Device to generate capsules, spheres, beads or filaments optionally with an enclosed compound using earth gravity, centrifugation forces or negative pressure or positive pressure

Abstract

The present invention relates to a device for the enclosure of a compound into capsules, spheres, beads or filaments, wherein the device comprises (A) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if earth gravity or centrifugal forces are applied, (iv) a tube between (i) and (ii) or (i) and (v) that transgresses the second tube, wherein the tube enables the exchange of air between (i) and (v), and (v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube; or (B) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross- linking solution of (i) if negative pressure forces are applied, (iv) a connection being configured to be connected to a vacuum pump, which connection is an extra opening in the first tube allowing to generate negative pressure forces, preferably from the bottom of the device via a connected vacuum pump, and (v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube; or (C) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if positive pressure forces are applied, and (iv) a lid positioned on top of the second tube, and wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube and which opening is also a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump; or wherein the second tube or the lid further comprises a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube.

Description

[0001] Device to generate capsules, spheres, beads or filaments optionally with an enclosed compound using earth gravity, centrifugation forces or negative pressure or positive pressure

[0002] The present invention relates to a device for the enclosure of a compound into capsules, spheres, beads or filaments, wherein the device comprises (A) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if earth gravity or centrifugal forces are applied, (iv) a tube between (i) and (ii) or (i) and (v) that transgresses the second tube, wherein the tube enables the exchange of air between (i) and (v), and (v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube; or (B) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the crosslinking solution of (i) if negative pressure forces are applied, (iv) a connection being configured to be connected to a vacuum pump, which connection is an extra opening in the first tube allowing to generate negative pressure forces, preferably from the bottom of the device via a connected vacuum pump, and (v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube; or (C) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one ortwo reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if positive pressure forces are applied, and (iv) a lid positioned on top of the second tube, and wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube and which opening is also a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump; or wherein the second tube or the lid further comprises a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube.

[0003] In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] The method known as immobilization consists in the generation of polymer-based scaffold which adopts a specific shape after being contacted to a solution containing a specific chemical compound crosslinking the polymers The method known as encapsulation consists in the generation of spheric polymer-based scaffolds, which adopt this shape after being contacted to a solution containing a specific chemical compound crosslinking the polymers. These beads are characterized by their shape, and the presence of micropores, enabling the exchange of material (i.e. gas, small particles, other compounds) between the bulk of the capsules, and their surroundings. One traditional example of polymer-based capsules is made of alginate. This polymer enables the generation of capsules, which can be used to entrap several molecules (i.e. drugs, proteins, DNA, RNA, etc.), or living organisms (bacteria, yeast, mammalian cells) which can be used, for example, in the food, pharmaceutical and / or biomedical industry.

[0005] Capsules can be made using several man-made or industrial devices. The easier strategy used to generate polymer-based capsules is by sequentially dropping small volumes of a solution containing a cross-linkable polymer, such as alginate, into a solution containing a crosslinker compound, such as CaCh. This can be done by using a syringe, a pipette, a micropipette or more complex devices, such as the commercial microencapsulator devices offered by the companies Biichi and Nisco. These above- mentioned devices use two main systems to drop the polymer-based solution into the crosslinking bath, using air pumps (Biichi devices), peristaltic pumps (Nisco devices), or gravity (Gravity VAR W10, Nisco).

[0006] While some devices and methods for the generation of capsules, spheres, beads or filaments are already available in the art, there is still an ongoing need for further devices and methods, in particular devices that have a simple format and can be easily used for the generation of capsules, spheres, beads or filaments and in particular for the enclosure of a compound in capsules, spheres, beads or filaments by applying gravity or centrifugal or negative pressure or positive pressure forces. This need is addressed by the present invention.

[0007] Accordingly the present invention relates in first aspect to a device for the enclosure of a compound into capsules, spheres, beads or filaments, wherein the device comprises (A) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if earth gravity or centrifugal forces are applied, (iv) a tube between (i) and (ii) or (i) and (v) that transgresses the second tube, wherein the tube enables the exchange of air between (i) and (v), and (v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube; or (B) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the crosslinking solution of (i) if negative pressure forces are applied, (iv) a connection being configured to be connected to a vacuum pump, which connection is an extra opening in the first tube allowing to generate negative pressure forces, preferably from the bottom of the device via a connected vacuum pump, and (v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube; or (C) (i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution, (ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one ortwo reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed, (iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if positive pressure forces are applied, and (iv) a lid positioned on top of the second tube, and wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube and which opening is also a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump; or wherein the second tube or the lid further comprises a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube.

[0008] A device is an object that has been produced for a particular purpose. This purpose is in accordance with the present invention the enclosure of a compound into capsules, spheres, beads or filaments or, in the absence of such a compound, the generation of capsules, spheres, beads or filaments (without an enclosed compound). The device according to (A) relies on earth gravity or centrifugation forces, the device of (B) relies on negative pressure forces and the device of (C) relies on positive pressure forces when being used for the indicated purposes.

[0009] The device according to (A) is preferred as compared to the device according to (B) and (C).

[0010] The compound that can be enclosed is not particularly limited as long as it fits into and can be taken up by the capsules, spheres, beads or filaments being formed when the claimed device is used for the enclosure of a compound into capsules, spheres or beads. The compounds can be an organic or inorganic compound. Fats, nucleic acids, carbohydrates, proteins (such as enzymes), and hydrocarbon fuels are examples of organic molecules. Non-metals, salts, metals, acids, and bases are examples of inorganic compounds. Further and preferred examples of compound s will be described herein below.

