Acoustic control of calibrated gas-filled microvesicles manufacturing

Abstract

The invention relates to a method for controlling the manufacturing process of calibrated gas-filled microvesicles (103'), particularly via microfluidic flow-focusing technique, by applying an ultrasound wave. In particular the applied ultrasound wave has a predetermined frequency and / or acoustic pressure, which allow to control the size and manufacturing rate of the microvesicles.

Description

[0001] Acoustic control of calibrated gas-filled microvesicles manufacturing

[0002] Technical field

[0003] The invention relates to a method for controlling the manufacturing process of calibrated gas-filled microvesicles, particularly via microfluidic flow-focusing technique, by applying an ultrasound wave.

[0004] Background of the invention

[0005] Calibrated-size gas-filled microvesicles (in short "calibrated microvesicles" or "CMV") are a new generation of gas-filled microvesicles (in short "GFV") having a relatively narrow and controlled size distribution (with mean diameter sizes which can be selected to range between 2 and 8 pm), as compared to commercially available polydisperse microbubble ultrasound contrast-agents (USCA). Calibrated microvesicles are in particular designed to enhance imaging sensitivity and improve efficiency in therapeutic treatments, e.g. in delivering drugs or genes to specific organs through different techniques, including, for instance, BBB (Blood Brain Barrier) disruption, thermal ablation, sonopermeabilisation or sonothrombolysis. Calibrated microvesicles can be produced using various techniques: decantation, mechanical filtration, centrifugation, bubble sorting and flow-focusing.

[0006] Flow-focusing devices typically comprise a chip (e.g. of polymeric material) on which microfluidic channels are provided. A gaseous phase and a liquid phase comprising an amphiphilic material are generally introduced through respective inlets; at the junction of the channels, the gaseous phase is focused into a narrow stream by the liquid phase; gas-filled microvesicles are thus formed at the exit (nozzle), the amphiphilic material forming a layer surrounding the inner gas core. This creates a highly controlled environment where microvesicles of uniform size can be formed. The size of the CMV can be adjusted by controlling parameters such as flow rates, viscosity, and surface tension of the fluids.

[0007] One drawback of this technique relates to the difficulties of scaling up the manufacturing process to industrial levels. In order to reach industrial scales, it would in fact be very expensive to operate many systems in parallel, each requiring its own flow controllers for gas and liquid.

[0008] To increase the manufacturing rate, chips with multiple flow-focusing structures arranged in parallel on a single microfluidic chip ("parallelized chips") have been proposed (see e.g. H.-H. Jeong, S. Yadavali, D. Issadore, and D. Lee, "Literscale production of uniform gas bubbles via parallelization of flow focusing generators," Lab on a Chip 17, 2667-2673, 2017}. By operating multiple flowfocusing structures in parallel, these chips can significantly increase the manufacturing throughput compared to single-channel devices. The main drawback of parallelized chips is usually an increased polydispersity in the population of gas- filled microvesicles, as compared to the microvesicles obtained with single channel chips. For instance, the above cited H.H. Jeong et al. describes a coefficient of variation of 5% for 400 parallelized chips.

[0009] The increased polydispersity is possibly related to very small differences of the flow-resistances between individual channels. Even though the various chip designs try to limit or eliminate these differences by completely symmetric designs, it is difficult to avoid them completely. As a consequence, manufacturing rates (and thus CMV mean sizes) are very similar but not identical, leading hence to an increased polydispersity.

[0010] Chinese patent application CN104923137 discloses applying ultrasounds on a flow focusing device to induce bubble cavitation for increasing fluid mixing and preventing clogging.

[0011] The Applicant has now found that by applying an ultrasound wave having a predetermined frequency on a flow focusing device comprising a plurality of nozzles, it is possible to obtain a substantially uniform manufacturing rate from the various nozzles.

[0012] The Applicant has further observed that the size of gas-filled microvesicles can be controlled by applying an ultrasound wave at a predetermined acoustic pressure.

[0013] Summary of the invention

[0014] The invention generally relates to a method for controlling the manufacturing process of calibrated gas-filled microvesicles said gas filled microvesicles being prepared by using a flow-focusing device having a respective manufacturing rate of microvesicles (expressed as number of microvesicles per second), said method comprising applying an ultrasound wave at a predetermined frequency on said flow-focusing device.

