Double emulsion structures for low-frequency ultrasound-induced drug delivery

Abstract

The present invention relates to methods and drug delivery systems for targeted drug delivery, and more particularly, to low frequency ultrasound induced drug delivery.

Description

[Technical Field]

[0001] The present invention relates to a method and drug delivery system for controlled drug delivery, and more particularly for low frequency ultrasound induced drug delivery. [Background technology]

[0002] Controlled drug delivery (CD) has been investigated as a solution to increasing the efficacy of treatments while reducing toxicity and side effects. Controlled drug delivery involves encapsulating an active substance into an object (e.g., a capsule, shell, etc.) and delivering it to the target site in a controlled manner. The drug is protected from numerous physiological barriers that would otherwise degrade it, resulting in a higher percentage reaching the target. The particles can then accumulate at the target site, either passively (by increasing vascular permeability and retention) or actively. Once the particles reach the target site, they can be destabilized by external stimuli, which can rapidly release their contents or induce diffusion of the drug from the particle into the surrounding environment. Liposomes that perform this function have been extensively studied, but only a few have been commercialized (e.g., Doxil, Onivyde).

[0003] The main disadvantage of liposomes is that they leak out before reaching the target location.

[0004] Other encapsulation methods include polymeric nanoparticles, which are difficult to produce in high throughput, or micelles, which can only accommodate lipophilic drugs.

[0005] Another encapsulation method involves the use of perfluorocarbon (PFC) oil emulsions. PFCs are biocompatible, immiscible with both water and organic oils, and possess the special ability to undergo a phase change when stimulated by ultrasound. Indeed, when exposed to acoustic pressures above a threshold, PFC droplets turn into gas bubbles. This process is called acoustic droplet vaporization (ADV). Because PFC oils are both hydrophobic and oleophobic, they can function as a shell surrounding a water or oil core that can solubilize drugs. The phase change caused by ADV is believed to be the mechanism responsible for ultrasound-induced delivery of active substances from PFC emulsions. A disadvantage of this mechanism is that acoustic pressure can also cause bubble implosion (called inertial cavitation). This is a powerful mechanism that can alter the encapsulated drug as well as cause harmful local side effects in surrounding tissues, especially when the drug is a protein such as an antibody.

[0006] Recently, it has been revealed that the use of non-perfluorocarbon oils that cannot be vaporized allows the diffusion mechanism to be induced using ultrasound without vaporization or thermal effects (Non-Patent Document 1). Although this paper describes the possibility of avoiding ADV release, it is only aimed at transporting hydrophobic bioactive compounds, while in the field of bioactive molecule control, there is still a need to be able to deliver hydrophobic and / or hydrophilic bioactive molecules. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] N. Al Rifai et al., Journal of Materials Chemistry B 8 (8) (2020) 1640-1648 Summary of the Invention

[0008] Surprisingly, the inventors have now been able to prepare new formulations that allow the targeted and localized release of any kind of compound, especially water-soluble compounds, by ultrasound.

[0009] The present invention relates to a double emulsion structure composed of a water-soluble compound contained in at least one aqueous droplet, said aqueous droplet being contained in an oily droplet, said structure allowing for on-demand release (responsive release) of said water-soluble compound when exposed to low frequency ultrasound, such release advantageously occurring without compromising the stability of the oil droplets.

[0010] That is, the present invention relates to a double emulsion structure composed of at least one aqueous droplet comprising at least one first biocompatible amphiphilic molecule and at least one water-soluble compound, said aqueous droplet being contained in an oily droplet comprising at least one biocompatible oil and at least one second biocompatible amphiphilic molecule, and said structure enabling responsive release of said water-soluble compound when exposed to low frequency ultrasound.

[0011] Thus, the double emulsion structure is composed of oil droplets with a size comprised between 100 nm and 1000 nm, and said oil droplets contain aqueous droplets with a size comprised between 50 nm and 800 nm.

[0012] The droplet size can be assessed as described in part I.5.3 of the Examples.

[0013] The oil droplets are composed of an aqueous phase (i.e., aqueous droplets) that occupies between 10% and 80% of the total volume of the oil droplets. Therefore, the oil phase of the oil droplets occupies between 20% and 90% of the total volume of the oil droplets. This ratio between the oil phase and the aqueous phase is selected according to the compound to be formulated in the double emulsion structure. For example, when only water-soluble compounds are formulated, the aqueous phase increases (for example, to 60% to 80% of the total volume of the oil droplets). When the compound to be formulated is a mixture of water-soluble compounds and lipophilic compounds, the ratio between the oil phase and the aqueous phase can be about 50:50.

[0014] The aqueous droplets are composed of a saline solution, at least one first biocompatible amphiphilic molecule, and at least one water-soluble compound, which is preferably a therapeutic active agent.

[0015] Preferably, the saline solution has a plasma osmolality and a concentration of NaCl in water of approximately 0.5% to 1.5% by weight.

[0016] The first biocompatible amphiphilic molecule may be a lipid, a protein (e.g., serum albumin), a polymer, or a surfactant (e.g., a dendrimer such as Dendri-TAC, F8TAC 13 oligomers such as FiTACn or HiTACn, TPGS 1000, TPGS 750M, Tween 20, Tween 80, amino acid-derived surfactants or sugar-derived surfactants), in the latter case surfactants having an HLB (hydrophilic-lipophilic balance) comprised between 3 and 6.

