WO2017199123A1

WO2017199123A1 - Device and methods for shell phase removal of core-shell capsules

Info

Publication number: WO2017199123A1
Application number: PCT/IB2017/052655
Authority: WO
Inventors: Antoine VIAN; Esther Amstad
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2016-05-17
Filing date: 2017-05-08
Publication date: 2017-11-23

Abstract

A device and method for the mechanical (partial or total) removal of the liquid phase of the external shell of a core-shell capsule is disclosed. The device comprises a main channel having at least one inlet, one outlet and a tubular element in between, adapted to be operably connectable with a source of core-shell capsules so that these latter can flow throughout said main channel upon application of a pressure or other driving forces; and at least one side channel intersecting the main channel along the tubular element. Upon injection of the capsules inside the device, the side channels peel off by mechanical action the liquid phase comprised within the external shell once brought into contact. The device can be used for production of thin-shelled core-shell capsules such as double emulsion drops for many applications such as food, cosmetic, coating or biomedical ones.

Description

Device and methods for shell phase removal of core-shell capsules Technical Field

[0001 ] The present invention relates to the field of microfluidics, in particular to a microfluidic device and methods to mechanically reduce the external layer of core-shell capsules.

Background Art

[0002] A microcapsule is a micrometer-scale particle such as for instance gas bubbles or liquid drops surrounded by a solid, liquid, or otherwise fluid shell. This shell acts as a barrier separating the core from the outer environment. Microcapsules are attractive candidates for encapsulating, transporting, or controllably releasing a wide variety of important active materials. These include surfactants, agricultural chemicals, food additives, pharmaceuticals, cosmetic components, cells or cell components such as nucleic acids or proteins, biochemical sensors, catalysts for chemical reactions, restorative agents for self-healing materials and inks. Ideally, capsules have a low permeability towards encapsulants during storage and a high permeability when the active ingredient needs to be delivered. In order to achieve this goal, capsules need to be designed to have an interchangeable permeability or enable triggered rupture.

[0003] Vesicles are one type of capsules that can be designed to storage active ingredients. Their membrane is generally composed of a bilayer of self- assembled amphiphiles that is often mechanically weak, providing a simple mechanism for triggered release of active agents. When block copolymers, with significant molecular weight, are used as amphiphilic molecular elements of the shell, a (sill limited) increase in mechanical and thermodynamic stability is achieved. This is the case of polymersomes, a class of artificial vesicles made using amphiphilic synthetic block copolymers to form a thinner vesicle membrane.

[0004] Robust capsules are often composed of thick shells, and therefore loaded with a limited amount of encapsulants as the shell accounts for a significant volume fraction of the capsule. Furthermore, upon rupture, they often decompose into large fragments that can easily introduce defects if used for example in coatings.

[0005] Many applications require a close control over the size, structure, and composition of these capsules. This level of control can be obtained if capsules are formed from drop templates. In particular, the use of double emulsion drops, which are drops contained in larger drops, dispersed in a third fluid, is attractive as it enables a good control over the composition and thickness of the capsule shell. Many applications require capsules with thin shells, i.e. wherein the volume fraction of shell material is very low. Thin capsules minimize the volume of capsule residues that remain in the material or the body, after the capsule is emptied. Moreover, by minimizing the shell thickness, the volume fraction of the capsule interior that can be loaded with encapsulants is maximized.

Suitable methods available to produce double emulsion capsules comprise spray drying, freeze drying, air drying, vacuum drying, fluidized-bed drying, milling, co-precipitation, solvent extraction or a combination thereof. However, at present, making double emulsions with very thin shells results very challenging. Capsules with thin shells can be assembled using microfluidic devices (A. S. Chaurasia et al., Chemphyschem, 16, 403, Feb 2, 2015; H. C. Shum et al., Langmuir, 24, 7651 , Aug 5, 2008; H. C. Shum et al., Journal of the American Chemical Society, 130, 9543, Jul 23, 2008; H. C. Shum et al., Journal of the American Chemical Society, 133, 4420, Mar 30, 201 1 ). For example, water-oil-water (W-o-W) double emulsion droplets can be produced using microfluidic devices, like glass capillary or PDMS-based devices. However, even in this case, the capsule shell is typically still between 1 μιτι and 10 μιτι. Even if double emulsions are made with microfluidic devices designed to generate thin shells, their shell cannot be made thinner than 1 μιτι (5.-Η. Kim et al., Lab on a Chip, 1 1 , 3162, 201 1 ; L. R. Arriaga et al., Small, 10, 950, Mar 2014; L. R. Arriaga et al., Lab on a Chip, 15, 3335, 2015). Double emulsion droplets can be converted into vesicles by removing the liquid phase of the external shell of the core-shell droplets, the oil phase in the case of a W-o-W emulsion. To minimize the volume of the capsule shell, the oil phase must be further removed after the double emulsions are assembled. The most employed and efficient techniques for reducing or otherwise regulating the size of a double emulsion capsule's external shell are evaporation and/or extraction of an oily fraction of the external shell, or its physical removal by mechanical separation.

