pipe to an engineering plastic adapter, and carefully packaging the inner pipe, the joint and the far end of the outer pipe on a transparent plastic or glass sheet by using quick-drying glue; the whole length of the micro-fluid platform is 8 cm, and the width of the micro-fluid platform is 1 cm; the joint of the far end of the inner pipe, the joint of the inner pipe and the outer pipe and the three adapter ports at the far end of the outer pipe are respectively a water phase inlet, an oil phase inlet and a product outlet.
The application of the prepared monodisperse freeze-dried microbubbles in ultrasonic development.
The improvement is that when the monodisperse freeze-dried microbubbles are applied, ventilation and aeration treatment are needed, namely, the monodisperse freeze-dried microbubbles are subjected to vacuum treatment, then target gas is introduced to serve as a bubble core, and the monodisperse freeze-dried microbubbles are hermetically packaged under the protection of the target gas, so that the practically usable monodisperse freeze-dried microbubbles are obtained.
In a further refinement, the step of ventilating and inflating includes the steps of:
step i, pipe making
Putting the freeze-dried micro bubbles into a vacuum bottle with the volume of 5-100 mL, sealing the bottle mouth with a medical rubber plug, and pumping the gas in the bottle by using a syringe needle connected with a vacuum pump for 1 minute;
step ii, aeration
Injecting core gas to be filled into the vacuum bottle by using a syringe needle;
step iii, repeating the evacuation and ventilation
Repeating steps i and ii three times to ensure that the ventilation process is sufficient and the interior of the microbubbles is sufficiently filled with the target gas.
In a further improvement, the core gas is one or a mixture of nitrogen, oxygen, fluorocarbon, carbon dioxide and the like.
Has the advantages that:
compared with the prior art, the invention relates to a method for preparing monodisperse freeze-dried microbubbles based on a microfluidics technology and application thereof, namely, a monodisperse liposome emulsion is prepared by using the microfluidics technology; freeze-drying the liposome emulsion to obtain hollow microspheres with liposomes with uniform sizes as shells; finally, the liposome microspheres are dissolved in aqueous solution to obtain the monodisperse microbubble ultrasonic developer. The concrete advantages are that:
(1) the liposome emulsion has uniform size, and the formed microbubbles have good monodispersity. The liposome emulsion prepared by using microfluid has narrow size distribution, the dispersity value (Polydispersity) is less than 0.05, and the hollow liposome microsphere formed after freeze-drying can still effectively maintain the monodispersity of the size. After dissolution in physiological saline, the microbubbles formed remain highly uniform in size. The size dispersion value (Polydispersity) of the common microbubble contrast agent in the market is generally larger than 0.5, and the size difference is obvious.
(2) The microemulsion and the microbubbles have high stability. The liposome has water-oil amphipathy, and can spontaneously form a self-assembled nano structure at a water-oil interface, thereby playing the roles of an encapsulating layer and a surfactant. And the microfluid emulsification technology can produce emulsion droplets with uniform size, and the Oswald ripening effect is greatly reduced. These advantages are not available in the commercial microbubble contrast agent products.
(3) The safety of monodisperse microbubbles is higher. Firstly, the liposome used by people belongs to safe and nontoxic biological materials, and does not bring harm to the blood circulation in vivo; and moreover, the size and the height of the micro bubbles are uniform, and compared with the traditional multi-dispersed micro bubble product, the micro bubbles are less prone to rupture and fusion, so that the risk of blocking capillary vessels is reduced.
(4) The microfluid production platform has high utilization rate (> 99%) of raw materials, low energy consumption, green and environment-friendly production process, and can realize accurate regulation and control of the size and the yield of products. And the preparation process of the microfluidic chip is simple and easy to repeat, and the method has strong potential of amplifying scale production.
(5) The microbubble freeze-dried powder obtained by the method is convenient to transport, store and use, and is not limited by the requirements of instruments, fields, technical backgrounds of users and the like.
(6) The size of the obtained micro-bubble is single and stable, the size of the micro-bubble can be adjusted through a micro-fluid technology, and the gas type of the micro-bubble core can be adjusted according to the requirement, which is the characteristic that the traditional commercialized product and the existing micro-fluid technology cannot be considered simultaneously.