[0017] The oil droplets comprise at least one biocompatible oil and at least one second biocompatible amphiphilic molecule.

[0018] The second biocompatible amphiphilic molecule may be a lipid, a protein (e.g., serum albumin), a polymer, or a surfactant (e.g., a dendrimer such as Dendri-TAC, F8TAC 13oligomers such as FiTACn or HiTACn, TPGS 1000, TPGS 750M, Tween 20, Tween 80, amino acid-derived surfactants or sugar-derived surfactants), where the surfactants in the latter case have an HLB (hydrophilic-lipophilic balance) comprised between 8 and 18.

[0019] To ensure droplet stability and prevent aggregation, the droplet surface area must be filled with biocompatible amphiphilic molecules. The number of amphiphilic molecules required to fill the droplet surface area is calculated by dividing the droplet surface area by the amphiphilic molecule surface area. This final value can be derived from interfacial tension measurements. This technique involves measuring the interfacial tension of millimeter-sized water droplets in bulk oil, or alternatively, millimeter-sized oil droplets in bulk water, for various concentrations of amphiphilic molecules solubilized either in oil or water (depending on the amphiphilic molecule solubilization). For example, the droplet spinning technique (e.g., using the following device: https: / / www.directindustry.fr / prod / kruess-gmbh / product-14849-1772556.html), or the sessile and hanging drop techniques can be used. The interfacial tension value increases with increasing surfactant concentration, eventually reaching a plateau at the critical micelle concentration (CMC). Below the CMC, most of the amphiphilic molecules are located at the water / oil interface. The surface area of ​​a single amphiphilic molecule can be obtained by dividing the droplet surface area in millimeters (derived from the droplet size) by the number of amphiphilic molecules.

[0020] According to a first embodiment, the at least one biocompatible oil is a non-vaporizable perfluorocarbon (PFC) oil such as perfluorohexane, perfluorooctyl bromide, or perfluoro-15-crown-5-ether, and the second biocompatible amphiphilic molecule is a telomeric FTAC surfactant (e.g., F8TAC, which is composed of an 8-carbon fluorocarbon chain and 13 telomer repeats). 13 ), and fluorinated surfactants such as dentriTAC surfactants.

[0021] Advantageously, when prepared with PFC, passive diffusion of water-soluble compounds from the double emulsion structure does not occur, and release of the water-soluble compounds is achieved only when the double emulsion structure is exposed to low-frequency ultrasound. This property is of great interest for therapeutic applications, as there is no loss of the active agent.

[0022] According to a second embodiment, the at least one biocompatible oil is selected from the group consisting of glycerol monoesters, diesters, or triesters, derivatives of glycerol, monoesters, diesters, triesters, or tetraesters of citric acid, derivatives of citric acid, fatty acids, fatty acid monoesters, steroids, sphingolipids, glycerophospholipids, polyketics, saccharolipids, terpenes, prenol-derived lipids, essential oils, grease substitutes, waxes (triglycerides), and combinations of these aforementioned oil compounds. A preferred example of such an oil is tributyl-o-acetylcitrate (ATBC).

[0023] The double emulsion structure according to the present invention is one in which the oily droplets contain one or more aqueous droplets.

[0024] According to certain embodiments, the therapeutic active agent is a chemical compound, a protein, a peptide, an antibody, a DNA construct, or an RNA construct.

[0025] According to certain embodiments, a therapeutic agent is contained within the aqueous droplets of the double emulsion structure of the present invention.

[0026] According to another particular embodiment, at least two therapeutic agents are contained in the same or different aqueous droplets.

[0027] According to yet another embodiment, the oily droplets contain another therapeutically active agent.

[0028] That is, the double emulsion structure is an oil droplet that does not contain any active agent and contains only one aqueous droplet, which may contain one or more water-soluble therapeutic active agents; or oily droplets composed of a non-perfluorocarbonated oil containing at least one hydrophobic therapeutic active agent and containing only one aqueous droplet, said aqueous droplets may contain one or more water-soluble therapeutic active agents; or an oil-based droplet that does not contain any active agent and that comprises a plurality of aqueous droplets, each aqueous droplet comprising one or more soluble therapeutic active agents, and according to one particular embodiment, each aqueous droplet comprising one soluble therapeutic active agent, but wherein the soluble therapeutic active agent can vary from one aqueous droplet to another; or oil droplets composed of non-perfluorocarbonated oil comprising at least one hydrophobic therapeutic active agent and comprising a plurality of aqueous droplets, each aqueous droplet comprising one or more soluble therapeutic active agents, and according to one particular embodiment, each aqueous droplet comprising one soluble therapeutic active agent, but wherein said soluble therapeutic active agent may vary from aqueous droplet to aqueous droplet; It may consist of:

[0029] According to certain embodiments, the double emulsion structure of the present invention is lyophilized, i.e., in a non-encapsulated, substantially water-free, dry form. Lyophilization is well known to those skilled in the art and can be performed as described in "Perfluorocarbon nanodroplets as potential nanocarriers for brain delivery" (C. Berard, et al. Pharmaceutics 14 (2022) 1498).

