WO2022120732A1 - Ultrasonic drug delivery experimental device and ultrasonic drug delivery experimental method - Google Patents

Abstract

An ultrasonic drug delivery experimental device and an ultrasonic drug delivery experimental method. The ultrasonic drug delivery experimental device comprises: a sample carrier (10), the sample carrier (10) having an accommodating cavity (11), and the accommodating cavity (11) containing a culture medium solution (12) for culturing experimental cells; a phononic crystal plate (20), the phononic crystal plate (20) being placed in the accommodating cavity (11), the phononic crystal plate (20) being immersed in the culture medium solution (12), and experimental cells growing on the wall of the phononic crystal plate (20); an ultrasound contrast agent, the ultrasound contrast agent being mixed in the culture medium solution (12); and an ultrasonic transmitting assembly (30), the ultrasonic transmitting assembly (30) being arranged at the bottom of the sample carrier (10), and the ultrasonic transmitting assembly (30) being configured to emit ultrasonic waves having a predetermined frequency to the phononic crystal plate (20). The problem in the prior art of low drug delivery efficiency due to the fact that a spatial position of microvesicles (40) cannot be effectively controlled, so that the distance between the microvesicles (40) and the cells is random and uncontrollable is solved.

Description

Ultrasonic drug delivery experimental device, ultrasonic drug delivery experimental method technical field

The application belongs to the technical field of biomedical experimental instruments, and in particular relates to an ultrasonic drug administration experimental device and an ultrasonic drug administration experimental method.

Background technique

Ultrasound delivery of drugs and genes (ie, ultrasound drug delivery technology) is mainly based on the biophysical process of ultrasound combined with ultrasound contrast agent microbubbles to perforate cells, which is also known as sonoporation: microbubbles The cavitation effect in the ultrasonic field produces repairable pores of tens of nanometers to hundreds of nanometers on the surface of the cell membrane, thereby enhancing the permeability of the cell membrane, allowing extracellular DNA, proteins and other biological macromolecules to pass through The pores enter the cell to function. The sonoporation effect is a short-range effect, and the distance between microbubbles and cells has an important impact on the efficiency of the sonoporation effect, thus directly affecting the efficiency of drug delivery. Currently, in ultrasonic drug delivery studies, cells are usually cultured on the surface of a petri dish, and the microbubble solution is added to the petri dish, followed by ultrasound. This method cannot effectively control the spatial position of the microbubbles, and due to the effect of buoyancy, the microbubbles mostly float on the surface of the culture medium and are far away from the surface of the culture dish where the cells grow adherently, so that the distance between the microbubbles and the cells is random. control, and is usually much larger than the effective interaction distance between microvesicles and cells, resulting in lower drug delivery efficiency.

technical problem

The purpose of this application is to provide an ultrasonic drug delivery experimental device and an ultrasonic drug delivery experimental method, aiming to solve the problem that the existing technology cannot effectively control the spatial position of the microbubble, so that the distance between the microbubble and the cell is random and uncontrollable, This leads to the problem of lower drug delivery efficiency.

technical solutions

In order to solve the above technical problem, the technical solution adopted in the embodiment of the present application is: an ultrasonic drug delivery experimental device, comprising: a sample carrier, the sample carrier has a accommodating cavity, and the accommodating cavity is filled with a culture medium solution for culturing experimental cells ; Phononic crystal plate, the phononic crystal plate is placed in the holding cavity, and the phononic crystal plate is immersed in the medium solution, and the experimental cells are grown on the phononic crystal plate; Ultrasonic contrast agent, ultrasonic contrast agent is mixed in the culture medium In the base solution; an ultrasonic emitting component, the ultrasonic emitting component is arranged at the bottom of the sample carrier, and the ultrasonic emitting component is used for emitting ultrasonic waves of a predetermined frequency to the phononic crystal plate.

Optionally, the phononic crystal plate includes a substrate and a plurality of resonant ridges, all the resonant ridges are distributed on one side of the substrate facing the ultrasonic emitting component, and the other side of the substrate away from the resonant ridges adheres to the wall. Grow with experimental cells.

Optionally, the spacing distance between any two adjacent resonance convex strips is equal.

Optionally, the thickness t of the substrate is micrometers, and the separation distance p between any two adjacent resonant ridges is micrometers.

Optionally, each resonant protruding strip is one of straight strip shape, curved shape or broken line shape.

Optionally, the cross section of each resonant ridge perpendicular to the tangential direction of the position at any position is a rectangular section, the width w of the rectangular section is micrometers, and the height h of the rectangular section is micrometers.

Optionally, the ultrasonic transmitting assembly includes a signal generator, a power amplifier and an ultrasonic transducer, the signal generator is electrically connected to the power amplifier, the power amplifier is electrically connected to the ultrasonic transducer, and the ultrasonic transducer is installed on the sample carrier. The bottom of the ultrasonic transducer emits ultrasonic waves of a predetermined frequency to the phononic crystal plate.