[0015] An aspect of the invention relates to a method for manufacturing a suspension of calibrated gas-filled microvesicles comprising a gas and an envelope comprising an amphiphilic compound, by using a flow-focusing device comprising a nozzle with a calibrated orifice having a respective natural manufacturing rate (microvesicles / s), said method comprising the steps of: providing (i) a gaseous flow and (ii) an aqueous liquid flow comprising an amphiphilic compound; directing said gaseous flow and said liquid flow through respective inlet channels towards a contact zone; directing said gaseous flow and said liquid flow from the contact zone through a nozzle to generate said gas-filled microvesicles, thus obtaining an aqueous suspension comprising said gas-filled microvesicles; directing said suspension of gas-filled microvesicles towards an outlet channel; and collecting said suspension of gas-filled microvesicles; wherein an ultrasound wave at a predetermined frequency is applied to said nozzle.

[0016] Preferably said gas filled microvesicles are prepared by using a device comprising at least two parallelized flow-focusing nozzles, each nozzle having a respective microvesicles manufacturing rate.

[0017] As observed by the Applicant, applying said ultrasound wave at said predetermined frequency results in particular in the possibility of locking the manufacturing rate of microvesicles to said predetermined frequency.

[0018] Another aspect of the invention relates to a method for controlling the manufacturing process of calibrated gas-filled microvesicles, said method comprising applying an ultrasound wave at a predetermined acoustic pressure.

[0019] As observed by the Applicant, applying said ultrasound wave at said predetermined acoustic pressure allows to control the size of microvesicles, in particular to reduce the size thereof with respect to the size where no acoustic pressure is applied.

[0020] Figures

[0021] Figure 1 schematically depicts a flow-focusing microfluidic device.

[0022] Figure 2 schematically depicts a parallelized flow-focusing device.

[0023] Figures 3 to 6 illustrate the effects on the manufacturing rate of a flow-focusing microfluidic device upon application of an USW at a predetermined frequency.

[0024] Figure 7 shows the effects of applying USW at a predetermined acoustic pressure for reducing the size of the gas-filled microvesicles.

[0025] Figure 8 schematically illustrates an experimental setup for practicing the invention.

[0026] Detailed description of the invention

[0027] The present method of manufacturing relies at least in part on the use of an ultrasound wave ("USW") having a predetermined frequency.

[0028] The present method may be used in combination with a microfluidic flow focusing device, as schematically illustrated in Figure 1. Figure 1 shows a schematic representation of the core portion 100 of a flow-focusing device ("microfluidic chip") useful in the process of the invention. The chip comprises a first feed channel 101 for feeding the gaseous flow 101' and two additional feed channels 102a and 102b for supplying the aqueous liquid flow containing the amphiphilic material.

[0029] The gas flow and the two liquid flows are directed towards the contact zone 103 and then through the calibrated orifice 104, shown as a dotted line in figure 1. The calibrated orifice is connected to a calibrated channel 104' (having preferably the same cross-section as the orifice), which is in turn connected to an output, here in the form of an outlet channel 105. In an alternative embodiment (not shown) the calibrated orifice 104 may be directly connected to the outlet channel 105 i.e. without the calibrated channel in-between. As used herein, the term nozzle corresponds substantially to the calibrated orifice 104; optionally, in some instances, such term may include the calibrated orifice 104 in combination with calibrated channel 104' and / or the initial portion of outlet chamber. The microvesicles 103' are formed at the exit of the calibrated orifice and directed, through calibrated channel 104', to the outlet channel 105. The hydraulic diameter of the outlet channel is generally larger than the hydraulic diameter of the calibrated orifice and typically increases, in its initial portion, from the initial diameter of the calibrated orifice to the final diameter of the outlet channel 105, corresponding substantially to the hydraulic diameter of a collecting tube (not shown), connecting the flow-focusing device to a container, e.g. a vial for collecting the suspension of microvesicles. The USW at a predetermined frequency (not shown) is applied to said nozzle, in particular in correspondence with the calibrated orifice 104.