[0030] Such a dry form of the double emulsion structure can be administered directly to a subject, for example, via topical, oral, intranasal routes, or a combination thereof. The dry form of the double emulsion structure can also be stored for a long period of time and then used in liquid form, for example, for intravenous administration, after adding water or an aqueous solution.

[0031] According to another particular embodiment, the double emulsion structure of the present invention is present in an aqueous continuous phase.

[0032] In such an embodiment, the present invention comprises: an aqueous continuous phase, a discontinuous phase of oily droplets containing aqueous droplets as defined above, This invention relates to a double emulsion for low frequency ultrasound induced drug delivery, comprising:

[0033] Advantageously, the double emulsion structure according to the present invention is capable of releasing at least one therapeutic active agent when exposed to ultrasound at a frequency of 0.5 MHz to 2 MHz for an ultrasonic exposure time of between 0.1 and 30 minutes, preferably between 0.1 and 5 minutes if the therapeutic active agent is water-soluble and between 0.1 and 20 minutes if the therapeutic active agent is hydrophobic.

[0034] The present invention therefore also relates to a method for the responsive release of water-soluble compounds, such as therapeutic agents, comprising exposing a double emulsion structure according to the invention to an acoustic signal at a frequency between 0.5 MHz and 2 MHz for an ultrasonic exposure time comprised between 0.1 and 30 minutes, preferably between 0.1 and 5 minutes, and if the double emulsion structure also comprises a hydrophobic therapeutic active agent, the ultrasonic exposure time is comprised between 0.1 and 20 minutes.

[0035] Preferably, the maximum peak negative pressure (expressed in absolute value) of the ultrasound to expel the contents of the droplet is between 0.2 MPa and 1 MPa.

[0036] By "responsive release of a compound" it is understood that the double emulsion structure of the present invention is capable of releasing said compound by stimulated diffusion when exposed to an acoustic signal, and such release ceases when said acoustic signal is gradually discontinued.

[0037] This control of the release of compounds contained in the double emulsion structure by acoustic signals allows for the targeting of very specific regions (organs or tissues) within the subject's body, which is therefore highly advantageous for therapeutic treatments, as it allows therapeutic active agents to be released only where they are needed, limiting loss of the therapeutic active agents and the potential for adverse effects without therapeutic benefit.

[0038] The present invention therefore relates to the double emulsion structure of the present invention for use as a medicament.

[0039] The choice of therapeutic active agent is not limited depending on the disease to be treated, for example, the therapeutic active agent may be an anti-cancer agent, an anti-inflammatory agent, an antioxidant agent, an anti-thrombotic agent, an antibiotic agent, an anti-bacterial agent, an anti-viral agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof.

[0040] Therapeutic uses of the double emulsion structure include administering it to a living organism, which may be by intravenous, oral, topical, intranasal, pulmonary, transmucosal routes, or a combination thereof. According to one or more embodiments, the double emulsion structure is administered intravenously.

[0041] The formulation of the double emulsion structure of the present invention is selected depending on its route of administration.

[0042] For example, in addition to dry and liquid forms, the double emulsion structure can be incorporated into implantable gels such as subcutaneous gels, skin patches or dressings, sprayable formulations, and the like.

[0043] In certain embodiments, the oil droplets of the emulsion further comprise a receptor-specific ligand for the organism to which the double emulsion structure is administered. In this way, control of drug delivery within the body and / or targeting of target tissues or organs, such as tumors, sites of infection or inflammation, can be enhanced. Preferably, the ligand is hydrophilic and may be attached at the surface of the oil droplets to amphiphilic molecules that encapsulate the oil droplets.

[0044] The present invention further relates to a process for preparing the double emulsion structures of the present invention, wherein said double emulsion structures are fabricated in at least one microfluidic device using a fluid focusing geometry, an example of such a process is detailed in part I.3 of the Examples.

[0045] The present invention also relates to a kit for treating a patient suffering from a disease, comprising a double emulsion structure according to the present invention and a focused ultrasound device (FUS). [Brief explanation of the drawings]

