System for producing a mixture of fluids in a microfluidic channel and associated method notably for formulating liposome-based medicaments through the alternating injection of two liquid phases

Abstract

The invention relates to a system (10) for producing a mixture of fluids, comprising: - a pressure source (11), - a pressure regulator (12), - at least a first container (16.1) containing a first fluid (15.1) and a second container (16.2) containing a second fluid (15.2), - a microfluidic mixer (20), and - a control unit (40) able to control a pressure level of the first fluid (15.1) and a pressure level of the second fluid (15.2) and an opening and closing of the first valve (23.1) and of the second valve (23.2) so as to inject the first fluid and second fluid successively into the microfluidic mixer (20) so as to generate a profile of fringes (F1, F2) of the first fluid (15.1) and of the second fluid (15.2) within the common outlet channel (34).

Description

DESCRIPTION TITLE: SYSTEM FOR PRODUCING A MIXTURE OF FLUIDS IN A MICROFLUID CHANNEL AND ASSOCIATED METHOD, PARTICULARLY FOR THE FORMULATION OF LIPOSOME-BASED MEDICINES BY THE CHOPPED INJECTION OF TWO LIQUID PHASES

[0001] The present invention relates to a system for producing a mixture of fluids in a microfluidic channel and an associated process, in particular for the formulation of liposome-based drugs by the chopped injection of two liquid phases.

[0002] Several systems for mixing organic-lipid solvent solutions and aqueous solutions in a microfluidic circuit have been described in the state-of-the-art literature. Existing methods most commonly involve passive mixers, such as hydrodynamic focusing or chaotic mixers, whose mixing performance is affected by variations in the flow rates of the mixed phases. This limits scalability for the production of high-volume liposomal drugs. Furthermore, existing devices present aggregation problems that can foul the microfluidic channels.

[0003] The invention aims to effectively overcome these drawbacks by proposing a system for producing a fluid mixture comprising: - a source of pressure, - a pressure regulator to which said pressure source is connected, - at least one first container containing a first fluid and a second container containing a second fluid, the pressurization of the first fluid and the second fluid being controlled by the pressure regulator, - a microfluidic mixer comprising at least one first inlet port and a second inlet port associated with at least one first valve and a second valve, - the first container being connected to the first inlet port via the first valve and the second container being connected to the second inlet port via the second valve, - said microfluidic mixer further comprising at least one first microfluidic conduit and one second microfluidic conduit, the first inlet microfluidic conduit being in fluidic communication with the first inlet orifice and the second inlet microfluidic conduit being in fluidic communication with the second inlet orifice, said first inlet microfluidic conduit and said second inlet microfluidic conduit intersecting at a non-zero angle to each other at the point of an intersection opening onto at least one common outlet channel, and - a control unit capable of controlling a pressure level of the first fluid inside the first container and a pressure level of the second fluid inside the second container, as well as an opening and closing of the first valve and the second valve to carry out successive injections of the first fluid and the second fluid into the microfluidic mixer, so as to generate a fringe profile of the first fluid and the second fluid inside the common outlet channel.

[0004] The invention thus enables the rapid and homogeneous mixing necessary for the formulation of small nanoparticles, particularly those smaller than 100 nm. Due to the absence of a static liquid-liquid interface and the ultra-rapid pressure variations, the invention drastically reduces nanoparticle aggregation and accumulation in the microfluidic channel. Furthermore, preventing fouling of the microfluidic circuit reduces the formation of fluidic instabilities. In addition, the invention reduces the dilution time of an organic-lipid phase in an aqueous phase to less than 1 ms through the alternating and optimized generation of pulsed organic-lipid and aqueous fringes, allowing for the formulation of liposomes with high size monodispersities. Preferably, the Polydispersity Index (PDI) is less than 0.1.

[0005] According to one embodiment of the invention, the control unit is configured to generate a fringe profile comprising an alternation of fringes of the first fluid and fringes of the second fluid.

[0006] According to one embodiment of the invention, the control unit is configured such that the fringes of the first fluid are narrower than the fringes of the second fluid.

