CN116897055A

CN116897055A - Cartridge for mixing phospholipid compositions intended for in vivo use

Info

Publication number: CN116897055A
Application number: CN202180093121.3A
Authority: CN
Inventors: 威廉·范·胡夫; 凯瑟琳·珍妮弗·詹纳西; 米格尔·德·巴尔加斯·瑟拉诺; 伦佐·鲍韦·范·德·普拉斯
Original assignee: Solstice Pharmaceutical Co ltd
Current assignee: Solstice Pharmaceutical Co ltd
Priority date: 2020-12-27
Filing date: 2021-12-23
Publication date: 2023-10-17

Abstract

The present invention relates to a cartridge. More particularly, the present invention relates to a cartridge by which a phospholipid composition held in the cartridge can be mixed with another liquid held in the cartridge or with a pressurized gas within the cartridge. The invention also relates to a cartridge system comprising such a cartridge and a device into which the cartridge can be releasably inserted, wherein the device is configured for providing a pressurized gas that serves as a propellant for moving the liquid inside the cartridge for mixing the liquid and/or as a gas to be mixed.

Description

Cartridge for mixing phospholipid compositions intended for in vivo use

The present invention relates to a cartridge. More particularly, the present invention relates to a cartridge by which a phospholipid composition held in the cartridge can be mixed with another liquid held in the cartridge or with a pressurized gas within the cartridge. The invention also relates to a cartridge system comprising such a cartridge and a device into which the cartridge can be releasably inserted, wherein the device is configured for providing a pressurized gas that serves as a propellant for moving a liquid inside the cartridge for mixing the liquid and/or as a gas to be mixed. The invention also relates to a device for releasably receiving the cartridge described above and a blister package comprising a phospholipid composition.

When using therapeutic or diagnostic fluids in vivo, it is important that sterility of the fluid be ensured. When such a liquid can be used without additional processing steps, such as mixing the liquid with another liquid, it is possible to supply the liquid into a sterile container intended for one person. Alternatively, a larger sterile liquid source may be used from which the desired amount may be withdrawn. However, in the latter case, stringent requirements are placed on the environment in which the reservoir is located and the manner in which the desired amount is obtained therefrom. The latter aspect makes the use of larger reservoirs impractical for environments such as hospitals or clinics.

However, when the liquid needs to be treated before use, different situations can occur. For example, therapeutic or diagnostic substances to be administered to a patient require a personalized composition or dose. Alternatively, it may occur that the therapeutic or diagnostic substance is inherently unstable and must be administered rapidly after preparation. In such cases, it is more difficult to ensure sterility of the final product.

It is an object of the present invention to provide a solution to the above-mentioned problems.

According to the invention, this object is achieved by a cartridge as defined in claim 1. The cartridge is configured for mixing the liquid held in the cartridge with another liquid held in the cartridge or with a pressurized first gas supplied to the cartridge within the cartridge, wherein the liquid is a phospholipid composition, wherein the concentration of phospholipids is at least 12mg/ml.

In one embodiment, the cartridge comprises a mixing unit configured for mixing the phospholipid composition with the pressurized first gas, the mixing unit comprising a microfluidic device configured for generating microbubbles in the phospholipid composition, the microbubbles being filled with the pressurized first gas.

Alternatively, the cartridge may comprise a barrel, one or more gas inlets formed in the barrel, and a fluid storage system formed in the barrel and comprising one or more fluid storage units, each configured for holding a respective fluid and for outputting the fluid in response to pressurized gas supplied to the fluid storage unit through the gas inlet in the one or more gas inlets by using the supplied gas as a propellant.

The cartridge may further comprise a mixing unit arranged in or mounted to the cartridge body and in fluid communication with the fluid storage unit using a fluid channel or channels formed in the cartridge body. The mixing unit may be configured for mixing the respective fluids output from the respective fluid storage units or for mixing the fluids output from the fluid storage units with a pressurized first gas received through a gas inlet of the one or more gas inlets. The at least one fluid held in the fluid storage system and configured to be mixed is a phospholipid composition. In a preferred embodiment, each fluid storage unit holds a liquid. When multiple fluid storage units are used, the liquids held by these units may be the same or different.

With the cartridge of the invention, the liquids to be mixed can be kept inside the cartridge in a sterile manner. Furthermore, using a phospholipid composition having a phospholipid concentration of at least 12mg/ml, bubbles that do not coalesce, for example, in a microfluidic chip, can be generated at high speed. Higher concentrations of phospholipids are advantageous for uniformity of bubble size. Higher concentrations are also advantageous for generating more than 1 million microbubbles per second if bubbles are generated, for example, using a chip by contacting the phospholipid composition with a suitable gas. Furthermore, advantageously, the phospholipid composition does not comprise dipalmitoyl phosphatidic acid (DPPA).

In addition, pressurized gas may be used to control the mixing process by using the gas (es) as propellant. Thus, the liquid may be mixed within the cartridge, avoiding a possible non-sterile transfer of the liquid between the mixing unit and the reservoir of the liquid. The only fluid exchanged with the cartridge is a gas, and the gas can be kept sterile in a more convenient manner than the liquid.

Preferably, the phospholipid composition comprises a hydrated phospholipid solvent mixture prepared by the method of:

-dissolving a first phospholipid in an organic solvent at a temperature above the phase transition temperature of the phospholipid to form a dissolved phospholipid solvent mixture;

-dissolving a second phospholipid in the dissolved phospholipid solvent mixture at a temperature above the phase transition temperature of the phospholipid to form a dissolved phospholipid solvent mixture;

-adding an aqueous phosphate buffer to the dissolved phospholipid solvent mixture to form a buffered phospholipid solvent mixture; and

-stirring the buffered phospholipid solvent mixture to form a hydrated phospholipid solvent mixture.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The term "microbubbles" as used herein includes bubbles that exhibit substantially the same resonance frequency, and are also referred to as monodisperse microbubbles.

The term "monodisperse" as used herein includes characterizing a collection of microbubbles and can be interpreted to mean the polydispersity index (PDI) is less than 10 x 10 ^-2 Preferably 5X 10 ^-2 PDI is mathematically defined as pdi=s/n, where n represents the average bubble radius and s represents the standard deviation of the bubble radius. Namely, PDI <The 10% collection of bubbles can be considered monodisperse. In the context of the present invention, microbubbles are bubbles having a diameter of less than 10 microns (inclusive), preferably in the range from 2 microns up to 5 microns (inclusive). Bubbles greater than 10 microns in diameter may not safely flow through the smallest capillaries of the patient's vascular system and may cause edema. On the other hand, smaller bubbles may have poor ultrasonic reflectance.

The term "dispersed phase fluid" as used herein includes one or more gases from the group consisting of: SF (sulfur hexafluoride) ₆ 、N ₂ 、CO ₂ 、O ₂ 、H ₂ He, ar, ambient air and perfluorocarbon gas (such as CF ₄ 、C ₂ F ₆ 、C ₂ F ₈ 、C ₃ F ₆ 、C ₃ F ₈ 、C ₄ F ₆ 、C ₄ F ₈ 、C ₄ F ₁₀ 、C ₅ F ₁₀ 、C ₅ F ₁₂ ) And mixtures thereof.

Microbubbles typically comprise a shell filled with a gaseous core. The combination of the gas core and shell determines the resonant frequency of the microbubbles. When the microbubbles are subjected to ultrasound at a suitable frequency (at or at least near the resonant frequency of the microbubbles), the bubbles will resonate at the resonant frequency of the microbubbles. Such resonance may be picked up by an ultrasound imaging device. In this way, a high contrast can be achieved between the areas rich in microbubbles and the areas lacking microbubbles.

From WO-A-2016118010A microbubble generating unit is known. The content of this patent application is incorporated herein by reference for all purposes.

The term "phase transition temperature of phospholipids" as used herein includes the temperature required to induce a change in the physical state of lipids from an ordered gel phase (in which the hydrocarbon chains are fully extended and closely packed) to a disordered liquid crystal phase (in which the hydrocarbon chains are randomly oriented and fluid).

The term "phospholipid" or "lipid" as used herein includes a lipid whose molecule has a hydrophilic "head" comprising a phosphate group and two hydrophobic "tails" derived from fatty acids linked by alcohol residues. The phosphate groups can be modified with simple organic molecules such as choline, ethanolamine or serine. Phospholipids are a key component of all cell membranes. Because of their amphiphilic nature, they can form lipid bilayers. In eukaryotes, the cell membrane also contains another lipid, sterols, interspersed with phospholipids. The combination provides two-dimensional flowability and mechanical strength against breakage.

The term "non-toxic solvent" as used herein includes a class of solvents that are not harmful to the health of living organisms (such as humans and animals). Examples are, but are not limited to, propylene glycol, ethylene glycol, water, various phosphate buffers, and the like.

The preparation of the phospholipid composition is preferably carried out via a new green process. This is a practical manufacturing method of lipid formulations, which is completely biocompatible, can be easily extended and, most importantly, results in the formation of a homogeneous filterable phospholipid solution. The solution is ready for formation of microbubbles using microfluidic flow focusing techniques. Preferably there is no coalescence during the bubble formation.

The dissolution of the lipids is preferably performed by weighing out the required amount, preferably at room temperature. If desired, the lipids are first thawed. The lipids are then dissolved one after the other in the flask at a temperature above the phase transition temperature of the phospholipids, preferably with a preheated organic solvent, more preferably with a preheated non-toxic organic solvent. Once the former lipid is preferably completely dissolved in the (non-toxic) organic solvent, only the next lipid is added to the mixture. Complete dissolution in a (non-toxic) organic solvent means at least 80 wt% lipid dissolution, preferably at least 90 wt% dissolution, more preferably at least 95 wt% dissolution, even more preferably at least 99 wt% dissolution. A temperature above the phase transition temperature of a phospholipid means a temperature above the phase transition temperature of a phospholipid having the highest phase transition temperature. The first phospholipid may be dissolved in the organic solvent at a temperature above the phase transition temperature of the first phospholipid to form a dissolved phospholipid solvent mixture, and the second phospholipid is then dissolved in the dissolved phospholipid solvent mixture at a temperature above the phase transition temperature of the second phospholipid to form a dissolved phospholipid solvent mixture. However, it is preferred to use the preheated organic solvent at a temperature above the phase transition temperature of the phospholipid having the highest phase transition temperature, and even more preferred to use the preheated non-toxic organic solvent. Preferably, the temperature of the preheated organic solvent is above 65 ℃, more preferably above 70 ℃.

Another option is to dissolve the lipids separately with solvents in separate flasks at a temperature above the phase transition temperature of the phospholipids and then adding the dissolved lipid solutions together to form a dissolved phospholipid solvent mixture. However, this is not a preferred route.

