An acoustofluidic micromixing platform

Abstract

The present invention relates to an acoustofluidic micromixing platform (1) used for the synthesis of ultra-small liposome nanoparticles and related nano-bio materials.

Description

[0001] DESCRIPTION

[0002] AN ACOUSTOFLUIDIC MICROMIXING PLATFORM

[0003] Technical Field

[0004] The present invention relates to an acoustofluidic micromixing platform used for the synthesis of ultra-small liposome nanoparticles and related nano-bio materials.

[0005] Background of the Invention

[0006] Nanoparticles (NPs) have gained profound attention in various applications in the fields of energy conversion, chemistry, nanomedicine, and biology. Liposome nanoparticles (LNPs) are employed excessively in nanomedicine, especially in cancer research for drug delivery applications, owing to their distinctive properties such as biodegradability, stability, high encapsulation efficiency and biocompatibility. The ability to tune the size of LipNPs is essential for optimizing their effectiveness in both cancer and adjuvant therapies. In adjuvant therapies, large liposomes (200-2500 nm) induce specific immune responses and in cancer therapy, smaller liposomes (<100 nm) can cross the blood-brain barrier, while those in the 30-40 nm range impact blood circulation time, cellular uptake and targeting efficiency, by enhancing transdermal transport. Liposomes are spherically shaped vesicles characterized by their unique lipid bilayer structure which allows for the encapsulation of both hydrophobic and hydrophilic drugs and this renders them highly favored as drug carriers. However, to harness these advantages, the synthesis process requires meticulous consideration in order to yield liposomes that are both size-tunable and monodisperse which are critical factors in pharmaceutical and biological applications. Conventional benchtop methods for NPs / LipNPs synthesis are based on mixing the reagents using specific approaches such as pipetting, stirring, vortexing, or shaking. Nevertheless, those methods frequently encounter challenges such as poly dispersity, high batch-to-batch variation, and low reproducibility stemming from the limited control over reaction conditions. Thus, it is crucial to choose a synthesis method that is able to govern reaction conditions in order to tackle the abovementioned limitations. Microfluidic platforms offer significant potential in addressing these challenges and enable the synthesis of high-throughput, reproducible, and size-controlled nanoparticles. Microfluidic platforms for NPs synthesis can be classified into two categories; namely, two phase droplet-based microfluidics and single phase mixing-based microfluidics. Droplet-based microfluidic platform is used to synthesize monodisperse and stable microparticles with larger sizes around a few microns and this, as a result, enhance their loading capacity for drug delivery.

[0007] However, this method suffers from additional downstream separation processes for purification. Additionally, the use of surfactants is required for droplet stabilization and this further complicates the process. On the other hand, mixing-based microfluidic platforms have been employed excessively in literature to produce much smaller NPs. In such platforms, the mixing element is one of the most critical and fundamental building blocks for NPs synthesis. Nevertheless, achieving complete mixing poses a significant challenge due to the microfluidic length scales that enforce a laminar flow regime characterized by low Reynolds numbers. Mixing-based microfluidic platforms can be divided into two subcategories: Passive and Active micromixers. Passive micromixers used in NPs synthesis, such as hydrodynamic flow focusing (HFF), grooved-based micromixers and staggered herringbone mixers rely solely on molecular diffusion and channel geometry to disrupt the fluid streamlines / interface and this results in acceptable mixing efficiency. Active micromixers employ external forces such as electrical, acoustic and magnetic forces, to disrupt the symmetry between fluids and induce disturbances in the flow; thereby mass transport is increased and consequently mixing efficiencies are elevated. While active micromixers provide rapid and controlled mixing, it is important to highlight the advantage of passive micromixers in terms of low energy consumption and high throughput and this makes them a more suitable choice for large-scale production. However, NP synthesis methods based on passive mixing frequently result in partial reagent mixing and cause unfavorable downstream reactions. Compared to passive, active micromixers used in NPs synthesis show dominant performances and controllable mixing in a shorter time and length and this is preferable. Among active methods, acoustically-integrated platforms have attracted considerable interest in the field due to their non-invasive characteristics, capability of achieving higher mixing rates, and ease of operation. Acoustofluidic micromixers (stirrers) consist of an integrated acoustic source with oscillating elements embedded within the microfluidic channel. In the literature, sharp edges and microbubbles are frequently employed as oscillating structures to induce acoustic microstreaming; nevertheless, they exhibit several limitations. One primary challenge associated with sharp-edge designs lies in the fabrication process, particularly shaping lower-angle sharp edges, which demands meticulous precision in the lithography procedure. In the context of microbubbles, the main concern revolves around the stability and robustness of the bubbles for long-term experiments.

