阅读说明：本技术 用于声驻波方法中的颗粒 (Particles for use in acoustic standing wave methods ) 是由 B·利普肯斯 K·库马尔 R·托斯通伊斯 T·J·肯尼迪 于 2018-12-03 设计创作，主要内容包括：公开了以各种配置使用的由各种材料制成的微粒和纳米颗粒。此类颗粒还可以含有各种类型的材料作为有效载荷,以用于系统或宿主解剖结构的分开、隔离、分化、修饰或过滤。微粒和纳米颗粒在各种过程中与声驻波或声行波结合利用。(Microparticles and nanoparticles made of various materials used in various configurations are disclosed. Such particles may also contain various types of materials as payloads for separation, isolation, differentiation, modification, or filtration of systemic or host anatomy. Microparticles and nanoparticles are utilized in conjunction with standing or traveling acoustic waves in a variety of processes.)

1. A method for concentrating particles in a primary fluid at a first location, comprising:

flowing a fluid mixture comprising the particulates and the primary fluid through an acoustophoresis device, the acoustophoresis device comprising:

an acoustic chamber through which the fluid mixture flows; and

an ultrasonic transducer comprising a piezoelectric material, the ultrasonic transducer being drivable to generate acoustic waves in an acoustic chamber; and is

Driving the ultrasonic transducer to generate sound waves;

wherein the acoustic wave concentrates particles at the first location.

2. The method of claim 1, wherein the acoustic wave is a multi-dimensional acoustic standing wave, a planar acoustic standing wave, a combination of a multi-dimensional acoustic standing wave and a planar acoustic standing wave, or an acoustic traveling wave.

3. The method of claim 1, wherein the particles contain a payload.

4. The method of claim 3, wherein the payload is a virus, a nucleic acid, a cytokine, a drug molecule, a liquid, a gas, or a mixture thereof.

5. The method of claim 3, further comprising releasing the payload from the particle at the first location.

6. The method of claim 1, wherein the particles are solid, porous, hollow, or foam.

7. The method of claim 1, wherein the particles are made of one or more polymeric materials, ionomers, ceramics, or glasses.

8. The method of claim 7, wherein the one or more polymeric materials are selected from the group consisting of: polyethylene, polypropylene, polystyrene, divinylbenzene, polymethylmethacrylate, polysaccharide, polylactic acid (PLA) and poly (lactic-co-glycolic acid) (PLGA).

9. The method of claim 1, wherein the particles are formed from multiple layers of polymeric material.

10. The method of claim 1, wherein the particles are hollow and made of glass and have an ablative polymer coating the outer surface of the glass.

11. The method of claim 10, wherein the ablative polymer is a polysaccharide functionalized with an antigen, an antibody, or a protein.

12. The method of claim 1, wherein the particles comprise:

a liquid core; and

a lipid shell encapsulating the liquid core.

13. The method of claim 12, wherein the liquid in the liquid core comprises a perfluorocarbon.

14. The method of claim 13, wherein the perfluorocarbon is perfluoropentane, perfluorohexane, perfluorooctane, perfluorobromooctane, perfluorodichlorooctane, or perfluorodecalin.

15. The method of claim 12, wherein the lipid shell is formed from Dipalmitoylphosphatidylcholine (DPPC), 1, 2-palmitoylphosphatidic acid (DPPA), a lipid-polyethylene glycol conjugate, or a complex of a lipid and albumin.

16. The method of claim 12, wherein the lipid shell is functionalized with streptavidin, biotin, avidin, or an antibody.

17. A particle, comprising:

a liquid core; and

a lipid shell encapsulating the liquid core.

18. The particle of claim 17, wherein the liquid in the liquid core comprises a perfluorocarbon.

19. The particle of claim 18, wherein the perfluorocarbon is perfluoropentane, perfluorohexane, perfluorooctane, perfluorobromooctane, perfluorodichlorooctane, or perfluorodecalin.

20. The particle of claim 17, wherein the lipid shell is formed from Dipalmitoylphosphatidylcholine (DPPC), 1, 2-palmitoylphosphatidic acid (DPPA), a lipid-polyethylene glycol conjugate, or a complex of a lipid and albumin.

21. The particle of claim 17, wherein the lipid shell is functionalized with streptavidin, biotin, avidin, or an antibody.

22. A method for separating target particles from a fluid, comprising:

receiving functionalized particles in a fluid in a chamber;

receiving target particles in a chamber;

binding the target particle to the functionalized particle;

applying sound waves to the chamber to affect the functionalized particles to be collected or blocked by the sound waves.

23. The method of claim 22, wherein the functionalized particles comprise a perfluorocarbon, the perfluorocarbon being one or more of perfluoropentane, perfluorohexane, perfluorooctane, perfluorobromooctane, perfluorodichlorooctane, or perfluorodecalin.

Background

The present disclosure relates to particles in the micro-or nano-scale that can be used with ultrasound generated acoustic waves (including traveling and standing waves) to achieve capture, concentration and/or transport of microparticles and nanoparticles to target locations.

Acoustophoresis is the separation of materials using acoustics such as acoustic standing waves. The acoustic standing wave may exert a force on the particles in the fluid when there is a difference in parameters of the particles and the fluid, which parameters may be influenced acoustically, including density and/or compressibility, also referred to as acoustic contrast factor. The pressure distribution in the standing wave contains regions of local minimum pressure amplitude at nodes of the standing wave and regions of local maxima at antinodes of the standing wave. Depending on its density and compressibility, the particles may be trapped at nodes or antinodes of the standing wave. Generally, the higher the standing wave frequency, the smaller the particles that can be captured.

On a microscopic scale, for example for structural dimensions on the order of micrometers, conventional acoustophoresis systems tend to use half-wavelength or quarter-wavelength acoustic chambers, which are typically less than one millimeter thick at frequencies of a few megahertz, and operate at very low flow rates (e.g., μ L/min). Such systems are not scalable as they benefit from extremely low reynolds numbers, laminar flow operation and minimal fluid dynamics optimization.

On a macroscopic scale, planar acoustic standing waves have been used in separation processes. However, a single plane wave tends to trap particles or secondary fluid, such that separation from the primary fluid is achieved by turning off or removing the plane standing wave. Plane waves also tend to heat the medium in which the waves propagate, due to energy dissipation into the fluid involved in generating the plane waves and the plane wave energy itself. Removal of the planar standing wave may prevent continuous operation. In addition, the amount of power used to generate the acoustic plane standing wave tends to heat the primary fluid through waste energy, which may be detrimental to the material being processed.

Disclosure of Invention

In various embodiments, disclosed herein are methods for moving particles within a bulk fluid or a primary fluid to a desired location using an acoustic standing wave. Particles are placed within the acoustophoretic device and an ultrasonic transducer is used to focus, capture, and/or move the particles as desired. The particles may also be used to interact or react with other particles or cells in the host fluid or primary fluid. Sometimes, the structure of the particles may change upon exposure to sound waves.

Disclosed herein, in various embodiments, are methods for concentrating particles in a primary fluid at a first location, comprising: a fluid mixture comprising particles and a primary fluid is flowed through an acoustophoresis device. The acoustophoresis device includes: an acoustic chamber through which the fluid mixture flows; and an ultrasonic transducer comprising a piezoelectric material, the ultrasonic transducer being drivable to generate acoustic waves in the acoustic chamber. The ultrasonic transducer is driven to generate acoustic waves, thus concentrating particles at nodes (nodes) and antinodes (antinodes) of the standing wave, with a negative contrast factor to the antinodes and positive contrast factor material accumulating at the nodes.

The acoustic wave may be a multi-dimensional acoustic standing wave, a planar acoustic standing wave, a combination of a multi-dimensional acoustic standing wave and a planar acoustic standing wave, or an acoustic traveling wave.