For example in US-B-9801959, the preparation of a lipid mixture differs from our first step in that the lipid mixture is dissolved in propylene glycol. Traditionally, liposome solutions of lipid mixtures were prepared according to the Bangham method, mostly known as the membrane hydration method (Bangham et al J.mol.biol.1965, 13:238). Briefly, the procedure involves dissolving a phospholipid solid mixture in an organic solvent (i.e., chloroform and methanol). The organic solvent is then removed by evaporation under reduced pressure, after which the film obtained is added to propylene glycol and hydrated with an aqueous buffer. The disadvantage of this procedure is that toxic solvents may be present in the end product. Post-treatment for removal of trace amounts of organic solvents is required and additional clinical trials to demonstrate the non-toxicity of the product.

Solvent systems used in lipid suspensions are classified as either aqueous or non-aqueous vehicles. The choice of typical solvent system depends on the solubility and long-term stability of the final formulation. The organic solvent used for dissolving the lipid in the present invention is preferably selected from the group of propylene glycol, ethylene glycol, polyethylene glycol 3000 and/or glycerin, more preferably the organic solvent is propylene glycol. These organic solvents are classified as non-aqueous water-miscible solvents and are used as co-solvents. In addition, they are non-toxic. Propylene glycol, also known as PG, 1, 2-propanediol or propane-1, 2-diol, having the formula C ₃ H ₈ O ₂ The use of organic compounds (diols or dialcohols) is most preferred becauseIt is a clear, colorless, viscous liquid, hygroscopic and water miscible. In this case, PG is most preferably used as a cosolvent to improve the solubility of the phospholipid compound. Clinically, the use of PG as an excipient in commercial products is generally well-tolerated. Preferably in the range of 5% V/V to 60% V/V.

In a next step, an aqueous phosphate buffer is added to the dissolved phospholipid solvent mixture to form a buffered phospholipid solvent mixture. The aqueous phosphate buffer is preferably Phosphate Buffered Saline (PBS), phosphate buffered saline containing glycerol, water, saline/glycerol and/or saline/glycerol/non-aqueous solutions, more preferably Phosphate Buffered Saline (PBS). Most preferably Propylene Glycol (PG) in combination with Phosphate Buffered Saline (PBS) is used as a combination of non-aqueous and non-toxic solvents to adjust and stabilize the pH of the mixture near physiological pH.

The ratio of solvent to buffer (PBS/PG in the most preferred case) is preferably in the range from 80% V/V/20% V/V, more preferably in the range from 90% V/V/10% V/V up to 98% V/V/2% V/V. Most preferred is a final liquid composition having 95% V/5% V/V PBS/PG ± 1.5V/VPBS/PG.

Preferred phospholipids according to the invention are selected from the group DPPC, DSPC, DSPG, DMPC, DBPC, DPPE, DPPE-mPEG5000, DMPE-PEG-2000 and DSPE-PEG 2000. More preferably, the phospholipid is a combination of at least one of the group of DPPC, DSPC, DSPG, DMPC, DBPC, DPPE with at least one of the group of DPPE-mPEG5000, DMPE-PEG-2000 and DSPE-PEG2000, even more preferably DPPC, DSPC, DPPE with one of the group of DPPE-mPEG5000 and DSPE, most preferably DPPC and DPPE-mPEG5000.DPPC is the most preferred lipid, as it was observed in e.g. single microbubble dissolution studies that DPPC coated microbubbles remained smooth. Furthermore, DPPC does not provide a measurable resistance to surface shear and oxygen permeation. Among the groups DPPE-mPEG5000, DMPE-PEG-2000 and DSPE-PEG2000, DPPE-mPEG5000 is preferred because it is an excellent lipopolymer emulsifier.

Advantageously, when 2 lipids are present in the hydrated phospholipid solvent mixture, the ratio of lipids is in the range from 95:5 to 70:30, more preferably in the range from 90:10 to 75:25, even more preferably in the range from 85:15 to 80:20.

Advantageously, one or more phospholipids may be sequentially dissolved in the dissolved phospholipid solvent mixture at a temperature above the phase transition temperature of the phospholipids. Thus, end products comprising more than two lipids are preferably contemplated in the present invention. As additional lipids, bifunctional pegylated lipids may be used.

Difunctional PEG acylated lipids include, but are not limited to, DSPE-PEG (2000) succinyl 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ succinyl (polyethylene glycol) -2000] (ammonium salt), DSPE-PEG (2000) PDP 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ PDP (polyethylene glycol) -2000] (ammonium salt), DSPE-PEG (2000) maleimide 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000] (ammonium salt), DSPE-PEG (2000) biotin 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000] (ammonium salt), DSPE-PEG (2000) cyano 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ cyano (polyethylene glycol) -2000] (ammonium salt), DSPE-PEG (2000) amine 1, 2-distearoyl; -sn-glycerol-3-phosphate ethanolamine-N- [ amino (polyethylene glycol) -2000] (ammonium salt), DPPE-PEG (5000) -maleimide, 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine-N- [ dibenzocyclooctyl (polyethylene glycol) -2000] (ammonium salt), 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine-N- [ azido (polyethylene glycol) -2000] (ammonium salt), 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine-N- [ succinyl (polyethylene glycol) -2000] (ammonium salt), 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine-N- [ carboxy (polyethylene glycol) -2000] (ammonium salt), 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine-N- [ maleimide (polyethylene glycol) -2000] (ammonium salt), 1, 2-distearoyl-sn-3-phosphate ethanolamine-N- [ PDP (polyethylene glycol) -2000] (ammonium salt), 1, 2-distearoyl-sn-3-phosphate ethanolamine-N- [ carboxyl (polyethylene glycol) -2000] (ammonium salt), 1, 2-distearoyl-sn-3-phosphate ethanolamine-N- [, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ biotin (polyethylene glycol) -2000 (ammonium salt), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ cyanogen (polyethylene glycol) 2000 (ammonium salt), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ folic acid (polyethylene glycol) -5000 (ammonium salt), N-palmitoyl-sphingosine-1- { succinyl [ methoxy (polyethylene glycol) 2000] }, and N-palmitoyl-sphingosine-1 { succinyl [ methoxy (polyethylene glycol) 5000] }. Bifunctional lipids can be used to attach antibodies, peptides, vitamins, glycopeptides, and other targeting ligands to microbubbles. The MW of the PEG chains in the lipids can vary from about 1000 daltons to about 5000 daltons.

According to the invention, it is preferred to perform all method steps at a temperature above the phase transition temperature of the phospholipids. This has the advantage that the lipids are then homogeneously mixed, since the lipids are in the liquid crystal phase during the whole process. The phase transition temperature is defined as the temperature required to induce a change in the physical state of the lipid from an ordered gel phase (where the hydrocarbon chains are fully extended and closely packed) to a disordered liquid crystal phase (where the hydrocarbon chains are randomly oriented and fluid).

Advantageously, the buffered phospholipid propylene glycol mixture is stirred for at least 1 hour, more preferably for at least 2 hours, even more preferably for at least 4 hours, most preferably for at least 8 hours. In this step, a large amount of hydration of the lipid occurs. The stirring step is an easy step when the procedure is extended to larger batches. Agitation can be performed using a standard baffled mixer reactor.

Advantageously, the hydrated phospholipid solvent mixture is filtered through a sterilizing filter to form a sterilized hydrated phospholipid solvent mixture. Contaminants are removed from the phospholipid solvent mixture. More preferably, the sterilizing filter has a pore size of 0.2 microns. To remove bacteria suspended in the solution, a pore size of 0.2 μm was considered to be effective. With such a filter, larger particles that may have an effect during the microfluidic process of generating microbubbles are also removed.

Advantageously, the hydrated phospholipid solvent mixture is filtered at least twice, more preferably at a mixture temperature above room temperature, more preferably above 50 ℃, and at a mixture temperature below room temperature, more preferably below 15 ℃. The second step is preferably carried out before storing the hydrated phospholipid solvent mixture, resulting in a more stable mixture.

According to the invention, the concentration of lipids in the hydrated phospholipid solvent mixture is in the range of from 5mg/ml up to 20mg/ml, preferably in the range of 10mg/ml up to 18 mg/ml. This higher concentration of lipids in the hydrated phospholipid solvent mixture is advantageous for microfluidic fabrication. These higher concentrations often create problems of coalescence and long settling times when using "standard" phospholipid compositions. Microbubbles typically require 24 hours or even more to stabilize. These problems have been overcome with the method of the present invention. When the method of the invention is applied, coalescence of the microbubbles resulting in polydisperse microbubbles is no longer observed. To maintain a monodisperse population of microbubbles, coalescence should still be avoided. Furthermore, with the phospholipid composition prepared via the process of the present invention, microbubbles prepared using microfluidics are virtually ready-to-use, meaning that the bubbles are ready-to-use and stable within minutes after production.

The invention also relates to a phospholipid composition obtainable by the method of the invention as described herein, wherein the total concentration of phospholipids is at least 12mg/ml. Advantageously, the total concentration of phospholipids is at least 15mg/ml. This higher concentration of phospholipids is advantageous for microfluidic fabrication. Higher concentrations create problems when dipalmitoyl phosphatidic acid (DPPA) is present. Advantageously, the phospholipid composition according to the invention does not comprise dipalmitoyl phosphatidic acid (DPPA). These higher concentrations present difficulties in coalescence when preparing phospholipid compositions via prior art methods. Coalescence of the microbubbles results in polydisperse microbubbles. To maintain a monodisperse population of microbubbles, coalescence should be avoided.

Both the size and shell properties of the phospholipids are important for the behavior of the microbubbles. Microbubbles of the same size but with different shell properties are known in the literature to behave acoustically differently. Advantageously, the phospholipid composition obtainable by the process of the invention as described herein comprises monodisperse microbubbles having an average particle size of between 1 μm and 10 μm, preferably between 2 μm and 5 μm. Alternatively defined, the phospholipid composition obtainable by the process of the invention as described herein preferably comprises microbubbles having a monodispersity PDI of 10% or less, which meets a Geometric Standard Deviation (GSD) of 1.1 or less. The method of preparing the phospholipid composition according to the invention facilitates a more uniform liposome distribution, resulting in a more uniform between the microbubble shells and thus a more uniform acoustic performance.