[0046] [Figure 1] Figure 1 shows a schematic representation of all droplet types used. A: Multicore PFH double emulsion. B: Single-core PFH double emulsion. C: Multicore ATBC emulsion. D: Plain ATBC emulsion. Droplets A to C are used to carry hydrophilic cargo, while droplet D is used to carry hydrophobic cargo. [Figure 2]A: Schematic of the fluid-focusing junction used to generate plain ATBC single emulsion droplets. The ATBC dispersed phase (pink) reaches the nozzle level and is sandwiched by the continuous phase to generate monodisperse droplets. The nozzle width is 40 μm. All channels are 20 μm deep and 100 μm wide. B: Schematic of the process for generating multi-core (PFH or ATBC) double emulsions. The core aqueous and oil phases are sonicated to generate primary nanoemulsions. C: Schematic of the two-nozzle device made of two fluid-focusing junctions (1-2-3 and 3-4-5) used to generate single-core double emulsions. The channel between the two junctions (channel 3) is hydrophobic. The flow rate was adjusted to encapsulate exactly one aqueous droplet in each double emulsion. All channels are 50 μm deep and 100 μm wide. The first nozzle (located at the confluence 1-2-3) is 35 μm, and the second nozzle (located at the confluence 3-4-5) is 70 μm. [Figure 3]A: Fluorescent probe emission from PFH and ATBC droplets normalized by R and n (where R is the radius and n is the number of droplets in the focal region of the transducer) plotted against acoustic energy. The meaning of the symbols is summarized on the right side of the figure. The first number specifies the radius of the droplet, the second number specifies the number of periods in the signal, MC indicates a multicore droplet, and PFH or ATBC indicates the oil used to create the droplet. Fitting the data for 20 μm radius droplets to equation (2) yields β = 5.33 × 10 μm (± 6.05 × 10) and α = 0.686 (± 0.006). Black dots: 20 μm 5c MC PFH, black and white dots: 20 μm 20c MC PFH, white dots: 20 μm 50c MC PFH, black squares: 20 μm 5c ATBC, white squares: 20 μm 5c ATBC, downward-pointing white triangles: 30 μm 5c ATBC, upward-pointing black triangles: 20 μm 5c MC ATBC, and upward-pointing white triangles: 20 μm 50c MC ATBC. B: Fitting of the release from 30 μm multicore PFH (MC PFH) droplets (shown as open blue circles) and plain ATBC (ATBC) droplets (shown as open blue squares) treated with 50 cycles of pulses to Eq. 1, where β = 8.85 × 10 μm (± 3.52 × 10) and α = 0.71 (± 0.03). Open dots: 30 μm 50c MC PFH and open squares: 30 μm 50c ATBC. [Figure 4]Figure 1 shows the fluorescent probe emission from single-core PFH droplets normalized by R3 and nf (where R is the radius and nf is the number of droplets in the focal region) plotted against acoustic energy. Droplets with a 20 μm radius are plotted as solid triangles, and droplets with a 30 μm radius are plotted as open triangles. Droplets treated with 5 pulses are shown in red, and droplets treated with 50 pulses are shown in blue. β = 8.47 × 10-7 μm-3 (± 5.38 × 10-8) and α = 1.60 (± 0.03). Black dots: 20 μm 5c SC PFH; white dots: 20 μm 50c SC PFH; black squares: 30 μm 5c SC PFH; and white squares: 30 μm 50c SC PFH. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0047] The following examples demonstrate that perfluorohexane (PFH) emulsions and tributyl-o-acetylcitrate (ATBC) emulsions can release their contents at high acoustic frequencies without an ADV.

[0048] We evaluate the release of the hydrophilic fluorescent probe, sodium fluorescein, from three types of water-core double emulsion droplets (Figure 1): Multiple water nanodroplets in PFH in water (called multi-core PFH droplets), A single water microdroplet in PFH in water (called a single-core PFH droplet), and Multiple water nanodroplets in ATBC in water (called multi-core ATBC droplets).

[0049] The release of Nile Red, a hydrophobic fluorescent probe, from a single emulsion of ATBC in water, called plain ATBC droplets, was also studied.

[0050] I. Materials and Methods I.1. Materials Unless otherwise specified, all chemicals were filtered using a 0.2 μm pore size Acrodisc syringe filter (Pall, France) and used without further purification. All aqueous solutions were prepared using a Milli-Q IQ 7000 Type 1 water purification system. PDMS Sylgard 184 was purchased from Neyco (France). Photoresist SU8 was purchased from Chimie Tech Services (France). Silicon wafers were obtained from BT Electronics (France). The MFCS-EZ pressure controller used to inject fluids into the microfluidic chip and the flow units (size S and size M) used to measure flow rates were purchased from Fluigent (Le Kremlin-Bicêtre, France). A 0.35 mm diameter biopsy puncher was purchased from World Precision Instruments (UK), while a plasma cleaner was purchased from Harrick Scientific (New York, USA).

[0051] Perfluorohexane and fluorinated surfactant Krytox FSL 157 were purchased from ABCR GmbH (Germany) and Costenoble GmbH (Germany), respectively. 13 and H 12 The TAC7 is home-made (see below).

[0052] Nile red was purchased from Merck (France) and sodium fluorescein was obtained from VWR (France).

[0053] Tributyl O-acetylcitrate, methanol, and NaCl were obtained from Sigma Aldrich (France).

[0054] I.2.F8TAC 13 Surfactants and H 12 Synthesis of TAC7 surfactant F-TAC and H-TAC surfactants are amphiphilic molecules composed of two structural units: [ka]

[0055] One of the units is a water-soluble oligomer of tris(hydroxymethyl)aminomethane (Tris) acrylamide units, which constitute the polar head of the surfactant. Depending on the conditions under which these syntheses are carried out, the average number of Tris-acrylamide units can be adjusted.

[0056] The second unit is a fluorinated tail (in the case of FTAC) or a hydrocarbonated tail (in the case of H-TAC), which acts as a fluorophilic or hydrophobic anchor, ensuring the stabilization of the PFC or ATBC droplets. The F-TAC used in this study is made of a perfluorooctyl tail with 13 Tris-acrylamide units (F8TAC). 13 The H-TAC used consists of a dodecane tail with seven Tris-acrylamide units.

[0057] I.3. Microfluidic Device for Generating Droplets Droplets were produced in a microfluidic device using a fluid focusing geometry (Figure 2).

[0058] Two types of chips were produced.