(7) In animal clinical experiments, the monodisperse freeze-dried microbubbles provided by the invention have stronger contrast effect than the traditional polydisperse microbubble contrast agents. The experiment of the invention has strong controllability, and the obtained microbubbles have wide application fields.
Drawings
FIG. 1 is a flow chart of a method of preparing PDMS microfluidic chips according to the present invention, wherein 1-pattern template, 2-UV exposure + photoresist curing, 3-uncured photoresist stripping, 4-preparing PDMS plate, 5-PDMS plate stripping, 6-well and outlet are reserved;
FIG. 2 is a schematic design diagram of a microfluidic chip channel;
FIG. 3 is a photograph of a microfluidic chip in actual production;
fig. 4 is a diagram of a microfluidic channel under a microscope for emulsion production in example 1, wherein (a) is a diagram of a microfluidic channel under a microscope, and a black reference scale is 100 μm; (B) is white liposome emulsion; (C) is monodisperse bubble ultrasonic developer freeze-dried powder; (D) the microbubble solution is obtained after the monodisperse bubble ultrasonic developer freeze-dried powder is soaked in water;
fig. 5 shows the bubble size distribution of different microbubble contrast agents, (a) is the monodisperse freeze-dried microbubbles prepared in example 1, and (B) is the existing commercial (Sonovue) microbubble ultrasound contrast agent;
fig. 6 is a comparison of clinical ultrasound contrast of mouse kidney, wherein (a) is a traditional commercialized (sononovue corporation) microbubble contrast agent, and (B) is a microfluid microbubble lyophilized powder contrast agent produced by the present invention;
fig. 7 is a glass capillary microfluidic chip of example 2.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Example 1
A method for preparing monodisperse freeze-dried micro-bubbles based on a microfluid technology comprises the following preparation steps:
(1) drawing: designing a microfluidic pipeline by using engineering drawing software AutoCAD, wherein the microfluidic pipeline is provided with a water phase inlet, an oil phase inlet and a product collecting outlet, the diameter of the inlet and the outlet is 1mm, the depth of the pipeline is 50 micrometers, and the width of the pipeline is 100 micrometers; the structure of the microfluidic channel is shown in fig. 2;
(2) molding: and (2) printing the photoetching template obtained in the step (1) on a silicon wafer substrate by using a soft lithography technology (soft lithography) in an equal proportion projection mode, wherein the size ratio is 1: 1. the pipeline is made of SU-82100 photoresist, the depth of the pipeline is 50 microns, and the width of the pipeline is controlled to be 100 microns;
(3) uncovering the mold: mixing dimethyl siloxane monomer and curing agent according to the weight ratio of 10: 1, and then pouring and coating the mixture on the silicon wafer template obtained in the step (2), wherein the thickness of the PDMS silica gel is 5 mm. Removing residual bubbles in the silica gel for 1 hour by using a vacuum pump, moving the silica gel into a baking oven at 70 ℃, heating for 1 hour, and removing the mold to obtain a PDMS pipeline;
(4) bonding: the PDMS tubing and a piece of a common microscope slide were placed in a plasma reaction chamber and the surface was irradiated with uv light for 1 minute. And (3) quickly taking out the PDMS and the glass substrate after the reaction is finished, and lightly contacting the surfaces of the PDMS and the glass substrate to finish bonding to obtain the microfluidic chip. The total size of the chip is 5cm multiplied by 2 cm;
(5) emulsification: 20mL of an aqueous solution containing a surfactant and 5mL of a chloroform solution containing liposomes (1mg/mL) were injected into the microfluidic chip obtained in step (4) from the aqueous phase inlet and the oil phase inlet, respectively. Wherein, the water: the volume flow rate ratio of the trichloromethane is 5mL/hr to 1.25mL/hr, and the generated liposome emulsion is monodisperse emulsion with uniform size of about 10 microns;
(6) freeze-drying: and (4) collecting the mixture of the liposome emulsion and water obtained in the step (5), standing and separating for 10 minutes, skimming the upper aqueous solution, transferring the rest liposome emulsion into a freeze dryer, and carrying out ultra-low temperature drying at-80 ℃ for 24 hours to obtain hollow microspheres with the liposome as the shell, namely monodisperse freeze-dried microbubbles.
