CN103372382B - A kind of preparation facilities of therapeutic type micro air bubble ultrasonic contrast medium and method - Google Patents

Abstract

The invention discloses a kind of runner, the device and method of preparing therapeutic type micro air bubble ultrasonic contrast medium, by by the flow control of described inner membrane material at 10 ~ 50 μ l/min, by the flow control of described outer membrane material at 30 ~ 60 μ l/min, and control the operating pressure of gas, by the microbubble diameter control of described therapeutic type micro air bubble ultrasonic contrast medium at 5 ~ 10 μm.Therapeutic type micro air bubble ultrasonic contrast medium prepared by the present invention shows a monodisperse distribution, and the Size Distribution centrality of microbubble is relatively good, and through checking, described therapeutic type micro air bubble ultrasonic contrast medium has good gene bearer properties.

Description

A preparation device and method for therapeutic microbubble ultrasound contrast agent

technical field

The invention relates to the field of medical engineering, in particular to a method and a device for preparing a microbubble ultrasonic contrast agent.

Background technique

Microbubble Ultrasound ContrastAgents (mUCA) is a suspension containing a high concentration of microbubbles with an average diameter of less than 8 microns (μm). After the microbubble ultrasound contrast agent is injected into the human body through the peripheral vein, the microbubble and the surrounding tissue present different acoustic characteristics, thereby enhancing the echo contrast between the blood flow and the surrounding tissue, and significantly improving the evaluation of tissue blood perfusion by ultrasound images. Microbubble ultrasound contrast agent can more accurately reflect the characteristics of blood flow, greatly improve the sensitivity and specificity of diagnosis, and further expand the field of ultrasound diagnosis, so that ultrasound diagnosis technology can be used in tissue perfusion and inflammation of various organs. In terms of detection and tumor detection, it has been widely used in research and clinical applications. In addition, specific molecules or drugs can be modified on the surface of microbubbles to realize molecular imaging or drug therapy.

Recent studies have shown that under ultrasonic irradiation, the microbubbles in tissues and body fluids can undergo cavitation effects, creating a passable gap on the cell membrane, thereby improving the permeability of the cell membrane, promoting the entry of genes into cells, and enhancing the ability of genes to enter cells. Intracellular transfection and expression. Therefore, the microbubble contrast agent can be used as a carrier of genes and drugs and can perform targeted release and tumor embolization therapy, providing a new direction for tumor gene therapy to break through the bottleneck problem. Therapeutic microbubble ultrasound contrast agent has become the current medical research. new hotspot.

At present, the preparation methods of microbubble contrast agents in clinical use mainly include acoustic cavitation, freeze drying, and hot air drying. For example, the microbubble ultrasonic contrast agent AF0150 produced by Alliance Pharmaceutical Company of the United States is a powder made by a drying method. The powder contains surfactants, salts, and buffer substances. The powder is saturated with perfluorocarbon gas and stored in a sealed vial. During use, injecting sterile water will form microbubbles.

The size distribution of the microbubbles prepared by the above method is not concentrated, and the microbubbles with a diameter greater than 8 microns must be removed to prevent blood vessel blockage, but the remaining microbubbles are still not well distributed, resulting in a very wide echo frequency. In addition, the thickness of each microbubble envelope is also very uneven, which also has a great influence on the echo response of the microbubbles. In order to realize the therapeutic function of the microbubble contrast agent, the above method cannot meet the requirements of the microbubble as a carrier of genes and drugs, and cannot produce a therapeutic microbubble ultrasound contrast agent.

Therefore, we need a new preparation method, which can prepare a therapeutic microbubble ultrasound contrast agent with good concentration of size distribution and good gene carrying properties.

Contents of the invention

The first object of the present invention is to provide a channel for preparing therapeutic microbubble ultrasound contrast agent.

The second object of the present invention is to provide a preparation device comprising the above flow channel.

The third object of the present invention is to provide a method for preparing the therapeutic microbubble ultrasound contrast agent by using the above preparation device.