[0030] Preferably, the flow-focusing device comprises a chip comprising at least two parallelized flow-focusing nozzles. Figure 2 shows a schematic example of a chip comprising two parallelized flow-focusing nozzles 201a and 201b. The chip comprises a feed channel 202 for the liquid flow, which is then divided in respective channels 203 and 204, which are then further divided into respective channels 203a / 203b and 204a / 204b. Similarly, the feed channel 205 for the gas is divided into respective channels 206 and 207.

[0031] An example of a microfluidic flow focusing device with multiple parallelized nozzles, is described for instance W02015 / 050960.

[0032] Typically, even if the respective elements of a parallelized microfluidic flowfocusing device (e.g. calibrated orifice and channel) have been designed to be as similar as possible, slightly different manufacturing rates (frequencies) can be observed among the different nozzles.

[0033] While the respective flow rates of the liquid and gas through the channels of the parallelized nozzles are set to be the same for each nozzle, the manufacturing process may induce small flow variations in the various nozzles (also due to the "ageing" of the various components), which may result in different dimension of the microvesicles generated by each nozzle.

[0034] For instance, as illustrated in the examples, two parallelized nozzles (with a same design) show respective nominal manufacturing rates of 620 kHz or 650 kHz (i.e. 620'000 or 650'000 bubbles per second).

[0035] As observed by the Applicant, by applying an USW at a predetermined frequency, it is possible to modify the manufacturing rate of a flow-focusing device (with respect to the nominal manufacturing rate) and lock it to said frequency. Preferably, the applied US wave has a frequency which ranges from a frequency corresponding to the nominal manufacturing rate of the nozzles or slightly higher than it (e.g. up to 10 % higher than the nominal manufacturing rate) down to frequencies of about 25% lower than those of the manufacturing rate. When the manufacturing rate of more than one nozzle has to be locked to the predetermined frequency of the applied USW, the above (and below) ranges of USW frequency are referred to the manufacturing rates of the nozzles showing the highest nominal manufacturing rate among those concerned.

[0036] In certain embodiments, the manufacturing rate can be locked at a frequency of from about the nominal manufacturing rate down to about 20% lower than the nominal manufacturing rate, preferably to about 15% lower than the nominal manufacturing rate.

[0037] In other embodiments, the manufacturing rate can be locked at a frequency corresponding to from about the nominal manufacturing rate up to about 10% higher than the nominal manufacturing rate, preferably up to about 5% higher than the nominal manufacturing rate.

[0038] For instance, (see e.g. figure 3), by applying an USW with a predetermined frequency between about 500 and 620 kHz (indicated as dots in fig. 3), it is possible to lock the manufacturing rate of the nozzles (originally ranging from 600-103to 620-103microvesicles / s, circles in fig. 3) to said respective predetermined frequency.

[0039] Similarly, as shown in figure 4, by applying an USW with a predetermined frequency between 750 kHz and 1050 kHz, it is possible to lock the manufacturing rate of the nozzles (originally ranging from 950-103microvesicles / s and 1000-103microvesicles / s) to said respective predetermined frequency.

[0040] According to alternative embodiments, the applied USW has a frequency of about twice the corresponding manufacturing rate. Fig. 5 shows such an embodiment, where the manufacturing rate of nozzles having a nominal manufacturing rate of from 540-103to 630- 103microvesicles / s can be locked to manufacturing rates of from 540- 103to 630- 103microvesicles / s, by applying USWs having respective frequencies of from 1080 kHz to 1260 kHz. Advantageously, the use of an USW at a frequency of about twice the nominal manufacturing rate allows using a transducer with smaller dimensions (as compared with a transducer adapted to emit USW at a frequency similar to the nominal manufacturing rate).

[0041] Preferably the applied frequency is of at least 500 kHz to allow a sufficiently high manufacturing rate. There is in principle no highest value of applied frequency, which will in any case be related to the natural manufacturing rate of the nozzle as illustrated before. For instance frequencies of 1, 2, 3, 4, or 5 MHz can be applied to nozzles having a natural manufacturing rate around those ranges of frequencies (preferably -10% to + 25% of the frequency of the applied USW).

[0042] Figure 6 shows a schematic representation of the effects of the application of an USW on two nozzles having different manufacturing rates. The left side (601) shows the natural manufacturing rates of the two nozzles, i.e. about 620 and 650 kHz respectively. Then (at time 9 JJS) an USW having a frequency of 580 kHz is applied on the two nozzles; after a transient period of less than 15 ps (602), the manufacturing rate of the two nozzles is locked at the frequency of the applied USW (603).