In some embodiments, the particle contains a payload (payload). The payload can be a virus, a nucleic acid, a cytokine, a drug molecule, a liquid, a gas, or a mixture thereof. After moving the particles to the first position, the payload may be released.

The particles may be microparticles or nanoparticles. The particles may be solid, porous, hollow, multi-layered, or foamed.

The particles may be made of one or more polymeric materials, ionomers, ceramics or glasses. Examples of polymeric materials include polyethylene, polypropylene, polystyrene, divinylbenzene, polymethylmethacrylate, polysaccharide, polylactic acid (PLA), and poly (lactic-co-glycolic acid) (PLGA).

Particles can also be produced from agarose and poly-hyaluronic acid. These particles will dissolve in the body and therefore do not pose a harmful problem to the patient.

The particles may be formed from multiple layers of polymeric material. In some embodiments, the particles may be hollow, made of glass, and/or have an ablative polymer coating the outer surface of the glass. The ablative polymer may be a polysaccharide functionalized with an antigen, antibody, or protein.

In other embodiments, the particles comprise: a liquid core; and a lipid shell encapsulating the liquid core. The liquid in the liquid core may comprise a perfluorocarbon. The perfluorocarbon may be perfluoropentane, perfluorohexane, perfluorooctane, perfluorobromooctane, perfluorodichlorooctane or perfluorodecalin.

The lipid shell may be formed from Dipalmitoylphosphatidylcholine (DPPC), 1, 2-palmitoylphosphatidic acid (DPPA), lipid-polyethylene glycol conjugates, or complexes of lipids and albumin. The lipid shell may be functionalized with streptavidin, biotin, avidin, or an antibody.

Also disclosed herein are particles comprising: a liquid core; and a lipid shell encapsulating the liquid core.

The liquid in the liquid core may comprise a perfluorocarbon. The perfluorocarbon may be perfluoropentane, perfluorohexane, perfluorooctane, perfluorobromooctane, perfluorodichlorooctane or perfluorodecalin. The lipid shell may be formed from Dipalmitoylphosphatidylcholine (DPPC), 1, 2-palmitoylphosphatidic acid (DPPA), lipid-polyethylene glycol conjugates, or complexes of lipids and albumin. The lipid shell may be functionalized with streptavidin, biotin, avidin, or an antibody.

A method known as acoustic droplet evaporation (ADV) may use sound waves for generating a phase shift of the liquid core of such particles from liquid to gas. The vapor pressure of a liquid is a function of temperature and is not necessarily based on liquid chemistry. Any liquid having a normal boiling point near or below body temperature may be used for these methods. Fluorocarbons can be used in these processes due to their low toxicity and high contrast factor.

Spacers may be placed between the particles and the antigen, antibody or protein. The spacers are typically polyethylene glycol (PEG) molecules that allow for less charged interference from the particles when the material is bound to functionalized molecules on the surface of the particles.

These materials may also be used for transduction of cells, for example by sonoporation. The bubbles are acoustically cavitated near the cell wall and produce oscillations that facilitate opening channels in the cell wall. Collapsing the bubbles via acoustically induced cavitation can produce a jet of fluid that facilitates opening the cell walls.

In another configuration, the gas bubbles can contain a therapeutic agent. Thus, when the bubble is broken via acoustic excitation, the ejected material is a therapeutic agent and enters the cell during this process. The therapeutic agent may be a small molecule, a large molecule or a piece of genetic material used to modify the DNA of the target cell.

More generally, the particles described herein may be used as an agent that causes a change in the second material when the particles are struck by sound waves. For example, the particles may be used to increase the contrast factor of the second material, which is a factor in increasing the efficiency of acoustophoresis. As another example, the liquid may be delivered through a particle to cause a change in the cell barrier in an operation such as sonoporation.

Techniques and apparatus for generating clusters of material that can be used to improve gravity or buoyancy separation and collection efficiency of the material are also discussed herein. Improved, continuous acoustophoretic devices using improved fluid dynamics, and device control with respect to desired performance, are also discussed. Depending on various parameters and characteristics of the acoustic wave and/or the material, including, for example, the contrast factor of the material, the material may be preferentially trapped in or released from/by the acoustic wave.

These and other non-limiting features are described in more detail below.

Drawings

The following is a brief description of the drawings, which are presented for the purpose of illustrating the exemplary embodiments disclosed herein and not for the purpose of limiting the same.

Fig. 1 is a photomicrograph of a particle according to the present disclosure.

Fig. 2A is a Scanning Electron Microscope (SEM) photograph of solid particles.

Fig. 2B is an SEM photograph of the cell particles.

Fig. 2C is a micrograph of the hollow particles.

Fig. 2D is an illustration of a particle having a solid core and an outer layer.

Fig. 2E is an illustration of a hollow particle having material within a core, and an outer layer that can be ablated to release the material within the core.

Fig. 2F is an illustration of a hollow particle having a payload and a shell surrounding the payload.

Fig. 3 is a schematic of a particle comprising a liquid core and a lipid shell.

Figure 4 is a schematic of several particles aligned/grouped with each other.

FIG. 5A is a graph showing the relationship between the initial droplet and the dropletPlot of particle number versus particle diameter for post-incubation droplets for diameters of 0.6 microns to 1.25 microns. The y-axis is linear and is at 1.0x10⁸Extend from 0 to 3.0x10⁸. The x-axis is in microns and extends from 0.6 to 1.2 at 0.2 intervals.

FIG. 5B is a graph showing the relationship between the initial droplet and the dropletThe number of particles relative to the diameter of the particles for a diameter of 1.25 to 2.25 microns of the droplet after incubationFigure (a). The y-axis is linear and is at 2.0x10⁶Extend from 0 to 8.0x10⁶. The x-axis is in microns and extends from 1.4 to 2.2 at 0.2 intervals.

Fig. 5B is a graph illustrating a droplet size distribution according to the present disclosure.

Fig. 6 illustrates a method for preparing a particle containing a payload and subsequent release of the payload in accordance with the present disclosure.

Fig. 7 is a depiction of a traveling wave in accordance with the present disclosure.

Fig. 8 is a depiction of a standing wave according to the present disclosure.

FIG. 9 is an elevational cross-section of an acoustophoretic device in which the methods of the present disclosure can be used.

Fig. 10 is an external perspective view of the acoustophoresis device of fig. 9.

Fig. 11 is a cross-sectional illustration of an ultrasound transducer of the present disclosure. There is an air gap within the transducer and no backing layer or wear plate is present.

Fig. 12 is a cross-sectional illustration of another ultrasound transducer suitable for use in the present disclosure. There is an air gap within the transducer and there is a backing layer and wear plate.

Detailed description of the preferred embodiments

The present disclosure may be understood more readily by reference to the following detailed description of the desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure selected for the embodiments shown in the drawings, and are not intended to define or limit the scope of the present disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used in the specification and claims, the term "comprising" may include embodiments "consisting of … … and" consisting essentially of … …. As used herein, the terms "comprising," "including," "having," "can," "containing," and variations thereof, are intended to be open-ended transition phrases, terms, or words that specify the presence of the stated ingredients/components/steps, and allow for the presence of other ingredients/components/steps. However, such description should be construed as also describing the compositions, articles or methods as "consisting of and" consisting essentially of the enumerated ingredients/components/steps, "which only allows for the presence of the named ingredients/components/steps along with any impurities that may result therefrom, and excludes other ingredients/components/steps.

Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement techniques for the type of measurement values described in this application.

All ranges disclosed herein are inclusive of the recited endpoints and independently combinable (e.g., a range of "2 grams to 10 grams" is inclusive of the endpoints 2 grams and 10 grams and all intermediate values).

The term "about" can be used to include any numerical value that can be varied without changing the basic function of the value. When used with a range, "about" also discloses the range defined by the absolute values of the two endpoints, e.g., "about 2 to about 4" also discloses the range "2 to 4". The term "about" may refer to plus or minus 10% of the indicated number.