[0011] In connection with capsules, spheres, beads “enclosed” or “enclosing” may also be further defined as “encapsulated” or “encapsulated”. The enclose of the compound into capsules, spheres, beads or filaments also immobilizes the compound in the capsules, spheres, beads or filaments.

[0012] A bead refers to a closed compact full sphere made of one or more polymers that can package in its inside the compound to be encapsulated.

[0013] A capsule refers to a closed shell or envelope that can package in its inside the compound to be encapsulated, if the compound is present.

[0014] A filament refers to a closed compact full cord or a closed shell cord or a enveloped cord made of one or more polymers that can package in its inside the compound to be enclosed.

[0015] A sphere is an object exhibiting a three-dimensional round morphology. Inside the ball-shaped sphere the compound can be encapsulated, if the compound is present. It is to be understood that the nature of capsules, spheres, beads may overlap, so that the same object may be classified, for example, at the same time as capsule and bead.

[0016] Among capsules, spheres, beads or filaments the three options capsules, spheres and beads are preferred and capsules are most preferred. The generation of capsules with an enclosed / encapsulated compound is shown in the appended examples.

[0017] The capsules, spheres or beads are preferably micro- or nano-capsules, micro- or nano-spheres or micro- or nano-beads. The term “micro- or nano-” designates that the capsules, spheres or beads have a size (e.g. largest diameter) in the range of micrometers (i.e. 1 pM to 1000 pm) or nanometers (i.e. 1 nm to 1000 nm). Preferred sizes of capsules, spheres or beads will be provided herein below.

[0018] The filaments are preferably micro- or nano-filaments. The term “micro- or nano-” designates that the filaments, have a diameter (e.g. shortest distance between the surface of this structures) in the range of micrometers (i.e. 1 M to 1000 pm) or nanometers (i.e. 1 nm to 1000 nm).

[0019] While the bottom side of the first tube preferably forms a flat footprint, so the device can stand alone it is also preferred that the bottom side preferably forms a cone or semi-circle and in particular a cone or semi-circle that fits into the centrifuge tube pick up structure of a centrifuge.

[0020] The second tube sits on top of the first tube.

[0021] The shape of the tubes (first and second tube) is not particularly limited and the tubes can be, for example, tringle, square-, hexagonal- or round shaped. Round shaped tubes are preferred, and they can also be referred to as cylindric tubes.

[0022] The second tube’s one or two reservoirs is / are configured to harbor a polymer solution that can be crosslinked by the cross-linking solution and to harbor the compound to be enclosed into the capsules, spheres, beads or filaments. In the case of one reservoir the polymer solution and the compound can be in one solution in one reservoir. In the case of two reservoirs the polymer solution can be in one reservoir and the compound in the form of a solution can be in the second reservoir. In the latter case the one or more outlets of each of the two reservoirs run into each other, so that the polymer solution and the compound to be enclosed can be in one solution before they come into contact with the crosslinking solution in the first tube.

[0023] Hence, in accordance with the invention in one alternative the reservoir will contain prior to the release into the cross-linking solution in the first tube, both the polymer solution and the compound to be enclosed in the same (one) reservoir. Alternatively, the polymer solution and the compound to be enclosed are retained in different (two) reservoirs prior to the release into the cross-linking solution in the first tube. As explained, in the that case they come into contact via the outlets. The option of one reservoir is preferred. Accordingly, the (one) reservoir is a receptacle that can take up and then comprises the polymer solution. The reservoir can also take up and then comprises the compound to be enclosed. As can be taken from the examples the device may also comprise more than one reservoir, in particular two reservoirs which enables the parallel enclosure of two or more compounds into distinct capsules, spheres, beads or filaments- In the latter case each of the two reservoirs comprises a polymer solution and a compound to be enclosed.

[0024] Whereas the reservoir(s) can be configured to comprise both, polymer solution and the compound to be enclosed, this does not require that the two items always need to be present in a reservoir at all or at the same time. Rather, it is preferred that the polymer solution is first added to a reservoir and the compound is added subsequently.

[0025] The one or two reservoirs and the one or more outlets are configured so that the solution(s) in the one or two reservoirs can only reach the polymer solution in the first tube via the one or more outlets.

[0026] The polymer solution can be cross-linked by the cross-linking solution. In this connection it is to be understood that when the cross-linking solution is in the first tube and the polymer solution is in the reservoir(s) of the second tube and earth gravity or centrifugal or negative pressure or positive pressure forces are applied to the device then drops or extrusions of the polymer solution pass through the one or more outlets of the reservoir(s) into the cross-linking solution resulting that the polymer solution is cross-linked thereby forming capsules, spheres, beads or filaments. In the case the reservoir(s) of the second tube also comprises the compound to be enclosed the formed capsules, spheres, beads or filaments enclose these compounds.

[0027] The polymer solution (with or without the compound) has a viscosity that ensures that it can drop or extrude into the cross-linking solution if earth gravity or centrifugal forces are applied. As long as this viscosity prerequisite is met the viscosity of the polymer solution is not particularly limited, The polymer solution may be non-crosslinked or already partially cross-linked.

[0028] The polymer solution comes into contact with the cross-linking solution in the form of drops or droplets or filaments. In this connection it is of note thar drops are larger than droplets. Other than that differing on both terms designate a globule or a filament of fluid accumulating enough volume and weight to fall. The drops of droplets can be oil drops or droplets or water- drops or droplets, to create emulsions (depending on which cross-linking and polymer solutions are used). It follows that both, the cross-linking solution and polymer solution can be water-based or oil-based or can be emulsions. Whether the polymer solution drops or extrudes into the cross-linking solution can be adjusted by distance between the one or more outlets of the reservoir(s) and the cross-linking solution. If the distance is larger, drops of polymer solution are formed that can fall into the cross-linking solution and if the distance is narrower extrusions of the polymer solution are formed. Drops are preferred over extrusions herein.