In order to achieve the above purpose, the main idea of the present invention is to use microfluidic technology to prepare microbubbles with composite encapsulation function as the carrier of genes and/or drugs to realize the therapeutic function of microbubble contrast agents. And disclose the following technical solutions:

A flow channel for preparing therapeutic microbubble ultrasound contrast agent, the flow channel has a main flow channel, symmetrical flow channels on both sides, two symmetrical fluid inlets and a fluid outlet;

The sprue has a point focus and an angle focus;

At the point focus position, the main flow channel is hourglass-shaped with a minimum width; at the angle focus position, the side channel is connected to the main flow channel at an angle.

Preferably, the ratio of the width of the main channel to the side channel is 2:1; the ratio of the width of the main channel to the minimum width of the hourglass shape is 100:7-100:20. Particularly preferably, the width of the main channel is 100 μm, the width of the side channel is 50 μm, and the minimum width of the hourglass shape is 7-20 μm.

Preferably, at the angular focusing position, the angle between the side channel and the main channel is in the range of 60°-90°.

The material of the flow channel may be polydimethylsiloxane (PDMS), which is prepared by etching.

The present invention is also a device for preparing therapeutic microbubble ultrasound contrast agent, said device comprising:

Any of the above runners;

a gas source connected to the point focus of the main flow channel;

Intima material sources on both sides of the symmetry, connected to the point focus of the main channel through side channels;

Symmetrical adventitia material sources on both sides are connected to the side channel at the angular focus of the main channel;

A regulating valve is provided on the side channel connected with the gas source for pressure regulation.

Preferably, a medical syringe pump is used to inject the material in the source of inner membrane material and the source of adventitia material into the side channel.

The present invention also provides a method for preparing a therapeutic microbubble ultrasonic contrast agent by using the above-mentioned device, by controlling the flow rate of the inner membrane material and the outer membrane material and the working pressure of the gas, the The microbubble diameter is controlled at 5~10μm.

Preferably, the flow rate of the inner membrane material is controlled at 10-50 μl/min, and the flow rate of the outer membrane material is controlled at 30-60 μl/min by a medical syringe pump.

Preferably, the other working pressure is controlled at 500-1000Pa through a regulating valve.

Some nouns involved in the present invention are explained below.

The hourglass shape refers to a shape in which both ends are wide and the middle is narrow, and the width changes gradually.

The medical syringe pump is a commercially available conventional device.

The point focus and angle focus belong to the category of flow focus.

The fluid is a general term for liquids and gases, and is composed of a large number of molecules that are constantly in thermal motion and have no fixed equilibrium position. Its basic characteristics are that it has no definite shape and has fluidity.

The present invention aims to provide equipment and methods for preparing therapeutic microbubble ultrasonic contrast agents. There are no special requirements for the inner membrane material, outer membrane material and gas. In specific use, the corresponding inner membrane material can be selected according to the required contrast agent. The material of the membrane and outer membrane, and the type of gas.

The therapeutic microbubble ultrasonic contrast agent prepared by the present invention has a monodisperse distribution, and the size distribution concentration of the microbubbles is relatively good, and it has been verified that the therapeutic microbubble ultrasonic contrast agent has good gene bearing characteristics.

Description of drawings

Describe the present invention in detail below in conjunction with accompanying drawing and specific embodiment:

Fig. 1 is a schematic diagram of a flow channel for preparing a therapeutic microbubble ultrasound contrast agent.

Fig. 2 is a schematic diagram of a device for preparing a therapeutic microbubble ultrasound contrast agent.

Fig. 3 is a microscope observation image of the flow channel 1 during the preparation process.

Figure 4 is an image of the prepared therapeutic microbubble ultrasound contrast agent observed on a microscope slide.

Fig. 5 is a microbubble distribution diagram of the prepared therapeutic microbubble ultrasound contrast agent measured by a laser particle analyzer.

Fig. 6 is an inverted fluorescence microscope image of the prepared therapeutic microbubble ultrasound contrast agent.

Fig. 7 is the ultrasound imaging effect of the prepared therapeutic microbubble ultrasound contrast agent.

detailed description

The present invention will be described in detail below in conjunction with the examples, and the examples are intended to explain rather than limit the technical solution of the present invention. Furthermore, the directional terms mentioned in the present invention, such as "up", "down", "front", "back", "left", "right", "inside", "outside", "side", etc. , are for orientation only with reference to the attached drawings. Therefore, the directional terms used are used to illustrate and understand the present invention, but not to limit the present invention.