[0007] According to one embodiment of the invention, a ratio between a volume of a fringe of the second fluid divided by a volume of a fringe of the first fluid is between 2 and 20, in particular between 8 and 15 and preferably between 9 and 11.

[0008] According to one embodiment of the invention, the first container contains a solution of lipids and / or polymers diluted in an organic solvent corresponding to the first fluid and the second container contains an aqueous solution corresponding to the second fluid.

[0009] According to one embodiment of the invention, the first valve and the second valve are solenoid valves with low dead volume, in particular less than 5 pL, and high responsiveness, in particular less than 5ms.

[0010] According to one embodiment of the invention, the common output channel is extended by a channel having a cross-sectional width greater than that of the common output channel.

[0011] According to one embodiment of the invention, said system includes an interchangeable flow sensor for measuring a fluid flow rate at the outlet of the microfluidic mixer.

[0012] According to one embodiment of the invention, the first inlet microfluidic conduit and the second inlet microfluidic conduit intersect at an angle equal to or less than 90 degrees.

[0013] According to one embodiment of the invention, the first inlet microfluidic conduit and the second inlet microfluidic conduit each have a cross-sectional height between 150 pm and 300 pm and a value of preferably on the order of 200 pm and a width between 150 pm and 300 pm, and preferably on the order of 200 pm wide

[0014] According to one embodiment of the invention, the pressure level of the first fluid inside the first container and the pressure level of the second fluid inside the second container are each between 0 and 8000 mbar.

[0015] According to one embodiment of the invention, an injection frequency equal to the inverse of the sum of an injection duration of the first fluid and an injection duration of the second fluid is between 0.1 Hz and 200 Hz and is preferably between 10 Hz and 100 Hz.

[0016] The present invention will be better understood and other features and advantages will become apparent upon reading the following detailed description, which includes embodiments given by way of illustration with reference to the accompanying figures, presented by way of non-limiting examples, which may serve to complete the understanding of the present invention and the explanation of its implementation and, where appropriate, contribute to its definition, on which:

[0017] [Fig. 1] Figure 1 is a schematic representation of a system for producing a mixture of two fluids according to the present invention;

[0018] [Fig. 2] Figure 2 is a cross-sectional view of a microfluidic conduit used in the system according to the present invention.

[0019] [Fig. 3] Figure 3 illustrates the process of generating alternating fringes in the static microfluidic mixer according to the present invention;

[0020] [Fig. 4] Figure 4 shows, during a phase of injection of alternating fringes of the first fluid and the second fluid, a time-dependent representation of the flow rate of the first fluid and the second fluid.

[0021] It should be noted that the structural and / or functional elements common to the different embodiments have the same references. Thus, unless otherwise stated, such elements have identical structural, dimensional, and material properties.

[0022] Figure 1 shows a fluid mixing production system 10 comprising a pressure source 11 which may, for example, take the form of an air compressor or a cylinder containing a pressurized gas, such as air or nitrogen.

[0023] The pressure source 11 is connected, via a conduit 13, to a pressure regulator 12, which controls the pressurization of a first fluid 15.1 and a second fluid 15.2 contained respectively inside a first container 16.1 and a second container 16.2. The pressure regulator 12 is connected to the first container 16.1 via conduit 17. The pressure regulator 12 is connected to the second container 16.2 via conduit 18. The pressure regulator 12 may be a PID (Proportional, Derivative, Integral) type regulator based on the use of highly sensitive piezoelectric sensors.

[0024] Advantageously, the first container 16.1 contains a solution of lipids and / or polymers diluted in an organic solvent corresponding to the first fluid 15.1 (organic phase). The second container 16.2 contains an aqueous solution corresponding to the second fluid 15.2 (aqueous phase).

[0025] A microfluidic mixer 20 has a first inlet port 21.1 to which the first container 16.1 is connected via a first valve 23.1 and a second inlet port 21.2 to which the second container 16.2 is connected via a second valve 23.2. The first valve 23.1 and the second valve 23.2 are preferably solenoid valves (also called electromagnetic valves) with low dead volume, in particular less than 5 pL, and high responsiveness, in particular less than 5ms.