During an ultrasound examination, an operator of the ultrasound imaging device determines a desired frequency of ultrasound waves at which the examination should be performed. The frequency is determined by the depth of the tissue or organ and the type of body structure to be analyzed and the ultrasound procedure. In order to obtain a suitable contrast, it is desirable that the resonance frequency of the microbubbles corresponds to the desired frequency. Furthermore, the variation in resonance frequency between microbubbles should be sufficiently low. This is an example, however, the insonification frequency may also be twice the resonance frequency of the microbubbles, for example. The change in acoustic behavior of the microbubbles is expected to be sufficiently low and predictable. For this reason, controlled fabrication of microbubbles is required.

The present invention thus relates to a system for the controlled production of microbubbles using the phospholipid composition described above. Brief explanation: in microfluidics, "flow focusing" air flow is used to flow through a narrow structure to create microbubbles. The inner gas is forced to flow through the narrow structure by an outer co-current liquid flow. In this configuration, the gas flow forms fine gas lines which break into uniform microbubbles. The size of these microbubbles is controlled by the gas-liquid flow rate ratio. Microbubbles are produced at typical production rates between 100,000 and 1,000,000 microbubbles per second. Once microbubbles are generated, they decelerate and collide. This is relevant to microfluidic methods of generating microbubbles. These collisions are severe and can cause coalescence (two bubbles merge). This can be avoided by increasing the lipid concentration (to about 15mg/ml, ten times higher than the "standard" lipid concentration). In general, increasing the lipid concentration causes problems in achieving uniform dispersion of the liposomes. This has been addressed by improving the preparation of phospholipid compositions and avoiding the use of DPPA. The presence of high lipid concentrations and DPPA can lead to the formation of aggregates. Aggregates hinder microfluidic production and lead to poor filterability. Aggregates (or poor homogeneity of the phospholipid composition) have a negative impact on the formation of the microbubble shell and lead to a higher probability of coalescence of the microbubbles. Coalescence should be avoided to obtain monodisperse microbubbles.

Another advantage of cartridges with phospholipid compositions is that it is possible to produce the cartridges and store for a considerable period of time before application, as the liquid can be kept in a fluid storage unit in a sterile manner.

The invention relates in particular to cartridges in which at least one liquid is held in a fluid storage system intended for use in the human or animal body, for example for intravenous use or for intra-luminal use. For example, the at least one liquid may include or may be used to form a diagnostic agent (such as a contrast agent for medical imaging) or a therapeutic agent (such as a pharmaceutical agent). However, the invention may also relate to other applications, where sterility of the liquid to be mixed and sterility of the mixed product are important.

Each fluid storage unit may include a fluid storage unit inlet and a fluid storage unit outlet, wherein the fluid storage unit is configured such that pressurized gas supplied to the fluid storage unit through the fluid storage unit inlet pushes fluid in the fluid storage unit through the fluid storage unit outlet. By using a gas as propellant, which can be kept sterile relatively easily, sterility of the liquid can be ensured during transport from the fluid storage unit to the mixing unit. In addition, the at least one fluid storage unit may include: a reservoir configured to receive a sealed container in which the liquid is held in a sterile and sealed manner; and a liquid reservoir in fluid communication with the storage chamber, wherein the fluid storage unit inlet is connected to one of the storage chamber and the liquid reservoir, and wherein the fluid storage unit outlet is connected to the liquid reservoir. The liquid reservoir may be configured to collect liquid released from the container after the container has been broken, ruptured, cut or pierced. The sealed container may for example comprise a blister pack. The sealed container may be fixedly held in the storage chamber, for example by means of an adhesive. Alternatively, the blister package is inserted into the storage compartment immediately prior to use. For example, the blister package may comprise an adhesive strip by means of which the blister package may be secured to the cartridge. Such adhesive tape may be covered by a cover tape that is to be removed when the blister package is to be secured to the cartridge. Thus, the present invention may relate to embodiments in which the cartridge and the sealed container are two separate items sold separately to the end user by a single entity or by different entities, or embodiments in which the sealed container is secured to the cartridge prior to being sold in combination to the end user.

For at least one fluid storage unit, the liquid reservoir may be formed as part of the storage chamber. In this case, the liquid released from the sealed container is at least partially collected in the reservoir itself. Alternatively, the at least one fluid storage unit may comprise a fluid channel formed in the cartridge, the fluid channel connecting the storage chamber and the liquid reservoir. Thus, after the liquid is released, the liquid will be transported through the fluid channel to the liquid reservoir.

For a fluid storage unit of the at least one fluid storage unit, the storage chamber may comprise a support surface for supporting the sealed container and at least one protruding pin or needle extending towards the sealed container so as to pierce the sealed container when a sufficient force is applied thereto. The sealed container may be deformable such that when a force is applied, a wall or segment of the container engages the pin or needle. When sufficient force is applied, the pin or needle will puncture the sealed container, releasing the liquid held by the container. It should be noted that the present invention does not exclude other means by which the container may be broken, cut, ruptured or otherwise opened in order to release the liquid. The pin or needle may protrude from the support surface. In other embodiments, the pin or needle may be disposed at a different surface such that the pin or needle does not engage the container when the container is supported by the support surface and no external force is applied to the container.

In addition, at least one fluid storage unit may be provided with a protection ring protruding farther from the cylinder than the sealing container when the sealing container is placed in the storage chamber. In this way, when the cartridge is placed onto the table and the container is facing downward toward the table, no significant force will be applied to the container.

The cartridge may comprise a plurality of fluid storage units, wherein the fluid storage unit inlets of at least two fluid storage units are in fluid communication with each other. In this case, the same gas may be used for at least two fluid storage units. In another embodiment, the gas used as propellant by the plurality of fluid storage units may be the same. Additionally or alternatively, the gas inlet through which the pressurized first gas is received may be the same as the gas inlet through which the pressurized gas used as propellant by the at least one fluid storage unit is received. The pressurized first gas may be different from the pressurized gas used as the propellant by the at least one fluid storage unit.

The invention relates in particular to a combination of gas and liquid as described in table 1. In this table, (m) indicates that a gas is to be used as the gas to be mixed, and (p) indicates that a gas is to be used as the propellant. For example, the second combination mentioned in the table indicates that the first gas is used as the pressurized first gas to be mixed and the second gas is used as a propellant for propelling different liquids (i.e. liquid 1 and liquid 2 held in the respective fluid storage units) towards the mixing unit. In the fifth combination, the first gas is supplied through the gas inlet 1 and serves as both the propellant and the gas to be mixed. Other combinations are not excluded.

Table 1: possible combinations of gas and liquid.

In these combinations, the invention relates in particular to embodiments wherein the cartridge has a single fluid storage unit and two gas inlets, wherein a first of the two gas inlets is in fluid communication with the mixing unit and wherein a second of the two gas inlets is in fluid communication with the fluid storage unit. This corresponds to the third combination in the table above.

The mixing unit may be configured to mix at least one liquid received from the fluid storage system with the pressurized first gas, wherein the mixing unit comprises a microfluidic device configured for providing a fluid to the fluid storage systemMicrobubbles are generated in the at least one liquid, the microbubbles being filled with a pressurized first gas. The microfluidic device may comprise two or more substrates, such as glass substrates, wherein at least one of the substrates comprises a groove structure made, for example, using wet etching, and the substrates are bonded together, for example, using fusion bonding, to form a channel structure having a cross-sectional dimension in the range of 1 to 1000 micrometers. The microfluidic device may be configured to generate microbubbles having diameters below 10 microns and preferably in the range of 2 microns to 5 microns. Such microbubbles may be used as contrast agents for ultrasound imaging or for therapeutic applications in combination with ultrasound, such as ultrasound guided drug delivery or focused ultrasound therapy. For such applications, the pressurized first gas may include one or more gases from the group consisting of: SF (sulfur hexafluoride) ₆ 、N ₂ 、CO ₂ 、O ₂ 、H ₂ He, ar, ambient air and perfluorocarbon gas (such as CF ₄ 、C ₂ F ₆ 、C ₂ F ₈ 、C ₃ F ₆ 、C ₃ F ₈ 、C ₄ F ₆ 、C ₄ F ₈ 、C ₄ F ₁₀ 、C ₅ F ₁₀ 、C ₅ F ₁₂ ) And mixtures thereof.

When used in the above-described application for generating contrast media for ultrasound imaging, the cartridge preferably comprises a gas inlet for receiving a pressurized first gas and a gas inlet for receiving a propellant gas to be used in a single fluid storage unit.

The microfluidic device may include: a first inlet for receiving a pressurized first gas; a second inlet for receiving the phospholipid composition; and a bubble forming channel for generating microbubbles based on the flow of the first pressurized gas received through the first inlet and the flow of the phospholipid composition received through the second inlet. Additionally, the cartridge may include a first opening, a second opening, and a third opening, wherein the first opening is in fluid communication with a gas inlet that receives the pressurized first gas, and wherein the second opening is in fluid communication with a fluid storage system to receive the phospholipid composition. In this case, the microfluidic device is positioned relative to the cartridge such that the first opening is aligned with the first inlet of the microfluidic device, the second opening is aligned with the second inlet of the microfluidic device, and the third opening is aligned with the outlet of the microfluidic device. The microfluidic device may be fixedly connected to the cartridge using an adhesive or an integral bond. Alternatively, the microfluidic device may be integrally formed with the cartridge.

The microfluidic device may include a flow focus junction; a first channel having one end connected to the second inlet and the other end connected to the flow focusing junction; a second passage having one end connected to the second inlet and the other end connected to the flow focusing junction; and a third channel having one end connected to the first inlet and the other end connected to the flow focusing junction. The bubble forming channel may be connected to the flow focus junction. Further, the flow focus joint may be configured to receive a flow of the phospholipid composition from two opposite directions via the first channel and the second channel, the flow of the phospholipid composition impinging on the flow of the first pressurized gas received via the third channel in a perpendicular manner. The flow of pressurized gas may be directed from the third channel into the bubble forming channel.

A microfluidic device as described above is known from WO 2013/141695 and WO 2016/118010. The contents of these publications are incorporated herein for all purposes.

The cartridge may include an outlet formed in the cartridge body for outputting the fluid mixed by the mixing unit. The outlet may be formed to allow a luer taper connection with the syringe.

The cartridge may comprise a buffer reservoir formed in the cartridge body and arranged between the mixing unit and the outlet, wherein the capacity of the buffer reservoir exceeds the volume of liquid held in the fluid storage system. Typically, the buffer reservoir is at least 30% greater than the volume of liquid held in the fluid storage system.