[0007] International Patent Application WO 201 1/087689 discloses the use of extraction and/or evaporation techniques for the removal of the fluid portion of the external shell in W-o-W droplets. According to the disclosure, the process involves adding W-o-W double-emulsion microcapsules to an extraction phase following an evaporative process, preferably while the microcapsules remain in the extraction phase. The formed microcapsules can then be collected, washed, dried, and packaged using techniques known in the art. Evaporation can be performed under atmospheric or reduced pressure conditions, and at ambient temperatures, or higher temperatures that do not harm the active agent. Other examples of phase removal by extraction and/or evaporation can be found in prior art documents US 4389330, US 5643605, WO 1990/013361 , WO 1995/013799, WO 2000/066087 and WO 201 1/087689.

[0008] Evaporation is another approach known in the art that allows the modulation of the external shell thickness of core-shell capsules. Emulsion template comprising oil-in-water or water-in-oil microcapsules can be subject to drying methods in order to control the microcapsule surface area and volume ratios. The method described in the International Patent Application WO 2012/162296 provides a drying bath comprising heating means and an impellor for agitating the emulsion. The drying rate is controlled by the temperature and/or the emulsion mixing and/or the dispersed vapour pressure. Other methods of similar approach using drying technique or heating bath can be found in prior art documents US 3891570, US 4384975, WO 2008/048271 , WO 2006/123359 and WO 2013/178802.

However, when W-o-W double emulsion microcapsules are employed, the evaporation of the external layer presents limitations in the choice of the organic solution composition (oil layer) and therefore of suitable block copolymers to be used. For instance, a mixture of chloroform and hexane is often chosen as the organic phase due to the limited efficiency in removing other types of solvent mixtures from the shell, thus preventing the conversion of double emulsion droplets into vesicles. However, chloroform is toxic and its complete removal from core-shell capsules has not been demonstrated to date. Its removal is achieved through an evaporation process during which it saturates the aqueous phase and evaporates through the liquid-air interface. Due to the fact that chloroform can persist in the aqueous layer where the active ingredient is present and to its high cytotoxicity, its use is limited in pharmaceutical, biomedical, cosmetic, and food applications. [0010] Alternatively, L. R. Ar aga et al. {Lab on a Chip, 15, 3335-3340, 2015) describe the use of a microfluidic device for the partial or total mechanical separation of the external shell of a core-shell capsule. A decrease in shell thickness is achieved by deforming (squeezing) the droplets passing through specific constrictions designed along the channels of the microfluidic device. Within these constrictions, the droplets accelerate, causing a difference in speed between the oily shell and the water core. The water core is less dense and will travel through the constriction faster compared to the external oil layer, which will be forced to the tail end of the squeezed drop. This accumulation of oil breaks up into droplets due to thermodynamic processes upon deformation. Subsequent constrictions (up to three in total per collecting tube) along the same collecting tube will induce via the same mechanism a progressive thinning of the external shell of a core-shell capsule and at a very low throughput. However, the method only results in a partial removal of the oily shell. Methods that enable a complete removal of the oil remain elusive.

[001 1 ] Notwithstanding the great amount of work in the field as described above, quick and reliable methods and devices that can efficiently remove via a mechanical approach, partially or totally, the liquid phase of the external shell of core-shell capsules are still lacking.

Summary of invention [0012] In view of the limitations of the prior art approaches to reduce or completely remove the liquid contained in the external shell of a core-shell capsule, the present inventors have designed a novel device, which allows to mechanically control the thickness of the external shell of a core-shell capsule. This device is simple and adaptable in its design to different scenarios and allows preparation of capsules with thin shells, including vesicles, of different kind by controlled removal of the external liquid shell. Accordingly, the invention provides for a microfluidic device for use in the removal of the liquid phase of the external shell of a core-shell capsule through a mechanical approach, said device comprising a main channel equipped with at least one inlet through which the core-shell capsules are injected. This main channel comprises a tubular element fluidically connected via its proximal portion with the inlet through which the capsules flow, as well as an outlet fluidically connected to the distal portion of the tubular element that allows the collection of the capsules. In order for the device to accomplish its function, at least one side channel of specific characteristics is intersecting the main channel along the tubular element. The capsules will flow through the main channel upon application of a driving force and/or a pressure (positive and/or negative) thereto.

[0013] In one embodiment, at least one dimension of the planar cross-section of the tubular element of the main channel is smaller than the diameter of the core-shell capsules. [0014] In a preferred embodiment, the planar cross-section of the intersecting side channel is comprised between about 3% and about 10% compared to the tubular element of the main channel cross sectional area.

[0015] In a preferred embodiment, at least one dimension of the planar cross- section of the intersecting side channel is between about 1 % and about 20%, preferably 3%, compared to the diameter of the core-shell capsule.