[0043] As it can be appreciated, the locking of the manufacturing rate of a plurality of parallelized nozzles to a same applied frequency advantageously allows to homogenize the manufacturing rate among the various nozzles; this in turn contributes to reducing the polydispersity of the obtained CMV, as compared to the polydispersity of the combined CMV obtained with the respective natural manufacturing rates of each nozzle (i.e. without applying the USW at the predetermined frequency).

[0044] Each channel of the parallelized device may be equipped with a detector, feedback loop and ultrasound source in order to fine-tune each channel and increase the overall monodispersity, as described for instance by B. van Elburg, G. Collado-Lara, G. -W. Bruggert, T. Segers, M. Versluis, and G. Lajoinie, Feedback-controlled microbubble generator producing lx 106monodisperse bubbles per second, Review of Scientific Instruments 92, 035110 (2021).

[0045] The USW can be applied on the parallelized nozzles (in particular on the respective calibrated openings) either with a multi-channel ultrasound transducer array adapted for sonicating the parallelized nozzles (either the totality or respective sub-groups thereof), or with a plurality of single-channel transducers (one for each respective nozzle). While the second option may by more cost-effective, it may nevertheless render the optical access more difficult, particularly for a high number of parallelized nozzles. The transducer (single or array) is positioned at a suitable distance from the nozzle(s), such that its focal spot is in correspondence with the nozzle(s) (in particular its respective calibrated opening), e.g. at a distance of from about 1 mm to about 1 cm from the nozzle(s). Alternatively, individual transducers may be positioned in direct contact with the respective nozzle, e.g. attached (for instance glued) to the chip or integrated in the chip structure.

[0046] Preferably, the parallelized nozzles should be sufficiently far away from each other (typically few millimeters) for allowing individual excitation thereof with the applied USW. The minimum distance is determined by the wavelength of the applied USW (e.g. about 1.5 mm for a 1 MHz wavelength).

[0047] The Applicant has further observed that by suitably modulating the acoustic pressure of an applied ultrasound wave, it is possible to control the size of the CMV manufactured according to the flow-focusing microfluidic technique. In particular, the Applicant has observed that the size of manufactured CMV can be reduced by increasing the acoustic pressure of the applied USW. Figure 7 shows the results of applying increasing acoustic pressures (from 150 to 300 kPa, gray scale on the right) associated with USWs at different frequencies (from 640 to 720 kHz, x axis) during the manufacturing of GFV; the resulting size reduction is expressed as the ratio between the mean size of the GFV obtained upon application of the USW with the predetermined acoustic pressure and the mean size of the GFV obtained without applying any USW during the manufacturing process (Rb,us / Rb,nous). The applied acoustic pressure depends, among other, on the manufacturing parameters and the materials forming the GFV and should be such that a reduction in size of the GFV is achieved by applying such acoustic pressure, as compared to GFV manufactured in the absence of such applied acoustic pressure; typically the applied acoustic pressure is of at least 10 kPa, preferably of at least 25 kPa, more preferably of at least 50 kPa, even more preferably of at least 100 kPa, particularly preferred being an acoustic pressure of at least 150 kPa, e.g. 200 kPa or 250 kPa or higher. On the other hand, such acoustic pressure should not be too high, in order not to negatively affect the stability of the forming GFV; typically, the applied acoustic pressure is not higher than 1 MPa, preferably not higher than 750 kPa, more preferably not higher than 500 kPa and even more preferbly not higher than 350 kPa. Such values of acoustic pressure are measured in water at the location of the nozzle. As observed by the applicant, by applying an USW with a predetermined acoustic pressure it is possible to reduce the size of the obtained GFV by a factor of about 0.90, depending on the frequency and the acoustic pressure of the applied USW. As above, the flowfocusing device may be provided with a feedback system (e.g. the above cited B. van Elburg et al.), in order to apply the suitable acoustic pressure for generating CMV having the desired size. Any technique (such as laser sheet, laser dot, electrodes and so on) providing information (on a sufficiently short timescale) on the size being generated from each nozzle may be used; this information is then used for adapting the acoustic pressure of applied USW to the value suitable for the manufacturing of the CMV with the desired size.