[0059] The first chip is suitable for generating plain ATBC single emulsion droplets and multicore double emulsion droplets (Figure 2A and B) and uses a single fluid-focusing junction. All channels are 20 μm deep, 100 μm wide, and have a nozzle size of 20 μm.

[0060] The second type of chip, shown in Figure 2C, was used to generate single-core PFH droplets and consisted of two fluid-focusing junctions arranged in series within a single chip. All channels were 50 μm deep and 100 μm wide, with nozzle sizes of 35 μm (the first nozzle between sections 1-2-3 in Figure 2C) and 70 μm (the second nozzle between sections 3-4-5 in Figure 2C). Both chips were initially designed in AutoCAD. They were then directly printed on a nitrogen-plasma-cleaned silicon substrate using a two-photon polymerization printer, Nanoscribe's GT Photonic Professional device, with a negative photoresist IP-S (Nanoscribe GmbH, Germany) and a 25x objective. To reduce printing time, a shell-writing strategy was applied. This strategy fabricates a dense shell that defines the boundary of the structure, while the inner part is only partially polymerized in the form of a scaffold.

[0061] After 30 minutes in propylene glycol methyl ether acetate (PGMEA) and development in isopropanol (5 minutes), batch polymerization was performed with UV exposure. Polydimethylsiloxane (PDMS) and its curing agent were used in a 10:1 ratio to create the microfluidic chip by pouring it onto a wafer, which served as a mold for the circuitry. This was then degassed in vacuum and baked at 70°C for 2 hours. Inlets and outlets were punched using a 0.35 mm diameter biopsy puncher. The chip was then cleaned with isopropanol and dried using nitrogen gas.

[0062] I.4. Surface Treatment I.4.1. Multicore Droplets and Plain Droplets A single-fluid focusing chip was used for a single emulsion, and the multicore droplets underwent a hydrophilic surface treatment. Both the circuit side of the chip and a microscope slide were activated with air plasma (18 W for 1 min). They were then brought into contact and bonded together, then placed in an oven at 70 °C for 30 min to strengthen the bond.

[0063] The bonded chip was cooled to room temperature and reactivated in air plasma for 1 minute. Water was injected into the chip using a Fluigent pressure controller to make the circuit walls hydrophilic.

[0064] I.4.2. Single-core droplet The dual fluid focusing chip used to create the single-core double emulsion was selectively treated. The section shown in dark gray in Figure 2C was kept hydrophobic because the phase in contact with the channel wall in this region was PFH.

[0065] Therefore, to avoid destabilization of the first emulsion, this region must be kept hydrophobic. The rest of the chip (blue) was treated to be hydrophilic because this is where water comes into contact with the channel walls (Figure 4C, 1, 4, and 5). Instead of a glass slide, a flat PDMS layer was used as the substrate for the chip. Following the procedure developed by Bodin-Thomazo et al. (2017) [N. Bodin-Thomazo, F. Malloggi, P. Guenoun, "Marker patterning: a spatially resolved method for tuning the wettability of PDMS," RSC Adv. 7 (73) (2017) 46514-46519. doi: 10.1039 / C7RA05654K], the hydrophobic regions were selectively patterned using a black permanent marker (Stabilo Superfine) to prevent their activation during subsequent plasma treatment. The mirror lines of this marker pattern were written onto the PDMS layer substrate. The chip and substrate were placed in air plasma (18 W for 1 minute), and then aligned to match the marker pattern. Two minutes after bonding the chip, the marker was removed by passing methanol at 20 mbar for 2 minutes using a pressure controller. This was followed by passing water for 30 minutes.

[0066] I.5. Generation of multicore double emulsion droplets Multicore double emulsions consist of three phases (Figure 1, top and bottom left): The dispersed phase consists of water containing 0.9 wt% NaCl and 1 wt% sodium fluorescein. The mesophase is made of PFH or ATBC and 5 wt% Krytox 157 FSL. The continuous phase is water and 0.1 wt% F8TAC 13 (When using PFH in the mesophase) or H 12 It is made of TAC7 (when using ATBC in the interphase).

[0067] First, primary nanoemulsions of saline (0.9 wt% NaCl in water) and fluorescein in oil were generated by surrounding nanodroplets within larger oil droplets with water. The pressures and flow rates used to generate these emulsions are shown in Table 1 below:

[0068] [Table 1]

[0069] I.5.1. Generation of Water Single-Core PFH Droplets Single-core double emulsion droplets consist of three phases: The dispersed phase consists of water containing 0.9 wt% NaCl and 1 wt% sodium fluorescein. The mesophase is made of PFH and 5 wt% Krytox 157 FSL. The continuous phase is water and 0.1 wt% F8TAC 13 It is made of.

[0070] These were generated using a double fluidic focusing and junction device, shown in Figure 2C, where section 1 represents the channel where the dispersed phase enters, section 2 represents the channel where the middle phase enters, section 3 represents the channel where a primary emulsion of water in PFH is formed, section 4 represents the channel where the continuous phase enters, and section 5 represents the channel where a double emulsion of single aqueous droplets in PFH in water is formed. After surface preparation, the various fluid phases were injected into the chip by applying 20 mbar pressure to the headspace of each inlet using a pressure controller. The flow rates were adjusted to ensure exactly one aqueous droplet was encapsulated in each double emulsion, as shown in Table 2:

[0071] [Table 2]

[0072] I.5.2. Generation of Plain Tributyl o-acetylcitrate Droplets A plain ATBC single emulsion was made using a dispersed phase of ATBC and 0.025 wt% Nile Red. The continuous phase was water and 0.1 wt% H 12 It was made of TAC7.