[0016] In a preferred embodiment, the core-shell capsule is a water-oil-water (W- o-W) or oil-water-oil (O-w-0) double emulsion capsule.

[0017] Another aspect of the invention relates to a system for use in the mechanical removal of the liquid phase of the external shell of a core-shell capsule, characterized in that it comprises the previously described device operably connected with means for applying a pressure or other driving forces adapted to allow core-shell capsules to flow through the main channel from a source of core-shell capsules.

[0018] Another aspect of the invention relates to methods for using the microfluidic device or the system of the invention to attain the control over the partial or total mechanical removal of the liquid phase of the external shell of a core- shell capsule. This method comprises the following steps:

[0019] a) providing the device or the system of the invention;

[0020] b) optionally adapting the main channel so to favour a high wettability of the liquid phase of the external shell of the core-shell capsules; c) providing a source of core-shell capsules in fluidic connection with the inlet of the main channel and with means for applying a pressure or another driving force to let the capsules flow through the channel; and

d applying a pressure or a driving force to the source of core-shell capsules in a way to allow said capsules to flow throughout the main channel.

[0021 ] In one preferred embodiment, step b) is achieved by injecting in the main channel a liquid phase of compatible polarity with the external shell of core- shell capsules.

[0022] In one embodiment, the method further comprises a step of collecting the obtained core-shell capsules into a reservoir connected with the outlet of the main channel.

Brief description of drawings

[0023] In the Figures:

[0024] Figure 1 depicts one embodiment of the device of the present invention;

[0025] Figure 2 depicts a microfluidic device for the production of double emulsion droplets comprising the device of the invention: a) Schematic overview: I: Inlet for outer phase; II: outlet for removal of the fluid contained in the shell of double emulsions; III: inlet for the injection of double emulsions; IV: outlet, b) Close-up of the central part of the device, where small side-channels intersect the main channel; [0026] Figure 3 depicts the fabrication of a device in PDMS using two masters (1 ) made using soft lithography. (2) PDMS and curing agent are mixed and poured onto the masters. (3) The elastomers are baked, and thereafter the two halves of the device are removed from the masters (4). The surface of the PDMS is activated using an 02-plasma. The two PDMS pieces can subsequently be bonded;

[0027] Figure 4a shows an optical micrograph of a double emulsion drop at rest; 4 b shows an optical micrograph of a double emulsion compressed in a channel. The direction of the flow is from top to bottom. The upper arrow points to the inner phase, the bottom arrow points to the middle phase. The scale bars are 50 μιτι;

[0028] Figure 5 shows time lapse optical micrographs of the transformation of double emulsion drops with relatively thick shells into capsules with much thinner shells. The time difference between each picture is 78ms. The oil is removed through the side channels (dotted arrows) resulting in a capsule with a thin shell that remains in the main channel (full arrows);

[0029] Figure 6 shows time lapse optical micrographs of the splitting of large double emulsion drops into many much smaller ones. The arrow at t=0 points to the compressed double emulsion drop. The full arrows point to the parts of the vesicles that are pushed/sucked into in the side channels. At the end of these side channels, small double emulsion drops form, as indicated with by the dotted arrows. The scale bar is 50μιτι. Description of embodiments

[0030] The present disclosure may be more readily understood by reference to the following detailed description presented in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

[0031 ] As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term "comprising", those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of."

[0032] A "microfluidic device", "microfluidic chip" or "microfluidic platform" is generally speaking any apparatus which is conceived to work with fluids at a micro/nanometer scale. Microfluidics is generally the science that deals with the flow of liquids inside channels of micrometer size. At least one dimension of the channel is of the order of a micrometer or tens of micrometers in order to consider it microfluidics. Microfluidics can be considered both as a science (study of the behaviour of fluids in micro- channels) and a technology (manufacturing of microfluidics devices for applications such as lab-on-a-chip). These technologies are based on the manipulation of liquid flow through microfabricated channels. Actuation of liquid flow is implemented either by external pressure sources, external mechanical pumps, integrated mechanical micropumps, hydrostatic pressures or by combinations of capillary forces and electrokinetic mechanisms.

[0033] The microfluidic technology has found many applications such as in medicine with the laboratories on a chip because they allow the integration of many medical tests on a single chip, in cell biology research because the micro-channels have the same characteristic size as the cells and allow such manipulation of single cells and rapid change of drugs, in protein crystallization because microfluidic devices allow the generation on a single chip of a large number of crystallization conditions (temperature, pH, humidity...) and also many other areas such as drug screening, sugar testers, chemical microreactor or micro fuel cells.

[0034] In the frame of the present invention, a microfluidic device can be easily adapted to work with fluid volumes spanning from millilitres down to femtoliters, and the dimensions can be adapted accordingly to have channels within the millimetre scale, without substantially departing from the teaching of the invention.