[0029] These drops or droplets or filaments are formed by applying earth gravity or centrifugal or negative pressure or positive pressure forces to the device. For applying earth gravity forces the device according to (A) just can be placed on a surface or a holder, so that the drops or droplets or filaments are formed. For applying centrifugal forces the device according to (A) is generally placed into a centrifuge. Also ultracentrifugation can be used with the purpose to create double emulsions (noting that double emulsions, so-called Water-in-Oil-in-Water emulsions (W / O / W emulsions), consist of water drops or droplets dispersed in oil or oil / polymer globules, themselves ultimately dispersed in an aqueous phase).

[0030] In addition, the device according to (A) comprises a tube transgressing the second tube. Through this tube the air can flow between the first tube and the lid. The options of the tube being between (i) and (ii) or (i) and (v) refer to the possible lengths of the tube. The length is not limited as long as the air can flow between the first tube and the lid. The tube is above the liquid level of the solutions) in the one or two reservoirs, so that no solution(s) can enter the liquid. The tube transgressing the second tube may therefore as be called ”air flow tube”. This air flow is important since otherwise pressure difference within the closed device may prevent the dropping or extruding of the polymer solution into the cross-linking solution when earth gravity or centrifugal forces are applied to the device according to (A).

[0031] For applying negative pressure (also referred to in the art as under pressure) forces the device according to (B) is generally connected to a vacuum pump. The negative pressure is to be applied to the first tube and preferably from the bottom of the device, so that the polymer solution is absorbed / soaked up / inspired into the first tube. For this purpose the first tube and preferably the bottom thereof comprises a connection to a vacuum pump which connection is or comprises an extra opening in the first tube containing the crosslinking solution.

[0032] For applying positive pressure forces the device according to (C) is generally connected to an air pump. The positive pressure is to be applied to the lid or second tube and preferably from the top of the device, so that the polymer solution is pressed from the one or more reservoirs into the first tube. For this purpose the lid or second tube comprises a connection to an air pump. The connection can either be the opening or an extra connection of the lid or second tube.

[0033] The lid sits on top of the second tube and closes the device, such that no impurities can enter the reservoir(s). The lid forms the top closing of the device. For this reason the device can also be a sterile device. For this purpose the device or the parts of the device (before its assembly) are sterilized and within the closed the device the capsules, spheres, beads or filaments can then be formed under sterile conditions. Means and methods for sterilization of devices or subparts thereof are well known in the art and comprise sterilization high-pressure steam (autoclave), dry heat (oven), chemical sterilants (ozone or ethanol or methanol) or physical agents (radiation).

[0034] Yet further, in the device the second tube (part (ii)) or the lid (part (v)) further preferably comprises an opening, optionally with a second lid on said opening. Through this opening the polymer solution and / or the compound can be placed into the reservoir(s) of the second tube. When the device is in use the lid can either be open or closed. The lid is preferably closed in order to avoid impurities that may enter via the opening, such as if working under sterile conditions.

[0035] The preferred opening is technically advantageous for the device according to (A) and (B) because thereby the polymer solution and / or the compound can be placed into the reservoir(s) in the completely assembled device. This is particularly advantageous for working under sterile conditions.

[0036] The opening can optionally be closed by a second lid. The opening may also be closed by a membrane. In addition, the opening may comprise a filter or mesh for retaining impurities or biological contaminants and only allowing the polymer solution and / or the compound to enter the reservoir(s).

[0037] Accordingly, the second lid preferably comprises a membrane with a pore size of equal or smaller than 0.45 pm and preferably of about 0.22 pm to avoid the entrance of contamination (virus, bacteria, yeast), keeping however, the possibility to exchange air when using the device for the enclosure of a compound into capsules, spheres, beads or filament or the formation of capsules, spheres, beads or filaments. The discussed membrane may be pierced for placing the polymer solution and / or the compound into the reservoir(s). Yet further the second lid, if present, may be opened when using the device for the enclosure of a compound or the formation of capsules, spheres, beads or filaments, in particular device in connection with the device according to (A).

[0038] In the device as illustrated in Figure 1 (i.e. a device according to (A)) the lid further comprises an opening with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the reservoir of the second tube.

[0039] In the device as illustrated in Figure 4 (i.e. a device according to (A)) the second tube comprises an opening (on its top) through which opening the polymer solution and / or the compound can be placed into the reservoir of the second tube. This opening can be closed by the lid and the lid in Figure 4 has no opening and is only for closing the second tube.

[0040] It can be taken from the above that the device of the invention offers a number of technical advantages. The device has a simple structure and the few components can be easily assembled to form the device. The device is flexible since any kind of polymer and corresponding cross-linking solution can be used to enclose a huge variety of possible compounds. The size of the device can be adapted as needed and in particular such that it fits into commercially available centrifuges for centrifuge tubes of different size, e,g from 0.1 ml to 250 mL tubes. The device is also easy to use since just earth gravity or centrifugal or negative pressure or positive pressure forces have to be applied in order initiate the formation of capsules, spheres, beads or filaments that can enclose the compound of interest. In addition and as discussed, the device enables to work under sterile conditions, if needed. The device can therefore be and is preferably a sterile device.

[0041] In accordance with a preferred embodiment of the first aspect, the compound to be enclosed is a cell, a small molecule, a micro- or nano-metallic compound, a micro- or nano-ceramic compound, micro- or nano-electronic device, virus, intracellular organelle, a (poly)peptide (e.g. enzyme, growth factor, hormone) or a nucleic acid molecule.