[0073] The droplets were made using the same tip geometry (Figure 2A) as for multicore double emulsion droplets, where the dispersed phase was sandwiched between the continuous phase at the nozzle to generate the droplets. The pressures and flow rates used are listed in Table 1.

[0074] I.5.3. Droplet Characterization The size and polydispersity of the droplets were determined from a series of images taken with an ultrafast camera (model SC1, Edgertronic, USA) during droplet formation.

[0075] The collected recordings were analyzed using an in-house MATLAB program, which uses an algorithm based on the Circular Hough Transform (CHT) to identify the droplet locations and then estimate their diameters as well as their polydispersity index (PDI).

[0076] The aqueous volume fraction in single-core double emulsion droplets is calculated by monitoring two parameters: the number of droplets produced per second, which can be captured via a high-speed camera, and the flow rates of each phase, which are recorded from the flow units of the pressure controller. The volume fraction is then predicted using an in-house MATLAB code.

[0077] The size and polydispersity of the nanosized droplets within the multicore droplets were determined by dynamic light scattering using an ALV / CGS-3 platform-based goniometer system (ALV GmbH). Measurements were performed on 1000-fold diluted emulsions at room temperature and at scattering angles θ ranging from 60° to 130° in 10° increments. At each angle θ, the device exhibited a decay rate Γ θ =q 2 (k B T / 6πηR), where k B is the Boltzmann constant, T is the temperature in K, η is the viscosity of the solvent, and q(θ) = 4πnsin(θ / 2) / λ is the magnitude of the scattering vector. The refractive index of the solvent is n = 1.33, and the wavelength of the laser is λ = 633 nm.

[0078] By fitting the curves using the cumulant method, it was possible to determine the polydispersity index (PDI) as well as the hydrodynamic droplet mean radius R [AG Mailer, PS Clegg, PN Pusey, "Particle sizing by dynamic light scattering: non-linear cumulant analysis", Journal of Physics: Condensed Matter 27 (14) (2015) 145102].

[0079] I.6.Interfacial tension The interfacial tensions between the three aqueous and PFH phases were measured at 20 °C with an error of 2 mN / m using a Tracker tensiometer (Teclis, France). The tensiometer derives the interfacial tension by analyzing the shape of a hanging or rising drop within the bulk using the Young-Laplace equation. 13 For measurements between water containing Krytox or between water and PFH containing 5% Krytox, the pendant drop method was used [JD Berry, MJ Neeson, RR Dagastine, DY Chan, RF Tabor, "Measurement of surface and interfacial tension using pendant drop tensiometry," Journal of Colloid and Interface Science 454 (2015) 226-237]. The volume of the water drop was set to 2 mm. 3 The temperature was kept constant at 100°C. The interfacial tension was allowed to reach an equilibrium value before extracting the interfacial tension value. 13 A different method was used when measurements were made between PFH and water containing 5% Krytox. The bulk was PFH and 5% Krytox on the bottom, and water containing F8TAC on top (because PFC is denser than water, the interface between the two was stable). A 0.6 mm diameter curved needle in a Hamilton 700 series syringe was used to form a rising water droplet in the PFH and Krytox phase. The rising water droplet was therefore covered in Krytox, creaming to the interface and releasing the F8TAC. 13 The droplets progressed halfway into the water containing the phase. The droplets remained stable at the interface for several minutes before collapsing. The interfacial tension was measured at the upper interface of droplets similar to the inner droplets of the single-core double emulsion droplets in the dewetting configuration. As a control test, the interfacial tension of a static air bubble in water was measured, yielding a value of 72 mN / m.

[0080] I.7. Determining the ADV Threshold ADV threshold of PFH droplets (P ADVTo determine the acoustic vaporization threshold, the intersect method has been used, as described by Aliabouzar et al., Osborn et al., and Fabiilli et al. [J. Osborn et al., "Acoustic Droplet Vaporization of Perfluorocarbon Droplets in 3D-Printable Gelatin Methacrylate Scaffolds," Ultrasound in Medicine & Biology 47 (11) (2021) 3263-3274; Aliabouzar et al., "Effects of droplet size and perfluorocarbon boiling point on the frequency dependence of acoustic vaporization threshold," The Journal of the Acoustical Society of America 145 (2) (2019) 1105-1116; M. Fabiilli et al., "The role of inertial cavitation in acoustic droplet vaporization," IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control 56 (5) (2009) 1006-1017. Briefly, the fast Fourier transform of the acoustic signal was analyzed, and the magnitude of the subharmonic peak at 0.5 MHz, an indicator of ADV, was normalized by pressure. This value was plotted against the acoustic pressure. P ADV was determined as the pressure at which the normalized magnitude began to increase (2.1 MPa). Alternatively, the probability of ADV occurring for each pressure was determined.