[0035] With reference to Figure 1 , a microfluidic device 1 for use in the removal of the liquid phase of the external shell of a core-shell capsule through mechanical separation according to one embodiment of the invention is shown. The structure of the device is such that it proves able to partially or totally remove by peeling the liquid phase of the external shell of a core- shell capsule. The device 1 comprises a main channel 2 having at least one inlet 3, an outlet 4 and a tubular element 5 in between, all these elements being fluidically connected between them. The inlet 3 is designed to allow the capsules to be introduced into the tubular element 5 preferably one by one and is adapted to be operably connectable with a source of core-shell capsules 7, for example with a microfluidic device designed to prepare the core-shell capsules or with a capsules' reservoir, being in turn operably connectable to means for allowing the flow of the said capsules throughout the main channel 2, by e.g. applying a pressure within device 1 and/or the above-mentioned associated structures. The inlet's 3 width is preferably similar in size to the diameter of the core-shell capsules used.

[0036] To efficiently remove the liquid phase of the external shell of the capsules, these latter should preferably be deformed. In preferred embodiments, to achieve this deformation, the planar cross-section of the tubular element 5 of the main channel 2 is smaller than the diameter of the core-shell capsules. To do so, in preferred embodiments of the invention the tubular element 5 is narrower in at least one dimension compared to the inlet 3.

[0037] The tubular element 5 may be designed to have any possible shape as long as the capsules are deformed therein and/or brought into a suitable contact with a side channel (described later on in more details), for instance, it can be a straight or a serpentine-like element. In a serpentine arrangement, the curves of the tubular element 5 may increase the efficiency of the removal of the shell liquid phase. In the curves of a serpentine tubular element 5, core-shell capsules are pushed towards one channel walls upon injection in the main channel 2, thereby favouring more the removal of the liquid phase of the external shell of a capsule than if the tubular element 5 was straight. At the end of the tubular element 5, the capsules regain their original shape. In some embodiments, the outlet 4 can be operably connected with additional device(s) or reservoir(s) for collecting the so-obtained capsules or for further analysis.

[0038] For the sake of clarity, as used herein, the wording "operably connected", "operably connectable" or even "operably connecting", reflects a functional relationship between two or more components of a device or a system, that is, such a wording means that the claimed components must be connected in a way to perform a designated function. The "designated function" can change depending on the different components involved in the connection; for instance, the designated function of pressure means operably connected to a reservoir is the ability to apply a positive or negative pressure within said reservoir in a way as to allow the flowing of its content, or at least a part of it, throughout the main channel 2. A person skilled in the art would easily understand and figure out what are the designated functions of each and every component of the device or the system of the invention, as well as their correlations, on the basis of the present disclosure.

One of the main features of the microfluidic device 1 of the invention is the design of the tubular element 5. This is intersected by at least one side channel 6. The side channel 6 intersecting the tubular element 5 is needed to perform the removal of the liquid phase of the external shell of the core- shell capsules. In a preferred embodiment, an array of intersecting side channels 6 is included in the device 1. A plurality of side channels 6 can allow a more efficient, tailored and controlled removal of the liquid phase of the external shell of the core-shell capsules. In order to avoid complete suction of the capsule and/or damage of its structure, the cross sectional area of the side channels 6 is much smaller than both the core-shell capsule diameter and the cross sectional area of the tubular element 5. It is important to have the dimensions of the side channels 6, i.e. the height and width thereof, much smaller than the diameter of the drops to prevent complete or partial removal of double emulsion capsules, and hence to ensure that only the middle fluid is removed. When a (deformed) core-shell capsule meets the side channels 6, the liquid phase of its outer shell is sucked out by capillary forces or an applied negative pressure, also referred to herein as "underpressure". To accelerate fluid removal from the capsules' shells, a negative pressure can be applied from the outlet of the side channels 6 (i.e., the extremity of the side channels not connected with the tubular element 5) through e.g. a (micro)pump to increase the removal driving force.

[0040] In some embodiments, to obtain an optimal mechanical removal of the liquid phase of the outer shell, the planar cross-sectional area of the intersecting side channel 6 is comprised between about 3% and about 10% compared to the tubular element 5 of the main channel 2 cross sectional area, and/or at least one dimension of the planar cross-section of the intersecting side channel 6 is between about 1 % and about 10%, preferably about 3%, compared to the diameter of the core-shell capsules used. The side channels 6 can be of any length, and can intersect the tubular element 5 at any point along its length and at any angle, such as for instance 20°, 40°, 60°, 75° or 90°. The choice of the incident angle can advantageously tailor the removal of the external shell of the core-shell capsules, working as a "razor blade" on this latter.