[0026] For this purpose, the first container 16.1 is in fluidic communication with the inlet of the first valve 23.1 via the conduit 25. The outlet of the first valve 23.1 is in fluidic communication with the first inlet port. 21.1 via conduit 26. The second container 16.2 is in fluidic communication with the inlet of the second valve 23.2 via conduit 27. The outlet of the second valve 23.2 is in fluidic communication with the first inlet port 21.1 via conduit 28.

[0027] The static microfluidic mixer 20 further comprises a first inlet microfluidic conduit 30.1 in fluidic communication with the first inlet port 21.1 and a second inlet microfluidic conduit 30.2 in fluidic communication with the second inlet port 21.2. As illustrated in Figure 2, the first inlet microfluidic conduit 30.1 and the second inlet microfluidic conduit 30.2 each have a cross-sectional height h between 150 pm and 300 pm, preferably on the order of 200 pm, and a width I between 150 pm and 300 pm, preferably on the order of 200 pm. "On the order of" means a variation of plus or minus 10% from the stated value.

[0028] The first inlet microfluidic conduit 30.1 and the second inlet microfluidic conduit 30.2 intersect at a non-zero angle to each other at a point of intersection 33 opening onto a common outlet channel 34. Advantageously, the first inlet microfluidic conduit 30.1 and the second inlet microfluidic conduit 30.2 intersect, that is, they cut across each other, at an angle of 90 degrees or less. The outlet ports of the first conduit 30.1 and the second conduit 30.2 open at the point of intersection 33.

[0029] The common outlet channel 34 is extended by a channel 35 having a cross-sectional width greater than that of the common outlet channel 34. In one embodiment, the common outlet channel 34 may have a width I approximately twice the width I of an inlet microfluidic conduit 30.1, 30.2, i.e., a width I of approximately 400 µm. The common outlet channel 34 is extended by another channel 35 having a cross-sectional width I between 1 and 5 mm, and preferably approximately 3 mm wide. The heights h of conduits 30.1, 30.2, 34, and 35 may be identical. Alternatively, the heights h may vary from one conduit 30.1, 30.2, 34, and 35 to another. The heights h can also be variable within the same microfluidic conduit / channel 30.1, 30.2, 34, 35.

[0030] An interchangeable flow sensor 37 is provided for measuring the fluid flow rate at the outlet of the microfluidic mixer 20.

[0031] A control unit 40 is capable of controlling the pressure level of the first fluid 15.1 inside the first container 16.1 and the pressure level of the second fluid 15.2 inside the second container 16.2, as well as the opening and closing of the first valve 23.1 and the second valve 23.2 to perform successive injections of the first fluid 15.1 and the second fluid 15.2 into the microfluidic mixer 20, so as to generate a fringe profile F1, F2 of the first fluid 15.1 and the second fluid 15.2 into the common outlet channel 34. The pressure levels of the first fluid 15.1 and the second fluid 15.2 are controlled by the control unit 40 via the pressure regulator 12. The control unit 40 is electrically connected to the valves 23.1 and 23.2 to control their opening and closing.

[0032] Figure 3 illustrates the process of generating alternating F1 and F2 fringes in the common outlet channel 34 of the microfluidic mixer 20 and an experimental model performed with a fluorophore diluted in ethanol (corresponding to the first fluid 15.1) and water (corresponding to the second fluid 15.2). In the figure, the fluorophore diluted in ethanol appears darker than the water. The mixing system 10 multiplies the liquid-liquid interfaces between the organic and aqueous phases, which promotes mixing between the two fluids.

[0033] It is observed that the control unit 40 is configured to generate a fringe profile F1, F2 comprising an alternation of fringes F1, F2 of the first fluid 15.1 and fringes F1, F2 of the second fluid 15.2; that is, a fringe F1 of the first fluid 15.1 (fluorophore + ethanol) is followed by a fringe F2 of the second fluid 15.2 (water), which is itself followed by a fringe F1 of the first fluid 15.1, and so on. A fringe F1, F2 corresponds to the quantity of fluid passing through a valve 23.1, 23.2 during the valve's opening time. By adjusting the opening time of a valve 23.1, 23.2 and the corresponding fluid pressure level, it is possible to adjust the width of the fringes F1, F2.