[0042] The cell can be a prokaryotic cell or a eukaryotic cell and is preferably a eukaryotic cell. The eukaryotic cell can be, for example, a plant cell, yeast cell or animal cell and is preferably an animal cell. The animal cell is preferably a human cell.

[0043] The "small molecule" as used herein is preferably an organic molecule. Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds. The original definition of the term organic related to the source of chemical compounds, with organic compounds being those carbon-containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources. Organic compounds can be natural or synthetic. The organic molecule is in one preferred embodiment an aromatic molecule and more preferably a heteroaromatic molecule. In organic chemistry, the term aromaticity is used to describe a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms. Aromatic molecules are very stable, and do not break apart easily to react with other substances. In a heteroaromatic molecule at least one of the atoms in the aromatic ring is an atom other than carbon, e.g. N, S, or O. For all above-described organic molecules the molecular weight is preferably in the range of 200 Da to 1500 Da and more preferably in the range of 300 Da to 1000 Da.

[0044] Alternatively, the "small molecule" in accordance with the present invention is an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates). Preferably, the small molecule has a molecular weight of less than about 2000 Da, or less than about 1000 Da such as less than about 500 Da, and even more preferably less than about Da. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry.

[0045] The small molecules may be designed, for example, based on the crystal structure of the target molecule, where sites presumably responsible for the biological activity can be identified and verified in in vivo assays such as in vivo high-throughput screening (HTS) assays.

[0046] The term “micro- or nano-” also designates for the compounds or devices that they have a size (e.g. largest diameter) in the range of micrometers (i.e. 1 pM to 1000 pm) or nanometers (i.e. 1 nm to 1000 nm).

[0047] A metallic compound is any compound that is made of or comprises a metal or an alloy. An alloy is a substance having metallic properties and which is composed of two or more elements at least one of which is a metal. Preferred are gold, silver, platin, aluminum, titanium, copper, tin, iron and alloy thereof. A ceramic compound is any compound that is made of or comprises a ceramic. A ceramic compound is any of the various hard, brittle, heat-resistant, and corrosion-resistant materials made by shaping and then firing an inorganic, nonmetallic material, such as clay, at a high temperature. Common examples are earthenware, porcelain, and brick.

[0048] Electronic devices, such as transistors, capacitors, and resistors, generally within an active chip, require some protection from the environment, as well as both electrical and mechanical connections to the surrounding components. The electronic device can include transistors, capacitors, inductors, resistors, diodes, insulators and / or conductors

[0049] The virus is generally in the form of viral particles. A virus particle, also called a virion, consists of the viral genome, which can be either DNA or RNA in a single- or double-stranded state, surrounded by a protein coat, the capsid.

[0050] An intracellular organelle is an organized structure of distinctive morphology and function, occurring within a cell. Intracellular organelles include the nucleus, mitochondria, plastids, vacuoles, vesicles, lysosomes, ribosomes, and chloroplasts .

[0051] The term “protein” as used herein interchangeably with the term “polypeptide” describes linear molecular chains of amino acids, including single chain proteins or their fragments, containing at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting of up to 49 amino acids, whereas the term “polypeptide” (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting with increased preference of at least 15 amino acids, at least 20 amino acids at least 25 amino acids, and at least 40 amino acids. The group of peptides and polypeptides are referred to together by using the term "(poly)peptide". (Poly)peptides may further form oligomers consisting of at least two identical or different molecules. The corresponding higher order structures of such multimers are, correspondingly, termed homo- or heterodimers, homo- or heterotrimers etc. Furthermore, peptidomimetics of such proteins / (poly)peptides where amino acid(s) and / or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention. Such functional analogues include all known amino acids other than the 20 gene-encoded amino acids, such as selenocysteine. The terms “(poly)peptide” and “protein” also refer to naturally modified (poly) peptides and proteins where the modification is affected e.g. by glycosylation, acetylation, phosphorylation and similar modifications which are well known in the art.

[0052] The (poly)peptide can be, for example, am enzyme, an antibody a growth factor, or hormone.

[0053] The term “nucleic acid molecule” in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA. The nucleic acid molecule may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2’-O- methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001 , 8: 1). LNA is a modified RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2’-oxygen and the 4’-carbon. Also included are nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil. A nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and / or polypeptides. The nucleic acid molecule may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.

[0054] The nucleic acid molecule can also be in the form of a vector. The term “vector” in accordance with the invention means preferably a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering which carries the nucleic acid molecule. The nucleic acid molecule may, for example, be inserted into several commercially available vectors. Non-limiting examples include prokaryotic plasmid vectors, such as ofthe pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCI neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1 , pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, pLXlN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen).

[0055] Nucleic acid molecules to be inserted into the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and / or to other amino acid encoding sequences can also be carried out using established methods. Transcriptional regulatory elements (parts of an expression cassette) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of transcription (e. g., translation initiation codon, promoters, such as naturally-associated or heterologous promoters and / or insulators; see above), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the polynucleotide encoding the polypeptide / protein or fusion protein is operatively linked to such expression control sequences allowing expression in prokaryotes or eukaryotic cells. The vector may further comprise nucleic acid sequences encoding secretion signals as further regulatory elements. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used, leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the invention. Such leader sequences are well known in the art.

[0056] Furthermore, it is preferred that the vector comprises a selectable marker. Examples of selectable markers include genes encoding resistance to neomycin, ampicillin, hygromycine, and kanamycin. Specifically-designed vectors allow the shuttling of DNA between different hosts, such as bacteria-fungal cells or bacteria-animal cells (e. g. the Gateway system available at Invitrogen). An expression vector is capable of directing the replication, and the expression, of the polynucleotide and encoded peptide or fusion protein.