[0081] Each time a subharmonic peak at 0.5 MHz was observed, it was counted as one event. The number of events in 100 pulses was counted to obtain the probability. The pressure value at which the probability p was 1 / 2 was the P obtained by the crossover method. ADV This probability method was then used to calculate the P values ​​of the multi-core PFH droplets and the single-core PFH droplets. ADV was chosen because this probability method is more accurate and can be fitted to a probability model.

[0082] I.8. ​​Delivery of fluorescent probes from droplets I.8.1. Sample preparation To remove free fluorescent probe from the sample, the droplet solution was pipetted into a tube with a membrane size of 100 kDa MWCO (Thermo Scientific, UK) and filtered. The sample was centrifuged at 12,000 g for 40 minutes at 4°C. The filtered solution was removed and replaced with fresh bulk solution. This filtration via centrifugation step was repeated five times to achieve complete removal (until the filtered solution contained no fluorescent probe detectable by spectrofluorometer).

[0083] For the fluorescein release experiment, a sample containing 1 mL of saline (9 g NaCl per L of water) and 5 μL of droplet solution collected from the bottom of the droplet sample tube where the droplets had settled was prepared in a 2 mL tube (MC 200, Fisherbrand). For the Nile Red release experiment, the sample contained 1.5 μL of droplet solution, 200 μL of saline, and 100 μL of ATBC. Because ATBC is less dense than water, it formed a layer on top of the water. Because Nile Red is hydrophobic, it was released from the droplet into the water but migrated to the oil layer.

[0084] I.8.2. Acoustic treatment of the sample The signal consisted of sinusoidal bursts at a fundamental frequency of 1.1 MHz. Duty cycles of 5% and 50% were used (i.e., the sample was insonified for 5% and 50% of the total time, respectively), and the pulse repetition frequency was 11 kHz. Peak negative acoustic pressures ranging from 0.2 MPa to 2.3 MPa were used. The total experimental time was 7 minutes, but the actual insonation time was either 0.35 minutes or 3.5 minutes, and the temperature difference did not exceed 1.5°C. For each sonicated sample, 1 mL of saline was pipetted into a 2 mL centrifuge tube, droplets were added, and the sample tube was placed on the sonicator for insonation. Two control samples were used at the beginning of each experimental set. One control sample served as a 100% release reference. The same volume of droplet solution was destabilized in 200 μL of methanol, followed by the addition of 800 μL of saline. The second control was a sample of 1 mL of saline and the same volume of droplet solution as the other samples. Passive release was assessed over a 7 minute period without acoustic treatment.

[0085] I.8.3. Determination of Percentage Release by Fluorescence Detection Emission was determined from the fluorescent energy of the probe detected in the sample supernatant. For experiments using fluorescein, 850 μL of the supernatant was pipetted into a 1 mm thick spectrophotometric cuvette (Hellma). The cuvette was placed in a spectrofluorometer (Jasco FP-8300, Germany). Fluorescence was analyzed using an excitation wavelength of 470 nm and an emission wavelength of 513 nm. To detect Nile Red, 70 μL of the top ATBC layer in the sample was pipetted into a 100 μL 1 mm thick spectrophotometric cuvette (Hellma). The excitation wavelength was 530 nm, and the emission wavelength was 568 nm. The released concentration was obtained from a standard curve for each fluorescent probe. The released concentration was compared to the initial concentration in the droplet to obtain a percentage.

[0086] I.8.4. Analysis of Emission Data For each sample, the pressure at which the experiment was performed was converted to an acoustic energy value. The acoustic energy at each pressure for each number of cycles was:

number

[0087] The percentage of emission is R 3 (where R is the radius of the droplet) and the number of droplets in the focal region n f was normalized by

[0088] The equation used to fit the data is

number

number

[0089] II. Results II.1. ADVICE OF MULTI-CORE PFH DOUBLE EMULSION AND SINGLE-CORE PFH DOUBLE EMULSION P of PFH droplets in multi-core double emulsions and single-core double emulsions ADV are 2.2 MPa and 1.8 MPa, respectively (Fig. 6).

[0090] II.2. More efficient release from larger droplets The pressure at the onset of ejection for both types of PFH droplets is ADV Below (Table 3):

[0091] [Table 3]

[0092] The percentage release from 30 μm droplets is higher than from 20 μm droplets at all pressures. The pressure at which ATBC-based droplets begin to release their contents and the maximum percentage release are comparable to those of PFH-based droplets of the same radius. Incorporation of a hydrophilic dispersed phase within the core does not affect the pressure at which release begins, but reduces the maximum percentage release by 8%. Overall, 30 μm droplets release a higher percentage of their contents than 20 μm droplets at all pressures and for all droplet types, but the pressure at which release begins is not significantly different.

[0093] II.3. Increasing acoustic energy increases emission ADV has been shown to occur during the first cycle of the first pulse of the acoustic signal, provided that the threshold pressure has been achieved. Increasing the number of cycles, and therefore the acoustic energy to the system (Equation 1), from a 5-cycle pulse to a 50-cycle pulse should not affect emission in any way, provided that the threshold pressure for ADV has been reached. The percentage of emission is calculated as R 3 and n f (where R is the radius of the droplet and n f The data is normalized by the number of droplets in the focal region (where σ is the number of droplets in the focal region) and plotted against the acoustic energy (Figure 3). As the acoustic energy increases, an increase in release is observed, thus confirming the absence of ADV. This behavior occurs for both PFH and ATBC droplets, suggesting that the release mechanism is the same.