Optionally, the ultrasonic probe of the ultrasonic transducer extends into the accommodating cavity through the bottom of the sample carrier, and the ultrasonic probe of the ultrasonic transducer is spaced apart from the phononic crystal plate.

Optionally, the ultrasonic probe of the ultrasonic transducer is abutted against the bottom of the sample carrier, and the ultrasonic probe of the ultrasonic transducer and the culture medium solution are isolated by the bottom of the sample carrier.

Optionally, the ultrasound contrast agent has a microbubble formed by enclosing a gas in an outer shell, the outer shell of the microbubble is composed of one of PLGA polymer material, phospholipid, and albumin material, and the gas of the microbubble is air, sulfur hexafluoride, all One of the fluoropropanes.

According to another aspect of the present application, an experimental method for ultrasonic drug delivery is provided. Specifically, the ultrasonic drug administration experimental method comprises the following experimental operation steps:

Step S10: containing the culture medium solution in the sample carrier, and mixing the ultrasonic contrast agent and the experimental drug into the culture medium solution;

Step S20: placing the phononic crystal plate with the experimental cells adherently growing on the plate surface in the sample carrier, and submerging the phononic crystal plate with the culture medium solution;

Step S30: transmitting an ultrasonic wave of a predetermined frequency to the phononic crystal plate, and the ultrasonic wave acts for a predetermined action time;

Step S40: Continue to incubate the experimental cells for a predetermined period of time.

Optionally, before performing the operation step 10, the ultrasonic drug administration experimental method further includes step 01: cultivating the experimental cells, and then transferring the cultivated experimental cells that meet the experimental use to the phononic crystal plate for continued cultivation so that the experimental cells adhere to the experimental cells. The walls are grown on the slab face of the phononic crystal slab.

Optionally, sterilization of the phononic crystal plate is performed before transferring the cultured experimental cells conforming to the experimental use to the phononic crystal plate.

Optionally, in the process of executing step S40, step S40 includes the following sub-steps:

Step S41: after step S30 is performed, continue to incubate the experimental cells with the original medium solution for a first predetermined period of time;

Step S42 : after reaching the first predetermined period of time, replace the original medium solution in the sample carrier with a brand new complete medium solution to continue culturing the experimental cells for a second predetermined period of time.

Optionally, in the process of performing step S40, the value range of the first predetermined duration is 4-5 hours, and the value range of the second predetermined duration is greater than or equal to 24 hours.

beneficial effect

This application has at least the following beneficial effects:

During the experimental operation using the ultrasonic drug delivery experimental device, the resonance frequency of the phononic crystal plate is excited by the emission of ultrasonic waves, so that the plate surface of the phononic crystal plate generates a local sound field to capture the microbubbles of the ultrasonic contrast agent mixed in the culture medium solution, Effectively manipulate the spatial position of the microvesicles relative to the adherent growing experimental cells, so that the microvesicles floating in the medium solution can approach and closely contact the experimental cells, and then induce sonoporation between the microvesicles and the experimental cells. The effect achieves the purpose of enhancing the permeability of the cell membrane, so that the drugs mixed in the culture medium solution can smoothly enter the experimental cells to produce effects, and the drug delivery efficiency is greatly improved.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only for the present application. In some embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

1 is a schematic structural diagram of an ultrasonic drug delivery experimental device according to Embodiment 1 of the application;

2 is a partial cross-sectional view of a phononic crystal plate of the ultrasonic drug delivery experimental device according to Embodiment 1 of the application;

3 is a schematic structural diagram of the ultrasonic drug delivery experimental device according to the second embodiment of the application;

4 is a schematic diagram of the effect of the local sound field (suction force) generated by the phononic crystal plate when the ultrasonic drug delivery experimental device of the application emits ultrasonic waves during the experiment;

5 is a schematic diagram of the effect of the local sound field (repulsion) generated by the phononic crystal plate when the ultrasonic drug delivery experimental device of the application emits ultrasonic waves during the experiment;

6 is a schematic diagram of the sound radiation force effect of the local sound field formed by the phononic crystal plate of the ultrasonic drug delivery experimental device of the present application to spherical particles according to Gor'kov's theoretical calculation and simulation;

FIG. 7 is a block diagram of the execution steps of the ultrasonic drug delivery experimental method of the present application.

Among them, each reference sign in the figure:

10. Sample carrier; 11. Accommodating cavity; 12. Culture medium solution; 13. Lug; 20. Phononic crystal plate; 21. Base plate; 32, power amplifier; 33, ultrasonic transducer; 40, microbubble.

Embodiments of the present invention

The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention and should not be construed as limiting the present invention.