[0057] In accordance with another preferred embodiment of the first aspect, the one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the crosslinking solution of (i) comprises or is connected to a syringe needle, hollow tube or mesh, wherein the connection is optionally via an adapter connecting the one or more outlets of the one or two reservoir and their syringe needle, hollow tube or mesh.

[0058] The syringe needle, hollow tube or mesh shall be configured such that polymer solution (optional with the compound to be enclosed) comes into contact with the cross-linking solution in the form of single drops or droplets or extrusions.

[0059] The diameter, length and shape of the syringe needle can be adjusted in order to adjust the size, formation speed and / or frequency of the drops or droplets or extrusions. Also the pores of the mesh can be adjusted in order to adjust the size, formation speed and / or frequency of the drops or droplets or extrusions.

[0060] The needle is preferably made of metal and the hollow tube or mesh is preferably made of a polymer, preferably a plastic polymer. In accordance with a more preferred embodiment of the first aspect, the syringe needle or hollow tube is curved or comprises a loop or comprises a helix or has different radii or has a conical shape.

[0061] These shapes of the syringe needle can be used in order to adjust the size, formation or extrusion speed and / or frequency of the drops or droplets or filaments. The loop is illustrated by the appended examples.

[0062] In accordance with a further preferred embodiment of the first aspect, the device has dimensions that fit into a centrifuge.

[0063] As discussed above, one option in order to initiate or accelerate the formation of the drops or droplets or filaments (as compared to earth gravity) is the appliance of centrifugation forces. In this connection a number of different centrifuges are commercially available. For such centrifuges also a series of centrifuge tubes with standardized dimensions are available; see, for example Table 1 from the supplier TEquipment and Table 2 from Chen et al., Aerosol Science and Technology 38(9):926-937.

[0064] Table 1 Centrifuge Tubes Series from l OmL to 250mL

[0065] Cat. No Packaging unit Materials Dimensions Capacity Qty

[0066] C2410 50 tubes per sterile bag PP 16 x 104 mm 10ml 1000 / cs

[0067] C2630 25 sterile bag PP 29x80 mm 30ml 500 / cs

[0068] C2603-B 25 tubes in notched bag PP 29x115 mm 50ml 500 / cs

[0069] C1250 14 bags of 5 tubes PP 59.8x160 mm 250ml 70 / cs

[0070] C2625-B 8 bags of 25 tubes PP 27x77 mm 25ml 200 / pack

[0071] C2625-R 8 foam racks of 25 tubes PP 27x77 mm 25ml 200 / pack

[0072] C2605 25 sterile bag PP 29x115 mm 50ml 500 / cs

[0073] Table 2 Microcentrifuge Tubes Series from 1 .0 mL to 2.0 mL

[0074] The tubes of Table 2 fit into a microcentrifuge. Microcentrifuges are devices for small tubes from 0.1 ml to 2.0 ml (micro tubes), up to 96 well-plates. They generally have a compact design, small footprint and can reach a up to 30,000 g

[0075] In accordance with a related preferred embodiment of the first aspect, the device has a length of between about 2.5 cm and about 16 cm and a diameter of between about 1 cm and about 6 cm.

[0076] As can be taken from Tables 1 and 2 these preferred dimensions of the device of the invention correspond to the dimensions of commercially available centrifuge tubes, including microcentrifuge tubes.

[0077] The term “about” as used herein is with increasing preference ±20%, ±10%, and ±5%.

[0078] In accordance with another preferred embodiment of the first aspect, the first cylindric tube, the second cylindric tube and the lid are connected with each other by plug connection^), bolted connection^), magnetic connection(s) and / or are molded into each other.

[0079] While in the appended example plug connection(s) and bolted connection^) are illustrated in order to assemble the parts of the device into the complete device, also magnetic connection^) and / or are molded connection(s) may be employed.

[0080] Molded connection(s) are in generally irreversible while plug connection^), bolted connection^), and magnetic connection(s) are reversible. In accordance with a further preferred embodiment of the first aspect, wherein the one ortwo reservoirs of the second cylindric tube is / are funnel shaped.

[0081] While the shape of the reservoir(s) is not particularly limited, the shape is preferably a funnel wherein the one or more tips of the funnel are as such the one or more outlets of the reservoir(s) or are connected to the one or more outlets of the reservoir(s).

[0082] In accordance with another further preferred embodiment of the first aspect, the first cylindric tube, the second cylindric tube and / or the lid are made of material that is selected from plastics, metal, Teflon, printed polymers, ceramic materials, glass or any combination thereof, wherein the plastics is preferably polypropylene, polyethylene terephthalate or polystyrene.

[0083] The surfaces of the device shall generally be flat, non-adhesive and durable. All materials that meet these criteria can be used for producing the parts of the device. Non-limiting but preferred examples are plastics, metal, Teflon (i.e. tetrafluoroethylene), printed polymers, ceramic materials, glass. The preferred material for the first tube, the cylindric tube and / or the lid is plastics and the plastic is preferably polypropylene, polyethylene terephthalate or polystyrene.

[0084] In accordance with a further preferred embodiment of the first aspect, at least one of the one or more outlets has two or more openings that are connected to the one or two reservoirs which empty into one opening through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i).

[0085] The option that at least one of the one or more outlets has two or more openings that are connected to the reservoir(s) which empty into one opening is a further means for adjusting the size, formation speed and / or frequency of the drops or droplets or extrusions next to the above discussed mesh and syringe needles.

[0086] It is understood that the polymer solution comprises the compound to be enclosed when dropping or extruding into the cross-linking solution.

[0087] In accordance with a further preferred embodiment of the first aspect, the first tube contains a crosslinking solution, wherein the cross-linking solution preferably comprises divalent or trivalent cations, such as Mg2+, Ca2+, Ba2+, Cu 2+, Fe2+, Zn2+, Cr3+, and / or Fe3+.