[0008] In accordance with the above-stated explanations, there is need for a novel acoustofluidic micromixing platform which utilizes the combined effects of oscillating thin elastic polydimethylsiloxane (PDMS) membrane and vibrating trapped microbubbles to induce acoustic streaming, and offers high mixing efficiency at high flow rates.

[0009] The Chinese patent document no. CN115193352, an application included in the state of the art, discloses an inertia and sound wave effect-based high-efficiency micromixer for nano-particle synthesis. The inertia and sound wave effect-based high- efficiency micro-mixer comprises a micro-fluidic chip, a sound wave control system and a flow control system; the micro-fluidic chip comprises a PDMS (Polydimethylsiloxane) micro-channel and a glass substrate; the PDMS microchannel is mainly composed of a side wall sharp corner and a contraction and expansion structure unit; the sound wave control system comprises a piezoelectric transducer, a signal generator and a signal amplifier; the fluid control system is composed of an injection pump, an injector and a catheter. In order to solve the problems that an existing micro-mixer is narrow in flow application range and difficult to integrate with a downstream analysis test platform, sound flow and inertia flow are introduced into a flow channel through a side wall sharp corner structure and a contraction-expansion array structure, so that the mixing process can be rapidly and efficiently carried out in a wide flow range. The method has high controllability and can be used for rapidly and continuously preparing nano-particles with controllable size and uniform particle size distribution. The device is simple in structure, easy to process and manufacture and wide in application flow range.

[0010] Summary of the Invention

[0011] An object of the present invention is to realize an acoustofluidic micromixing platform which is used for the synthesis of ultra-small liposome nanoparticles and related nano-bio materials.

[0012] Another object of the present invention is to realize an acoustofluidic micromixing platform which utilizes the combined effects of oscillating thin elastic polydimethylsiloxane (PDMS) membrane and vibrating trapped microbubbles to induce acoustic streaming.

[0013] Detailed Description of the Invention

[0014] “An Acoustofluidic Micromixing Platform” realized to fulfil the objectives of the present invention is shown in the figures attached, in which:

[0015] Figure 1. It is A) an exploded, B) a combined, and C) a side view of the inventive micromixing platform.

[0016] Figure 2. Mixing characterization of the acoustofluidic micromixing platform with deionized water and fluorescein reagents. A: Characterization of the mixing performance at different flow rates with an applied voltage of Vpp95 V and a frequency of 4.1 kHz. B: Microscopic snapshots showing the mixing profile for acoustics OFF and acoustics ON conditions at different flow rates. C: Characterization of the mixing performance under different applied voltages at a flow rate of 4000 pL / h and a frequency of 4.1 kHz. D: Characterization of the mixing performance at different resonance frequencies with a flow rate of 4000 pL / h and an applied voltage of Vpp95 V. E: Conductance analysis of the piezoelectric transducer showing the resonance frequency of 4.1275 kHz matching the highest mixing performance achieved by the micromixer.

[0017] Figure 3. DLS analysis for the synthesized LNPs at different glycerol concentrations. A: Size distribution comparison of the synthesized LNPSs for GPC, PC and GPC: PC (1 : 1) phospholipids, Acoustics OFF and Acoustics ON. B: Mean size of the synthesized LNPS for GPC, PC and GPC: PC (1 : 1) phospholipids. C: Poly dispersity index (PDI) of the synthesized LNPS for GPC, PC and CPC: PC (11) phospholipids. Note: Phospholipid: sn-Glycer-3- phosphocholine (GPC), Phosphatidylcholine (PC).

[0018] Figure 4. Mean size of the synthesized LNPs for different phospholipid precursor compounds, namely A: GPC phospholipid and B: PC phospholipid. Poly dispersity index (PDI) of the synthesized LNPs for different phospholipid precursor compounds, namely C: GPC phospholipid and D: PC phospholipid. Figure 5. Mean size of the synthesized LNPs at different total flow rates for A: GPC phospholipid and B: PC phospholipid. Poly dispersity index (PDI) of the synthesized LNPs at different total flow rates of C: GPC phospholipid and D: PC phospholipid.

[0019] Figure 6. DLS analysis for the solvent-free synthesized LNPs at different glycerol concentrations. A: Size distribution comparison of the synthesized LNPS for GPC, PC and GPC: PC (1 : 1) phospholipids. Acoustics OFF and Acoustics ON. B: Mean size of the synthesized LNPS for GPC, PC and GPC: PC (11) phospholipids. C: Poly dispersity index (PDI) of the synthesized LNPS for GPC, PC and GPC: PC (1 : 1) phospholipids.