[0034] Advantageously, the control unit 40 is configured such that the fringes F1 of the first fluid 15.1 are narrower than the fringes F2 of the second fluid 15.2 in order to promote the dilution of the first fluid 15.1 in the second fluid 15.2.

[0035] Figure 4 shows, during an injection phase of alternating fringes of the first and second fluids, a representation as a function of time (in seconds) of a flow rate D of the first and second fluids expressed in microlitres / s. Preferably, a ratio between a volume V2 of a fringe F2 of the second fluid divided by a volume V1 of a fringe F1 of the first fluid 15.1 is between 2 and 20, in particular between 8 and 15 and preferably between 9 and 11.

[0036] An injection frequency f equal to the inverse of the sum of an injection duration dt1 of the first fluid and an injection duration dt2 of the second fluid is between 0.1 and 200 Hz and is preferably between 10 Hz and 100 Hz.

[0037] The injection duration dt1 of the first fluid and the injection duration dt2 of the second fluid correspond respectively to the opening duration of the first valve 23.1 and the opening duration of the second valve 23.2. For equal injection flow rates of the first fluid and the second fluid, the ratio dt2 / dt1 is equal to the ratio V2 / V1.

[0038] The pressure level of the first fluid 15.1 inside the first container 16.1 and the pressure level of the second fluid 15.2 inside the second container 16.2 are each between 0 and 8000 mbar, in particular between 500 mbar and 7500 mbar.

[0039] As can be seen in Figure 3, the intensity of the F1 fringes decreases rapidly with distance from the exit of channel 34. The graph showing the intensity level of the pixels as a function of the distance from the exit of channel 34 shows that the organic phase dilutes after traveling a little over 5 mm in channel 35.

[0040] The invention makes it possible to reduce the dilution time of an organic phase—lipids (and / or polymers)—in an aqueous phase to less than 1 ms. The invention further enables the formulation of liposomes with high monodispersities of size (PDI < 0.1). The invention also makes it possible to optimize the nucleation rate of lipid nanoparticles.

[0041] These data (fluid pressure level, fluid injection frequency and corresponding duty cycles) are provided as input parameters to the control unit 40. For this purpose, a human-machine interface, such as a keyboard, a touch screen, or any other device suitable for the application, may be used.

[0042] The control unit 40 may include memory storing software instructions to control the pressure regulator 12 and the valves according to the input parameters received. The control unit 40 may, for example, take the form of a computer or a microcontroller dedicated to the application.

[0043] This precise control of the duration and amplitude of the pulsed injections determines the profile of the F1, F2 fringes of organic solvent-lipids and / or polymers and aqueous solution, and consequently, the final size of the liposomes.

[0044] The invention also relates to the process of producing a mixture of fluids implemented by system 10.

[0045] Alternatively, system 10 is devoid of conduit 35 and includes only conduit 34.

[0046] Alternatively, system 10 is used to create a gas mixture.

[0047] Alternatively, the microfluidic mixer 20 may have more than two inlet ports 21.1, 21.2, in particular N inlet ports associated with N microfluidic conduits and N valves (N being an integer). The fringe profile inside the common outlet channel 34 can then be a combination of N fluids injected sequentially or any possible combination of the fluids present. The number of containers may also be greater than two.

[0048] Alternatively, it is possible to use several common output channels, each corresponding to a specific fringe profile.

[0049] Alternatively, it is also possible to use 3 / 2 distributors, rotary valves, or any other means of alternately injecting fluids into a common outlet channel.

[0050] Of course, the different features, variants and / or embodiments of the present invention can be combined with each other in various ways as long as they are not incompatible or mutually exclusive.

[0051] Furthermore, the invention is not limited to the embodiments described above and provided solely by way of example. It encompasses various modifications, alternative forms, and other variations that a person skilled in the art may envision within the scope of the present invention, and in particular all combinations of the different modes of operation described above, which may be considered separately or in combination.