[0088] The most common method for cross-linking polymers is through the use of divalent or trivalent cations to establish a large network of coordination bonds in conjunction with the lone pairs from the oxygen atoms of the alginic acid chains. For example, the actual cross-linking process varies in regard to the cation in question, whereby varied cations have different bonding preferences based on the different combination of residue blocks present within the alginate molecule. Calcium ions are preferably used to cross-link alginate. All cations are preferably used at a concentration of about 0.05M to about 1 ,5M.

[0089] In accordance with a further preferred embodiment of the first aspect, the reservoir of the second tube comprises a polymer solution, wherein the polymer solution preferably comprises alginate, or gelatin and alginate, or chitosan and gelatin, or silk and polyethylene glycol and gelatin, or alginate and fibronectin.

[0090] In the case of two (or three) polymers all these different polymers are used preferably in a ratio of about 1 :1 (:1) (w / v) and / or preferably at a concentration of between about 0.3% w / v and about 2.0% w / v for each polymer. This applies in particular to alginate and gelatin.

[0091] In accordance with a further preferred embodiment of the first aspect, the capsules, spheres, beads or filaments to be produced have a diameter of 1 nm to 1 cm, preferably 5 nm to 5 mm, and most preferably 50 nm to 1000 pm.

[0092] As discussed above, the size of the entire device and in particular the size and / or shape of the various discussed means for adjusting the drop or droplet or extrusion size as discussed herein above can be adjusted in order to achieve a size of the drops or droplets or extrusions that results in capsules, spheres, beads or filaments within the range of the above preferred embodiment.

[0093] In the case of the most preferred size in diameter of 50 nm to about 1000 pm the capsules, spheres, beads or filaments can also be designated nano- or micro- capsules, spheres or beads, or filaments.

[0094] The present invention relates in a second aspect to method for the enclosure of a compound into capsules, spheres beads, or filaments by the device of the first aspect, wherein the first tube contains a cross-linking solution and the compound to be enclosed, and the one or two reservoirs of the second tube comprises a polymer solution that can be cross-linked by the cross-linking solution, wherein the method comprises (a) dropping or extruding the polymer solution with the compound to be enclosed into the cross-linking solution by applying earth gravitational forces or centrifugation forces or negative pressure preferably from the bottom or positive pressure preferably from the top.

[0095] The present invention relates in a related third aspect to a method for the generation of capsules, spheres, beads or filaments by the device of the first aspect, wherein the first tube contains a crosslinking solution, and the reservoir of the second tube comprises a polymer solution that can be crosslinked by the cross-linking solution, wherein the method comprises (a) dropping or extruding the polymer solution into the cross-linking solution by applying earth gravitational forces or centrifugation forces or negative pressure preferably from the bottom or positive pressure preferably from the top.

[0096] The definitions and preferred of the first aspect of the invention apply mutatis mutandis to the second and third aspect as far as being amenable therewith. In accordance with the second aspect the one or two reservoirs contain a cross-linking solution and the compound to be enclosed, so that the method results in the formation capsules, spheres, beads or filaments wherein the compound is enclosed.

[0097] In accordance with the third aspect the one or two reservoirs contain a cross-linking solution and but no compound to be enclosed, so that the method results in the formation of capsules, spheres, beads or filaments (i.e. capsules, spheres, beads or filaments not enclosing a compound) devoid of the compound. Such capsules, spheres beads or filaments are, for example, of interest to study polymerization processes, as well as the properties of several materials in a spherical geometry, like gel strength or diffusion.

[0098] In accordance with a further preferred embodiment of the second and third aspect, wherein the method further comprises (b) isolating the capsules, spheres, beads, or filaments that are formed in step (a) from the first tube.

[0099] Means and method for the isolation of the capsules, spheres or beads are well-known and comprise, for example, centrifugation, filtration and sedimentation.

[0100] In accordance with another preferred embodiment of the second and third aspect, the method further comprises prior to step (a) (a’) adding the cross-linking solution and / or, if present, the compound to be enclosed to the first tube, and / or adding the polymer solution that can be cross-linked by the crosslinking solution to the one or two reservoirs of the second tube.

[0101] As discussed above, the cross-linking solution and / or, if present (e.g., in the case of the second aspect), the compound to be enclosed as well as the polymer solution can be placed into the device during the assembly of the device or into the assembled device though the discussed openings. On the other hand, the crosslinking solution cannot be placed into the first tube using this opening but the cross-linking solution can only be placed into the first tube when the device is assembled.

[0102] In accordance with a further preferred embodiment of the second and third aspect, the method further comprises prior to step (a) (a’) adding the cross-linking solution and / or, if present, the method further comprises prior to step (a) or (a’) sterilizing, preferably autoclaving the device.

[0103] As discussed above, means and methods for sterilization of devices or subparts thereof are well known in the art and comprise sterilization high-pressure steam (autoclave), dry heat (oven), chemical sterilants (-ozone, ethanol, methanol) or physical agents (radiation).

[0104] The present invention relates in a fourth aspect to a kit of parts comprising parts (i) to (iv) of one or more devices of the first aspect as single parts or partially preformed devices. The definitions and preferred of the first aspect of the invention apply mutatis mutandis to the fourth aspect as far as being amenable therewith.

[0105] In particular for the discussed case where the cross-linking solution and / or, if present, the compound to be enclosed as well as the polymer solution are placed into the device during the assembly the kit may be provided to the users instead of the assembled device.

[0106] The kit also allows the separate sterilization of the single parts or partially preformed devices.

[0107] In accordance with a preferred embodiment of the fourth aspect, the kit further comprises the crosslinking solution and / or the polymer solution that can be cross-linked by the cross-linking solution.