The cartridge may further include a vent formed in the barrel that is in fluid communication with the buffer reservoir. A vent is provided to prevent over-pressurization in the buffer reservoir. In addition, the vent holes allow purging the buffer reservoir with a predetermined gas prior to mixing. For example, in embodiments having the microfluidic device described above, the buffer reservoir may be purged with the same gas as the gas filling the microbubbles. By filling the buffer reservoir with this gas, the stability of the microbubbles can be improved, e.g. the dissolution rate of the microbubbles can be reduced compared to embodiments in which other gaseous medium is present in the buffer reservoir. The vent holes also allow for pressure equalization in the buffer reservoir when liquid is extracted from the buffer reservoir, for example using a syringe. Without a vent, a negative pressure will be generated inside the buffer reservoir, which will destroy the microbubbles and complicate the extraction of the liquid inside the buffer reservoir.

The cartridge may further comprise: a fluid channel formed in the barrel between the one or more gas inlets and the mixing unit; a fluid passageway formed in the cartridge between the one or more gas inlets and the fluid storage system; a fluid channel formed in the barrel between the fluid storage system and the mixing unit; a fluid channel formed in the cartridge between the mixing unit and the buffer reservoir; and a fluid channel formed in the cartridge between the buffer reservoir and the vent. These fluid channels may extend substantially in the same fluid channel plane. In some embodiments, the cartridge may include a top surface and a bottom surface that are relatively close together, and wherein the top surface and the bottom surface extend substantially in a plane parallel to the plane of the fluid passage. Such a cartridge may be rod-shaped.

The cartridge may be configured to be in a first orientation during said mixing, wherein in the first orientation: the normal to the plane of the fluid passage is substantially horizontal; the mixing unit is positioned at a lower position relative to the fluid storage system and relative to the gas inlet through which the pressurized first gas is received; and in this orientation the fluid channel between the mixing unit and the buffer reservoir is located in the buffer reservoir at the lower end of the buffer reservoir, preferably at or near the lowest point of the buffer reservoir. Hereinafter, the terms horizontal and vertical will be used in relation to the direction of earth's gravity unless otherwise indicated. For example, vertical shall be used to refer to a direction perpendicular to the earth's surface, and horizontal shall be used to refer to a direction parallel to the earth's surface.

Generally, the gas filling the microbubbles is heavier than ambient air. When the cartridge is held in the first orientation, and when the buffer reservoir has been flushed with the gas prior to mixing, liquid exiting the microfluidic device will engage with the gas and/or liquid that has been output upon entering the buffer reservoir. In this way, the stability of the microbubbles can be improved, as contact between the output liquid and ambient air that may be present in the buffer reservoir can be minimized.

The cartridge may be configured to be in a second orientation, for example during storage, wherein in the second orientation a normal to the fluid passage plane is substantially vertical; and the fluid channel between the mixing unit and the buffer reservoir is output in the buffer reservoir at a location above the liquid-air interface of the buffer reservoir to prevent liquid output by the mixing unit into the buffer reservoir from flowing back into the fluid channel between the mixing unit and the buffer reservoir when the cartridge is in the second orientation. Thus, after mixing with the cartridge held in the first orientation, the cartridge may be arranged flat on the table. This configuration generally corresponds to the second orientation. By arranging the fluid channel between the mixing unit and the buffer reservoir in the described manner, it is achieved that liquid in the buffer reservoir will not flow back through the fluid channel towards the mixing unit.

When the cartridge is in the second orientation, the fluid channel between the vent and the buffer reservoir may be output at a location above a liquid-air interface located in the buffer reservoir. Thus, when the cartridge is arranged flat on the table (i.e., corresponding to the second orientation), liquid in the buffer reservoir may be prevented from flowing into the fluid channel toward the vent hole.

The buffer reservoir may be vertically elongated when the cartridge is held in an orientation (e.g., a first orientation) in which the normal to the plane of the fluid channel is horizontal. In this way, the liquid output by the mixing unit into the buffer reservoir only experiences a relatively small area in which the liquid may be exposed to the gas in the buffer reservoir. Thus, by having an elongated shape, the stability of microbubbles can be improved.

The buffer reservoir may be transparent to allow the user to visually inspect its contents or its level.

It should be noted that the above-described features associated with the buffer reservoir are equally advantageous for applications other than microbubble formation.

The cartridge may further comprise a filter arranged in each of the one or more gas inlets formed in the cartridge body and, where applicable, in the vent. These filters are configured to prevent bacteria and/or other pathogens from entering the cartridge. These filters are preferably hydrophobic. The at least one filter may comprise a filter membrane, a first filter support and a second filter support, wherein the first filter support and the second filter support are fixedly attached to or integrally formed with the cartridge, and wherein the filter membrane is arranged between the first filter support and the second filter support. The first and second filter supports prevent rupture of the filter membrane in the event of sudden changes in negative or overpressure, removal or tearing of the filter membrane from the cartridge. The first and second filter supports may include ribs extending from the outside of the cylinder or from the inside of the cylinder over the openings for the gas inlet and exhaust holes.

The cartridge may be formed by fixedly attaching the first cartridge portion and the second cartridge portion using ultrasonic welding, each cartridge portion including a base layer. Prior to fixedly attaching the portions, one of the first and second barrel portions may include a ridge and/or protruding portion extending from the base layer, the ridge and/or protruding portion configured to cooperate with a protruding portion and/or ridge extending from the base layer of the other of the first and second barrel portions during ultrasonic welding. After ultrasonic welding, the protruding portions are integrally connected to the corresponding ridges. The integrally connected ridge and protruding portions and the base layers of the first and second barrel portions may together define at least one of: a mixing unit, a fluid storage system, one or more gas inlets, outlets, a buffer reservoir and a fluid channel for connecting them. Preferably, all of these structures are formed in this manner. The first barrel portion and the second barrel portion may be made from one or more materials of the group of thermoplastic materials such as polycarbonate using injection molding.

According to a second aspect, the present invention provides a cartridge system comprising an apparatus comprising a housing having an opening into which a cartridge can be releasably inserted, and a cartridge as described above. The apparatus comprises one or more nozzles for introducing respective pressurized gases into the one or more gas inlets, respectively, for said mixing by means of the mixing unit of the cartridge.

The cartridge may be configured to include the sealed container described above. In this case, the apparatus may further comprise an engagement unit for engaging a sealed container arranged in the storage chamber of the at least one fluid storage unit so that the sealed container is broken, ruptured or cut or pierced.

The apparatus may comprise a drive system for engaging and disengaging the nozzle and/or the engagement unit with the one or more gas inlets and the sealed container, respectively. The apparatus may include a controller for controlling the drive system. Further, at least one of the one or more nozzles may be connected to a controllable valve, wherein the controller is configured to control the flow of pressurized gas through the at least one of the one or more nozzles via the controllable valve. The apparatus may include one or more reservoirs for holding pressurized gas, the one or more reservoirs being connected to one or more nozzles. Alternatively, the device may comprise one or more further gas inlets connected to one or more nozzles, respectively, wherein the one or more further gas inlets are configured to be connected to one or more gas reservoirs external to the device. Thus, the device may rely on internal storage of the desired gas or may be connected to an external infrastructure that provides the desired gas. In both cases, the controllable valve is preferably arranged in the device.

The controller may be configured to control the device to be operable in a first state in which the nozzle and the engagement unit are positioned at a distance from the cartridge. The system is further operable in a second state in which the controller controls the drive system to bring the nozzle into contact with the one or more gas inlets, and in which the controller controls the controllable valves such that pressurized gas is supplied to the cartridge via the respective nozzles and gas inlets of the cartridge. In this way, the various fluid passages and optional buffer reservoirs inside the cartridge may be purged with one or more specified gases.

The apparatus is further operable in a third state in which the controller controls the drive system to cause the engagement unit to engage the sealed container so as to cause the sealed container to be broken, ruptured or cut or pierced, and subsequently controls the controllable valve to provide pressurized gas to the cartridge via the respective nozzle and gas inlet of the cartridge for said mixing by the mixing unit of the cartridge. Typically, some time remains between the time the propellant gas is applied and the time the sealed container releases its liquid to allow all or most of the liquid to be collected in the liquid reservoir of the associated fluid storage unit prior to the mixing process.

The controller may be configured to control the controllable valve to stop supplying pressurized gas to the cartridge when changing from the second state to the third state. In this way, the flow of liquid from the sealed container to the liquid reservoir is not affected.

The drive system may comprise a first unit in which the one or more nozzles are movably mounted and in which the engagement unit is mounted. The drive system may further comprise an actuator for moving the first unit relative to the cartridge when the cartridge is inserted into the apparatus.

The nozzle may be spring-biased mounted in the first unit to permit movement of the nozzle relative to the first unit in a first direction toward and perpendicular to the barrel. As described above, the first unit is movable toward the cartridge to allow the nozzle to engage the cartridge. At this time, the engagement unit does not engage the sealed container. Such engagement is obtained when the first unit is moved even further towards the cartridge. By having the nozzle mounted in the first unit in a spring-biased manner, the nozzle can be moved back towards the first unit during this last movement of the first unit, while maintaining engagement with the gas inlet. In this way, damage to the nozzle and/or gas inlet of the cartridge may be prevented.

The engagement unit may be movably mounted to the first unit. The movement of the engagement unit relative to the first unit may be used to accommodate non-zero tolerances in the relative positions and/or shapes of the sealed container and the gas inlet. For example, the engagement unit can translate in one or more directions other than the direction toward the cartridge and/or rotate about these directions. Similar degrees of freedom may apply to one or more nozzles. In both cases, degrees of freedom are used to accommodate tolerances in the manufacturing process of the cartridge. In other words, the degrees of freedom allow the nozzle and the engagement unit to be aligned relative to the cartridge.

The drive system may further comprise a second unit coupled to the first unit, wherein the first unit is movable in at least one degree of freedom relative to the second unit, and wherein the actuator is configured to move the second unit in a first direction (i.e. towards the cartridge). In this way, the movement of the second unit is entirely controlled by the actuator. However, movement of the first unit may also generally depend on the alignment of the nozzle and engagement unit with the cartridge. This is achieved by the fact that the first unit is movable relative to the second unit. The mutual movement between the first and second units may be achieved by coupling the second unit to the first unit using one or more curved leaf springs. Thus, the actuator will drive the first unit indirectly (i.e. via the second unit).

The first unit is movable relative to the second unit in a second direction orthogonal to the first direction and in a third direction orthogonal to both the first direction and the second direction. The first direction toward the cartridge is referred to as the z-direction, and the first direction and the second direction may correspond to the x-direction and the y-direction, respectively. The first unit is also rotatable about a first direction relative to the second unit. Preferably, the first unit is only movable relative to the second unit by translation in the x-direction and/or the y-direction and by rotation about the z-direction.