A statement that a value exceeds (or exceeds) a first threshold is equivalent to a statement that the value meets or exceeds a second threshold that is slightly greater than the first threshold, e.g., the second threshold is a value that is higher than the first threshold in the resolution of the associated system. A statement that a value is less than (or within) a first threshold is equivalent to a statement that the value is less than or equal to a second threshold that is slightly lower than the first threshold, e.g., the second threshold is a value that is lower than the first threshold in the resolution of the associated system.

It should be noted that many of the terms used herein are relative terms. For example, the terms "upper" and "lower" are positionally relative to one another, e.g., the upper component is positioned at a higher elevation than the lower component in a given orientation, but these terms may change if the device is flipped. The terms "inlet" and "outlet" relate to a fluid flowing therethrough with respect to a given structure, e.g., a fluid flows into a structure through an inlet and exits the structure through an outlet. The terms "upstream" and "downstream" are relative to the direction in which fluid flows through various components, e.g., fluid flows through an upstream component before flowing through a downstream component. It should be noted that in a ring, a first component may be described as being upstream and downstream of a second component.

The terms "horizontal" and "vertical" are used to indicate directions relative to an absolute reference, such as ground plane. However, these terms should not be construed as requiring structures to be absolutely parallel or absolutely perpendicular to each other. For example, the first and second vertical structures need not be parallel to each other. The terms "top" and "bottom" or "base" are used to refer to surfaces in which the top is always higher than the bottom/base relative to an absolute reference, such as the earth's surface. The terms "upward" and "downward" are also relative to absolute reference; the upward flow is always against the earth's gravity.

The present application relates to "same order of magnitude". If the quotient of the larger number divided by the smaller number is a value of at least 1 and less than 10, the two numbers are of the same order of magnitude.

The term "virus" refers to an infectious agent that can only replicate inside another living cell, otherwise in the form of a virion formed from a capsid surrounding and containing DNA or RNA, and in some cases a lipid envelope surrounding the capsid.

The term "crystal" refers to a single crystal material or a polycrystalline material used as a piezoelectric material.

The present disclosure relates to "microparticles. The term refers to particles having an average particle size of 1 micrometer (μm) to 1000 μm.

The present disclosure relates to "nanoparticles. The term refers to particles having an average particle size of 1 nanometer (nm) to less than 1000 nm.

Some of the materials discussed herein are described as having an average particle size. The average particle size is defined as the particle size at which a cumulative percentage of 50% (by volume) of the total number of particles is reached. In other words, 50% of the particles have a diameter above the average particle size, while 50% have a diameter below the average particle size. The particle size distribution of the particles may comprise a gaussian distribution with upper and lower quartiles at 25% and 75% of the average particle size and all particles being less than 150% of the average particle size. Any other type of distribution may be provided or used. Note that the particles need not be spherical. For non-spherical particles, the particle size is the diameter of a spherical particle having the same volume as the non-spherical particle.

Particles may be described herein as having a "core" and "shell" structure. In such particles, the core is made of a liquid or gas and the shell is made of one or more layers of a relatively strong material (relative to the core). The shell and the core may be distinguished by their material phases. The term "particle" means any type of individual structure that can be suspended in a fluid, such as a liquid or a gas, and can be in any phase, such as a solid, liquid, or gas, and combinations thereof.

This document relates to "organic" and "inorganic" materials. For purposes of this disclosure, "organic" materials are composed of carbon atoms (often along with other atoms), while "inorganic" materials do not contain carbon atoms.

The present disclosure may relate to the temperature of certain method steps. In the present disclosure, temperature generally refers to the temperature reached by the material in question, and not the temperature below which the heat source (e.g., furnace, oven) is set. The term "room temperature" refers to the range from 68 ℃ F. (20 ℃) to 77 ℃ F. (25 ℃).

The present disclosure relates to particles for use in conjunction with an acoustophoresis device. Acoustophoresis devices generate sound waves that can be used in a variety of ways. For example, acoustic waves may be used to move particles to a desired location, or to alter certain characteristics of particles, or to enhance the reaction of particles with other particles (e.g., biological cells). The particles may be microparticles or nanoparticles, as desired. The particles will be discussed first, followed by the acoustophoresis device itself. Various methods and reactions that can be performed using the particles in conjunction with an acoustophoresis device are also discussed.

Granules

As discussed above, the particles are generally microparticles or nanoparticles. The particles may be spherical in shape, as shown by reference numeral 100 in fig. 1. However, their shape may vary. For example, the particles may be elliptical or elongated along a longitudinal axis.

The particles may be, for example, solid, porous, hollow or foam. The solid particles do not contain any voids or cavities and the solid particles 200 are shown in fig. 2A. Porous particles contain voids/cavities in their interior and have passages from the exterior of the particle to those voids/cavities (similar to open-cell foams). In fig. 2B, a porous particle 204 is shown, with voids/cavities 206 visible from the outside. The hollow particles are shown in fig. 2C. The hollow particles 210 have one or more large voids or cavities 212 within the solid outer surface 214. The foam contains multiple voids/cavities, each of which is completely surrounded by solid material (also known as closed cell foam).

In particular embodiments, the particles may be made of inorganic materials, organic materials, or combinations thereof. Such materials may include polymers, ionomers, ceramics, glass, and other materials.

Polymers that may be used to make the particles discussed herein include polyolefins, such as polyethylene and polypropylene. The polyethylene may be linear low density polyethylene, high density polyethylene, low density polyethylene or ultra high molecular weight polyethylene. The polyethylene or polypropylene material may be polymerized with a catalyst such as a peroxide catalyst, a ziegler-natta catalyst or a metallocene catalyst.

Other polymers that can be used to make the particles include polystyrene, divinylbenzene, Polymethylmethacrylate (PMMA), polysaccharides such as agarose and agar, polylactic acid (PLA) and poly (lactic-co-glycolic acid) (PLGA).

These polymers can be used to form a majority of particles, microparticles or nanoparticles. The polymers may also be utilized in various combinations to produce particles from multiple layers (e.g., multilayer particles). Different polymers may be used to obtain the desired effect on the particles. For example, preparing particles from multiple different layers may be used to obtain a desired density and a desired acoustic contrast factor, or to obtain a desired behavior or interaction for the particles.

As an example, polystyrene beads may be produced in an aqueous suspension and then freeze-dried to obtain foam particles. When freeze-dried foam particles are suspended in water, small bubbles may form on the surface thereof, resulting in foam particles having a relatively strong core, and nanobubbles trapped in cavities on the surface of the foam particles.

As another example, the polymethylmethacrylate core may be coated with a PLA or PLGA polymer that forms the outer surface for specialized drug delivery or interaction with biological cells. The resulting particles may be considered solid particles or foam particles (depending on the configuration of the polymethylmethacrylate core) and may have a negative or positive contrast factor depending on the density of the composite particles and the speed of sound in the composite particles. This example is shown in fig. 2D. The particles 220 have a PMMA core 222 with a PLA or PLGA coating 224.

As another example, the outer layer of the particle may be used to cause a biological interaction/reaction of the particle. For example, the outer layer may allow the particles to be used for affinity binding. As another example, the particles may be hollow particles having an outer layer made of an ablative material (e.g., a melted or dissolved material). This configuration allows the material held in the core of the hollow particle to be released over a period of time or after exposure to sufficient heat or other energy, which allows the particle to travel to a desired target or location. This example is shown in fig. 2E. The particle 230 has a core 232 with an outer layer 234 of ablative material. Material 236 is present within the core.

In some embodiments, the acoustic contrast factor of the particles may be varied. For example, hollow glass particles may be coated with an ablative polymer, such as a polysaccharide functionalized with an antigen or antibody or other protein or biological moiety. The particles may begin the process with a first acoustic contrast factor and then change to a second acoustic contrast factor by removing the ablative polymer.