[0048] CMV size reduction by application of USW with a predetermined acoustic pressure may for instance allow using channel with larger dimensions, which are generally less prone to possible clog issues, as opposed to channels of smaller dimensions.

[0049] Suitable devices, methods and materials for preparing CMV are disclosed for instance in W02019 / 170606, here incorporated by reference. A suitable microfluidic flow-focused device typically comprises a chip (e.g. as illustrated in figures 1 and 2) comprising a first fluid channel for the feeding of a gas and a second fluid channel (divided in two respective feeding channels) for the feeding of a solution (e.g. an aqueous solution) comprising the amphiphilic material forming the envelope of the microvesicles for entrapping the gas; at the junction (e.g. a T-junction) between the channel with the gas and the two channels with the liquid flow, the microvesicles are formed through a nozzle and collected as a (aqueous) suspension of microvesicles.

[0050] Typically, the CMVs are manufactured at a manufacturing rate of at least 1.0-106microvesicles / s; preferably the manufacturing rate is of at least 2.5- 106microvesicles / s, more preferably 5.0- 106microvesicles / s. The highest manufacturing rate is determined by the geometry of the chip, the gas pressure / liquid flow rates and the materials forming the microvesicles; manufacturing rate of 1.0-107microvesicles / s can easily be achieved, up to e.g. 5.0-107microvesicles / s. Typical liquid flow rate may vary from about 100 pljmin to about 500 pL / min, preferably from about 120 pljmin to about 400 pL / min, more preferably from 150 pL / min to about 300 pL / min. Typical gas (over) pressures (with respect to normal 1 bar ambient pressure) may be of at least 1.0 bar, preferably at least 1.3, more preferably at least 1.5 bar, even more preferably at least 1.8 mbar, up to e.g. 4.0 bar, preferably up to 3.5 bar, more preferably up to 3.0 bar, even more preferably up to 2.5 bar.

[0051] Suitable materials for preparing the CMV include amphiphilic materials known in the art such as those disclosed in W02019 / 170606. Preferred amphiphilic materials include phospholipids and / or non-ionic surfactants.

[0052] Phospholipids typically include esters of glycerol with one or preferably two (equal or different) residues of fatty acids and with phosphoric acid, wherein the phosphoric acid residue is in turn bound to a hydrophilic group, such a, for instance, choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine (phosphatidylethanolamines - PE), inositol (phosphatidylinositol). Fatty acids residues present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated. Examples of suitable fatty acids included in the phospholipids are, for instance, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Preferably, saturated fatty acids such as myristic acid, palmitic acid, stearic acid and arachidic acid are employed. Further examples of phospholipids are phosphatidic acids, i.e. the diesters of glycerol-phosphoric acid with fatty acids; sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid; glycolipids such as gangliosides GM1 (or GM2) or cerebrosides; glucolipids; sulfatides and glycosphingolipids. Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer, such as polyethyleneglycol (PEG) or polypropyleneglycol (PPG), thereto. Preferred polymer-modified phospholipids include "pegylated phospholipids", i.e. phospholipids bound to a PEG polymer. Examples of pegylated phospholipids are pegylated phosphatidylethanolamines ("PE-PEGs" in brief) i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g. from 300 to 20000 daltons, preferably from 500 to 5000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPE-PEG or DOPE-PEG). For example, DPPE-PEG2000 refers to DPPE having attached thereto a PEG polymer having a mean average molecular weight of about 2000.

[0053] Examples of non-ionic surfactants include, for instance, polyoxyethylene fatty acid esters (such as polyoxyethylene stearates), polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol ethers and polyoxyethylated sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monopalmitate or polyoxyethylene sorbitan monooleate, such as Tween®).

[0054] Gases suitable for preparing CMV are those known in the art. They may include for instance, nitrogen, air, carbon dioxide, fluorinated gases and mixtures thereof. The fluorinated gas is preferably a perfluorinated gas, such as perfluoropropane or perfluorbutane.

[0055] Suitable methods (e.g. temperature control, mixture of gases) for controlling the manufacturing of the CMV are disclosed for instance in the above cited W02019 / 170606.