In the application process, the micro-bubble solution can be obtained only by adding the monodisperse bubbles into the physiological saline and slightly oscillating. The sizes of the microemulsion, the liposome microspheres and the microbubbles are measured by using a method of taking a picture by using a microscope to compare with a scale, and test results show that the sizes of the microbubbles provided by the invention are about 3 microns, the monodispersity is greatly improved compared with that of a traditional commercialized product, and the stability of the liposome hollow microspheres under vacuum drying packaging exceeds 3 months.
In animal experiments, a 6-week-old CD1 mouse is used as an animal model, 100 μ L of microbubble solution is injected from the caudal root vein at a flow rate of 10 μ L/s, and the kidney part is imaged by contrast under an ultrasonic detector at 18 MHz. Through comparison, the microfluidic microbubble produced by the invention has obviously improved contrast quality compared with the traditional commercialized microbubble contrast agent (figure 6), can fully and clearly present the deep tissue structure of the mouse kidney, and the traditional product has a large amount of shadows and is difficult to distinguish the focus and the boundary of the peripheral tissue in practical application.
Example 2
A glass capillary microfluidics technology method for preparing monodisperse freeze-dried microbubbles based on microfluidics technology comprises the following preparation steps: (1) pipe making: we used a round glass capillary (CM Scientific) having a length of 2CM, an inner diameter of 0.5 mm and an outer diameter of 0.7 mm as an inner tube, and stretched one end thereof using a capillary stretcher (P-97, Sutter Instrument Company) to form a streamlined constriction port having a port size of 0.1 mm; we used another square glass capillary (CM Scientific) with a length of 5CM, an inner side of 0.7 mm and an outer side of 1mm as the outer tube. The inner and outer tubes are fully cleaned and dried before use.
(2) Assembling: carefully inserting the inner pipe obtained in the step (1) into the outer pipe by about 5mm, respectively connecting the far end of the inner pipe, the joint of the inner pipe and the outer pipe and the far end of the outer pipe to engineering plastic adapter ports (Nanoports), and carefully packaging the inner pipe and the outer pipe on a transparent plastic or glass thin plate by using quick-drying glue. The microfluidic platform has an overall length of about 8 cm and a width of 1 cm. The joint of the far end of the inner pipe, the joint of the inner pipe and the outer pipe and the three adapter ports at the far end of the outer pipe are respectively a water phase inlet, an oil phase inlet and a product outlet. The structure is shown in fig. 7.
(3) Emulsification: 20mL of an aqueous solution containing a surfactant and 5mL of a chloroform solution containing liposomes (1mg/mL) were injected into the microfluidic chip obtained in step (4) from the aqueous phase inlet and the oil phase inlet, respectively. Wherein, the water: the volume flow rate ratio of the trichloromethane is 5mL/hr to 1.25mL/hr, and the generated liposome emulsion is monodisperse emulsion with uniform size of about 15 microns;
(4) freeze-drying: collecting the mixture of the liposome emulsion and water obtained in the step (3), standing and separating for 10 minutes, skimming the upper aqueous solution, transferring the rest liposome emulsion into a freeze dryer, and carrying out ultra-low temperature drying at-80 ℃ for 24 hours to obtain hollow microspheres with the liposome as a shell, namely monodisperse freeze-dried microbubbles;
in the application process, the micro-bubble solution can be obtained only by adding the monodisperse bubble ultrasonic developer into physiological saline and slightly oscillating. The sizes of the microemulsion, the liposome microspheres and the microbubbles are measured by using a method of taking a picture by using a microscope to compare with a scale, and test results show that the sizes of the microbubbles provided by the invention are about 7 microns, the monodispersity is greatly improved compared with that of a traditional commercialized product, and the stability of the liposome hollow microspheres under vacuum drying packaging exceeds 3 months.
It should be noted that, in this example, the suitable size of the inner tube may be between 0.1 mm and 2 mm, and the suitable size of the outer tube may be between 0.5 mm and 1 cm, and the specific values described in this example are all optimized experimental schemes.