In some embodiments, the particles of the present disclosure have a positive acoustic contrast factor. Such particles may be trapped at nodes of the acoustic standing wave. In other embodiments, the particles of the present disclosure have a negative acoustic contrast factor. These particles are trapped at the antinodes of the acoustic standing wave. If the acoustic contrast factor of the particle changes while in the processing system or in vivo, the particle may subsequently migrate from a node to an antinode if the particle changes from a positive contrast factor to a negative contrast factor, and vice versa if the particle changes from a negative contrast factor to a positive contrast factor.

In some embodiments, the particles of the present disclosure contain a payload. The payload may include a primary material, a secondary material, a tertiary material, and/or more materials that are delivered by the particles to a specific area or cell population. Examples of materials that can be delivered as payloads include viruses, nucleic acids, cytokines (e.g., interleukins), drug molecules, liquids or gases, or mixtures of such materials. These payloads can be delivered to the desired target or location (by acoustic co-localization) and then the payload released. The payload will affect the target at the desired location, for example causing a change in the morphology, biochemistry or other properties of the targeted material. This example is shown in fig. 2F. The particles 240 are hollow with a solid shell 242 surrounding a core 244, the core 244 containing a payload 246.

Additionally, the particles of the present disclosure may also be affected by external forces such as magnetic, electromagnetic, dielectric, ultrasonic, or other types of energy. By affecting the particles with an external energy source, the particles can be activated upon reaching certain method steps (e.g., affinity binding) or specific parts of the host anatomy (e.g., to destroy a tumor located in the patient).

In some further embodiments, the particles have a core-shell structure, wherein the liquid core is encapsulated by a lipid shell. In a more particular embodiment, the liquid in the liquid core is a Perfluorocarbon (PFC). As used in this disclosureThe term "perfluorocarbon" refers to a molecule in which all hydrogen atoms have been replaced with halogens, and a majority of the halogen atoms are fluorine atoms. For purposes of this disclosure, "halogen" refers to fluorine, chlorine, and bromine. Specific examples of PFCs include perfluoropentane (PFP), Perfluorohexane (PFH), Perfluorooctane (PFO), perfluorobromooctane (PFOB, C)₈F₁₇Br), perfluorodichlorooctane (PFDCO, C)₈F₁₆Cl₂) Or perfluorodecalin (PFD, C)₁₀F₁₈)。

These PFC liquids have unique characteristics. PFC liquids are denser than water, have low surface tension, and have low viscosity. PFC liquids also have a high capacity to absorb oxygen and nitrogen. Perfluorocarbon liquids have low acoustic speed, are highly chemically inert and are biocompatible. Table 1 below shows various physical and acoustic properties of various PFC liquids that may be used in the particles, along with other polymers for comparison. Note that the compressibility of PFC liquids is very high compared to biological cells.

Table 1.

Specific examples of lipids that can be used to form the lipid shell include Dipalmitoylphosphatidylcholine (DPPC), 1, 2-palmitoyl phosphatidic acid (DPPA), 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE). These lipids may also be used in lipid-polyethylene glycol conjugates or complexes of lipids with albumin (e.g., bovine serum albumin or human serum albumin). The lipid shell may be functionalized with streptavidin, biotin, avidin, antibodies or other functionalized moieties.

This configuration is shown in fig. 3. The particle 300 is made of a lipid shell 302, which lipid shell 302 surrounds a liquid core 304, in this example perfluorohexane. The shell may be made of DPPA, DPPC or functionalized lipid-ethylene glycol conjugates, herein labeled DSPE-PEG 5000-BIOTIN. Avidin derivatives 306 bound to biotin of the lipid shell are also shown.

The lipid shell serves to attach the particle to another molecule, as well as to protect the liquid core. These lipid-PFC particles are believed to be capable of producing transient changes in cell membrane permeability upon ultrasound-induced cavitation, while reducing cell damage. They can result in tissue-specific or site-specific intracellular delivery of genetic material both in vitro and in vivo. They can be used as non-viral vector systems for enhancing the efficacy of gene delivery.

Generally, PFC liquids and lipid solutions are combined to prepare a liquid core with a lipid shell. The PFC liquid is dispersed in another solution to form droplets. Emulsifiers may be added to the solution to prevent coalescence of the droplets. In some embodiments, phospholipids are used as emulsifiers/surfactants. PFC liquids are dispersed by different methods depending on the droplet size required for the application. To produce small nanometer-sized droplets, ultrasonic agitation may be used. To produce larger droplets, a vial shaker may be used to agitate the liquid mixture.

In some embodiments, the lipid solution consists of several different lipid materials in solution. The obtained lipids were stored in a refrigerator at about-20 ℃. At this temperature, the lipids are in a solid state. The lipids can be removed from the refrigerator prior to use and left to stand at room temperature for about 20 minutes. This is done to bring the lipid to a gel state. Since lipids are generally insoluble in water, propylene glycol can be used to solubilize them. It is desirable not to dissolve all of the lipids in propylene glycol at once, as putting all of the lipids into solution at once may result in the formation of white clots in the solution. The solubility of each lipid material should be compared and the lipid material with the greatest solubility should be dissolved in propylene glycol first, and so on. Since the solubility of lipids is a function of the temperature of the solution, the solution should be maintained at a temperature above the lipid transition temperature. Table 2 is an example of a lipid composition.

Table 2.

One representative method for producing a lipid solution is as follows. First, the propylene glycol is heated to the maximum transition temperature of the lipid blend for mixing. Next, the lipid material with the greatest solubility is added to the heated propylene glycol. The lipid material and propylene glycol were then mixed in a bath sonicator. Sequentially, while in the bath sonicator, a lower solubility lipid was added to the propylene glycol mixture.

A mixture of glycerol and buffer solution may be prepared simultaneously. The glycerol and buffer solution are heated to the maximum transition temperature. Once the lipid-propylene glycol solution was translucent (no white clot) in the sonicator, the lipid-ethylene glycol solution was mixed with the glycerol buffer solution. The resulting mixture was homogenized with a homogenizer operating at 3000 rpm. Homogenization is carried out for about one hour. During the homogenization process, the temperature is maintained at the maximum transition temperature of the lipids.

The prepared lipid solution is filtered to remove any possible contaminants, such as dust, undissolved lipid clots, etc. The filtration process may be performed with a hydrophilic syringe filter. Prior to use, the filters were soaked in batches at the same temperature. In some embodiments, a 2.0 micron filter is used. In other embodiments, a 0.8 micron filter is used. In still other embodiments, a 0.45 micron filter is used. In some embodiments, a combination of filters may be used.

The lipid solution is then mixed with the PFC liquid in a narrow vessel to produce core-shell particles. The PFC liquid was first placed in a container and then the lipid solution was poured on top. In order to produce smaller sized droplets, the amount of PFC liquid in the vessel should be minimal. As the ratio of PFC liquid volume to lipid solution volume increases, the size of the droplet formed increases until it reaches a plateau for a given sonication power. It should be noted that because PFC liquids have low surface tension values, they are of low strength. Therefore, the sonication amplitude should be appropriately selected, and the input of the ultrasonic waves should be done in a pulsed mode rather than a continuous mode. The tip of the horn sonicator assembly should be placed at the interface of the two liquid solutions. To avoid the formation of bubbles/foam, the horn should be located well inside the solution. Here, the aim is to prepare a droplet solution in order to immerse a narrow container in a transparent, low-temperature bath. A clear cryogenic bath is prepared, for example, by preparing a supersaturated solution of the salt, and then storing the salt solution in a refrigerator at-20 ℃. Sonication produces smaller beads.