[0056] The following examples will help to further illustrate the invention. EXAMPLES

[0057] Experimental setup

[0058] Figure 8 depicts a schematic drawing of the experimental setup. The same setup was previously used and described in [S. Cleve, C. Diddens, T. Segers, G. Lajoinie, and M. Versluis, Time-resolved velocity and pressure field quantification in a flow-focusing device for ultrafast microbubble production, Physical Review Fluids 6, 114202 (2021 ).]. For the sake of simplicity, details on the optical path and on the supply of the liquid / gas flows have been omitted. With reference to figure 8, the setup comprises (a) a microfluidic flow-focusing chip 801 immersed in a water tank 802; (b) a high-speed camera 803, coupled to a microscope objective 604 and respective light source 805; and (c) an ultrasound transducer 806.

[0059] The microfluidic chip was a custom parallelized (two nozzles) PDMS (polydimethylsiloxane) chip bound to a glass-slide, with calibrated channels of each nozzle having a width of 20 pm, a height of 15.4 pm and a length 30 pm. The inlet channels (gas and liquid) were 80 pm wide.

[0060] After the microfluidic chip was immersed in the water tank, it was placed in a bright-field microscope (Olympus, BXF) and imaged with an ultra-high-speed camera (Shimadzu HPV-X2). Two imaging modes were used. The first mode aimed at analyzing the general dynamics of the chip, and in particular the manufacturing rate. There, recordings were taken with a frame rate of 5 Mfps and with a low magnification (lOx or 20x). Illumination was provided by a continuous light source (KL2500LED, Schott) for the duration of the recording (256 frames, i.e., 51.2 ps). The second recording mode was used to analyze the detailed bubble and gas cusp dynamics. This required a frame rate of 10 Mfps and high magnification (80x). Illumination was provided by a xenon flash light (Vision Light Tech).

[0061] Water with a weight fraction of 2% Tween 80 (Sigma-Aldrich) was pumped into the chip with a flow rate of from about 200 pl / min to 350 pl / min (per nozzle) and air was supplied via a pressure regulator (IMI Norgren) at a pressures around 2 bar (1.8-2.5 bar) The manufacturing rate was evaluated from the pixel grey value by performing a wavelet transform.

[0062] The US transducer (Olympus C302-SU, 1.64 inch, 1MHz center resonance frequency) was then arranged so that its focal spot was in correspondence of the nozzle.

[0063] Example 1

[0064] Effect of applied ultrasound wave on manufacturing rate of two parallelized nozzles The effect of applied USW on the manufacturing rate is shown in fig. 6 for the two parallel nozzles (liquid flow rate of 500 pl / min, i.e. 250 pl / min for each nozzle; gas pressure of about 2 bar). Initially, the measured natural manufacturing rates fb,nous of the two channels are different, i.e. 620 kHz and 650 kHz, respectively (601 in fig. 6). Then, at t « 9 ps the US transducer is switched on, for generating an USW at a driving frequency (fac) of 580 kHz (i.e. slightly lower than the natural manufacturing rates of each nozzle); this results in a transient regime with irregular bubble detachment (602 in fig. 5) until t « 22 ps. At t « 22 ps (603 in fig. 6) the manufacturing rate (frequency) of both nozzles then locks onto the applied driving frequency.

[0065] Example 2

[0066] Effect of applied ultrasound wave on manufacturing rate of a microfluidic nozzles The experiment illustrated in example 1 was repeated on a single nozzle.

[0067] For this purpose, one of the two parallelized nozzles was clogged. The liquid flow rate was varied between 200 pl / min and 350 pl / min, while gas pressure was varied between 1.8 and 2.5 bar, to obtain the respective natural manufacturing rates illustrated in figure 3.

[0068] Figure 3 illustrates the results of applying an USW with a predetermined driving frequency (from 500 to 720 kHz) to nozzles having a manufacturing rate around 600 kHz. As observable in figure 2, upon turning on the US transducer at the selected frequency fac, the natural manufacturing rate of a nozzle (fb,nous, represented by the black dots in fig. 2) is shifted to a desired manufacturing frequency fb by locking it to the selected frequency fac, represented by circles in correspondence with respective dots.