Comparative example 1
At present, the types of microbubble commercialized products can be divided into two types, namely microbubble freeze-dried powder and instantly produced microbubble. The microbubble lyophilized powder product has the defects that the size homogenization of microbubbles is difficult to realize, so that the actual ultrasonic development effect is low; the micro-bubble technology for instant production is mainly based on micro-fluid technology, and Tide Microfluidics company (http:// microfluidic. nl/applications/medical-imaging) in the Netherlands develops a micro-fluid production platform capable of instantly producing micro-bubbles, but the operation and maintenance of micro-fluid equipment have high requirements on professional knowledge background of users, and are difficult to develop and popularize in ordinary hospitals, and the platform has low production capacity, long waiting time for preparing bubbles, and is not suitable for large demands of hospitals and patients.
Compared with the two patents mentioned in the background technology, the invention firstly does not directly use the complex two-chip bonding, only uses the single chip and the glass substrate bonding, simplifies the manufacturing steps and reduces the bonding difficulty; secondly, the invention does not need to use gas to directly manufacture bubbles, but only uses oil/water fluid to produce precursor emulsion, compared with the method for directly manufacturing bubbles, the method for producing precursor emulsion does not need to additionally operate a gas compression device; in addition, the microbubbles obtained in the patent are produced and used as required, are difficult to store, carry and transport, and have higher technical knowledge requirements on users, and the finally obtained product is freeze-dried bubble powder which is convenient to store, carry and transport; third, the microbubble core gas of the present invention can be any combination of gases or mixtures of gases, which is an advantage that conventional commercial products and existing microfluidic technologies cannot meet.
In summary, the present patent successfully prepares monodisperse liposome microbubbles on microfluidic platforms (e.g., PDMS, glass capillary) of different materials by using microfluidic emulsification techniques. Compared with the traditional commercialized microbubble product, the microbubble prepared by the invention has the important advantages of uniform and controllable size, better ultrasonic development effect in vivo experiments and the like; compared with microbubbles produced by other existing microfluidics technologies, the product is simpler and easier to operate in use, has better stability of freeze-dried microbubbles, and is easier to store, transfer and transport, and the core of the microbubbles can be any gas or a mixture of a plurality of gases. This patent provides reliable technical scheme for exploring the wide application of microbubble in the medical product field.
Claims (9)
1. A method for preparing monodisperse freeze-dried microbubbles based on a microfluidics technology is characterized by comprising the following steps:
step 1, preparing a microfluidic chip
Assembling a droplet generation device assembled by a microfluidic chip or a glass capillary;
step 2, preparing a monodisperse emulsion
Injecting an aqueous solution containing a surfactant and a trichloromethane solution dissolved with liposome into a microfluidic chip from a water phase inlet and an oil phase inlet respectively by using a mechanical pump or a pneumatic pump as a drive, wherein the aqueous solution contains Pluronic-F68 surfactant with the volume fraction of 2.5%, the trichloromethane solution contains 0.1-1% of liposome with the mass fraction, the flow rate of the water phase is 1-50ml/hr, the flow rate of the oil phase is 0.1-10ml/hr, the ratio of the flow rate of the water phase to the flow rate of the oil phase is 1/5-1/20, and oil/water emulsion with the diameter of 5-50 microns and uniform distribution can be prepared by adjusting the flow rate ratio of the flow rate of the water phase to the flow rate of the oil phase; wherein, the liposome is distributed at the interface of the oil/water emulsion due to the self amphipathy;
step 3, freeze drying
And (3) collecting and standing the oil/water emulsion obtained in the step (2) for separation, skimming most of the upper aqueous solution, transferring the rest emulsion into a freeze dryer, and carrying out ultra-low temperature drying at-80 ℃ to obtain hollow microspheres wrapped by the liposome, namely monodisperse freeze-dried microbubbles.