In one example, the lipid solution may comprise about 1mL of propylene glycol +1mL of glycerol +8mL of buffer solution +10mg of lipid blend. 9mL of the lipid solution can be combined with about 1mL of the PFC solution. The lipid-PFC solution may be sonicated. For a 0.5 inch probe and a 750 watt sonicator, sonication with a PFC solution of 30% PFP was allowed to proceed for about 3 seconds, then stopped for about 10 seconds until a total sonication time of about 15 seconds was reached. Sonication with a PFC solution of 40% PFH for about 3 seconds was followed by a stop for about 10 seconds until a total sonication time of about 15 seconds was reached. Sonication with a PFC solution of 50% PFOB for about 3 seconds was then stopped for about 10 seconds until a total sonication time of about 15 seconds was reached. Sonication produces a solution of droplets.

To produce larger sized droplets, the amount of PFC liquid is increased and the power input to the sonicator is significantly reduced. In another non-limiting exemplary embodiment, 500 microliters of PFC and 2mL of lipid solution can be placed in a 3mL vial. The vial may then be shaken in a vial mixer at 4800rpm for 30 seconds. The prepared droplet suspension may have some microbubbles. In case microbubbles are present therein, the solution may be centrifuged.

Can be prepared by mixingWas added to the droplet solution to test the binding efficiency of these PFC-lipid particles.Is a deglycosylated form of avidin with a mass of about 60,000 daltons. As with the avidin itself,is a tetramer with strong affinity for biotin (Kd 10-15M). Although unwanted lectin binding is reduced to undetectable levels due to carbohydrate removal, biotin binding affinity is retained.It also has a near neutral pI (pH 6.3) to minimize non-specific interactions with negatively charged cell surfaces or DNA/RNA.Still have lysine residues, which remain available for derivatization or conjugation. Alternatively, if a binding complex (e.g., avidin-biotin) is present, aggregates may form. This aggregation phenomenon may be one way to bias the droplet population to larger sizes. This mechanism is shown in fig. 4. Nine PFC-lipid particles 300 are shown on the left, with a lipid shell surrounding a liquid PFH core. The lipid comprises biotin complex 306. Upon exposure to avidin or similar molecules, the particles aggregate into larger particles 310.

In one experiment, 5mL of a drop solution was taken and mixed with 100 microliters of 5mg/mLThe solutions were incubated together. The combined solution was allowed to stand for one hour and was prepared from the original droplet solution andthe drop solutions incubated together complete the size measurement.

Fig. 5A is a graph showing the particle size distribution of droplets having a size of 0.6 to 1.25 microns. Fig. 5B is a graph showing the particle size distribution of droplets having a size of 1.25 to 2.25 microns. Fine wire for not addingA droplet solution of (a). The thicker line being used forIncubated drop solution together. As can be seen here, when addingIn time, the number of particles of a given size is greater, or in other words, the line moves to the right (e.g., a larger particle size).

The polymer particles may also be produced by continuous and discontinuous phase emulsions, in which an aqueous phase and a discontinuous monomer phase are present. The reaction vessel of the emulsion may also contain a surfactant and a free radical initiator. While stirring the emulsion, it is heated and a free radical initiator is introduced into the emulsion. This causes the monomer particles to polymerize and thus gives a particulate mixture of polymerized particulates in the aqueous phase. This method allows for uniformly sized particles. An example of such a process is styrene monomer dispersed in an aqueous phase with octylphenol ethoxylate (nonionic surfactant) wherein benzoyl peroxide is introduced into the reaction vessel while stirring and heating the emulsion.

Particulates may also be produced using electrohydrodynamic spraying (EHDS) techniques in which a polymer fluid is sprayed into a gas mixture such that atomization upon liquid flow spraying allows for the generation of very fine particle sizes. The polymer may be placed prior to introduction into the nozzle. Alternatively, the polymer may be the result of the reaction of a two-component or multi-component mixture that is mixed prior to the nozzle and polymerizes as it moves through the nozzle and into the gas or gas mixture. The gas may be an inert gas such as nitrogen or argon. The gas mixture may be air or other gas blends, such as helium/oxygen and nitrogen/oxygen mixtures. EHDS systems are typically physical processes caused by the application of electricity to the surface of a liquid.

Microparticles and nanoparticles can also be produced by simple spray drying of polymer liquids or polymer liquids carried in an aqueous or solvent matrix.

The medium or primary fluid in which the particles are used may also be modified to increase the distinction between the particles and the primary fluid.

FIG. 6 shows an exemplary process 600 for generating and loading a payload into a microparticle/nanoparticle and releasing the payload, which is described in more detail in Xu et al "Hollow regenerative hydrophilic/Au/polymeric microparticles for multi-reactive drug delivery," J.Mater.Chem.B.2014, 2, 6500-. First, at 602, Na is added₂CO₃And Ca (NO)₃)₂Combined to form CaCo₃Template particles 603. Next, a (Ca10(PO4)6(OH)2, HAP) ("HAP") coating 606 is applied to the CaCo in the hydrothermal reaction at 604₃A core 603. HAPs are widely used in the biomedical field due to their biocompatibility and biodegradability. After the HAP layer 606 is created, the CaCo is provided₃The particles of the core 603 and the HAP coating 606 are subjected to a layer-by-layer (LbL) technique to incorporate the polyelectrolyte 608. Such polyelectrolytes include (aliphatic poly (urethane-amine) (PUA) and sodium poly (styrenesulfonate) (PSS) — after LbL coating 605, gold nanoparticles (aunps) 610 are loaded into the microparticles via electrostatic interactions the aunps 610 help slow the release of the payload loaded into the hollow particles.

Removal of CaCo by using a chemical etching solution step 611 (e.g., acetic acid)₃Core 603 to form hollow HAP particles 612. The hollow HAP particles 612 are then loaded with a payload 614 for payload delivery. Once the loaded particles 616 reach the desired destination, the payload 614 may be released from the hollow particle carrier 612. The release/activation 620 of the payload 614 may be facilitated by changes in ambient temperature, pH, or response to Near Infrared Radiation (NIR).

Device and system

Generally, acoustic waves can be used to manipulate the particles of the present disclosure. The acoustic waves that may be used to manipulate the microparticles and nanoparticles may be acoustic standing waves, such as multi-dimensional acoustic standing waves, planar standing waves, or a combination of multi-dimensional acoustic standing waves and planar waves.

Fig. 7 shows an acoustic traveling wave 700. Acoustic waves are a type of longitudinal waves that propagate by means of adiabatic compression and decompression in a medium. Wave 700 includes a peak 702. The peak 702 moves in the propagation direction 704.

The acoustic traveling wave 700 may change the contrast factor of microparticles and nanoparticles as they are processed in an acoustic system. In other words, the contrast factor of particulate nanoparticles processed by a traveling acoustic wave may be different from the particulates and nanoparticles as they are processed by the acoustic standing wave.

The combination of multiple traveling waves can generate an acoustic standing wave when each wave travels in opposite directions, creating a superposition of the waves. FIG. 8 shows an acoustic standing wave system 800 that produces an acoustic standing wave 801. The system includes a reflector plate 804 and an ultrasound transducer 802. An excitation frequency, typically in the range of hundreds of kHz to tens of MHz, is applied by the transducer 802. One or more standing waves are generated between the transducer 802 and the reflector 804. A standing wave is the sum of two propagating waves that are equal in frequency and intensity and travel in opposite directions, e.g., from the transducer to the reflector and back again. The propagating waves destructively interfere with each other and thus generate a standing wave. Point a on the medium moves from a maximum positive displacement to a maximum negative displacement over time. The illustration shows only half the period of the standing wave pattern motion. The motion will continue and continue with point a returning to the same maximum positive displacement and then continuing its oscillation back and forth between the up and down positions. The position a with the largest displacement is called the antinode. Note that point B on the medium is a point that never moves. Point B is a point without displacement. Such points are called nodes.