[0108] The cross-linking solution and / or the polymer solution are preferably provided in separate containers or vials. They may also be provided in unit dosage forms wherein the volumes of the cross-linking solution and / or the polymer solution are, for example, such that the volumes fit into one device.

[0109] As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0110] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1 . In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0111] The Figures show.

[0112] Figure 1 : General design of the Pocket Micro-Encapsulator. (I) Picture of one Pocket Micro- Encapsulator made by 3D printing. (II) Draw of the four main parts of the Pocket Micro-Encapsulator. (Ill) Internal dimensions of Pocket Micro-Encapsulator device. Figure 2: Details of pieces “b” and “e” of the Pocket Micro-Encapsulator device.

[0113] Figure 3: Details of pieces “a” and “d” of the Pocket Micro-Encapsulator device.

[0114] Figure 4: General design of the S-Pocket Micro-Encapsulator. The left image shows the new device. The pictures in the center and on the left side depict the dimensions of each section of this microencapsulator.

[0115] Figure 5: General design of the sections “a” and “b” of the S-Pocket Micro-Encapsulator. The image B1 shows an overview of the piece “b”, the image B1 depicts the inner sections of this device. The cartoons B2, B3 and B4 describe three options for the section “b”, differing in the angle to allow the runoff of the hydrogel from the section “b” to the section “c”.

[0116] Figure 6: General design of the 2X-Pocket Micro-Encapsulator. The left image shows a view of the 2X- Pocket Micro-Encapsulator from the outside. The picture on the right side, describe the inner sections of the pieces “a” and “b” of this new device.

[0117] Figure 7: Design of sections “b” and “g” of the 2X-Pocket Micro-Encapsulator. The image “I” shows a top view of the section “b” used in the 2X-Pocket Micro-Encapsulator. The image “II” depicts the inner sections of this piece “b”. The picture “III” shows the inner section of 2X-Pocket Micro-Encapsulator, which is described in the Figure “IV”.

[0118] Figure 8: Coil syringe designed to improve the performance of the Pocket Microencapsulator.

[0119] Certain features in the figures are summarized in Table 3.

[0120] TABLE 3 The examples illustrate the invention.

[0121] Example 1 : Pocket Micro-Encapsulator: concept design and technical details

[0122] Operational concept

[0123] As already mentioned, one alternative to drop the alginate solution into a CaCI2 solution, is mediated by the use of gravitational forces. The Pocket Micro-Encapsulator has been designed to produce microcapsules, either using the normal earth gravitational forces (or G-forces; 1.0 G), or enhanced gravitational forces, mediated by the use of a centrifuge (i.e. laboratory centrifuge).

[0124] General design

[0125] The Pocket Micro-Encapsulator has a length of 10.5 mm and a diameter of 28 mm. These dimensions enable it fit into commercial laboratory centrifuges.

[0126] The edges of the Pocket Micro-Encapsulator are flat, to enable their use it in laboratory centrifuges without touching their lid, as well as enabling to keep this device stand, allowing to work with this device on any flat surface (i.e. sterile hood).

[0127] The Pocket Micro-Encapsulator is constituted by 5 main sections (named as “a”, “b”, “c”, “d” and “e”). (Figure 1).

[0128] The piece “c” is a cylindric tube, opened on one of its sides. This part is used as reservoir for the solution containing crosslinking compounds (i.e. CaCI2). The section “b” is a cylindric tube, opened on both sides, having a funnel-like feature in its interior. This accomplishes the function to be the reservoir for the polymer-based solution (i.e. alginate), enabling its dropping or extruding onto the piece “e” by runoff.

[0129] Additionally, the piece “b” contains a hollow tube (in blue color, please see the Figure 1-111), which cross the piece “b”. This thin tube enables the exchange of the air between the sections “b” and “c”. This is needed to allow the equilibrium of pressures between these two compartments (Figure 2).

[0130] Concerning the piece “e”, this is a hollow conic cylinder, fitting into a hole located on the bottom of the piece “b”. It allows the runoff of the polymer-based solution between the sections “b” and “c”. Additionally, the piece “e” allows the generation of single polymer-based drops or droplets, which fall (by simple gravity or by centrifugation forces) into the section “c”, containing the crosslinking solution.

[0131] Finally, the bottom of the piece “e” has been designed to fit commercial syringes (which are named as “f” in Figure 1). This accomplishes the role to allow the users / customers to change the diameter of the needle, and therefore, the size of the capsules to be performed, according to their needs. With respect the piece “a”, this piece corresponds to the main lid of the pocket microencapsulator, and it has a cylinder form. This piece is closed on one of it sides. When this lid is opened, it allows to load the polymer-based solution from the top of the section “b”.

[0132] On the top of the piece “a”, there is a small circular conduct, acting as upper aperture for this piece. This small aperture accomplishes the function to keep a constant pressure within the whole Pocket Micro- Encapsulator, avoiding the generation of vacuum, or zones with negative pressure within the device. Additionally, it is a second alternative to load the polymer-based solution into the piece “b, using a commercial syringe.

[0133] The size of this aperture fits the size of the piece “d”. The piece “d” corresponds to a small circular lid, fitting into the conduct found on the top of the piece “a”. Additionally, the piece “d” is aimed to keep the microencapsulator sealed, avoiding their contamination (i.e. bacteria, yeast, virus). A detailed description of the pieces “a” and “d” can be found in Figure 3.

[0134] The sections “a”, “b”, and “c” can be bound and locked using a screw system. The coils can be seen on the bottom of the piece “a”, the top of the piece “c”, and on both edges of the piece “b”.