2. The method of claim 1, wherein the microfluidic chip is a PDMS microfluidic chip, and the method for preparing the PDMS microfluidic chip comprises the following steps:
designing a micro-fluid pipeline with the depth of 50 microns and the width of 50 microns by using engineering drawing software AutoCAD, and printing the micro-fluid pipeline as a photoetching template on a transparent plastic sheet, wherein the micro-fluid pipeline is provided with a water phase inlet, an oil phase inlet and a product collecting outlet, and the calibers of the water phase inlet and the oil phase inlet are 1 mm;
secondly, printing a photoetching template on a silicon wafer substrate by using a soft photoetching technology, wherein the projection size ratio of patterns on the photoetching template on the silicon wafer substrate is 1: 1;
thirdly, uncovering the mould
Uniformly coating PDMS silica gel on a silicon wafer substrate, wherein the thickness of the layer is 5mm, removing residual bubbles in the silica gel by using a vacuum pump, moving the silica gel into a 70 ℃ oven to heat for 1 hour, and removing the mold to obtain a pipeline, wherein the silicon wafer substrate can be repeatedly used for removing the mold;
the fourth step, bonding
And placing the microfluid pipeline and the glass substrate into a plasma reaction box, irradiating the surfaces with ultraviolet light for 1 minute to ensure that the surfaces of the material A and the glass substrate are fully hydroxylated, and quickly taking out the microfluid pipeline and the glass substrate after the reaction is finished to ensure that the surfaces of the microfluid pipeline and the glass substrate are lightly contacted to complete bonding, thereby obtaining the microfluid chip.
3. The method for preparing monodisperse freeze-dried microbubbles based on the microfluidics technology according to claim 1, wherein the specific components of the liposomes in the step 2 and the step 3 are as follows: the liposome contains 60mg of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1, 2-hexacosanyl-sn-glycero-3-phosphocholine) per 100mg of liposome; 10mg of 1, 2-dipalmitoyl-sn-glycerol-3-phosphate, sodium salt; 20mg of 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanomine (1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine); and 10mg of 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ carboxy (polyethylene glycol) -2000 ].
4. The method of claim 1, wherein the improvement is that the mass fraction of liposomes in the chloroform solution is 0.5%.
5. The method for preparing monodisperse freeze-dried microbubbles based on the microfluidics technology as claimed in claim 1, wherein the microfluidic chip is a droplet generation device assembled by glass capillaries, and the method for preparing the droplet generation device comprises the following steps:
step i, pipe making
Taking a round capillary as an inner tube, and stretching one end of the round capillary by using a capillary stretcher to form a streamline contraction port, wherein the size of the port is 0.1 mm; taking another square glass capillary as an outer tube; the outer diameter of the inner pipe is not larger than the inner diameter of the outer pipe, the difference is not larger than 0.5 mm, and the inner pipe and the outer pipe are fully cleaned and dried before use;
step ii, assembling
Carefully inserting the inner pipe obtained in the step i into the outer pipe by about 5mm, respectively connecting the far end of the inner pipe, the joint of the inner pipe and the outer pipe and the far end of the outer pipe to an engineering plastic adapter, and carefully packaging the inner pipe, the joint and the far end of the outer pipe on a transparent plastic or glass sheet by using quick-drying glue; the whole length of the micro-fluid platform is 8 cm, and the width of the micro-fluid platform is 1 cm; the joint of the far end of the inner pipe, the joint of the inner pipe and the outer pipe and the three adapter ports at the far end of the outer pipe are respectively a water phase inlet, an oil phase inlet and a product outlet.
6. Use of monodisperse freeze-dried microbubbles prepared according to claim 1 in medical ultrasound imaging.
7. The use of claim 5, wherein the monodisperse lyophilized microbubbles are subjected to ventilation and aeration treatment during the use, that is, the monodisperse lyophilized microbubbles are subjected to vacuum treatment, then the target gas is introduced as the inner core of the microbubbles, and the target gas is sealed and packaged under the protection of the target gas, so as to obtain the monodisperse lyophilized microbubbles for practical use.
8. Use according to claim 6, wherein said step of ventilation inflating comprises the steps of:
step i, pipe making
Putting the freeze-dried micro bubbles into a vacuum bottle with the volume of 5-100 mL, sealing the bottle mouth with a medical rubber plug, and pumping the gas in the bottle by using a syringe needle connected with a vacuum pump for 1 minute;
step ii, aeration
Injecting core gas to be filled into a vacuum bottle by using a syringe needle;
step iii, repeating the evacuation and ventilation
Repeating steps i and ii three times to ensure that the ventilation process is sufficient and the interior of the microbubbles is sufficiently filled with the target gas.
9. The use of claim 8, wherein the core gas is a mixture of one or more of nitrogen, oxygen, fluorocarbons, and carbon dioxide.
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