The fluid medium carrying particles 806 (microparticles or nanoparticles) disclosed above may flow in direction 805 through acoustic chamber/acoustic standing wave system 800. The resulting standing wave 801 may cause particles 806 to be trapped against the fluid flow 805. Particles with a positive contrast factor are captured at the pressure node, while particles with a negative contrast factor are captured at the antinodes. In other words, the particles are concentrated at a first or desired location. If the particles carry a payload, the payload may be released. Such release may occur after a period of time (e.g., dissolution or melting of the shell), or after exposure to an external energy source, or as previously described herein.

The acoustic devices discussed herein may operate in multiple modes or planar modes. Multiple modes refer to sound waves generated by an acoustic transducer that produces acoustic forces in three dimensions. Multiple modes of acoustic waves, which may be ultrasonic waves, are generated by one or more acoustic transducers and are sometimes referred to herein as multi-dimensional or three-dimensional acoustic standing waves. A planar mode refers to an acoustic wave generated by an acoustic transducer that generates an acoustic force substantially in one dimension, e.g., along a propagation direction. Such acoustic waves, which may be ultrasonic waves, generated in planar modes are sometimes referred to herein as one-dimensional acoustic standing waves.

Acoustic devices can be used to generate bulk acoustic waves (bulk acoustic wave) in a fluid/particle mixture. Bulk acoustic waves propagate through a volume of fluid and, unlike surface acoustic waves, tend to operate on the surface of a transducer and do not propagate through a volume of fluid.

The acoustic transducer may be comprised of a piezoelectric material. Such acoustic transducers may be electrically excited to generate planar or multi-mode acoustic waves. The three-dimensional acoustic forces generated by the multiple modes of acoustic waves include radial or lateral forces that are not aligned with the direction of acoustic wave propagation. The lateral force may act in two dimensions. The transverse force is a complement to the axial force in the multi-mode acoustic wave, which is substantially aligned with the direction of acoustic wave propagation. The transverse force may be of the same order of magnitude as the axial force of such a multi-mode acoustic wave. An acoustic transducer excited in multi-mode operation may exhibit a standing wave on its surface, thereby generating multi-mode acoustic waves. Standing waves at the surface of the transducer may be related to the operating mode of the multi-mode acoustic wave. When an acoustic transducer is electrically excited to generate a planar acoustic wave, the surface of the transducer may exhibit a piston-like motion, thereby generating a one-dimensional acoustic standing wave. Multimode acoustic waves exhibit significantly greater particle capture activity on a continuous basis for the same input power compared to planar acoustic waves. One or more acoustic transducers may be used to generate planar and/or multi-dimensional acoustic standing waves. In some modes of operation, multimodal acoustic waves generate interface effects that can block or retain particles of a certain size, while smaller particles can flow through the multimodal acoustic waves. In some modes of operation, plane waves can be used to deflect particles at an angle that is characteristic of particle size.

Acoustophoresis is the separation of materials using sound waves. Embodiments discussed herein provide a low power, non-pressure drop, non-clogging solid state process for particle separation from fluid dispersions. The sound field produces secondary acoustic forces that pull the particles together from scattering on the particles. Multimode operation results in three-dimensional acoustic radiation forces, which act as three-dimensional trapping fields. When the particles are small relative to the wavelength, the acoustic radiation force is proportional to the particle volume (e.g., the cube of the radius). The acoustic radiation force is proportional to the frequency and the acoustic contrast factor. Acoustic radiation force is proportional to acoustic energy (e.g., the square of the acoustic pressure amplitude). For harmonic excitation, the sinusoidal spatial variation of force drives the particles to stable positions within the standing wave. Particles are trapped within the acoustic standing wave field when the acoustic radiation force applied to the particles is stronger than the combined effect of the fluid drag and buoyancy/gravity forces. The effect of the transverse and axial acoustic forces on the captured particles results in the formation of tightly packed clusters by concentration, clustering, agglomeration, coagulation and/or coalescence of the particles, which clusters, when reaching a critical size, continuously settle by enhanced gravity for particles heavier than the bulk fluid or rise by enhanced buoyancy for particles lighter than the bulk fluid. In addition, secondary interparticle forces, such as the Bjerkness force, contribute to particle agglomeration.

The following discussion is directed to biological cells, which may be considered particles for the purpose of acoustophoresis. Most biological cell types exhibit higher density and lower compressibility than the fluid medium in which they are suspended, so that the acoustic contrast factor between the cells and the medium has a positive value. As a result, the axial Acoustic Radiation Force (ARF) drives the cells towards the standing wave pressure node. The axial component of the acoustic radiation force drives cells with positive contrast factors to the pressure node, while cells or other particles with negative contrast factors are driven to the antinodes. The radial or lateral component of the acoustic radiation force is the force that captures the cell. The radial or lateral component of the ARF is greater than the combined effect of fluid resistance and gravity.

For a cell to be captured in a multi-dimensional ultrasonic standing wave, it can be assumed that the force balance on the cell is zero, and thus, the lateral acoustic radiation force F_LRFIs expressed as F_LRF＝F_D+F_BIn which F is_DIs a resistance, and F_BIs buoyancy. For cells of known size and material properties, and for a given flow rate, this equation can be used to estimate the magnitude of the transverse acoustic radiation force.

One theoretical model for calculating acoustic radiation force is based on the formula developed by Gor' kov. Primary acoustic radiation force F_ADefined as a function of the field potential U,the field potential U is influenced by the sound pressure p, the fluid particle velocity U and the cell density rho_pAnd fluid density ρ_fRatio of the cell acoustic velocity c_pSpeed of sound c of fluid_fRatio of the amounts of the two components, and volume V of the biological cell_oThe influence of (c).

The theory of Gor' kov may be limited to a small particle size relative to the wavelength of the acoustic field in the fluid and particles, and it may also not take into account the effect of the viscosity of the fluid and particles on the radiation force. Additional theoretical and numerical models have been developed for calculating the acoustic radiation force of particles without any limitation regarding the particle size with respect to wavelength. These models also include the effects of fluid and particle viscosity and are therefore more accurate calculations of acoustic radiation force. The model implemented was based on the theoretical work of Yurii Ilinskii and Evgenia Zabolotskaya, as described in AIP Conference Proceedings, Vol.1474-1, p.255-258 (2012). Additional internal models have also been developed to calculate the acoustic trapping force of cylindrical objects, such as the "hockey" trapping particles in standing waves, which closely resemble a cylinder.

Desirably, the ultrasonic transducer generates a multi-dimensional standing wave in the fluid that exerts a lateral force on the suspended particles to accompany the axial force. Common results published in the literature state that transverse forces are two orders of magnitude smaller than axial forces. In contrast, the technology disclosed in the present application provides a lateral force that is of the same order of magnitude as the axial force. However, in certain embodiments further described herein, the device uses both a transducer that produces a multi-dimensional acoustic standing wave and a transducer that produces a planar acoustic standing wave. The lateral force component of the total Acoustic Radiation Force (ARF) generated by the ultrasound transducer of the present disclosure is significant and sufficient to overcome fluid resistance at linear velocities up to 1cm/s and produces close-packed clusters and is of the same order of magnitude as the axial force component of the total acoustic radiation force.

An acoustic standing wave is a three-dimensional acoustic field that, when excited by a rectangular transducer, can be described as occupying a generally rectangular parallelepiped fluid volume. The transducer may be configured to face the reflector or boundary to allow standing wave generation therebetween. The transducer may be configured to face the other transducer, both of which operate to generate a standing wave therebetween. The transducer may be configured to face the sound absorbing material to allow for the generation of a traveling wave.

In some examples, the cuboid includes two opposing faces defined by the transducer and the reflector, an adjacent pair of opposing faces comprised by the walls of the device, and a final pair of opposing faces that may define the flow channel inlet and outlet. The acoustic waves generated by the transducer and reflector create an interface or barrier region interface near the flow channel entrance (e.g., near the upstream face of the acoustic standing wave field), creating a "sound barrier or edge effect". This location is also referred to as the upstream interface region. The sound barrier may prevent particles with certain characteristics (e.g., high acoustic contrast factor) from passing through the sound waves generated by the transducer and reflector.