In the description of the present invention, it should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", The orientations or positional relationships indicated by "horizontal", "top", "bottom", "inside", "outside", etc. are based on the orientations or positional relationships shown in the accompanying drawings, which are only for the convenience of describing the present invention and simplifying the description, rather than An indication or implication that the referred device or element must have a particular orientation, be constructed and operate in a particular orientation, is not to be construed as a limitation of the invention.

In addition, the terms "first", "second", etc. are used for descriptive purposes only, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature defined as "first", "second", etc., may expressly or implicitly include one or more of that feature. In the description of the present invention, "plurality" means two or more, unless otherwise expressly and specifically defined.

In the present invention, unless otherwise expressly specified and limited, the terms "installed", "connected", "connected", "fixed" and other terms should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection , or integrated; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the internal connection of the two elements or the interaction relationship between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

Example 1:

As shown in FIG. 1 , it shows a schematic structural diagram of the ultrasonic drug delivery experimental device according to the first embodiment of the present invention. In the first embodiment, the ultrasonic drug delivery experimental device includes a sample carrier 10, a phononic crystal plate 20, an ultrasonic contrast agent (not shown) and an ultrasonic emission component 30, which are the components of the ultrasonic drug delivery experimental device Parts, through the mutual use of these components, and operating according to the experimental process, so as to carry out the ultrasonic drug administration experiment on the cultured cells. During a specific experimental operation, the sample carrier 10 has an accommodating cavity 11, and the accommodating cavity 11 is filled with a medium solution 12 for culturing experimental cells. The phononic crystal plate 20 is placed in the accommodating cavity 11, and the phononic crystal plate 20 is immersed in the culture medium solution 12, and experimental cells are grown on the phononic crystal plate 20, that is, the culture medium solution 12 immerses the experimental cells to The adherent cells can continue to absorb nutrients to survive and grow. The ultrasonic contrast agent is mixed in the medium solution 12, the ultrasonic contrast agent contains microbubbles 40, and the microbubbles 40 contain gas. Generally, after the ultrasonic contrast agent is mixed in the medium solution 12, most of the microbubbles 40 float in the culture medium. On the surface of the base solution 12 , the distance between these microvesicles 40 and the experimental cells that grow adherently is greater than the effective distance between the microvesicles 40 and the experimental cells. Therefore, the ultrasonic emitting assembly 30 is disposed at the bottom of the sample carrier 10 , and during the ultrasonic drug administration experiment, the ultrasonic emitting assembly 30 is used to emit ultrasonic waves of a predetermined frequency to the phononic crystal plate 20 , so as to reach the phononic crystal plate 20 The predetermined frequency of the ultrasonic wave is close to (ideally equal to) the resonant frequency of the phononic crystal plate 20 itself, so that after the ultrasonic wave of the predetermined frequency reaches the phononic crystal plate 20, the working frequency of the phononic crystal plate 20 is excited and the An ultrasonic field is generated on the surface of the phononic crystal plate 20, and a local sound field is generated on the plate surface of the phononic crystal plate 20. As shown in Figure 4, the arrow in the figure indicates the direction of the ultrasonic radiation force, and the resonance frequency of the local sound field of the phononic crystal plate 20 The resonant frequency less than the microbubble 40 has suction (correspondingly, as shown in FIG. 5, the arrow in the figure represents the direction of the ultrasonic radiation force, and the resonant frequency of the local sound field of the phononic crystal plate 20 is greater than the resonant frequency of the microbubble 40 has a repulsive force. ), so the local sound field attracts most of the microbubbles 40 close to the experimental cells, and then induces the cavitation effect of the microbubbles 40 to rupture, producing high-efficiency sonoporation for the large-scale experimental cells that adhere to the phononic crystal plate 20 Therefore, repairable pores of tens of nanometers to hundreds of nanometers are generated on the surface of the cell membrane of the experimental cells, thereby enhancing the permeability of the cell membrane. Then, biomacromolecules such as drug molecules, DNA, and proteins mixed in the culture medium solution 12 can pass through the pores and enter the experimental cells to play a role.