A nozzle of the one or more nozzles is movable in a first direction and rotatable about the first direction relative to the first unit, and wherein another nozzle of the one or more nozzles is movable in the first direction, the second direction, and the third direction and rotatable about one of the second direction and the third direction. For example, in the case of using two nozzles, the first nozzle can move only in the z direction and rotate around that direction. In this case, the other nozzle can move only in the x-direction, the y-direction, and the z-direction, and rotate around the y-direction. Other degrees of freedom of the nozzles may be fixed.

The drive system may include a threaded shaft driven by the actuator and extending in a first direction, and wherein the second unit is coupled to the threaded shaft to cause translation of the second unit in the first direction upon rotation of the threaded shaft. Preferably, rotation of the shaft will cause movement of the second unit in the first direction only.

The device may further comprise a second frame fixedly connected to the housing, wherein the threaded shaft is rotatably received in the second frame, the second frame preferably comprises a wall section extending substantially perpendicular to the first direction, and the threaded shaft is rotatably received in the wall section.

The apparatus may comprise a force sensor for sensing a force of the engagement unit against the sealed container, wherein the controller is configured to control the drive system in dependence of the sensed force.

The second frame may include a first portion and a second portion coupled to the first portion, wherein the first portion and the second portion are movable relative to each other when a force is applied to the cartridge by the engagement unit. In this case, the force sensor may be configured to determine the force from a mutual displacement between the first portion and the second portion. For example, the first and second portions may be connected using a structure compressible in a first direction, such as a curved rod or lattice of rods. The compression may be determined in a known manner using a position sensor. The output of the position sensor in combination with known mechanical properties of the compressible structure may then be used to calculate a force or a parameter indicative of and/or corresponding to such force applied to the cartridge.

According to a third aspect, the present invention provides a cartridge configured for mixing a liquid held in the cartridge with another liquid held in the cartridge or with a pressurized gas supplied to the cartridge within the cartridge, wherein the liquid is intended for use in a human or animal body, for example for intravenous use or for intracavity use, wherein the liquid held in the cartridge comprises or is used to form a diagnostic agent (such as a contrast agent for medical imaging) or a therapeutic agent (such as a medicament). The cartridge may be configured as a cartridge as defined above.

According to a fourth aspect, the present invention provides a cartridge system comprising a cartridge according to the third aspect of the present invention and a device into which the cartridge is releasably insertable, wherein the device is configured for providing a pressurized gas which serves as a propellant for moving a liquid inside the cartridge for mixing the liquid and/or as a gas to be mixed. The device may be configured as described above.

The invention also relates to a blister package comprising a phospholipid composition, wherein the concentration of phospholipids is at least 12mg/ml, preferably at least 15mg/ml. The main advantage of the blister package is that the phospholipid composition remains sterile for a long period of time. Preferably, the phospholipid composition does not comprise dipalmitoyl phosphatidic acid (DPPA).

The invention will be described in more detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows a perspective view of an embodiment of a cartridge system according to the present invention;

FIGS. 2 and 3 illustrate different views of a cartridge in the cartridge system of FIG. 1;

FIG. 4 shows details regarding the construction of the cartridge of FIG. 2;

FIG. 5 shows a microfluidic device for use in the cartridge of FIG. 2;

FIG. 6 illustrates the engagement between the sealed container and the cartridge of FIG. 2;

FIGS. 7A and 7B show details regarding a filter used in the cartridge of FIG. 2;

FIG. 8 shows the interior of the device shown in FIG. 1;

figures 9 to 14 show different detailed views of different parts of the interior of the device shown in figure 8; and is also provided with

Fig. 15 schematically illustrates the cartridge system of fig. 1.

FIG. 16 shows the size distribution of different microbubble populations; and is also provided with

Fig. 17 shows normalized attenuation for different microbubble samples.

Fig. 1 shows an embodiment of a cartridge system according to the invention. The system includes a cartridge 100 and an apparatus 200. The latter device comprises a housing 201 and an opening 202 in the housing 201 into which the cartridge 100 can be releasably inserted. In practice, the device 200 is shown with the cartridge 100 inserted therein.

The cartridge 100 comprises a barrel 101 and is configured for mixing a liquid held in a sealed container 120 with a gas supplied through a gas inlet 111 within the cartridge. To this end, the cartridge 100 comprises a mixing unit in the form of a microfluidic device, which generates microbubbles filled with a gas supplied via the gas inlet 111. The mixing unit will be discussed in more detail later in connection with fig. 5.

Because the microfluidic device is a passive device, i.e. it requires energy to operate, pressurized gas must be supplied to the gas inlet 111 and towards the microfluidic device. Furthermore, once the liquid is released from the container 120, pressurized gas will be supplied to the gas inlet 110 to push the released liquid towards the microfluidic device. In other words, the gas supplied to the gas inlet 110 will act as a propellant for pushing the liquid released from the container 120 towards the microfluidic device.

The liquid in the container 120 may be released by breaking the container 120. For this, a sufficient force should be applied to the container 120, as will be explained later.

The device 200 is configured to perform the actions described above. In other words, when cartridge 100 is inserted into opening 202, device 200 will ensure that the proper gas is supplied to cartridge 100 and that container 120 will be destroyed. This function will be explained later with reference to fig. 9 to 15.

The output of the microfluidic device is connected to a buffer reservoir 140 in which the mixed liquid may be temporarily stored. The reservoir, which will be discussed in more detail in connection with fig. 3, is connected to the vent 112 to prevent excessive pressure from developing in the reservoir 140. Furthermore, a buffer reservoir 140 is connected to the output 113 of the cartridge 100, which in fig. 1 is covered by a closing cap 114.

As shown in fig. 1, sealed container 120 comprises a blister package. The package is configured such that when a force is applied on the top side of the package in fig. 1, the back side of the package will flex towards the barrel 101. The container 120 may contain the above-described phospholipid composition having a phospholipid concentration of at least 12 mg/ml. An exemplary volume of liquid held in the container 120 is about 2ml.

The gas to be used as a propellant supplied to the gas inlet 110 may be SF ₆ Or C ₃ F ₈ . The same gas may be supplied to the gas inlet 111. Based on these fluids, the microfluidic device will generate a filling with SF, as will be explained later ₆ Or C ₃ F ₈ Is a suspension of microbubbles in an aqueous solution. Such suspensions may be used as contrast agents for ultrasound imaging.

SF of microbubbles ₆ Or C ₃ F ₈ The combination of the size of the core and the shell formed from the phospholipids determines the resonant frequency of the microbubbles. When the microbubbles are subjected to ultrasound at a suitable frequency (at or at least near the resonant frequency of the microbubbles), the bubbles will resonate at the resonant frequency of the microbubbles. Such resonance may be picked up by an ultrasound imaging device. In this way, a high contrast can be achieved between the areas rich in microbubbles and the areas lacking microbubbles.

Fig. 2 shows an exploded view of the cartridge 100. Here, an adhesive 121 can be seen with which the container 120 is fixedly attached to the cylinder 101. In addition, the barrel 101 includes a first portion 101A and a second portion 101B that are fixedly connected to each other using ultrasonic welding or some other form of integral bonding.

Both the first portion 101A and the second portion 101B include ridges and/or protrusions extending from the base layer. The base layer may include structures, such as recesses, for forming structures in a final cartridge, such as buffer reservoir 140. An example of the protrusion 102 and the ridge 103 is shown in fig. 4. Here, each ridge 103 includes a pair of ridges 103A, 103B with an opening formed therebetween. In this opening, the protrusion 102 of the other body portion may extend once the first portion 101A and the second portion 101B are properly aligned with each other. Thereafter, the portions 101A and 101B are connected to each other using ultrasonic welding, after which the tip of the protrusion 102 will be melted to form an integral connection with the ridges 103A and 103B. In fig. 4, the two ridges are connected in the manner described above. Thus, a fluid channel, indicated by a rectangle "C", is formed between the two ridges.

Returning now to fig. 2, gas inlets 110 and 111 are each formed using cylindrical protrusions 110C and 111C, respectively. The protrusion extends from the body portion 101A. Further, between the body portions 101A, 101B, a hydrophobic filter membrane 115 is arranged at the positions of the inlets 110, 111 and the vent 112. These filters are supported on both sides by suitable support structures. Fig. 2 shows the first filter support 116 connected to the body portion 101A using ultrasonic welding prior to attachment of the body portions 101A, 101B. On the other side of the filter membrane 115 a second filter support 117 is provided in the shape of a star, see cross-sectional and top view of the gas inlet 111 of fig. 7A and 7B, respectively.

Returning again to fig. 2, the microfluidic device 130 may be mounted to the cartridge 101, and more specifically to the body portion 101B, using a suitable adhesive, represented in fig. 2 as component 131.

Fig. 2 also shows that a cover 150 may be mounted on the rear side of the cartridge 100, covering the microfluidic device 130. On this cover 150, data about the cartridge 100 may be printed.

Fig. 3 shows a schematic top view of the cartridge 100. In this view, the various components of the cartridge 100 are shown in more detail. In this figure, various gas-liquid interfaces 126 and 141 are shown to indicate the boundary between the liquid medium (L) and the gas medium (G). The interfaces 126, 141 indicate that the configuration shown in fig. 3 corresponds to the cartridge 100 still inside the device 200. In other words, earth gravity acts in the right-to-left direction in fig. 3.

As shown in fig. 3, the fluid channel 110A extends from the gas inlet 110 to the liquid reservoir 125. In which liquid from the container 120 will be collected as will be explained later. At a bottom portion of the reservoir 125, a fluid channel 125A extends toward an opening 125B in the barrel 101. The fluid channel 111A extends from the gas inlet 111 to an opening 111B in the cylinder 101.

The openings 125B, 111B are used to deliver liquid and gas, respectively, to the microfluidic device 130, as shown in fig. 5. In this figure, it can be seen that opening 125B is connected to a first channel 132A and a second channel 132B in the microfluidic device 130, and that opening 111B is connected to a relatively short channel 133 in the microfluidic device 130.

The first and second channels 132A and 132B and the channel 133 exit in the flow focus junction 134. At this junction, the SF inside the channel 133 ₆ Or C ₃ F ₈ The flow of gas is restricted by the flow of liquid in the channels 132A, 132B so that a fill SF is generated in the bubble forming channel 135 ₆ Or C ₃ F ₈ Is a microbubble of (a). The liquid with microbubbles is output from the microfluidic device 130 through the opening 130B in the cartridge 101. It is noted that the operation of the microfluidic device 130 is known from WO 2013/141695 and WO 2016/118010. The microfluidic device 130 is typically made of a glass substrate (such as borosilicate glass) or polymeric material.