Example 1 is used to prepare the flow channel of therapeutic microbubble ultrasound contrast agent

Please refer to FIG. 1 . FIG. 1 shows a non-limiting example of a flow channel for preparing a therapeutic microbubble ultrasound contrast agent. In this embodiment, the flow channel 1 has a main flow channel 11 and several side flow channels 12 .

The flow channel 1 comprises a point focus 13 and an angle focus 14 .

At the point focus 13, the main flow channel 11 is hourglass-shaped and has a minimum width b ₃ ; at the angle focus 14, the side channel 12 is connected to the main flow channel 11 at an angle within the range of α Between 60°~90°.

In this embodiment, the width b ₁ of the main channel 11 is 100 μm, the width b ₂ of the side channel 12 is 50 μm, and the minimum width b ₃ of the hourglass shape is 7 μm. The material of the flow channel may be polydimethylsiloxane (PDMS), which is prepared by etching.

Example 2 Device and preparation method for preparing therapeutic microbubble ultrasound contrast agent

Please refer to FIG. 2 . FIG. 2 shows a non-limiting example of a device for preparing a therapeutic microbubble ultrasound contrast agent.

The right end in the figure is the outlet of the fluid, and the flow direction of the gas, inner membrane material and outer membrane material in the flow channel can be seen in the direction of the arrow in Figure 2.

In this embodiment, the gas source is connected to the point focus part 13 of the main channel; the two inner membrane material sources are respectively connected to the point focus part connection 13 of the main channel through the side channel 12;

Two adventitia material sources are respectively connected to the side channel 12 of the angular focus 14 of the main channel.

The width b ₁ of the main channel 11 is 100 μm, the width b ₂ of the side channel 12 is 50 μm, and the minimum width b ₃ of the hourglass shape is 7 μm. The material of the flow channel is polydimethylsiloxane (PDMS), which is prepared by etching.

In this embodiment, the flow channel connected to the gas source is provided with a regulating valve (not shown in the figure) for regulating the pressure. The regulating valve may be a high-precision regulating valve.

In this embodiment, a medical syringe pump (not shown in the figure) is used to inject the materials in the inner membrane material source and the outer membrane material source into the side channel.

In this embodiment, sulfur hexafluoride is used for the gas, calf thymus DNA is used for the inner membrane material, and glycerin and polyethylene glycol 2000 (PEG2000) are used for the outer membrane. The flow rate of the inner membrane material was controlled at 10 μl/min and the flow rate of the outer membrane material at 30 μl/min by a medical syringe pump, and the working pressure of the gas was controlled at 500 Pa through a regulating valve. In specific operation, after adjusting the medical syringe pump and the regulating valve to the specified parameters, all the fluids can be injected into the device at the same time.

Example 3

In this embodiment, the width b ₁ of the main channel 11 is 100 μm, the width b ₂ of the side channel 12 is 50 μm, and the minimum width b ₃ of the hourglass shape is 10 μm. The material of the flow channel is polydimethylsiloxane (PDMS), which is prepared by etching.

The gas uses sulfur hexafluoride, the inner membrane material uses calf thymus DNA, and the outer membrane uses glycerin and polyethylene glycol 2000 (PEG2000). Through a medical syringe pump, the flow rate of the inner membrane material was controlled at 30 μl/min, the flow rate of the outer membrane material was controlled at 50 μl/min, and the working pressure of the gas was controlled at 800 Pa through a regulating valve. The preparation steps are the same as in Example 2.

Example 4

In this embodiment, the width b ₁ of the main channel 11 is 100 μm, the width b ₂ of the side channel 12 is 50 μm, and the minimum width b ₃ of the hourglass shape is 20 μm. The material of the flow channel may be polydimethylsiloxane (PDMS), which is prepared by etching.

The gas uses sulfur hexafluoride, the inner membrane material uses calf thymus DNA, and the outer membrane uses glycerin and polyethylene glycol 4000 (PEG4000). The flow rate of the inner membrane material was controlled at 50 μl/min and the flow rate of the outer membrane material at 60 μl/min by a medical syringe pump, and the working pressure of the gas was controlled at 1000 Pa through a regulating valve. The preparation steps are the same as in Example 2.