Claims

CLAIMS System (10) for producing a mixture of fluids characterized in that it comprises: - a source of pressure (11), - a pressure regulator (12) to which said pressure source (11) is connected, - at least one first container (16.1) containing a first fluid (15.1) and a second container (16.2) containing a second fluid (15.2), the pressurization of the first fluid (15.1) and the second fluid (15.2) being controlled by the pressure regulator (12), - a microfluidic mixer (20) comprising at least a first inlet port (21.1) and a second inlet port (21.2) associated with at least a first valve (23.1) and a second valve (23.2), - the first container (16.1) being connected to the first inlet port (21.1) via the first valve (23.1) and the second container (16.2) being connected to the second inlet port via the second valve (23.2), - said microfluidic mixer (20) further comprising at least one first microfluidic conduit (30.1) and a second microfluidic conduit (30.2), the first inlet microfluidic conduit (30.1) being in fluidic communication with the first inlet orifice (21.1) and the second inlet microfluidic conduit (30.2) being in fluidic communication with the second inlet orifice (21.2), said first inlet microfluidic conduit (30.1) and said second inlet microfluidic conduit (30.2) intersecting at a non-zero angle to each other at the point of an intersection (33) opening onto at least one common outlet channel (34), and - a control unit (40) capable of controlling the pressure level of the first fluid (15.1) inside the first container (16.1) and the pressure level of the second fluid (15.2) inside the second container (16.2), as well as the opening and closing of the first valve (23.1) and the second valve (23.2) to carry out successive injections of the first fluid (15.1) and the second fluid (15.2) into the mixer microfluidic (20), so as to generate a fringe profile (F1, F2) of the first fluid (15.1) and the second fluid (15.2) inside the common outlet channel (34). System according to claim 1, characterized in that the control unit (40) is configured to generate a fringe profile (F1, F2) comprising alternating fringes (F1) of the first fluid (15.1) and fringes (F2) of the second fluid (15.2). System according to claim 2, characterized in that the control unit (40) is configured such that the fringes (F1) of the first fluid (15.1) are narrower than the fringes (F2) of the second fluid (15.2). System according to claim 3, characterized in that a ratio between a volume (V2) of a fringe (F2) of the second fluid divided by a volume (V1) of a fringe (F1) of the first fluid (15.1) is between 2 and 20, in particular between 8 and 15 and preferably between 9 and 11.A system according to any one of claims 1 to 3, characterized in that the first container (16.1) contains a solution of lipids and / or polymers diluted in an organic solvent corresponding to the first fluid (15.1) and the second container (16.2) contains an aqueous solution corresponding to the second fluid (15.2). A system according to any one of claims 1 to 4, characterized in that the first valve (23.1) and the second valve (23.2) are solenoid valves with low dead volume, in particular less than 5 pL, and high responsiveness, in particular less than 5 ms. A system according to any one of claims 1 to 5, characterized in that the common outlet channel (34) is extended by a channel (35) having a cross-sectional width greater than that of the common outlet channel (34). A system according to any one of claims 1 to 6, characterized in that it comprises an interchangeable flow sensor (37) for measuring the flow rate of fluid at the outlet of the microfluidic mixer (20). A system according to any one of claims 1 to 7, characterized in that the first inlet microfluidic conduit (30.1) and the second inlet microfluidic conduit (30.2) intersect at an angle of 90 degrees or less. A system according to any one of claims 1 to 8, characterized in that the first inlet microfluidic conduit (30.1) and the second inlet microfluidic conduit (30.2) intersect at an angle of 90 degrees or less.2) each have in cross-section a height (h) between 150 pm and 300 pm and preferably of the order of 200 pm and a width (I) between 150 pm and 300 pm and preferably of the order of 200 pm wide. System according to any one of claims 1 to 10, characterized in that the pressure level of the first fluid (15.1) inside the first container (16.1) and the pressure level of the second fluid (15.2) inside the second container (16.2) are each between 0 and 8000 mbar. System according to any one of claims 1 to 12, characterized in that an injection frequency (f) equal to the inverse of the sum of an injection duration (dt1) of the first fluid and an injection duration (dt2) of the second fluid is between 0.1 and 200 Hz and is preferably between 10 Hz and 100 Hz.

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