[0041 ] As previously stated, the device 1 is connectable with a source of core-shell capsules, in such a way that these latter can flow throughout the main channel 2 upon application of a suitable pressure and/or other driving forces, such as magnetic or electric fields, surface acoustic waves and the like. For instance, a positive or negative pressure can be applied through the main channel 2 via the inlet 3 or the outlet 4, respectively. For "positive pressure" is herein meant an applied pressure that increases the main channel 2 internal fluid pressure, whereas for "negative pressure" it is herein meant an applied pressure that diminishes the main channel 2 fluid pressure, as in case of a suction. Means to apply a pressure will usually be coupled with the inlet 3 and/or the outlet 4 reservoir, either directly or indirectly (via e.g. a connection tube). Suitable means of altering the pressure within the device are external or integrated pumps or micropumps, combinations of capillary forces and electrokinetic mechanisms, hydrostatic pressure or simply a syringe. As will be evident for a person skilled in the art, for what said above, the invention is intended to cover also a system comprising a microfluidic device as defined above and pressure means operably connected to an inlet 3 and/or an outlet 4 adapted to generate a pressure within said inlet 3 and/or outlet 4, in a way as to allow the flow of its (their) content or at least a part of it.

[0042] In a particular, preferred embodiment, the core-shell capsule is a substantially spherical micro- or nanocapsule. Said structures are characterized in that they are hollow in their inner core, and comprise in said core a fluid, non-gaseous material.

[0043] Core-shell capsules are vesicular systems that are made up of polymeric membranes, which encapsulate an inner fluid core at the nanoscale or micron level. Core-shell capsules have a myriad of uses, which include medical promising applications for drug delivery, food enhancement, nutraceuticals, and for the self-healing of materials. The benefits of encapsulation methods are for protection of these substances to protect in the adverse environment, for controlled release, and for precision targeting. They can improve the stability of active substances and can be biocompatible with tissue and cells when synthesized from materials that are either biocompatible or biodegradable. Other advantages of micro encapsulated systems as active substance carriers include for instance high substance encapsulation efficiency due to optimized substance solubility in the core, or polymeric shell protection of the inner core against degradation factors like pH and light. A skilled person will appreciate that the present disclosure is meant to include vesicular and micellar core-shell structures such as microcapsules, microspheres or double emulsion capsules, i.e. hollow core-shell structures, in a nanometric scale rather than in a micrometric scale, or conversely in a millimetre scale. The capsules' size can be tailored based on specific needs by, e.g., reducing or augmenting the inner core fluid content or by altering the condition for producing them. In preferred embodiments of the invention, the inner core of core-shell capsules comprises a fluid material. Such fluid material comprises gases or preferably liquids such as e.g. aqueous solutions or non-polar solutions, gellike materials, composite hydrogels and the like. An "aqueous solution" is a solution in which the solvent is substantially made of water. In the frame of the present disclosure, the term "aqueous" means pertaining to, related to, similar to, or dissolved in water. The expression aqueous solution in the frame of the present disclosure also includes highly concentrated and/or viscous solutions such as for instance hydrogels, syrups (i.e., saturated water/sugars solutions) and the like, in which the water content is e.g. less than 5% weight of the total solution weight. A "non-polar solution" is a solution in which the solvent is a non-polar compound. Non-polar solvents are intended to be compounds having low dielectric constants and that are not miscible with water. A non-exhaustive list of non-polar solutions can comprise for example solutions comprising oils, benzene, carbon tetrachloride, chloroform, diethyl ether, xylene, toluene, ethanol, hexanol, heptanol, decanol, dodecanol, hydrocarbon-based solutions (e.g. hexane, cyclohexane, n-octane, isooctane, decane, hexadecane and the like), fluorophilic solvents, ethyl acetate, silicon oils, mineral oils, oils used for food and so forth. An "oil" is any non-polar chemical substance that is a viscous liquid at ambient temperatures and is both hydrophobic and lipophilic. A fluid material is also intended to comprise any fluid material comprising a gas dispersed within, such as e.g. liquid-gas solutions.

In a preferred embodiment of the present invention, the core-shell capsule is a double emulsion capsule. Double emulsion capsules are particles composed of a drop (the core) contained in a second, larger drop (the shell membrane) made of an immiscible, or partially miscible fluid. The second, larger drop typically contains the chemicals that are used to optionally solidify the shell. However, certain chemicals can also be contained in the smaller drop, which forms the core of the double emulsion, or in the outermost phase, where the drops are dispersed in. These double emulsion drops can for example be used for forming two monolayers of self- assembled amphiphiles that form a bilayer of amphiphilic molecules upon removal of the liquid contained in the shell of double emulsions. The core of the capsule or double emulsion is substantially made of an aqueous solution. These capsules or double emulsions are dispersed in a continuous aqueous phase (the water-in-oil-in-water type, or W-o-W). Alternatively, the core of the capsule or double emulsion is composed of an oil, the larger drop is made of an aqueous phase and the double emulsion drops are dispersed within a continuous oil (the oil-in-water-in-oil type, or O-w-0). Hollow core-shell capsules according to the invention offer the possibility of including within the inner core one or more active agents. This is of particular interest for what concerns their application, such as e.g. biomedical, cosmetic, agriculture, coating or food ones. In the frame of the present disclosure, an "active agent" is any agent capable of altering, modifying or otherwise interacting with the surrounding environment once brought into direct or indirect contact with it. An active agent can be any agent having the ability to bring about chemical reactions or physical state changes. Suitable agents to be used in the frame of the present invention are for instance imaging or contrast agents, bioactive agents, magnetically or optically active substances, organic compounds, inorganic compounds and/or elements (such as e.g., gold particles), coating substances and/or precursors thereof or food substances including seeds or probiotics.