[0069] Similarly, as illustrated in figure 4, upon turning on the US transducer at the selected frequency fac(from 750 to 1050 kHZ), the natural manufacturing rate of a nozzle (fb,nous) of about 1000- 103GFV / s (black dots) can be shifted to a desired manufacturing frequency fb by locking it to the selected frequency fac.

[0070] Alternatively, as illustrated in figure 5, the manufacturing rate fb of the nozzle can be locked to half the selected frequency fac.

[0071] Example 3

[0072] Effect of applied ultrasound wave on the size of manufactured GFV

[0073] The same setup of example 2 was used while varying acoustic pressure between 150 and 300 kPa at applied frequencies of from 640 to 720 kHz (natural manufacturing rate of the nozzle being around 700 kHz). Acoustic pressure is measured in water at the spot of the flow focusing nozzle (in the absence of the chip).

[0074] Results are collected in figure 7, showing that and increase of applied acoustic pressure generally results in a reduction of GFV size. In particular, it can be observed that when an USW having a frequency of 640 kHz is applied, the dimensions of the obtained GFV can be reduced already by applying an acoustic pressure of 150 kPa; the dimensions can then be further reduced (down to a ratio Rb,us / Rb,nous of 0.90 by applying an acoustic pressure of 250 kPa. When an USW at a frequency of 720 kHZ is applied, the size of the GFV can be further reduced below 0.90 by applying an acoustic pressure of 300 kPa.

Claims

CLAIMS1. A method for manufacturing a suspension of calibrated gas-filled microvesicles (103') comprising a gas and an envelope comprising an amphiphilic compound, said method comprising : providing (i) a gaseous flow (101') and (ii) an aqueous liquid flow comprising said amphiphilic compound to a flow-focusing device comprising a nozzle with a calibrated orifice (104) having a respective natural manufacturing rate (microvesicles / s); directing said gaseous flow and said liquid flow through respective inlet channels (101, 102a, 102b) towards a contact zone (103); directing said gaseous flow and said liquid flow from the contact zone (103) through said calibrated aperture (104) to generate said gas-filled microvesicles (103'), thus obtaining an aqueous suspension comprising said gas-filled microvesicles; directing said suspension of gas-filled microvesicles towards an outlet channel (105); and collecting said suspension of gas-filled microvesicles; wherein an ultrasound wave at a predetermined frequency is applied to said nozzle.

2. The method according to claim 1 wherein applying said ultrasound wave at said predetermined frequency results in the manufacturing rate of said nozzle being locked to said predetermined frequency.

3. The method according to claim 1 or 2, wherein said ultrasound wave is applied at a frequency of from +10% to -25% of the natural manufacturing rate.

4. The method according to claim 1 or 2 wherein said ultrasound wave is applied at a frequency of from twice of the natural manufacturing rate to -25% of twice the natural manufacturing rate.

5. The method according to any of the preceding claims, wherein said flow-focusing device comprises at least two parallelized flow-focusing nozzles (201a, 201b), each nozzle having a calibrated orifice with a respective natural manufacturing rate.

6. The method according to any of the preceding claims wherein said ultrasound wave is applied at a predetermined acoustic pressure.

7. The method according to claim 6, whereby applying said ultrasound wave at said predetermined acoustic pressure results in a reduction of the size of the manufactured microvesicles.

8. The method according to claim 6 or 7 wherein said acoustic pressure is of least 10 kPa.

9. The method according to claim 8 wherein said acoustic pressure is of at least 50 kPa.

10. The method according to claim 8 wherein said acoustic pressure is of at least 100 kPa.

11. The method according to any of the preceding claims wherein the gas-filled microvesicles are manufactured at a manufacturing rate of at least 1.0- 106microvesicles / s.

12. The method according to any of the preceding claims wherein the liquid flow is at a rate is from 100 pL / min to 500 pL / min.

13. The method according to any of the preceding claims wherein the gaseous flow is at a pressure of at least 1.0 bar above ambient pressure.

14. The method according to any of the preceding claims wherein said amphiphilic material comprises a phospholipid, a non-ionic surfactant or mixtures thereof.

15. The method according to any of the preceding claims wherein said gas is selected from nitrogen, air, carbon dioxide, fluorinated gases and mixtures thereof.

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