[0020] The components illustrated in the figures are individually numbered, where the numbers refer to the following:

[0021] 1. Micromixer platform

[0022] 2. Upper layer 2.1. Inlet pipe

[0023] 2.2. Outlet pipe

[0024] 2.3. Groove

[0025] 2.4. Air microbubbles

[0026] 2.5. Membrane layer

[0027] 3. Middle layer

[0028] 4. Lower layer

[0029] 4.1. Microchannel

[0030] 5. Table

[0031] 6. Transducer

[0032] The inventive acoustofluidic micromixing platform (1) which is used for the synthesis of ultra-small liposome nanoparticles and related nano-bio materials, and offers high mixing efficiency at high flow rates comprises at least one upper layer (2) which is a plate-shaped structure; includes the grooves (2.3) embedding the air microbubbles (2.4), and the inlet / outlet pipes (2.1, 2.2) providing the entry of components to be used in the synthesis of micro-nano-bio-material and nanoparticle and the exit of the synthesized product; at least one middle layer (3) in the form of a thin plate which is connected such that it will contact the surface of the upper layer (2) where the grooves (2.3) and the inlet / outlet pipes (2.1, 2.2) are not present; and has an opening such that it will allow a component to pass through the pipe at the connection level of the inlet / outlet pipe (2.1, 2.2); at least one lower layer (4) which has a curved microchannel (4.1) whereon the synthesis of micro-nano-bio-material and nanoparticle is performed and at least one opening such that it will allow a component to pass through the inlet / outlet pipes (2.1, 2.2) at the inlet and outlet level of the microchannel (4.1); and is configured such that the microchannels (4.1) are connected to the surface of the middle layer (3) that is not in contact with the upper layer (2); at least one table (5) made of glass which is the area whereon the upper, middle and lower layers (2, 3, 4) are placed after being connected; and at least one piezoelectric transducer (6) which is positioned next to the upper, middle and lower layers (2, 3, 4) placed on the table (5); and provides the mixing process necessary for the components received from the inlet pipes (2.1) in order to synthesize micro-nano-bio-materials and nanoparticles in the microchannel (4.1) of the lower layer (4) by converting the electrical current into acoustic waves that cause mechanical vibrations.

[0033] The upper layer (2) included in the inventive micromixing platform (1) has the same plate size as the middle and lower layers (3, 4) and comprises two inlet pipes (2.1) in the form of ‘V’ that extend outwards from the plate and are in connection with the input terminal of the microchannel (4.1), and an outlet pipe (2.2) in the form of “I” that is connection with the output terminal thereof. The air microbubbles (2.4) of the upper layer (2) are isolated from the external environment by the membrane layer (2.5) made of any elastic material in the form of PDMS (polydimethylsiloxane). The upper layer (2) contributes to the mixing of components streaming in the microchannel (4.1) of the lower layer (4) by means of the oscillation effect, that results from the conversion of the electric current supplied to the transducer (6) into the mechanical vibration, on the air microbubbles (2.4). The upper layer (2) enables the compression and movement of the bubbles (2.4) by expanding and contracting in the form of volumetric vibrations due to the pressure nodes and antinodes that apply force imbalance on the surface of the air microbubbles (2.4) as a result of the acoustic waves generated by the transducer (6).

[0034] The middle layer (3) included in the inventive micromixing platform (1) is configured to be in contact with both layers when it is placed between the upper layer (2) and the lower layer (4) and then the upper layer (2) and the lower layer (4) are combined. The middle layer (3) has a membrane made of any elastic material in the form of PDMS that is selected as the material for the membrane layer due to its exceptional elastic properties exhibiting significant deformation capability and that has a thickness of approximately 0.1-5000 pm. The middle layer (3) is triggered so as to stretch by means of the acoustic waves generated by the transducer (6) and then to oscillate. The lower layer (4) included in the inventive micromixing platform (1) is the area where the synthesis of micro-nano-bio-material and nanoparticle is performed, and has a lower layer (4) with a snake-like curved microchannel (4.1). The lower layer (4) ensures that input components such as lipid in the organic solvent and glycerol in the deionised water -that are required for the synthesis of liposome nanoparticle, micro- nano-bio-material, and nanoparticle- are provided into the microchannel (4.1) separately from the inlet pipes (2.1) connected to one end of the microchannel (4.1), and then the liposome nanoparticle synthesized in the microchannel (4.1) is received from the outlet pipe (2.2) connected to the other end thereof. The lower layer (4) is configured to ensure that the input components progressing in the microchannel (4.1) are mixed with a mixing efficiency exceeding 98% at high flow rates that reach up to 1-80000 pL / hour, by means of the effect of the vibration caused by the acoustic waves generated by the converter (6), the pressure change, the oscillation of the middle layer (3), and the vibration of the air microbubbles (2.4) in the upper layer (2); and to synthesize ultra-small and size-tunable micro-nano-bio-materials and nanoparticles. In the lower layer (4), the synthesis of micro-nano-bio-material and nanoparticle is performed in the curves close to the inlet of the curved microchannel (4.1), whereas the stabilization of the micro-nano-bio-material and nanoparticle synthesised is performed on the side close to the outlet pipe (2.2).