The ultrasonic drug administration experimental device provided by the present invention is used to conduct experiments on the experimental cells to perform ultrasonic drug administration operations. The pre-cultured experimental cells are placed on the plate surface of the phononic crystal plate 20, and a culture medium solution 12 is provided to make the experimental cells. The phononic crystal plate 20 is adherently grown on the plate surface, and then the phononic crystal plate 20 is placed into the sample holder 10 together with the adherent growth experimental cells and the culture medium solution 12 is immersed, wherein the culture medium solution 12 The ultrasonic contrast agent and the desired drug to be delivered into the experimental cells are mixed in the medium, and then the ultrasonic wave transmitting assembly 30 is operated to generate ultrasonic waves of a predetermined frequency to the phononic crystal plate 20, and the ultrasonic frequency excites the resonance frequency of the phononic crystal plate 20, so that the acoustic The plate surface of the sub-crystal plate 20 generates a local sound field, thereby attracting the microbubbles 40 floating in the culture medium solution 12 to approach and closely contact the experimental cells growing on the wall, and induce the cavitation effect of the microbubbles 40 to achieve acoustic effects on the experimental cells. The perforating effect produces repairable pores of tens of nanometers to hundreds of nanometers on the surface of the cell membrane of the experimental cells, thereby enhancing the permeability of the cell membrane. Macromolecular drugs can pass through the pores and enter the experimental cells to play a role, so as to achieve the experimental purpose of ultrasonic drug delivery. Therefore, during the experimental operation using the ultrasonic drug delivery experimental device, the resonance frequency of the phononic crystal plate 20 is excited by the emission of ultrasonic waves, so that a local sound field (attraction force) is generated on the surface of the phononic crystal plate 20 to capture the mixed solution in the culture medium. The microbubble 40 of the ultrasonic contrast agent in Then, the sonoporation effect between the microbubbles 40 and the experimental cells is induced to enhance the permeability of the cell membrane, so that the drugs mixed in the medium solution 12 can smoothly enter the experimental cells to produce effects, which greatly improves the drug delivery efficiency. .

As shown in FIG. 2 , the phononic crystal plate 20 of the ultrasonic drug delivery experimental device includes a substrate 21 and a plurality of resonant ridges 22 , and all the resonant ridges 22 are distributed on the side of the substrate 21 facing the ultrasonic emitting component 30 . , these resonant ridges 22 have a resonating effect on the ultrasonic waves reaching the phononic crystal plate 20, so that resonance is generated under the excitation of the acoustic wave frequency of the ultrasonic wave, so that on the other side of the phononic crystal plate 20 where the resonant ridges 22 are not arranged A local sound field is generated on the surface of the substrate 21 , and experimental cells are grown on the other side of the substrate 21 on which the resonant ribs 22 are not arranged. In this way, the suction force generated by the local sound field on the microbubbles 40 will attract the microbubbles 40 floating in the medium solution 12 to the experimental cells growing close to the wall, and then rupture under the excitation of ultrasonic waves to form a sonoporation effect. The generated force acts on the cell membrane of the experimental cells, so that the cell membrane produces repairable pores of tens of nanometers to hundreds of nanometers, thereby enhancing the permeability of the cell membrane.

In the phononic crystal plate 20 , the distance between any two adjacent resonance ridges 22 is the same. Specifically, the thickness t of the substrate 21 is (200±20) microns, preferably t=200 microns, and the separation distance p between any two adjacent resonant ridges 22 is (800±80) microns, preferably p= 800 microns. Wherein, each resonant ridge 22 is one of straight, curvilinear, or zigzag shape, and the phononic crystal plate 20 in this embodiment is preferably a straight resonant ridge 22 . Further, the cross section perpendicular to the tangential direction of the position at any position of each resonant convex strip 22 is a rectangular section, and the width w of the rectangular section is (100±10) microns, preferably w=100 microns, the rectangular The height h of the section is (100±10) microns, preferably h=100 microns. Optionally, the cross-section perpendicular to the tangential direction of the position at any position of each resonance ridge 22 can also be a triangle, a polygon or a semicircle. When the cross-sectional shape of the resonance ridge 22 is selected as a triangle, a polygon or a In the case of a semicircle, the size of the cross-section can be prepared with reference to the size of the cross-section with a rectangular cross-section.

When preparing the phononic crystal plate 20, it is necessary to determine the operating frequency of the phononic crystal plate 20 to be prepared, and then perform targeted preparation and molding. According to the required structural geometry and material parameters of the phononic crystal plate 20, theoretically predict the ultrasonic operating frequency of the localized sound field mode generated on the surface of the phononic crystal plate 20. After the sample of the phononic crystal plate 20 is prepared, the experimental test is carried out. , so as to experimentally measure the ultrasonic working frequency of the local sound field mode generated on the surface of the phononic crystal plate 20. During the experimental work, the phononic crystal plate 20 can be placed in water, and the resonance frequency can be obtained by measuring the transmission spectrum.

For a spherical particle with a diameter much smaller than the wavelength of the acoustic wave (that is, the diameter of the microbubble 40 is much smaller than the wavelength of the ultrasonic wave), the acoustic radiation force in the acoustic field is mainly caused by the gradient of the acoustic potential, and the acoustic radiation force can be calculated by the classical Gor'kov's theory, the sound radiation force F _R is given by the following formula:

Among them, u ₁ and p ₁ are the velocity and sound pressure of the incident sound wave in the surrounding fluid medium, respectively; ρ ₀ and c ₀ are the density and sound velocity of spherical particles (ie, microbubbles 40 ) in the sound field, respectively; is the time mean value of the physical quantity in parentheses (for example, <p ₁ ² > is the time mean value of sound pressure),

is the Hamiltonian operator, which represents the gradient of U, and a represents the particle size of the particle. According to this theory, solid particles such as spherical particles (ie, microbubbles 40 ) or experimental cells are attracted in the resonant local sound field of the phononic crystal plate 20 and are trapped at two specific positions within a cycle, as shown in FIG. 6 . The arrows shown point to the A and B positions.