Embodiment 5 detection experiment

During the preparation process, the flow channel 1 was observed with a microscope, and the observation results are shown in FIG. 3 . Fig. 4 is the prepared therapeutic microbubble ultrasound contrast agent observed on a microscope glass slide. Fig. 5 is a microbubble distribution diagram of the prepared therapeutic microbubble ultrasonic contrast agent measured by a laser particle analyzer, the abscissa indicates the radius of the microbubble in nanometers; the ordinate indicates the percentage of particle size distribution. It can be seen from Fig. 4 and Fig. 5 that the contrast agent prepared by applying the present invention is monodispersely distributed, and the size distribution of microbubbles is more concentrated.

Please refer to FIG. 6, which is an inverted fluorescence microscope image of the prepared therapeutic microbubble ultrasound contrast agent. It can be seen from FIG. 6 that the contrast agent prepared by applying the present invention has better gene carrying properties. After the microbubbles were destroyed by ultrasound, all the DNA was dissolved in the solution, and the gene carrying rate was calculated by formula 1.

Formula 1

load rate = , where DNA _w refers to the DNA content of the solution after the microbubbles are destroyed by ultrasonic; DNA ₁ refers to the DNA content in the solution before the microbubbles are destroyed by ultrasonic.

In this example, the measured DNA _w is 88.2 μg/μl, DNA ₁ is 60 μg/μl, and the calculated gene carrying rate is 32%.

Please refer to FIG. 7 . FIG. 7 is the ultrasonic imaging effect of the prepared contrast agent. It can be seen from FIG. 7 that the contrast agent prepared by applying the present invention has a better ultrasonic imaging effect.

Based on the above, it can be seen that the therapeutic microbubble ultrasonic contrast agent prepared by the present invention is monodispersely distributed, the size distribution of the microbubbles is relatively concentrated, and it has been verified that the therapeutic microbubble ultrasonic contrast agent has good gene bearing properties .

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be considered Be the protection scope of the present invention.

Claims (8)

1., for the preparation of a runner for therapeutic type micro air bubble ultrasonic contrast medium, described runner has a sprue, two groups of side runners of symmetry, the fluid intake of two kinds of fluids and fluid outlet, it is characterized in that,

Described sprue has some focusing position and an angle and focuses on position;

At described point focusing position, described sprue is hour-glass in shape, has a minimum widith; Focus on position in described angle, described side runner is angularly connected with sprue.

2. runner as claimed in claim 1, is characterized in that, described sprue is 2:1 with the width ratio of side runner; The width of described sprue is 100:7 ~ 100:20 with the ratio of hourglass-shaped minimum widith.

3. runner as claimed in claim 2, it is characterized in that, the width of described sprue is 100 μm, and the width of described side runner is 50 μm, and described hourglass-shaped minimum widith is 7 ~ 20 μm.

4. runner as claimed in claim 2, is characterized in that, focus on position in described angle, the angular range between side runner and sprue is between 60 ° ~ 90 °.

5. for the preparation of a device for therapeutic type micro air bubble ultrasonic contrast medium, it is characterized in that, described device comprises:

As the runner as described in arbitrary in claim 1-4;

Gas source, is connected with the point focusing position of described sprue;

Symmetrical inner membrane material source, is connected with the point focusing position of described sprue by side runner;

Symmetrical outer membrane material source, the side runner focusing on position with the angle of described sprue is connected;

The described sprue be connected with gas source is provided with control valve, for regulating pressure.

6. device as claimed in claim 5, is characterized in that, use a medical injection pump that the material in described inner membrane material source and outer membrane material source is injected described side runner.

7. for the preparation of a method for therapeutic type micro air bubble ultrasonic contrast medium, it is characterized in that, apply device as claimed in claim 6, prepare described therapeutic type micro air bubble ultrasonic contrast medium;

By described medical injection pump, by the flow control of described inner membrane material at 10 ~ 50 μ l/min, by the flow control of described outer membrane material at 30 ~ 60 μ l/min, and control the operating pressure of gas, by the microbubble diameter control of described therapeutic type micro air bubble ultrasonic contrast medium at 5 ~ 10 μm.

8. method as claimed in claim 7, be is characterized in that, controlled at 500 ~ 1000Pa by the operating pressure of control valve by described gas.