[0035] The table (5) included in the inventive micromixing platform (1) serves the purpose of creating a support ground, after being combined to the upper, middle and lower layers (2, 3, 4). The table (5) is a plate-shaped structure larger than the upper, middle and lower layers (2, 3, 4) in terms of size, and has a size sufficient to enable the placement of the converter (6) close these layers.

[0036] The transducer (6) included in the inventive micromixing platform (1) is a piezoelectric structure that is positioned near the upper, middle and lower layers (2, 3, 4) on the table (5); and converts the electrical current into acoustic waves that generate mechanical vibration, by generating a radio frequency signal at the resonance frequency. The transducer (6) can generate acoustic waves propagating in all directions by relocating on the table (5) and can even generate waves propagating perpendicular to its axis.

[0037] With the inventive micromixing platform (1), provision of the components to generate the micro-nano-bio-material and nanoparticle -that is desired to be synthesized- into the microchannel (4.1) separately from the inlet pipes (2.1) in the structure of the combined upper, middle and lower layer (2, 3, 4) placed on the table (5); oscillation of the middle layer (3) caused by the acoustic waves generated by the components streaming at ultra-high speed in the microchannel (4.1) by means of the converter (6); synthesis of micro-nano-bio-materials and nanoparticles at high-efficiency by mixing with the vibration of the micro air bubbles in the upper layer (2) and the lower layer (4) and receiving the product synthesized from the outlet pipe (2.2) are basically ensured. It is possible to develop various embodiments of the inventive “Acoustofluidic Micromixing Platform (1)”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.

Claims

CLAIMS1. An acoustofluidic micromixing platform (1) which is used for the synthesis of ultra-small liposome nanoparticles and related nano-bio materials, and offers high mixing efficiency at high flow rates; characterized by at least one upper layer (2) which is a plate-shaped structure; includes the grooves (2.3) embedding the air microbubbles (2.4), and the inlet / outlet pipes (2.1, 2.2) providing the entry of components to be used in the synthesis of micro-nano-bio-material and nanoparticle and the exit of the synthesized product; at least one middle layer (3) in the form of a thin plate which is connected such that it will contact the surface of the upper layer (2) where the grooves (2.3) and the inlet / outlet pipes (2.1, 2.2) are not present; and has an opening such that it will allow a component to pass through the pipe at the connection level of the inlet / outlet pipe (2.1, 2.2); at least one lower layer (4) which has a curved microchannel (4.1) whereon the synthesis of micro-nano-bio-material and nanoparticle is performed and at least one opening such that it will allow a component to pass through the inlet / outlet pipes (2.1, 2.2) at the inlet and outlet level of the microchannel (4.1); and is configured such that the microchannels (4.1) are connected to the surface of the middle layer (3) that is not in contact with the upper layer (2); at least one table (5) made of glass which is the area whereon the upper, middle and lower layers (2, 3, 4) are placed after being connected; and at least one piezoelectric transducer (6) which is positioned next to the upper, middle and lower layers (2, 3, 4) placed on the table (5); and provides the mixing process necessary for the components received from the inlet pipes (2.1) in order to synthesize micro-nano-bio- materials and nanoparticles in the microchannel (4.1) of the lower layer (4) by converting the electrical current into acoustic waves that cause mechanical vibrations.

2. A micromixing platform (1) according to Claim 1 ; characterized by the upper layer (2) which has the same plate size as the middle and lower layers (3, 4) and comprises two inlet pipes (2.1) in the form of ‘V’ that extend outwards from the plate and are in connection with the input terminal of the microchannel (4.1), and an outlet pipe (2.2) in the form of “I” that is connection with the output terminal thereof.

3. A micromixing platform (1) according to Claim 1 or 2; characterized by the upper layer (2) the air microbubbles (2.4) of which are isolated from the external environment by the membrane layer (2.5) made of any elastic material in the form of PDMS.