Further, for the microbubble 40, Gor'kov's theory does not consider the volume change of the microbubble 40 in the sound field with time, and the influence of the resonant frequency of the microbubble 40. Therefore, the acoustic radiation force of the microbubble 40 in the local sound field is numerically solved based on the nonlinear dynamic equation of the microbubble, as shown in the following formula:

(1) Microbubble nonlinear dynamic equation

in,

represents the pressure of the gas filled inside the microbubble, R is the instantaneous radius of the microbubble vibration, c is the sound speed of the fluid medium around the microbubble,

is the first derivative of the instantaneous radius of the microbubble vibration,

is the second derivative of the instantaneous radius of the microbubble vibration, R ₀ is the initial particle size of the microbubble, ρ _l is the density of the fluid medium around the microbubble, κ is the polytropic gas index of the gas filled inside the microbubble, P ₀ is the ambient pressure,

is the surface tension of the microbubble surface,

is the initial surface tension of the microbubble surface,

is the force acting on the viscosity of the fluid around the microbubble, μ is the viscosity of the fluid around the microbubble,

is the force acting on the viscosity of the microbubble envelope, κ _s is the swelling viscosity of the microbubble envelope, and P _ac (r,t) is the incident sound pressure.

(2) The acoustic radiation force received by the microbubbles

sound radiation

Instantaneous volume of microbubbles

P _ac (r,t)=P(r)cos[ωt+φ(r)] The sound pressure at the location of the microbubble

It can be seen that in the calculation process of the above equation, it is necessary to calculate the curve R(t) of the radius of the microbubble 40 changing with time, and further calculate the instantaneous volume V(t) of the microbubble 40, and then the acoustic radiation received by the microbubble 40 can be obtained. force F _B , and the acoustic radiation force F _B experienced by the microbubble 40 is only related to the gradient of the sound pressure

related. Based on the above equation, the acoustic radiation force of the vibrating microbubble 40 in the local sound field is calculated and obtained. From the two acoustic radiation forces shown in FIGS. 4 and 5 , it can be seen that the resonance frequency of the microbubble 40 resonates with the phononic crystal plate 20 The relative magnitude of the frequency has a significant effect on the distribution of the acoustic radiation force. That is, when the size of the microbubble 40 is relatively large, causing the resonant frequency of the microbubble 40 to be lower than the resonant frequency of the phononic crystal plate 20, the acoustic radiation force is away from the surface of the phononic crystal plate 20, so the microbubble 40 is subjected to a repulsive force. , as shown in FIG. 5, the microbubbles 40 will not be trapped on the surface of the phononic crystal plate 20; when the size of the microbubbles 40 is small, causing the resonant frequency of the microbubbles 40 to be greater than the resonant frequency of the phononic crystal plate 20, the acoustic The radiation force is directed to the surface of the phononic crystal plate 20 , so the microbubbles 40 are attracted by an attractive force. As shown in FIG. 4 , the microbubbles 40 will be trapped on the surface of the phononic crystal plate 20 . The microbubbles 40 contained in the ultrasonic contrast agent usually have a wide size distribution, and the particle size distribution range is 0.1-10 microns. Therefore, the resonant frequency of the phononic crystal plate 20 is designed to be in the commonly used ultrasonic frequency range of 1-10 MHz, and there are microbubbles 40 . The resonant frequency is larger than the particle size space of the resonant frequency of the phononic crystal plate 20 , so these smaller microbubbles 40 can be trapped on the surface of the phononic crystal plate 20 . These theoretical calculations are in good agreement with our experimental observations. The previous capture results for polystyrene microspheres and cells showed that solid particles were always attracted by the acoustic radiation force and were captured at two specific positions within a structural period, as shown in Figure 6, position A and position B, By comparison, it can be seen that the acoustic radiation force effect of the phononic crystal plate 20 on solid particles such as polystyrene is significantly different from the acoustic radiation force effect on the microbubbles 40 .