[0047] A "bioactive agent", as well as "bioactive molecule", "bioactive compound", or "therapeutic agent", is any active agent that is biologically active, i.e. having an effect upon a living organism, tissue, or cell. The expression is used herein to refer to a compound or entity that alters, inhibits, activates, or otherwise affects biological or chemical events. Bioactive compounds according to the present disclosure can be small molecules or macromolecules, including recombinant ones. One skilled in the art will appreciate that a variety of bioactive compounds can be used depending upon the needs, e.g. a condition to be treated when the polymeric structure of the invention is intended for prophylactic, therapeutic or even diagnostic purposes. A non-exhaustive list of suitable bioactive agents includes pharmacologically active substances, drugs such as antibiotics or chemotherapeutics, peptides, enzymes, antibodies, vitamins, spores, pesticides and the like.

[0048] Exemplary therapeutic agents further include, but are not limited to, a growth factor, a protein, a peptide, an enzyme, an antibody or any derivative thereof (such as e.g. multivalent antibodies, multispecific antibodies, scFvs, bivalent or trivalent scFvs, triabodies, minibodies, nanobodies, diabodies etc.), an antigen, any type of nucleic acid, such as e.g. DNA, RNA, siRNA, miRNA and the like, a hormone, an anti-inflammatory agent, an anti-viral agent, an anti-bacterial agent, a cytokine, a transmembrane receptor, a protein receptor, a serum protein, an adhesion molecule, a lipid molecule, a neurotransmitter, a morphogenetic protein, a differentiation factor, an analgesic, organic molecules, polysaccharides, a matrix protein, a spore, a cell, and any functional fragment or derivative of the foregoing, as well as any combinations thereof. A "functional fragment" is herein meant any portion of an active agent able to exert its physiological/pharmacological activity. For example, a functional fragment of an antibody could be an Fc region, an Fv region, a Fab/F(ab')/F(ab')2 region and so forth.

[0049] In one embodiment, the double emulsion capsule is characterized in that the total capsule diameter is at least 5 times bigger than the thickness of the shell.

[0050] The invention further relates to a method for the partial or total mechanical removal of the liquid phase of the external shell of a core-shell capsule by using the microfluidic device 1 or a system including it. The method comprises a step of adapting the surface of the main channel 2 to the external shell of the core-shell capsule so to favour a high wettability of the liquid phase of the external shell of the core-shell capsules. This can be obtained by wetting the internal surface of the main channel 2 with a liquid phase of compatible polarity with the external shell of the core-shell capsule; for instance, if the external shell is substantially composed of a hydrophilic material, the main channel 2 will be wetted with a hydrophilic liquid. Alternatively, all or part of the device of the invention can be e.g. coated with suitable materials in order to match the polarity of the external shell of the core-shell capsule, thus improving the wettability of the internal surface of the device, and particularly of the main channel 2. However, this step(s) is (are) considered optional in the method of the present invention, since the materials used for the manufacturing of the device 1 can already be compatible with the liquid portion of the capsules' shell in terms of polarity and wettability.

According to the method of the invention, a source of core-shell capsules is operably (fluidically) connected with the inlet 3 of the main channel 2 and a pressure or another driving force is applied to allow the capsules to engage the inlet 3 and to subsequently flow through the main channel 2. In some embodiments, when entering the tubular element 5 of the main channel 2, the capsules get deformed, and this deformation allows the external shell of the core-shell capsule to be even more efficiently removed upon its passage through the tubular element 5. The removal of the external shell is carried out by the mechanical scraping of the intersecting side channels 6 through the generation of capillary forces or because of a difference in pressure between intersecting side channels 6 and the main channel 2. To enhance this mechanical separation, additional external forces can be applied such as vacuum and/or other sucking systems to the side channels 6 towards the outlet of these latter.

[0052] Among other parameters, the number of side channels 6 placed along the tubular element 5 will determine the degree of liquid removal from the external shell of the core-shell capsule. By adjusting this number one can efficiently modulate the thickness of the external shell and eventually remove it entirely.

[0053] At the end of the tubular element 5, the capsules pass through the outlet 4 and are collected in a reservoir and/or the microfluidic device 1 is operably connected through the outlet 4 to an external device.