4. A micromixing platform (1) according to any one of the preceding claims; characterized by the upper layer (2) which contributes to the mixing of components streaming in the microchannel (4.1) of the lower layer (4) by means of the oscillation effect, that results from the conversion of the electric current supplied to the transducer (6) into the mechanical vibration, on the air microbubbles (2.4).

5. A micromixing platform (1) according to any one of the preceding claims; characterized by the upper layer (2) which enables the compression and movement of the bubbles (2.4) by expanding and contracting in the form of volumetric vibrations due to the pressure nodes and antinodes that apply force imbalance on the surface of the air microbubbles (2.4) as a result of the acoustic waves generated by the transducer (6).

6. A micromixing platform (1) according to any one of the preceding claims; characterized by the middle layer (3) which is configured to be in contact with both layers when it is placed between the upper layer (2) and the lower layer (4) and then the upper layer (2) and the lower layer (4) are combined.

7. A micromixing platform (1) according to any one of the preceding claims; characterized by the middle layer (3) which has a membrane made of any elastic material in the form of PDMS that is selected as the material for the membrane layer due to its exceptional elastic properties exhibiting significant deformation capability and that has a thickness of approximately 0.1-5000 pm. The middle layer (3)8. A micromixing platform (1) according to any one of the preceding claims; characterized by the middle layer (3) which is triggered so as to stretch by means of the acoustic waves generated by the transducer (6) and then to oscillate.

9. A micromixing platform (1) according to any one of the preceding claims; characterized by the lower layer (4) which is the area where the synthesis of micro-nano-bio-material and nanoparticle is performed, and has a lower layer (4) with a snake-like curved microchannel (4.1).

10. A micromixing platform (1) according to any one of the preceding claims; characterized by the lower layer (4) which ensures that input components such as lipid in the organic solvent and glycerol in the deionised water -that are required for the synthesis of liposome nanoparticle, micro-nano-bio-material, and nanoparticle- are provided into the microchannel (4.1) separately from the inlet pipes (2.1) connected to one end of the microchannel (4.1), and then the liposome nanoparticle synthesized in the microchannel (4.1) is received from the outlet pipe (2.2) connected to the other end thereof.

11. A micromixing platform (1) according to any one of the preceding claims; characterized by the lower layer (4) which is configured to ensure that the input components progressing in the microchannel (4.1) are mixed with a mixing efficiency exceeding 98% at high flow rates that reach up to 1-80000 pL / hour, by means of the effect of the vibration caused by the acoustic wavesgenerated by the converter (6), the pressure change, the oscillation of the middle layer (3), and the vibration of the air microbubbles (2.4) in the upper layer (2); and to synthesize ultra-small and size-tunable micro-nano-bio- materials and nanoparticles.

12. A micromixing platform (1) according to any one of the preceding claims; characterized by the lower layer (4) wherein the synthesis of micro-nano-bio- material and nanoparticle is performed in the curves thereof close to the inlet of the curved microchannel (4.1), whereas the stabilization of the micro-nano- bio-material and nanoparticle synthesised is performed on the side close to the outlet pipe (2.2).

13. A micromixing platform (1) according to any one of the preceding claims; characterized by the table (5) which serves the purpose of creating a support ground, after being combined to the upper, middle and lower layers (2, 3, 4).

14. A micromixing platform (1) according to any one of the preceding claims; characterized by the table (5) which is a plate-shaped structure larger than the upper, middle and lower layers (2, 3, 4) in terms of size, and has a size sufficient to enable the placement of the converter (6) close these layers.

15. A micromixing platform (1) according to any one of the preceding claims; characterized by the transducer (6) which is a piezoelectric structure that is positioned near the upper, middle and lower layers (2, 3, 4) on the table (5); and converts the electrical current into acoustic waves that generate mechanical vibration, by generating a radio frequency signal at the resonance frequency16. A micromixing platform (1) according to any one of the preceding claims; characterized by the transducer (6) which can generate acoustic waves propagating in all directions by relocating on the table (5), and can even generate waves propagating perpendicular to its axis.

17. A micromixing platform (1) according to any one of the preceding claims; which is used by following the process steps of basically provision of the components to generate the micro-nano-bio-material and nanoparticle -that is desired to be synthesized- into the microchannel (4.1) separately from the inlet pipes (2.1) in the structure of the combined upper, middle and lower layer (2, 3, 4) placed on the table (5); oscillation of the middle layer (3) caused by the acoustic waves generated by the components streaming at ultra-high speed in the microchannel (4.1) by means of the converter (6); synthesis of micro-nano- bio-materials and nanoparticles at high-efficiency by mixing with the vibration of the micro air bubbles in the upper layer (2) and the lower layer (4) and receiving the product synthesized from the outlet pipe (2.2).

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