As shown in FIG. 1 , the ultrasonic transmitting assembly 30 includes a signal generator 31, a power amplifier 32 and an ultrasonic transducer 33. The signal generator 31 is electrically connected to the power amplifier 32, and the power amplifier 32 is electrically connected to the ultrasonic transducer 33. , the ultrasonic transducer 33 is installed at the bottom of the sample carrier 10. In this embodiment, the ultrasonic transducer 33 can be a single-vibration-element ultrasonic transducer, a phased array ultrasonic transducer, or a linear array ultrasonic transducer One of a convex array ultrasonic transducer and an interdigital transducer, the ultrasonic wave emitted by the ultrasonic transducer 33 used in the present invention has a relatively low entering power, which can greatly improve the survival rate and activity of the experimental cells. Wherein, the ultrasonic transducer 33 transmits ultrasonic waves of a predetermined frequency to the phononic crystal plate 20, and in fact, the resonant frequency of the phononic crystal plate 20 determines the driving frequency of transmitting ultrasonic waves. Specifically, the ultrasonic transducer 33 of this embodiment adopts a single-vibration-element ultrasonic transducer, the ultrasonic probe of the ultrasonic transducer 33 is attached to the bottom of the sample carrier 10, and the ultrasonic probe of the ultrasonic transducer 33 is connected to the bottom of the sample carrier 10. The medium solution 12 is isolated by the bottom of the sample carrier 10 . During the experiment, after the specific signal generated by the signal generator 31 is amplified by the power amplifier 32 , the ultrasonic transducer 33 is excited to emit ultrasonic waves to the phononic crystal plate 20 .

When the signal generator 31 is set to generate a specific signal, it can be set that what the signal generator 31 generates is a continuous sinusoidal signal or a pulse sinusoidal signal. In this embodiment, the signal generator 31 may be a programmable signal generator (AFG3021, Tektronix), and correspondingly, the power amplifier 32 may be a 50 dB linear power amplifier (325LA, ENI). Preferably, during the experiment, the signal generator 31 generates a sinusoidal signal, and the sinusoidal signal passes through the power amplifier 32 to excite the ultrasonic transducer 33 to generate ultrasonic waves.

In addition, the ultrasonic transmitting assembly 30 can also use the Sonovitro FUAUTO, an automatic ultrasonic transfection instrument of Shengxiang Hi-Tech, which integrates an ultrasonic transducer 33, a signal generator 31, a power amplifier 32 and a three-dimensional displacement stage, to transmit ultrasonic waves. At this time, the setting parameters are sound intensity 1W/cm ² , duty cycle 20%, and action time 3 minutes.

The ultrasonic contrast agent has a microbubble 40 composed of a gas-encapsulated shell, and the shell of the microbubble 40 is a PLGA polymer material (PLGA, poly lactic-co-glycolic acid, polylactic-co-glycolic acid copolymer, which is composed of two monomers - Lactic acid and glycolic acid are randomly polymerized. It is a degradable functional polymer organic compound with good biocompatibility, non-toxicity, good encapsulation and film-forming properties), phospholipids, and albumin materials. The gas of the microbubble 40 is one of air, sulfur hexafluoride and perfluoropropane.

As shown in FIG. 3 , it shows a schematic structural diagram of the ultrasonic drug delivery experimental device according to the second embodiment of the present invention. Compared with the first embodiment, the ultrasonic drug delivery experimental device of the second embodiment has the following differences.

The ultrasonic probe of the ultrasonic transducer 33 extends into the accommodating cavity 11 through the bottom of the sample carrier 10 . During assembly, it is necessary to align the side wall of the ultrasonic probe of the ultrasonic transducer 33 and the edge of the through hole at the bottom of the sample carrier 10 . Sealing is performed to ensure the sealing effect to prevent leakage of the culture medium solution 12, and the ultrasonic probe of the ultrasonic transducer 33 is spaced from the phononic crystal plate 20, so the inner wall of the sample carrier 10 is provided with lugs 13 for receiving the sample The phononic crystal plate 20 can be suspended in the accommodating cavity 11 of the sample holder 10 when the surrounding edges of the carrier 10 are placed. At this time, when ultrasonic waves are emitted, the culture medium solution 12 will act as a propagation medium to propagate the ultrasonic waves to the phononic crystal plate 20 .

Compared with the ultrasonic drug delivery experimental device of the second embodiment, except for the above structures, the rest of the structures are the same, and thus will not be repeated here.

As shown in FIG. 7 , the present invention also provides an ultrasonic drug administration experimental method using the ultrasonic drug administration experimental device provided above. Specifically, the ultrasonic drug administration experimental method includes the following experimental operation steps of the main part of the experiment:

Step S10: containing the culture medium solution 12 in the sample carrier 10, and mixing the ultrasonic contrast agent and the experimental drug into the culture medium solution 12;

Step S20: placing the phononic crystal plate 20 with the experimental cells attached to the plate surface in the sample holder 10, and immersing the phononic crystal plate 20 with the culture medium solution 12;

Step S30: transmitting ultrasonic waves of a predetermined frequency to the phononic crystal plate 20, and the ultrasonic waves act for a predetermined action time;

Step S40: Continue to incubate the experimental cells for a predetermined period of time.