Particles retained or blocked by the sound barrier may be captured in a chamber, such as a column, or returned to a holding device, such as a bioreactor. The circulating flow motion may be generated by a primary recirculation flow against the sound barrier and may be optimized with acoustic chamber geometry changes to improve system efficiency.

Fig. 9 and 10 are views of an acoustophoretic device that can be used with particles of the present disclosure. Fig. 9 is an elevational sectional view, and fig. 10 is an external perspective view. Notably, this embodiment is specifically designed so that it can be manufactured using class VI materials (e.g., medical device grade HDPE) with clean machining techniques, even as a single or welded injection molded part. In this manner, this embodiment is an example of a gamma-stabilized single-use device. The apparatus is flushed to remove the bioburden and then gamma irradiated (typically 25-40kGy) to sterilize any potential contamination that may destroy the healthy cell culture, such as contamination present in perfusion bioreactors.

Referring first to fig. 9, in this device 700, the inlet port 710 and the collection port 770 are both located at the top end 718 of the device, or on the top wall 776 of the device. The outlet port 730 is located at the bottom end 716 of the device. Here, both the inlet 710 and the outlet 730 are on the first side 712 of the device. The inlet flow path 751 is in the form of a channel 755 extending from an inlet port down to a bottom end and through an outlet port, the channel being separated from the acoustic chamber 750 (where separation occurs through the inner wall 756). The fluid will flow downward in the channel and then rise upward into the acoustic chamber 750. The bottom wall 720 of the acoustic chamber is an inclined flat surface that slopes downward toward the outlet port 730. The position of the ultrasound transducer 760 is shown here as two squares between the top and bottom ends of the device. The collection flow path 753 is located above the transducer.

Referring now to fig. 10, device 700 is shown formed within a three-dimensional rectangular housing 706. It can be seen that the outlet port 730 at the bottom end 716 of the device is located on the front wall 775. Again, the collection port 770 and the inlet port 710 are located on the top wall 776. A viewing window 708 made of a transparent material is present in the front wall. Through the viewing window, the ultrasonic transducer can be seen mounted in the back wall 778 of the device housing 706. The viewing window acts as a reflector to generate a multi-dimensional acoustic standing wave.

The apparatus 700 may be used to cause cells and particles to react with each other, where the particles deliver a payload to the cells in a region generally around the transducer 760 where acoustic waves are present. The cells may then exit through outlet port 730, while the other fluids exit through collection port 770.

The particles may also interact with cells and, depending on the surface of the particles and the functionalization on the surface of the desired cells to be selected, either a negative selection or a positive selection is performed. The functionalized portion of the particle binds to a receptor on the surface of the target cell, allowing the cell to be removed or retained in the system.

Fig. 11 is a cross-sectional view of an ultrasonic transducer 81 according to an example of the present disclosure, for use in an acoustic filtering device of the present disclosure. The transducer 81 is shaped as a disk or plate and has an aluminum housing 82. The aluminum housing has a top end and a bottom end. The transducer housing may also be composed of plastic such as medical grade HDPE or other metals. The piezoelectric elements are perovskite ceramic masses, each made of a mixture of a relatively large divalent metal ion (usually lead or barium) and O^2-The smaller tetravalent metal ion (usually titanium or zirconium) in the lattice of the ion. In this example, a PZT (lead zirconate titanate) piezoelectric element 86 defines the bottom end of the transducer and is exposed from the outside of the bottom end of the case. The piezoelectric element is supported on its periphery by a small resilient layer 98, such as epoxy, silicone or similar material, said resilient layer 98 being located between the piezoelectric element and the housing. In other words, there is no wear plate or backing material present. However, in some embodiments, there is a layer of plastic or other material separating the piezoelectric element from the fluid in which the acoustic standing wave is generated. The piezoelectric element/crystal has an outer surface (which is exposed) and an inner surface. In certain embodiments, the piezoelectric element/crystal is an irregular polygon, and in further embodiments, an asymmetric irregular polygon.

The screw 88 attaches the aluminum top plate 82a of the housing to the main body 82b of the housing via threads. The top plate includes a connector 84 for powering the transducer. The top surface of the PZT piezoelectric element 86 is connected to positive 90 and negative 92 electrodes separated by an insulating material 94. The electrodes may be made of any conductive material, such as silver or nickel. Power is supplied to the PZT piezoelectric element 86 through electrodes on the piezoelectric element. Note that the piezoelectric element 86 does not have a backing layer or epoxy layer. In other words, in the transducer, there is an internal volume or air gap 87 between the aluminum top plate 82a and the piezoelectric element 86 (e.g., the housing is empty). In some embodiments, a minimal backing 58 (on the inner surface) and/or wear plate 50 (on the outer surface) may be provided, as seen in fig. 12.

The design of the transducer can affect the performance of the system. A typical transducer is a layered structure in which a ceramic piezoelectric element is bonded to a backing layer and a wear plate. Conventional design guidelines for wear plates (e.g., half-wavelength thickness for standing wave applications or quarter-wavelength thickness for radiation applications) and manufacturing methods may be inadequate due to the high mechanical impedance presented by the standing wave loaded transducer. In contrast, in one embodiment of the present disclosure, the transducer has no wear plate or backing, allowing the piezoelectric element to vibrate in a combination of one or several of its high-Q factor eigenmodes. The vibrating ceramic piezoelectric element/disc is directly exposed to the fluid flowing through the fluid cell.

Removing the backing (e.g., air backing the piezoelectric element) also allows the ceramic piezoelectric element to vibrate in higher order vibration modes (e.g., higher order mode displacements) with little damping. In a transducer having a piezoelectric element with a backing, the piezoelectric element vibrates like a piston with a more uniform displacement. Removing the backing allows the piezoelectric element to vibrate in a non-uniform displacement mode. The higher the mode shape of the piezoelectric element, the more nodal lines the piezoelectric element has. Higher order mode displacements of the piezoelectric element produce more trapping lines, although the correlation between trapping lines and nodes is not necessarily one-to-one, and driving the piezoelectric element at higher frequencies does not necessarily produce more trapping lines.

The reflector may be of a non-planar type, such as a polygon mirror. The reflector may also be another transducer, which may have a flat surface or a non-flat surface. In some examples, two opposing transducers are used to generate an acoustic wave, such as an acoustic standing wave, therebetween.

In some embodiments of the acoustic filtering devices of the present disclosure, the piezoelectric element can have a backing that minimally affects the Q factor of the piezoelectric element (e.g., less than 5%). The backing may be made of a substantially acoustically transparent material, such as balsa wood, foam, or cork, which allows the piezoelectric element to vibrate in a high order mode shape and maintain a high Q factor while still providing some mechanical support for the piezoelectric element. The backing layer may be a solid or may be a lattice with holes through the layer such that the lattice follows the nodes of the vibrating piezoelectric element in certain higher order vibration modes, providing support at the node locations while allowing the remaining piezoelectric element to vibrate freely. The purpose of the lattice or acoustically transparent material is to provide support without reducing the Q factor of the piezoelectric element or interfering with the excitation of specific mode shapes.

Placing the piezoelectric element in direct contact with the fluid also contributes to a high Q factor by avoiding the damping and energy absorbing effects of the epoxy and wear plates. Other embodiments of the transducer may have a wear plate or wear surface to prevent PZT containing lead from contacting the bulk fluid. This may be desirable, for example, in biological applications such as separation of blood, biopharmaceutical perfusion, or fed-batch filtration of mammalian cells. Such applications may use wear resistant layers such as chromium, electrolytic nickel or electroless nickel. Chemical vapor deposition may be used to apply a layer of poly (p-xylene) (e.g., Parylene) or another polymer. Organic and biocompatible coatings (e.g., silicone or polyurethane) may also be used as wear resistant surfaces. Thin films such as PEEK films can also be used as a covering for the transducer surface exposed to fluid, with the advantage of a biocompatible material. In one embodiment, the PEEK film is adhered to the surface of the piezoelectric material using a Pressure Sensitive Adhesive (PSA). Other membranes may also be used.