[0054] EXAMPLES

[0055] Fabrication of the device

[0056] A microfluidic device for the production of single-core double emulsions was fabricated from poly(dimethyl siloxane) (PDMS) using soft lithography, as schematically shown in Figure 2. However, the device can also be made out of any other elastomer or even hard materials such as thermoplasts, thermosets or even glass. The device has a main channel through which the produced double emulsions flow and at least one side channel that intersects the main channel; the side channels enable removal of the oil from the double emulsion shells. In the embodied example, the height of the side channels is at least 2 times lower than the height of the main channel. To fabricate channels with different heights within the same device, multiple layers of photoresists have been used. Other suitable ways of manufacturing the device known in the art can be however envisaged, such as 3D printing, hot embossing and so forth. The first layer contains the main channel as well as all the side channels and is 10μιτι thick. The second layer contains only the main channel and is 30 μιτι thick. Hence, the total height of the main channel is 70 μιτι, as schematically shown in Figure 3.

[0057] Device surface functionalization

[0058] To efficiently remove fluid from the shell of the double emulsion, this fluid must wet the channel walls. In operative examples, when the solvent in the shell was a hydrocarbon-based oil, the channel walls were rendered hydrophobic by treatment with e.g. dodecyl trichlorosilane. If the solvent in the shell was a perfluorinated oil, the channel walls were rendered fluorophilic for example by treating them with a perfluorinated trichlorosilane. If the solvent in the shell was water, the channel walls were rendered hydrophilic for example by treating them with polyelectrolytes.

[0059] Design

[0060] The device contains two inlets and two outlets, as can be seen in Figure 2a.

Double emulsions dispersed in a continuous phase are injected through inlet I. The middle phase is removed through the side-channels that are combined into a larger channel further apart from the main channel. This oil is removed through outlet II. To stabilize the obtained vesicles and to space them apart, additional outer phase, optionally containing surfactants is injected through inlet III. The resulting vesicles or double emulsions with thinner shells are collected through outlet IV.

[0061 ] The initial part of the main channel is 90 μιτι wide and 70 μιτι tall. To efficiently remove the middle phase, double emulsion droplets are deformed; this is accomplished by narrowing the main channel to 40 μιτι while the height of this channel, h, is maintained at 70 μιτι. The narrowed main channel is intersected by multiple small side-channels, which are 10 μιτι wide and 10 μιτι tall; the center of these side channels intersects the main channel at h/2. An important consideration related to the dimensions of these side channels relates to the height and the width thereof, that shall be much smaller than the diameter of the drops to prevent complete or partial removal of double emulsion drops and hence, to ensure that only the middle fluid is removed.

[0062] After the middle fluid is removed, the width of the main channel is increased to 120 μιτι while h is kept constant at 70 μιτι. The widened main channel is intersected by a pair of channels used for injection of additional outer phase to space the vesicles or double emulsions further apart, and to optionally stabilize them for example by injecting additional aqueous solution containing additional surfactants.

[0063] Operation of the device [0064] As-produced double emulsion drops are injected into the main channel of the device. The initial width and height of the main channel is similar to the diameter of the double emulsion such that it is nearly spherical, as seen in the optical micrograph in Figure 4a. When the width of the main channel is reduced, the double emulsion drop becomes deformed, resulting in an inhomogeneous shell thickness: the shell of the double emulsion drop becomes much thicker at the leading end of the drop and much thinner on the sides and at the tailing end of the drop, as shown in the optical micrograph in Figure 4b. When the deformed double emulsion drops meet the side channels, the liquid phase present in the shell is sucked out of it by capillary forces, as indicated by the formation of small single emulsion oil drops at the end of the side channels, shown by the black arrows in Figure 5. To accelerate fluid removal from the double emulsion shells, an underpressure can be applied to outlet II to increase the driving force for the oil to be sucked out of the shell.

[0065] As the main channel widens, the resulting vesicle or double emulsion with a thinner shell attains again a nearly spherical shape. Once the main channel meets the pair of inlets used to inject additional outer phase, vesicles or double emulsion drops can be spaced apart and optionally stabilized with the injection of additional fluid optionally containing a surfactant such as polyvinyl alcohol). The resulting vesicles or double emulsion drops exit the device through outlet IV and are collected in a collection vial.

[0066] Removal ofperfluorinated oil from water-oil-water double emulsion drops

[0067] Setup

[0068] Water-oil-water double emulsions are produced using a microfluidic device previously described (Arriaga, L. R. et al., Lab Chip, 473-0197). The shell of the double emulsion is formed from HFE7500, a perfluorinated oil, containing 48 mg/mL surfactant composed of a poly(ethylene glycol)-FSH (Dupont Krytox 157FS oil) di-block copolymer. The resulting water-oil-water double emulsion is injected into the main channel of the device at a flow rate fn/ic that is varied between 200 μί/h and 1600 μ ι. The oil removal is accelerated from the double emulsion shell by applying an underpressure to the outlet via a withdrawal pump, with a rate that varies from 0 to 1500 μΙ_/Ι"ΐ. An external phase is injected in the entire system through inlet III, at a rate, f₀, kept constant at 800 μΙ_/Ι"ΐ.