Referring now again to fig. 3, liquid output by the microfluidic device 130 is supplied to the buffer reservoir 140 via the opening 130B and the fluid channel 130A. The reservoir is elongated in the vertical direction. Thus, when liquid enters buffer reservoir 140, it is less likely that the liquid will mix with the gaseous medium in reservoir 140 than if buffer reservoir 140 were elongated in the horizontal direction.

Via a fluid channel 112A extending between the buffer reservoir 140 and the vent 112, the overpressure may be released.

A buffer reservoir 140 is connected to the outlet 113. The buffer reservoir 140 may be withdrawn through the outlet 113 using a syringe. A luer taper connection is used for outlet 113 to provide a sealed connection between the syringe and outlet 113. It should be noted that when liquid is pumped from buffer reservoir 140, ambient air may be drawn through vent 112 to achieve pressure equalization, thereby preventing severe negative pressure in buffer reservoir 140 that would destroy microbubbles and complicate liquid pumping.

After the liquid is collected in the buffer reservoir 140, the cartridge 100 may be removed from the device 200. Typically, the user then places the cartridge 100 on a support surface (such as a table). To prevent accidental flow of liquid from reservoir 140 into fluid channel 130A, it is ensured that in this orientation of cartridge 100, fluid channel 130A and fluid channel 112A exit to a position above the gas-liquid interface in buffer reservoir 140. Thus, to achieve this advantage, the capacity of the reservoir 140 should be selected according to the volume of liquid in the container 120.

Referring now to fig. 6, the container 120 is disposed in a storage chamber 122 having a support surface including an edge portion 122A and a center portion 122B. The container 120 may be fixedly attached to the rim portion 122A using a suitable adhesive, indicated as component 121 in fig. 6.

Three needles 124 extend from the central portion 122B toward the container 120. When sufficient force is applied to the top side of the container 120, the rear side thereof will flex downwardly, thereby engaging the needle 124. Thus, the rear side of the container 120 will be pierced and the liquid inside the container 120 will be released. The liquid will flow through the opening 123 at the upper portion of the central portion 122B. Once flowing through the opening 123, the liquid will be collected in the liquid reservoir 125. Thus, when the cartridge 100 is inside the device 100, a small amount of liquid will remain in the reservoir 122 below the opening 123. The positioning of the opening 123 should be such that the opening 123 remains above the gas-liquid interface 126 when the cartridge 100 is inside the device 200. In this way, the gas supplied through the gas inlet 110 cannot push the liquid inside the liquid reservoir back into the storage chamber 122.

Next, the function of the apparatus 200 will be described with reference to fig. 8 to 15. First, in fig. 8, the most relevant components of the device 200 are shown. For example, the apparatus 200 includes an actuator 210 (e.g., a motor) that drives a threaded shaft 214 via a first gear 211 and a second gear 212 coupled using a belt 213.

The shaft 214 is rotatably mounted in the wall section 221. The wall section is part of a frame 220 fixedly connected to the housing 201. It should be noted here that fig. 8 shows only a part of the frame 220 for illustrative purposes.

As shown in fig. 9, the frame 220 includes a first portion 220A and a second portion 220B that are connected using a plurality of parallel and spaced apart component bars 222. The provision of the lever 222 allows the first portion 220A to move toward the second portion 220B and vice versa. As will be explained later, this feature will be used to create a force sensor.

Figure 9 also shows SF for introducing pressurization through gas inlet 110 ₆ Or C ₃ F ₈ Nozzle 242A for gas and SF for introducing pressure through inlet 111 ₆ Or C ₃ F ₈ A nozzle 242B for gas. It should be noted that different inlets 110, 111 are used, although they carry the same gas, as the function of the gas is different, i.e. as a mixed gas or with a propellant. Fig. 9 also shows an engagement unit 241 for applying a force to the reservoir 120. The nozzles 242A, 242B and the engagement unit 241 are each movably mounted in the unit 240.

Referring now to fig. 10, threaded shaft 214 is connected to U-shaped frame 230. More specifically, as the shaft 214 rotates, the frame 230 will only move in a direction toward or away from the cartridge 100. This direction will be referred to as z-direction hereinafter. In addition, the x-direction will correspond to a direction orthogonal to the z-direction and to the direction along which the cartridge 100 is inserted into the device 200. The remaining y-direction is orthogonal to the z-direction and the x-direction.

Fig. 11 shows various folded leaf springs 232A to 232C connecting the U-shaped frame 230 to the unit 240 using screws 231. The springs 232A-232C are configured to enable the unit 240 to move relative to the U-shaped frame 230. More specifically, the unit 240 is only movable in the x-direction, the y-direction, and rotates about the z-direction relative to the U-shaped frame 230.

Fig. 12 shows a cross section of the unit 240. The figure shows nozzles 242A, 242B mounted in a spring biased manner in unit 240 using springs 243A, 243B, respectively. Gas is supplied to nozzles 242A, 242B through nozzle inlets 244A, 244B, respectively. Typically, these inlets are connected to a pipe (not shown).

In fig. 12, nozzle 242A is movable in the z-direction and is rotatable about that direction relative to unit 240. Nozzle 242B is mounted in a slot allowing nozzle 242B to move in the x-direction and the y-direction relative to unit 240 and rotate about the y-direction.

Referring now to fig. 13, the engagement unit 241 is fixedly connected to the tripod-like structure 247. The engagement unit 241 is arranged to pass through the connection ring 246. The connecting ring is connected to the bottom of structure 247 by a rod 245.

Referring now to fig. 12 and 13, the attachment ring 246 is fixedly mounted in the unit 240. However, the provision of the lever 245 allows the engagement unit 241 to rotate about the x-direction and the y-direction with respect to the unit 240. In this way, the engagement unit 241 may adjust its position relative to the cartridge 100 to optimally engage the container 120. Further, in order to avoid leakage, an O-ring (not shown) may be placed in the groove 248 on the contact area of the engagement unit 241.

Fig. 14 presents a partial cross-section showing how nozzle 242A engages gas inlet 110 of cartridge 100. Nozzle 242A may be provided with an O-ring 249 to provide a sealed connection with gas inlet 110.

Next, a possible operating cycle of the cartridge system of fig. 1 will be described with reference to the schematic diagram of the system in fig. 15.

As a first step, cartridge 100 is installed in device 200 through opening 202 in fig. 1. Thereafter, the controller 250 in the apparatus 200 may optionally perform various checks, such as gas leak testing or checking whether the cartridge 100 is properly positioned.

When cartridge 100 is properly placed in opening 202, the system and thus device 200 is in a first state,in this first state, the unit 240 and the U-shaped frame 230 are positioned relatively far from the cartridge 100. Thereafter, the controller 250 will operate the actuator 210 to move the U-shaped frame 230 toward the cartridge 100. As a result, the nozzles 242A, 242B will engage the inlets 110, 111. Due to the degrees of freedom of these nozzles and the degrees of freedom of the unit 240 with respect to the U-shaped frame 230, an alignment of the unit 240 with respect to the cartridge 100 will be obtained. In addition, when properly engaged, the controller 250 will control the SF that is arranged with pressurization ₆ Or C ₃ F ₈ Controllable valves 251, 252 between the container 1000 and the nozzles 242A, 242B. More specifically, the valves 251, 252 are controlled to allow SF for the gas inlets 110, 111 and the components of the cartridge 100 to which they are connected ₆ Or C ₃ F ₈ And (5) flushing. This is referred to as the second state of the system and device 200. When the flushing action is completed, the controller controls the actuator 210 to move the U-shaped frame 230 and thus the unit 240 closer to the cartridge 100 so that the engagement unit 241 can engage the container 120. During this movement, the nozzles 242A, 242B will move rearward relative to the unit 240 against the spring bias. In fact, the spring constant of springs 243A, 243B largely determines the force with which nozzles 242A, 242B press against inlets 110, 111, respectively. It should also be noted that a single container 1000 may also be used to supply the same gas to the inlets 110, 111.

During movement of the unit 240, the controller 250 checks the force applied by the engagement unit 241 to the reservoir 120 using the force sensor 253. The latter sensors may have sensed forces caused by the engagement of nozzles 242A, 242B with inlets 110, 111, respectively. Specifically, the reaction forces exerted by inlets 110, 111 on nozzles 242A, 242B, respectively, are transferred to wall segment 221 via unit 240, U-shaped frame 230 and shaft 214. Thus, as can be seen in fig. 9, frame portion 220B has a tendency to move away from frame portion 220A. This displacement is achieved by the lever 222 and is measured by a position sensor. The force sensor 253 calculates a resultant force based on the observed displacement. It should be noted that the force sensor 253 may be integrated in the controller 250. In addition, there is no need to calculate the actual force, as a parameter representing the force is sufficient.

When the engagement unit 241 is pressed against the reservoir 120 with sufficient force, the reservoir will be broken and its liquid released to the liquid reservoir 125. Thereafter, the valves 251, 252, which are normally closed during the release of liquid from the container 120, are controlled to start the mixing process. The valves 251, 252 are preferably controlled after the liquid has reached the liquid reservoir 125.

During the mixing process, SF is added ₆ Or C ₃ F ₈ To the inlet 110 to act as a propellant to move liquid from the liquid reservoir 125 out to the microfluidic device 130. At the same time SF ₆ Or C ₃ F ₈ Is supplied to the inlet 111 as a gas to be used by the microfluidic device 130 for generating microbubbles.

After a predetermined amount of time, the valves 251, 252 are closed by the controller 250 and the unit 240 is removed from the cartridge 100. Thereafter, the cartridge 100 may be removed from the apparatus 200, with the microbubble suspension held in the buffer reservoir 140.

The last part of the process, i.e. moving the unit 240 towards the cartridge 100, allows the engagement unit 241 to engage the reservoir 120 and perform a subsequent mixing process when the system and thus the device 200 is in the third state.

Hereinabove, the invention has been explained using embodiments aimed at producing microbubble suspensions intended for use as contrast agents in vivo. However, those skilled in the art will appreciate that the present invention is not limited to such applications. Other applications are also possible in which the sterile liquid held in the cartridge will be mixed within the cartridge with another sterile liquid held in the cartridge or with a gas supplied to the cartridge.

Using the system of the present application, a user can repeatedly generate a mix of specific cases or people that can be generated immediately prior to use. Because the critical components (i.e., the liquid) remain in the container, and because the mixing process also occurs in the cartridge, sterility of the final product is ensured. Contact with an external device (i.e., device 200) involves only the exchange of gaseous media. The exchange may be performed in a sterile manner using off-the-shelf filters.

Thus, an advantage of the present application is that the device 200 may be arranged in a non-sterile environment, such as an examination room of a hospital, while still allowing a sterile mixed product to be obtained.