After performing step S40, use a microscope to observe the experimental cells after the experiment is completed. First, it is necessary to observe and determine whether the effect is successful, then record and analyze the experimental data, and finally summarize the experiment as a whole. If it is judged that the experimental effect is a failure, for example, the experimental cells are completely or basically inactivated, or the biological activity performance characteristics of the experimental cells are completely inconsistent with the expected results of the experiment, etc., the experiment can be determined to fail, and the overall operation process of the experiment needs to be reviewed and summarized, and Record and organize the experimental parameter settings during the experimental operation, then re-plan and adjust the experimental parameters and re-run the experiment. If it is judged that the experimental effect is successful, the parameter settings in the experimental process should be sorted and determined, and the experimental effect data such as the biological activity performance characteristics of the experimental cells that have been successfully tested should be carefully observed, and a written experimental report will be formed by summarizing the results.

Before performing the operation step 10, the ultrasonic drug administration experimental method further includes the preparatory step 01 before performing the experimental operation of the main part of the experiment: that is, placing the experimental cells on the plate surface of the phononic crystal plate 20 and making the experimental cells in the phononic crystal plate 20. Before the adherent growth is performed on the plate surface of the phononic crystal plate 20, the experimental cells need to be cultivated. In the process of this experiment, C6 cell glioma cells are used to culture in a culture flask until passage. Then, put the phononic crystal plate 20 into the sterilization box for 20 minutes at high temperature and autoclave, and then use sterile tweezers to put the phononic crystal plate 20 into a petri dish of a 6-well plate or a 24-well plate with 3* The phononic crystal plate 20 in the petri dish was seeded at a density of 10 ⁴ /well, and the experimental cells were grown by plating overnight.

During the experimental operation, the phononic crystal plate 20 on which the experimental cells are grown adherently is placed into the accommodation chamber 11 of the sample carrier 10 and immersed in the culture medium solution 12 , and the culture medium solution 12 is injected with microbubbles 40 containing microbubbles 40 . The ultrasonic contrast agent solution and the injection of 12 μg of plasmid DNA make the ultrasonic contrast agent solution and the plasmid DNA evenly mixed in the medium solution 12 as much as possible, and then use the ultrasonic wave transmitting assembly 30 to transmit ultrasonic waves to the phononic crystal plate 20 for about 3 minutes, Thus, the ultrasonic field at the resonance frequency of the working frequency of the phononic crystal plate 20 is excited, that is, a local sound field is generated on the plate surface of the phononic crystal plate 20 to generate an acoustic attraction force to capture the microbubbles 40 to the plate surface of the phononic crystal plate 20, so that The microbubble 40 is in close contact with the experimental cells, and induces a cavitation effect to achieve a sonoporation effect, thereby producing repairable pores of tens of nanometers to hundreds of nanometers on the surface of the cell membrane of the experimental cells, thereby enhancing the permeability of the cell membrane sex. Then, the plasmid DNA biomacromolecule drug mixed in the medium solution 12 can pass through the pores and enter into the experimental cells to perform gene transfection. After transfection, put the experimental cells into the cell culture incubator and continue to incubate the experimental cells for 4-5 hours under suitable conditions, that is, perform step S41: after performing step S30, maintain the original medium solution 12 to continue the experiment. The cells are incubated for a first predetermined period of time, wherein the value range of the first predetermined period of time is 4-5 hours. After incubation, the transfection buffer is discarded, that is, the culture medium solution in the accommodating chamber 11 of the sample carrier 10 is cleaned up, and replaced with a new complete culture medium solution 12, and the culture is continued for 24 hours or the culture time is slightly longer than 24 hours, that is, the steps are performed. S42: After reaching the first predetermined period of time, replace the original medium solution 12 in the sample carrier 10 with a brand new complete medium solution 12 to continue culturing the experimental cells for a second predetermined period of time, wherein the value of the second predetermined period of time The range is greater than or equal to 24 hours. Then, observe EGFPEGFP, Enhanced Green Fluorescent Protein under a fluorescence microscope, that is, enhance the expression of green fluorescent protein, and record relevant experimental data to complete the experimental results.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (15)

1. A kind of ultrasonic drug delivery experimental device, is characterized in that, comprises:

   a sample carrier (10), the sample carrier (10) has an accommodating cavity (11), and the accommodating cavity (11) is filled with a culture medium solution (12) for culturing experimental cells;

   a phononic crystal plate (20), the phononic crystal plate (20) is placed in the accommodating cavity (11), and the phononic crystal plate (20) is immersed in the culture medium solution (12), The experimental cells are adherently grown on the phononic crystal plate (20);

   an ultrasound contrast agent, which is mixed in the culture medium solution (12);

   An ultrasonic emitting assembly (30), the ultrasonic emitting assembly (30) is disposed at the bottom of the sample carrier (10), and the ultrasonic emitting assembly (30) is used for emitting predetermined signals to the phononic crystal plate (20) frequency of ultrasound.
2. The ultrasonic drug delivery experimental device according to claim 1, wherein,