In some embodiments, the ultrasonic transducer has a nominal 2MHz resonant frequency for applications such as oil/water emulsion splitting and others such as irrigation. Each transducer may consume approximately 28W of power for droplet capture at a flow rate of 3 GPM. This translates into 0.25kW hr/m³The energy cost of (a). This is an indication that the energy cost of this technique is extremely low. Each transducer may be powered and controlled by a dedicated driver, which may include an amplifier, or multiple transducers may be driven by a single driver. In other embodiments, the ultrasound transducer uses square piezoelectric elements, for example having a1 "x 1" size. Alternatively, the ultrasonic transducer may use a rectangular piezoelectric element, e.g. havingThere are 1 "x 2.5" dimensions. The power consumption of each transducer was 10W per 1 "x 1" transducer cross-sectional area and per inch of acoustic standing wave span in order to obtain sufficient acoustic capture force. For a 4 "span for a medium scale system, each 1" x1 "square transducer consumes 40W. The larger 1 "x 2.5" rectangular transducer uses 100W in a medium scale system. An array of three 1 "x 1" square transducers consumes a total of 120W, while an array of two 1 "x 2.5" transducers consumes about 200W. Closely spaced transducer arrays represent an alternative potential implementation of this technique. The size, shape, number and location of the transducers may be varied as desired to generate a desired multi-dimensional acoustic standing wave pattern.

The size, shape and thickness of the transducer determine the transducer displacement at different excitation frequencies, which in turn affects the separation efficiency. Typically, the transducer operates at a frequency near the thickness resonance frequency (half wavelength). The gradient of transducer displacement generally results in more capture locations for cells/biomolecules. High order mode displacements generate three dimensional acoustic standing waves with strong gradients in the acoustic field in all directions, resulting in equally strong acoustic radiation forces in all directions, resulting in a number of trapping lines, where the number of trapping lines is associated with a particular mode shape of the transducer.

In contrast to the vibration mode where the crystal effectively moves as a piston with uniform displacement, the lateral force of the acoustic radiation force generated by the transducer can be increased by driving the transducer in a higher order mode shape. The sound pressure is proportional to the drive voltage of the transducer. Power is proportional to the square of the voltage. The transducer is typically a thin piezoelectric plate with an electric field in the z-axis and a dominant displacement in the z-axis. The transducer is typically coupled on one side by air (e.g., an air gap within the transducer) and on the other side by a fluid mixture of cell culture media. The type of wave generated in the plate is called a complex wave. The subset of the composite waves in the piezoelectric plate resemble leaky symmetric (also called compression or expansion) lamb waves. The piezoelectric properties of the plate typically result in the excitation of a symmetric lamb wave. These waves are leaky because they radiate into the water layer, which results in the generation of acoustic standing waves in the water layer. Lamb waves exist in an infinite range of thin sheets with stress-free conditions on their surfaces. Because the transducer of this embodiment is limited in nature, the actual mode displacement is more complex.

The transducer is driven such that the piezoelectric element vibrates in a higher order mode of the general formula (m, n), where m and n are independently 1 or greater. Generally, the transducer vibrates in a higher order mode than (2, 2). Higher-order modes produce more nodes and antinodes, resulting in a three-dimensional standing wave in the water layer, characterized by a strong gradient in the acoustic field in all directions, not only in the direction of the standing wave, but also in the lateral direction. As a result, the acoustic gradient results in a stronger trapping force in the lateral direction.

In embodiments, the voltage signal driving the transducer may have a sinusoidal, square, sawtooth, pulsed, or triangular waveform; and has a frequency of 50kHz to 10 MHz. The voltage signal may be driven with pulse width modulation, which produces any desired waveform. The voltage signal may also have amplitude or frequency modulated start/stop capability to eliminate flow.

The transducer is used to generate a pressure field that generates an acoustic radiation force orthogonal to and of the same order of magnitude as the direction of the standing wave. When the forces are approximately the same order of magnitude, particles of sizes 0.1 to 300 microns will move more effectively toward the "capture line" so that the particles do not pass through the pressure field and continue to exit through the collection port of the filtration device. Instead, the particles will remain in the acoustic chamber and be recycled back to the bioreactor.

In biological applications, all parts of the system (e.g., the bioreactor, the acoustic filtering device, the tubing fluidly connecting them, etc.) may be separate from one another and disposable. By allowing better separation of CHO cells without reducing cell viability, the acoustophoretic separator can provide improved performance over centrifuges and filters. The transducer may also be actuated to produce rapid pressure changes to prevent or clear blockages due to the coagulation of CHO cells. The frequency of the transducer may also be varied to obtain optimum effectiveness for a given power.

The techniques and embodiments described herein may be used for integrated, continuous automated bioprocessing. By way of non-limiting example, CHO mAb processing may be performed using the techniques and instruments described herein. Control may be distributed to some or all of the units involved in bioprocessing. Feedback from the unit may be provided to allow for a summary of the biological process, which may be in the form of screen displays, control feedback, reports, status reports, and other information transfer. Distributed processing allows a high degree of flexibility in achieving the desired process control, for example by coordinating the steps in a unit and providing batch execution control.

Bioprocessing can be achieved with commercially available components and 100% cell retention is achieved. Cell density can be controlled via external cell drainage based on the capacitance signal. Perfusion devices utilizing acoustic wave systems may be implemented with biocompatible materials and may include gamma sterilized and disposable components. The processing system also allows for ultrasonic flow measurements, which are non-invasive and capable of operating with high viscosity fluids. The system may be implemented with a single-use sterile connector and a simple Graphical User Interface (GUI) for control.

The acoustic wave system includes a sweeping flow induced below the acoustic chamber. The acoustic standing wave may act as a barrier to particulate matter in the fluid to allow passage and extraction of the clarified stream. The recirculation loop can be implemented with high fluid velocity and low shear rate. The fluid velocity through the acoustic field may be lower than the fluid velocity through the recirculation loop, which may help improve separation at low shear.

Various particles can also be used for positive and negative selection of cells. For example, negative selection of TCR-positive T cells is a process in which the functionalized particles bind to TCR-positive T cells, such that the TCR-positive T cells are removed from the system. TCR positive T cells are detrimental to processes such as chimeric antigen receptor T cell therapy (CAR-T).

Positive selection procedures can also be used for specific cells, where modified T cells are selected by appropriately functionalized particles so that they are selected from cell culture for subsequent use in cell therapy.

The methods, systems, and apparatus discussed above are examples. Various configurations may omit, substitute, or add various programs or components as appropriate. For example, in alternative configurations, the methods may be performed in a different order than described, and various steps may be added, omitted, or combined. In addition, features described with respect to certain configurations may be combined in various other configurations. Different aspects and elements of the configurations may be combined in a similar manner. In addition, technology is evolving and, thus, many elements are examples and do not limit the scope of the disclosure or claims.

Specific details are given in the description to provide a thorough understanding of example configurations, including embodiments. However, configurations may be practiced without these specific details. For example, well-known methods, structures and techniques have been shown without unnecessary detail in order to avoid obscuring the arrangement. This description provides example configurations only, and does not limit the scope, applicability, or configuration of the claims. Rather, the previous description of the configurations provides a description for implementing the techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure.

Additionally, a configuration may be described as a method depicted as a flowchart or a block diagram. Although each may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be rearranged. The method may have further stages or functions not included in the figures.

Having described several example configurations, various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the disclosure. For example, the above elements may be components of a larger system in which other structures or processes may take precedence over or otherwise modify the application of the invention. In addition, many actions may be taken before, during, or after the above elements are considered. Accordingly, the above description does not limit the scope of the claims.