[0094] II.4. More efficient release from single-core droplets compared to multi-core droplets Ejection from single-core PFH droplets occurs more rapidly compared to multi-core droplets (Figure 4). Similarly, for multi-core PFH droplets, nearly 50% of ejection occurs from both 20 μm and 30 μm droplets before the ADV threshold.

[0095] III. Conclusion We investigated the ultrasonic release of fluorescent molecules from droplets made of biocompatible oils. The droplets were generated by microfluidics technology and stabilized with surfactants and dispersed in water. The hydrophobic dye was sequestered within the ATBC droplets, while the hydrophilic dye was present in either many water nanodroplets or a single microdroplet dispersed in ATBC or PFH oil droplets.

[0096] The pressure (P) required to acoustically vaporize droplets using 1.1 MHz waves ADV Dye release occurs at pressures significantly lower than P = 0.1 P (approximately 2 MPa). ADV A pressure of approximately 0.3 MPa releases approximately 30% of the dye after only 2.3 minutes of ultrasound exposure. Wave-induced temperature rises of no more than 1.5°C were observed.

[0097] The diffusion model can describe the release from any droplet type.

[0098] One of the main advantages of the double emulsion of the present invention is that it ensures the most efficient delivery of biologically active substances. For example, when a mixture of antibiotics, one hydrophilic and the other hydrophobic, is delivered using a tablet, the antibiotics must be concentrated so that an effective dose of the mixture can pass through the intestinal barrier and act against bacteria in the body. By using the double emulsion structure of the present invention, the antibiotic mixture can be targeted directly to the location where it is needed (e.g., directly at the lung level in the case of treating chronic obstructive pulmonary disease (COPD)) with a reduced amount of active substance than in a tablet. One hydrophobic one is located in the water of the droplet, and the other hydrophobic one is located in the biocompatible oil droplet.

Claims

1. A double emulsion structure composed of at least one aqueous droplet comprising at least one first biocompatible amphiphilic molecule and at least one water-soluble compound, said aqueous droplet being contained in an oily droplet comprising at least one biocompatible oil and at least one second biocompatible amphiphilic molecule, said structure enabling responsive release of said water-soluble compound when exposed to low frequency ultrasound.

2. 2. The double emulsion structure of claim 1, wherein the size of the oily droplets is comprised between 100 nanometers and 1000 nanometers.

3. 3. A double emulsion structure according to claim 1 or 2, wherein the size of the aqueous droplets is comprised between 50 nanometers and 800 nanometers.

4. The double emulsion structure of any one of claims 1 to 3, wherein the aqueous droplets comprise a saline solution.

5. 5. The double emulsion structure of claim 4, wherein the saline solution has a plasma osmolality and a concentration of NaCl in water of approximately 0.5% to 1.5% by weight.

6. A double emulsion structure according to any one of claims 1 to 5, wherein said at least one water-soluble compound is a therapeutically active agent.

7. A double emulsion structure according to any one of claims 1 to 6, wherein the lipid droplets comprise at least one biocompatible oil and at least one second biocompatible amphiphilic molecule.

8. 8. The double emulsion structure according to any one of claims 1 to 7, wherein the biocompatible oil is selected from the group consisting of PFC or ATBC.

9. 9. A double emulsion structure according to any one of claims 1 to 8, wherein the lipid droplets comprise one or more aqueous droplets, and the aqueous droplets comprise one or more different therapeutically active agents.

10. A double emulsion structure according to any one of claims 1 to 9, wherein the therapeutically active agent is a chemical compound, a protein, a peptide, an antibody, a DNA construct, or an RNA construct.

11. 11. A double emulsion structure according to any one of claims 1 to 10, wherein said structure releases said therapeutic active agent when exposed to an acoustic signal at a frequency between 0.5 MHz and 2 MHz for an ultrasonic exposure time comprised between 0.1 and 5 minutes.

12. 12. The double emulsion structure of claim 11, wherein the maximum peak negative pressure to expel the contents of the droplets is between 0.2 MPa and 1 MPa.

13. A double emulsion structure according to any one of claims 1 to 12, wherein the lipid droplets comprise a hydrophobic therapeutic active agent.

14. A double emulsion structure according to any one of claims 1 to 13, wherein the structure is a liquid structure or a freeze-dried structure.

15. A double emulsion comprising the double emulsion structure of any one of claims 1 to 13 in an aqueous continuous phase.

16. The double emulsion structure according to any one of claims 1 to 14 for use as a medicine.

17. A method for the responsive release of water-soluble compounds, comprising exposing the double emulsion structure of any one of claims 1 to 14 to an acoustic signal at a frequency between 0.5 MHz and 2 MHz for an ultrasonic exposure time comprised between 0.1 and 30 minutes.

18. A process for preparing the double emulsion structure according to any one of claims 1 to 15, wherein said double emulsion structure is fabricated in at least one microfluidic device using a fluid focusing geometry.

19. A kit for treating a patient suffering from a disease, comprising the double emulsion structure of any one of claims 1 to 14 and a focused ultrasound device (FUS).

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