In view of the above, it must be concluded that the application is not limited to the embodiments shown. Rather, various modifications are possible and other applications may be implemented without departing from the scope of the application, which is defined by the appended claims.

The following non-limiting examples are provided to illustrate the application.

Example 1

To prepare 30ml of phospholipid solutions of DPPC and DPPE-mPEG5000K in a molar ratio of 85:15, respectively, with a total mass lipid concentration of 15mg/ml, dissolved in a liquid solution of PG and PBS in a volume ratio (V/V%) of 5:95, the following ingredients were weighed out:

0.189g of DPPC

0.261g DPPE-mPEG5000K

1.5g of PG

28.4g of PBS.

PG and PBS were preheated to 74℃in separate round bottom flasks. In this case DPPC is first added and dissolved in pre-heated PG, and after it is completely dissolved DPPE-mPEG5000k is added to the pre-heated PG solution containing dissolved DPPC. After complete dissolution of the lipids in PG, pre-warmed PBS was added. The resulting solution was stirred at 74 ℃ overnight and filtered using a 0.22 μm cellulose acetate membrane.

The final phospholipid solution was stored and cooled to room temperature for use.

Use of this phospholipid preparation, C ₃ F ₈ Gas and flow focused microfluidic devices, using different gas-to-liquid flow ratios, produced seven microbubble samples. Microbubbles are collected in a collection reservoir designed for this purpose. The size of each microbubble sample was characterized using a particle size standard analyzer Counter (Beckman) to obtain the results as summarized in table 1.

TABLE 1

	Mould diameter (mu m)	PDI(％)	Resonant frequency (MHz)
				Sample 1	1.9	5.6	8.4
Sample 2	2.7	7.5	5.3
				Sample 3	3.1	7.2	4.3
Sample 4	3.5	6.8	3.8
				Sample 5	4.2	7.2	2.9
Sample 6	4.5	7.8	2.4
				Sample 7	5.8	6.7	1.7

Further, attenuation measurements are made to measure the resonant frequency. For monodisperse microbubbles, the resonant frequency corresponds to the peak frequency in the decay curve. The results are given in fig. 16 and 17.

Figure 16 shows the size distribution of different microbubble populations. As can be seen from the figure, the size distribution of the microbubbles is narrow and no coalescence of the microbubbles occurs which results in polydisperse microbubbles.

Fig. 17 shows normalized attenuation for different microbubble samples. The resonance frequency corresponds to the peak in the decay curve. The resonant frequency is linear with the inverse of the microbubble diameter.

In summary, the method of producing a phospholipid composition of the present invention has proven successful and phospholipid compositions can be prepared with high concentrations of phospholipids, which can be suitable for use in systems for the controlled manufacture of microbubbles.

Hereinabove, the present invention has been disclosed using embodiments thereof. However, it will be appreciated by those skilled in the art that the invention is not limited to these embodiments and that further embodiments are possible without departing from the scope of the invention as defined by the appended claims and their equivalents.

Claims

1. A cartridge configured for mixing a liquid held in the cartridge with another liquid held in the cartridge or with a pressurized first gas supplied to the cartridge within the cartridge, wherein the liquid is a phospholipid composition, wherein the concentration of phospholipids is at least 12mg/ml.

2. The cartridge of claim 1, the cartridge comprising:

a cylinder;

one or more gas inlets formed in the cylinder;

a fluid storage system formed in the barrel and comprising one or more fluid storage units, each fluid storage unit configured to hold a respective fluid and to output the fluid in response to pressurized gas supplied to the fluid storage unit through a gas inlet of the one or more gas inlets by using the supplied gas as a propellant; and

a mixing unit arranged in or mounted to the barrel and in fluid communication with the fluid storage unit using a fluid channel or channels formed in the barrel, the mixing unit being configured for mixing respective fluids output from the respective fluid storage units or for mixing fluids output from the fluid storage units with the pressurized first gas received through a gas inlet of the one or more gas inlets;

wherein at least one fluid held in the fluid storage system and configured to be mixed is the phospholipid composition.

3. The cartridge of claim 1, comprising a mixing unit configured to mix the phospholipid composition with the pressurized first gas, the mixing unit comprising a microfluidic device configured to generate microbubbles within the phospholipid composition, the microbubbles filled with the pressurized first gas.

4. A cartridge according to any one of claims 1 to 3, wherein the phospholipid composition comprises a hydrated phospholipid solvent mixture prepared by the method of:

5. The cartridge of claim 4, wherein the hydrated phospholipid solvent mixture is filtered through a sterilizing filter.

6. The cartridge of claim 4 or 5, wherein the phospholipid is a combination of at least one of the group of DPPC, DSPC, DSPG, DMPC, DBPC, DPPE with at least one of the group of DPPE-mPEG5000, DMPE-PEG-2000 and DSPE-PEG2000, preferably DPPC, DSPC, DPPE with at least one of the group of DPPE-mPEG5000 and DSPE-PEG2000, more preferably DPPC and DPPE-mPEG5000.

7. The cartridge of any one of claims 4 to 6, wherein the organic solvent is selected from the group comprising propylene glycol, ethylene glycol, polyethylene glycol 3000 and/or glycerol, preferably the organic solvent is propylene glycol.

8. The cartridge of any one of claims 4 to 7, wherein the aqueous phosphate buffer is Phosphate Buffered Saline (PBS), phosphate buffered saline containing glycerol, water, saline/glycerol and/or saline/glycerol/non-aqueous solution, preferably Phosphate Buffered Saline (PBS).

9. A cartridge according to any one of claims 4 to 8, wherein the concentration of the phospholipid in the hydrated phospholipid solvent mixture is in the range from 12mg/ml up to 20mg/ml, preferably in the range from 15mg/ml up to 18 mg/ml.

10. The cartridge according to any one of claims 4 to 9, wherein the ratio of phospholipids is in the range from 95:5 to 70:30, preferably in the range from 90:10 to 75:25, more preferably in the range from 85:15 to 80:20.

11. A cartridge according to any one of the preceding claims when dependent on claim 2, wherein each fluid storage unit comprises a fluid storage unit inlet and a fluid storage unit outlet, wherein the fluid storage unit is configured such that the pressurized gas supplied to the fluid storage unit through the fluid storage unit inlet pushes the fluid in the fluid storage unit through the fluid storage unit outlet.

12. The cartridge of claim 11, wherein at least one fluid storage unit comprises:

a storage chamber configured to receive a sealed container in which a liquid is held in a sterile and sealed manner; and

a liquid reservoir in fluid communication with the storage chamber, wherein the fluid storage unit inlet is connected to one of the storage chamber and the liquid reservoir, and wherein the fluid storage unit outlet is connected to the liquid reservoir;

Wherein the liquid reservoir is configured for collecting liquid released from the container after the container has been broken, ruptured, cut or pierced.

13. The cartridge of claim 12, wherein the sealed container comprises a blister package.

14. A cartridge according to claim 12 or 13, wherein the sealed container is fixedly held in the storage chamber, for example by means of an adhesive.

15. The cartridge of claims 12 to 14, wherein for at least one fluid storage unit, the liquid reservoir is formed as part of the storage chamber.

16. The cartridge of claims 12-14, wherein the at least one fluid storage unit comprises a fluid channel formed in the barrel connecting the storage chamber and the liquid reservoir.

17. A cartridge according to any one of claims 12 to 16, wherein for a fluid storage unit of the at least one fluid storage unit, the storage chamber comprises a support surface for supporting the sealed container and at least one protruding pin or needle extending towards the sealed container so as to pierce the sealed container when sufficient force is applied thereto.

18. The cartridge of claim 17, wherein at least one fluid storage unit is provided with a protective ring that protrudes farther from the barrel than the sealed container when the sealed container is placed in the storage chamber.

19. A cartridge according to any one of the preceding claims when dependent on claim 2, comprising a plurality of the fluid storage units, wherein the fluid storage unit inlets of at least two fluid storage units are in fluid communication with each other.

20. The cartridge of claim 19, wherein the gas used as propellant by the plurality of fluid storage units is the same.

21. A cartridge according to any preceding claim when dependent on claim 2, wherein the gas inlet through which the pressurised first gas is received is the same as the gas inlet through which the pressurised gas used as a propellant by at least one fluid storage unit is received.

22. A cartridge according to any preceding claim when dependent on claim 2, wherein the pressurised first gas is different to the pressurised gas used as propellant by the at least one fluid storage unit.

23. The cartridge of claim 22, wherein the cartridge has a single fluid storage unit and two gas inlets, wherein a first of the two gas inlets is in fluid communication with the mixing unit, and wherein a second of the two gas inlets is in fluid communication with the fluid storage unit.

24. A cartridge according to any one of the preceding claims when dependent on claim 2, wherein the mixing unit is configured to mix at least one liquid received from the fluid storage system with the pressurised first gas, the mixing unit comprising a microfluidic device configured to generate microbubbles within the at least one liquid, the microbubbles being filled with the pressurised first gas.

25. A cartridge according to claim 23 or claim 3, wherein the microfluidic device is configured to generate microbubbles having a diameter below 10 microns and preferably in the range 2 to 5 microns.

26. The cartridge of claim 24 or 25, wherein the pressurized first gas comprises one or more gases from the group consisting of: SF (sulfur hexafluoride) ₆ 、N ₂ 、CO ₂ 、O ₂ 、H ₂ He, ar, ambient air and such as CF ₄ 、C ₂ F ₆ 、C ₂ F ₈ 、C ₃ F ₆ 、C ₃ F ₈ 、C ₄ F ₆ 、C ₄ F ₈ 、C ₄ F ₁₀ 、C ₅ F ₁₀ 、C ₅ F ₁₂ And mixtures thereof.

27. The cartridge of any one of claims 22 to 26, wherein the microfluidic device comprises:

a first inlet for receiving the pressurized first gas;

a second inlet for receiving the phospholipid composition;

a bubble forming channel for generating the microbubbles based on the flow of the first pressurized gas received through the first inlet and the flow of the phospholipid composition received through the second inlet.

28. The cartridge of claim 27, wherein the cartridge body comprises a first opening, a second opening, and a third opening, wherein the first opening is in fluid communication with the gas inlet that receives the pressurized first gas, and wherein the second opening is in fluid communication with the fluid storage system so as to receive the phospholipid composition;

wherein the microfluidic device is positioned relative to the cartridge such that the first opening is aligned with the first inlet of the microfluidic device, the second opening is aligned with the second inlet of the microfluidic device, and the third opening is aligned with the outlet of the microfluidic device;

Wherein the microfluidic device is fixedly connected to the cartridge using an adhesive or integral bond, or wherein the microfluidic device is integrally formed with the cartridge.