[0069] The device can be operated in three different regimes, depending on the ratio of fMc fw and the initial shell thickness of the double emulsion drop, wherein f_w is the withdrawal rate of the liquid phase of the external shell and fMc is the injection flow rate of the double emulsion drops. At very low ratios of fMc fw the oil is completely removed and vesicles, herein referred to also as polymersomes, are formed. At intermediate ratios of fMc/fw, the shell thickness is reduced, yet the resulting double emulsion still contains oil in its shell. At high ratios of fMc fw not only oil but also some of the inner aqueous phase is sucked through the side channels, resulting also in a split of the initial double emulsion drops into many much smaller double emulsion drops.

In the embodied device, for obtaining the formation of polymersomes a fMc fw ratio should be kept < 2, with an fw < 700 μΙ_/Ι"ΐ; for decreasing the shell thickness, a fMc fw ratio > 2 and fw < 700 μ ι is the optimum, while for reducing the size of double emulsion drops a fMc fw ratio < 2 and fw > 700 μί/h should be used. In this last case, the shear stress acting on the drop is sufficiently high to push the drop into the side channel. The strong deformation of the double emulsion, caused by the small side channels, causes it to break into many much smaller double emulsion drops whose diameter is similar to the width of the side channel (in the present case, 10 μιτι; Figure 6). This operation mode closely resembles the extrusion process often used to reduce the size vesicles formed through re-hydration techniques, yet on a much larger length scale. However, here the diameters of starting double emulsion drops are in the order of 100 μιτι, and they are reduced to double emulsions with diameters of order of 1 μιτι - 10 μιτι (1 to 10% of the starting diameter size). By contrast, extrusion processes used to reduce the size of vesicles typically reduce their size to values varying between 50 nm and 200 nm: the device of the invention enables production of much smaller vesicles than those which can currently be produced using microfluidics.

The device of the invention enables the mechanical removal of fluid from the shell of double emulsions. It allows conversion of double emulsion drops into vesicles without the need for fluid to evaporate. This enables to use microfluidic techniques to produce vesicles that can be used for biomedical applications, which are sensitive to traces of toxic solvents such as chloroform. Hence, this device significantly broadens the use of the microfluidic assembly techniques. In addition, the device offers the possibility to only partially remove fluid from the shell of double emulsions, thereby allowing accurate control over the shell thickness down to very small values. Finally, it can be used to break large double emulsion drops into many much smaller double emulsion drops, which have very thin shells and can hence again be used as templates for the production of vesicles. Small vesicles are of high interest especially for the biomedical industry. However, thus far, small vesicles could not be made using microfluidic technologies. Hence, this device has the potential to further broaden the use of microfluidically assembled vesicles to biomedical applications that have thus far be hampered by the large size of these vesicles.

Claims

1 . A device for use in the mechanical removal of the liquid phase of the external shell of a core-shell capsule, characterized in that said device comprises: a) a main channel (2) having at least one inlet (3), one outlet (4) and a tubular element (5) in between, adapted to be operably connectable with a source of core-shell capsules (7) so that these latter can flow throughout said main channel (2) upon application of a pressure or other driving forces; and

b) at least one side channel (6) intersecting the main channel (2) along the tubular element (5).

2. The device of claim 1 , characterized in that at least one dimension of the planar cross-section of the tubular element (5) of the main channel (2) is smaller than the diameter of the core-shell capsules.

3. The device of claims 1 or 2, characterized in that the planar cross-section of the intersecting side channel (6) is comprised between about 3% and about 10% compared to the cross sectional area of the tubular element (5) of the main channel (2).

4. The device of any previous claim, characterized in that at least one dimension of the planar cross-section of the intersecting side channel (6) is between about 1 % and about 20%, preferably 3%, compared to the diameter of the core-shell capsule.

5. The device of any previous claim, characterized in that the core-shell capsule is a water-oil-water (W-o-W) or oil-water-oil (O-w-0) double emulsion capsule.

6. A system for use in the mechanical removal of the liquid phase of the external shell of a core-shell capsule, characterized in that it comprises the device of claims 1 to 5 operably connected with means for applying a pressure or other driving forces adapted to allow core-shell capsules to flow through the main channel (2) from a source of core-shell capsules (7).

7. A method for mechanically removing the liquid phase of the external shell of a core-shell capsule, said method comprising the steps of:

a) providing the device of claims 1 to 5 or the system of claim 6;

b) optionally adapting the main channel (2) so to favour a high wettability of the liquid phase of the external shell of the core-shell capsules; c) providing a source of core-shell capsules (7) in fluidic connection with the inlet (3) of the main channel (2) and with means for applying a pressure or another driving force to let the capsules flow through the channel; and

d) applying a pressure or a driving force to the source of core-shell capsules in a way to allow said capsules to flow throughout the main channel.

8. The method of claim 7, characterized in that step b) is achieved by injecting in the main channel (2) a liquid phase of compatible polarity with the external shell of core-shell capsules.

9. The method of claims 7 or 8, further comprising a step of collecting the obtained core-shell capsules into a reservoir connected with the outlet (4) of the main channel (2).