   The phononic crystal plate (20) includes a substrate (21) and a plurality of resonant ridges (22), all of the resonant ridges (22) are distributed on the substrate (21) toward the ultrasonic emitting component (30) ) on one side of the board, and on the other side of the board (21) facing away from the resonance protruding strip (22), the experimental cells are adhered to the wall.
3. The ultrasonic drug delivery experimental device according to claim 2, wherein,

   The distance between any two adjacent resonance convex strips (22) is the same.
4. The ultrasonic drug delivery experimental device according to claim 3, wherein,

   The thickness t of the substrate (21) is (200±20) microns, and the separation distance p between any two adjacent resonant convex strips (22) is (800±80) microns.
5. The ultrasonic drug delivery experimental device according to claim 4, wherein,

   Each of the resonance protruding strips (22) is one of a straight strip, a curve or a broken line.
6. The ultrasonic drug delivery experimental device according to claim 5, wherein,

   The cross section perpendicular to the tangential direction of the position at any position of each of the resonant protruding strips (22) is a rectangular section, the width w of the rectangular section is (100±10) microns, and the height h of the rectangular section is (100 ±10) microns.
7. The ultrasonic drug delivery experimental device according to any one of claims 1 to 6, wherein,

   The ultrasonic transmitting assembly (30) comprises a signal generator (31), a power amplifier (32) and an ultrasonic transducer (33), the signal generator (31) is electrically connected with the power amplifier (32), The power amplifier (32) is electrically connected to the ultrasonic transducer (33), the ultrasonic transducer (33) is installed at the bottom of the sample carrier (10), and the ultrasonic transducer ( 33) Sending ultrasonic waves of a predetermined frequency to the phononic crystal plate (20).
8. The ultrasonic drug delivery experimental device according to claim 7, wherein,

   The ultrasonic probe of the ultrasonic transducer (33) extends into the accommodating cavity (11) through the bottom of the sample carrier (10), and the ultrasonic probe of the ultrasonic transducer (33) is connected to the The phononic crystal plates (20) are arranged at intervals.
9. The ultrasonic drug delivery experimental device according to claim 7, wherein,

   The ultrasonic probe of the ultrasonic transducer (33) is abutted against the bottom of the sample carrier (10), and the ultrasonic probe of the ultrasonic transducer (33) and the culture medium solution (12) are held together. The bottom of the sample carrier (10) is isolated.
10. The ultrasonic drug delivery experimental device according to claim 7, wherein,

    The ultrasound contrast agent has microbubbles (40) formed by encapsulating gas in a shell, and the shells of the microbubbles (40) are composed of one of PLGA polymer materials, phospholipids and albumin materials, and the microbubbles (40) The gas is one of air, sulfur hexafluoride and perfluoropropane.
11. An ultrasonic drug delivery experimental method, characterized in that, comprising the following experimental operation steps:

    Step S10: containing a culture medium solution (12) in the sample carrier (10), and mixing an ultrasound contrast agent and an experimental drug into the culture medium solution (12);

    Step S20: placing the phononic crystal plate (20) on which the experimental cells are grown adherently on the plate surface in the sample carrier (10), and making the culture medium solution (12) immerse the phononic crystal plate (20) );

    Step S30: transmitting an ultrasonic wave of a predetermined frequency to the phononic crystal plate (20), and the ultrasonic wave acts for a predetermined action time;

    Step S40: Continue to incubate the experimental cells for a predetermined period of time.
12. Ultrasonic drug administration experimental method according to claim 11, is characterized in that,

    Before performing operation step 10, the ultrasonic drug administration experimental method further includes step 01: cultivating the experimental cells, and then transferring the cultivated experimental cells that meet the experimental use to the phononic crystal plate (20) for continued cultivation In order to make the experimental cells adhere to the plate surface of the phononic crystal plate (20).
13. Ultrasonic drug delivery experimental method according to claim 12, is characterized in that,

    The phononic crystal plate (20) is sterilized and sterilized before the cultured experimental cells conforming to the experimental use are transferred to the phononic crystal plate (20).
14. Ultrasonic drug administration experimental method according to claim 11, is characterized in that,

    In the process of executing the step S40, the step S40 includes the following sub-steps:

    Step S41: after performing the step S30, maintaining the original medium solution (12) to continue incubating the experimental cells for a first predetermined period of time;

    Step S42: after the first predetermined time period is reached, replace the original culture medium solution (12) in the sample carrier (10) with a brand new complete culture medium solution (12) and continue to test all the samples. The experimental cells are cultured for a second predetermined period of time.
15. Ultrasonic drug delivery experimental method according to claim 14, is characterized in that,

    In the process of executing the step S40, the value range of the first predetermined duration is 4-5 hours, and the value range of the second predetermined duration is greater than or equal to 24 hours.

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