29. The cartridge of claim 27 or 28, wherein the microfluidic device comprises:

a flow focusing junction;

a first channel having one end connected to the second inlet and the other end connected to the flow focusing junction;

a second channel having one end connected to the second inlet and the other end connected to the flow focusing junction;

a third channel having one end connected to the first inlet and the other end connected to the flow focusing junction;

wherein the bubble forming channel is connected to the flow focus junction;

wherein the flow focus joint is configured to receive a flow of the phospholipid composition from two opposite directions via the first channel and the second channel, the flow of the phospholipid composition impinging on a flow of the first pressurized gas received via the third channel in a perpendicular manner, wherein the flow of the pressurized gas is directed from the third channel into the bubble forming channel.

30. A cartridge according to any one of the preceding claims when dependent on claim 2, further comprising an outlet formed in the barrel for outputting the fluid mixed by the mixing unit.

31. The cartridge of claim 30, wherein the outlet is formed to allow a luer taper connection with a syringe.

32. The cartridge of claim 30 or 31, further comprising a buffer reservoir formed in the barrel and arranged between the mixing unit and the outlet, the buffer reservoir having a capacity exceeding the volume of liquid held in the fluid storage system.

33. The cartridge of claim 32, further comprising a vent formed in the barrel, the vent in fluid communication with the buffer reservoir.

34. The cartridge of claim 33, wherein the cartridge comprises:

a fluid channel formed in the barrel between the one or more gas inlets and the mixing unit;

a fluid passageway formed in the cartridge between the one or more gas inlets and the fluid storage system;

a fluid channel formed in the barrel between the fluid storage system and the mixing unit;

A fluid channel formed in the cartridge between the mixing unit and the buffer reservoir; and

a fluid channel formed in the cartridge between the buffer reservoir and the vent hole;

wherein the fluid channels extend substantially in the same fluid channel plane.

35. The cartridge of claim 34, wherein the cartridge is configured to be in a first orientation during the mixing, wherein, in the first orientation:

the normal to the plane of the fluid passage is substantially horizontal;

the mixing unit is positioned at a lower position relative to the fluid storage system and relative to the gas inlet through which the pressurized first gas is received;

the fluid channel between the mixing unit and the buffer reservoir is located in the buffer reservoir at the lower end of the buffer reservoir, preferably at or near the lowest point of the buffer reservoir, buffer reservoir output.

36. The cartridge of claim 34 or 35, wherein the cartridge is configured to be in a second orientation during storage, wherein in the second orientation:

The normal to the fluid passage plane is substantially vertical;

the fluid channel between the mixing unit and the buffer reservoir is output in the buffer reservoir at a location above a liquid-air interface of the buffer reservoir to prevent liquid output into the buffer reservoir by the mixing unit from flowing back into the fluid channel between the mixing unit and the buffer reservoir when the cartridge is in the second orientation.

37. The cartridge of claim 36, wherein the fluid channel between the vent and the buffer reservoir is output in the buffer reservoir at a location above the liquid-air interface of the buffer reservoir when the cartridge is in the second orientation.

38. The cartridge of any one of claims 32 to 37, wherein the buffer reservoir is vertically elongated when the cartridge is held in an orientation in which a normal to the fluid channel plane is horizontal.

39. The cartridge of any one of claims 32 to 38, wherein the buffer reservoir is transparent.

40. A cartridge according to any preceding claim, further comprising a filter arranged in each of the one or more gas inlets formed in the cartridge body and, where applicable, in the vent.

41. The cartridge of claim 40, wherein at least one filter comprises a filter membrane, a first filter support, and a second filter support, wherein the first filter support and the second filter support are fixedly attached to or integrally formed with the cartridge, and wherein the filter membrane is disposed between the first filter support and the second filter support.

42. A cartridge according to any one of the preceding claims when dependent on claim 2, wherein the cartridge is formed by fixedly attaching the first and second cartridge parts using integral bonding, for example using ultrasonic welding, each cartridge part comprising a substrate layer;

wherein, prior to the fixedly attaching, one of the first and second barrel portions comprises a ridge and/or a protruding portion extending from the base layer, the ridge and/or protruding portion being configured to cooperate with a protruding portion and/or ridge extending from the base layer of the other of the first and second barrel portions during ultrasonic welding, the protruding portion being integrally connected to a corresponding ridge after the ultrasonic welding;

The integrally connected ridge and projection portions and the base layers of the first and second barrel portions together define at least one of: the mixing unit, the fluid storage system, the one or more gas inlets, the outlet, the buffer reservoir and the fluid channel for connecting them.

43. The cartridge of claim 42, wherein the first cartridge portion and the second cartridge portion are made from one or more materials of the group of thermoplastic materials such as polycarbonate using injection molding.

44. A cartridge system, the cartridge system comprising:

a cartridge as defined in any one of the preceding claims when dependent on claim 2;

an apparatus comprising a housing having an opening into which the cartridge is releasably insertable;

wherein the apparatus comprises one or more nozzles for introducing respective pressurized gases into the one or more gas inlets, respectively, for said mixing by the mixing unit of the cartridge.

45. The system of claim 44, wherein the cartridge is configured according to at least the definition of claim 12, wherein the apparatus further comprises an engagement unit for engaging a sealed container arranged in the reservoir of the at least one fluid storage unit so as to cause the sealed container to be broken, ruptured or cut or pierced.

46. The system according to claim 44 or 45, wherein the apparatus comprises a drive system for engaging and disengaging the nozzle and/or the engagement unit with the one or more gas inlets and the sealed container, respectively.

47. The system of claim 46, wherein the apparatus further comprises a controller for controlling the drive system.

48. The system of claim 47, wherein at least one of the one or more nozzles is connected to a controllable valve, wherein the controller is configured to control the flow of pressurized gas through the at least one of the one or more nozzles via the controllable valve.

49. The system of claim 48, wherein the apparatus comprises one or more reservoirs for holding pressurized gas, the one or more reservoirs being connected to the one or more nozzles, or wherein the apparatus comprises one or more additional gas inlets respectively connected to the one or more nozzles, the one or more additional gas inlets configured to be connected to one or more gas reservoirs external to the apparatus.

50. A system according to claim 48 or 49 when dependent on claim 45, wherein the controller is configured to control the apparatus to be operable in:

a first state in which the nozzle and the engagement unit are positioned at a distance from the cartridge;

a second state in which the controller controls the drive system to bring the nozzle into contact with the one or more gas inlets, and wherein the controller controls the controllable valves such that pressurized gas is supplied to the cartridge via the respective nozzles and gas inlets of the cartridge;

a third state in which the controller controls the drive system to cause the engagement unit to engage the sealed container so as to cause the sealed container to be broken, ruptured or cut or pierced, and then controls the controllable valve to provide pressurized gas to the cartridge via its respective nozzle and gas inlet to effect the mixing by the mixing unit of the cartridge.

51. The system of claim 50, wherein the controller is configured to control the controllable valve to stop supplying pressurized gas to the cartridge when changing from the second state to the third state.

52. The system of any one of claims 46 to 51 and 45, wherein the drive system comprises:

a first unit in which the one or more nozzles are movably installed, and the engagement unit is installed;

an actuator for moving the first unit relative to the cartridge when the cartridge is inserted into the device.

53. The system of claim 52, wherein the nozzle is spring-biased mounted in the first unit to allow the nozzle to move relative to the first unit in a first direction toward and perpendicular to the barrel.

54. The system of claim 52 or 53, wherein the engagement unit is movably mounted to the first unit.

55. The system of any one of claims 52-54, further comprising a second unit coupled to the first unit, wherein the first unit is movable in at least one degree of freedom relative to the second unit, and wherein the actuator is configured to move the second unit in the first direction.

56. The system of claim 55, wherein the second unit is coupled to the first unit using one or more curved leaf springs.

57. The system of claim 55 or 56, wherein the first unit is movable relative to the second unit in a second direction orthogonal to the first direction and in a third direction orthogonal to both the first direction and the second direction.

58. The system of claim 57, wherein the first unit is further rotatable about the first direction relative to the second unit.

59. The system of claim 57 or 58, wherein a nozzle of the one or more nozzles is movable in and rotatable about the first direction relative to the first unit, and wherein another nozzle of the one or more nozzles is movable in and rotatable about one of the second direction and the third direction.

60. The system of any one of claims 55-59, wherein the drive system includes a threaded shaft driven by the actuator and extending in the first direction, and wherein the second unit is coupled to the threaded shaft to cause translation of the second unit in the first direction when the threaded shaft rotates.

61. The system of claim 60, wherein the drive system further comprises a second frame fixedly connected to the housing, wherein the threaded shaft is rotatably received in the second frame, the second frame preferably comprises a wall segment extending substantially perpendicular to the first direction, and the threaded shaft is rotatably received in the wall segment.

62. The system of any one of claims 48 to 61 and 45, wherein the apparatus further comprises a force sensor for sensing a force of the engagement unit against the sealed container, wherein the controller is configured to control the drive system in dependence of the sensed force.

63. The system of claims 61 and 62, wherein the second frame comprises a first portion and a second portion coupled to the first portion, wherein the first portion and the second portion are movable relative to each other when a force is applied to the cartridge by the engagement unit, the force sensor being configured to determine the force from a mutual displacement between the first portion and the second portion.

64. The system of claim 63, wherein the first and second portions are connected using a structure compressible in the first direction, such as a curved rod or lattice of rods.

65. A cartridge system comprising a cartridge as defined in any one of claims 1 to 43 and a device into which the cartridge is releasably insertable, wherein the device is configured for providing a pressurized gas that acts as a propellant for moving the liquid inside the cartridge for mixing the liquid and/or as the pressurized first gas.

66. The cartridge system of claim 65, wherein the apparatus is further configured as defined in any one of claims 44 to 64.

67. An apparatus adapted to releasably receive a cartridge as defined in any one of claims 1 to 43, wherein the apparatus is configured for providing a pressurized gas that serves as a propellant for moving the liquid inside the cartridge for mixing the liquid and/or as the pressurized first gas, wherein the apparatus is further preferably configured as an apparatus as defined in any one of claims 44 to 64.

68. A blister package comprising a phospholipid composition, wherein the concentration of phospholipids is at least 12mg/ml, preferably at least 15mg/ml.

69. The blister package of claim 68, wherein the phospholipid composition does not comprise dipalmitoyl phosphatidic acid (DPPA).

70. A blister package according to claim 68 or 69, wherein the blister package is configured to be inserted into a cartridge as defined in any one of claims 1 to 43 when dependent on claim 12.