[0135] Dropping conditions

[0136] The formation of microcapsules occurs when single drops of the polymer-based solution come in contact the crosslinking solution. Depending on the chemical compounds used to generate the polymer-based microcapsules, the crosslinking process may take from millisecond to several minutes, and the generation of microcapsules with spheric shapes depends on four main parameters:

[0137] 1) The concentration of the polymer-based solution used to make the microcapsules.

[0138] 2) The distance between the dropping (i.e. needle) and surface of the crosslinking solution, or the volume of the crosslinking solution placed in the section “c” of the Pocket-Micro Encapsulator.

[0139] 3) The centrifugation speed or the g-forces utilized to drop the polymer-based solution on the crosslinking solution.

[0140] 4) The internal diameter of the dropping section (piece “e”) or the commercial needle (piece “f”) used to drop the polymer-based solution on the crosslinking solution.

[0141] 5) The concentration of the crosslinking solution

[0142] Concerning the sizes of the capsules to be generated by this device, these can be as smaller as 20 pm (in diameter), and as bigger as 1.0 cm (in diameter).

[0143] Table 4 shows examples of the parameters used with different blends alginate gelatin.

[0144] Table 4: Experimental working parameters use to perform polymer-based microcapsules made of a blend alginate gelatin (1 :1) as polymer-based solution and CaCI2 as crosslinking solution.

[0145] Pocket-microencapsulator: production

[0146] The Pocket Microencapsulator can be made of different materials. Among others, we have been made a version of Teflon and one 3D printed version, using the ink BioMedClear V1 (FormLabs industries).

[0147] The same prototype can be done, in metal and / or glass as well.

[0148] Example 2 - Small version of the Pocket Micro-Encapsulator: concept design and technical details.

[0149] Operational concept

[0150] In order to decrease the size of the pocket-microencapsulator, and to allow the use of even lesser amount of biological / chemical material, a smaller version of this device was designed and performed. It will be named as S-Pocket-Microencapsulator (i.e. S-device).

[0151] The S-device is constituted by 3 sections (named as “a”, “b” and “c”), as shown in Figure 4. The S- Pocket-Microencapsulator have a high of circa 5 cm, and a width of 1 .5 cm. it has been designed to be used in table centrifuges (i.e. those used with small tubes, having a maximum volume of 2.0 mL volume).

[0152] This device has a small lid (shown in the Figure 4 in red color), which is closed by a two-click seal system (Figure 5). The reservoir for the hydrogel is circular and, similar to the Pocket-Microencapsulator, it contains two conducts in its interior. One enables the exchange of air, and the second enables the runoff of the polymer from the section “b” to the “c”. This device uses no needle, since the part “b” incorporate its own needle in the design.

[0153] Regarding the section “b”, 3 different designs are exposed in Figure 5. These 3 options differ in the angle of the reservoir used to keep the hydrogel, enabling its runoff from the section “b” to the section “c” of this device.

[0154] With respect to the section “c”, this corresponds to the reservoir for the crosslinking solution, used to collect the polymer-based capsules, after their crosslinking. In order to fit into the table centrifuge. Example 3: Double needle version of the Pocket Micro-Encapsulator: concept design and technical detail

[0155] In order to enable the use of this device with two different solutions, and / or two different compositions, a combined system has been designed and performed. This device differs in the section “b” of the original device, as shown in Figure 6. This new device will be named as 2X- Pocket Micro-Encapsulator.

[0156] Differently to the traditional version of the Pocket Micro-Encapsulator, the 2X-Pocket Micro- Encapsulator consists on a combined system, having two small reservoirs in the section “b”, as shown in Figure 6-1 and Figure 6-11. These two reservoirs are used to contain two different hydrogels I solutions which are mixed in the section “g”, as shown in Figures 6-111 and 6-IV. Similar to the previous designs, the system has a tube, crossing the section “b” to keep the pressure equal, in the different sections of this device.

[0157] In this case, two different syringes converge to a single point, to generate a drop, which combines both solutions, and which crosslinks in contact with the solution placed in the section “c”, as shows in Figure 7. The dispositive “g” is removable, and can combine different syringe sizes, enabling the blend of solutions having different physical and / or rheological properties.

[0158] Example 4: Twisted syringe

[0159] Besides working on refining the performance of the Pocket microencapsulator, another option to improve the device was new kind of dropping system, consisting in the use of a coil syringe (Figure 8), also named as twisted syringe. Since this has the capability to slow down the movement of the fluids during the dropping, this was used to drop solutions having very low viscosity, or those having low surface tensions. This coiled syringe was tested with the polymer-based solutions, as shown in table 4.

Claims

CLAIMS1 . A device for the enclosure of a compound into capsules, spheres, beads or filaments, wherein the device comprises(A)(i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution,(ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed,(iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of(ii) can drop or extrude into the cross-linking solution of (i) if earth gravity or centrifugal forces are applied,(iv) a tube between (i) and (ii) or (i) and (v) that transgresses the second tube, wherein the tube enables the exchange of air between (i) and (v), and(v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube; or(B)(i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution,(ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed,(iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if negative pressure forces are applied,(iv) a connection being configured to be connected to a vacuum pump, which connection is an extra opening in the first tube allowing to generate negative pressure forces preferably from the bottom of the device via a connected vacuum pump, and(v) a lid positioned on top of the second tube, and preferably wherein the second tube or the lid further comprises an opening, optionally with asecond lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube;(C)(i) a first tube, which is opened on the top side and closed on the bottom side, wherein the bottom side preferably forms a flat footprint, and is configured to collect the capsules, spheres, beads or filaments to be produced and to contain a cross-linking solution,(ii) a second tube positioned on top of the first tube, which is opened on both, the top and bottom sides, and comprises one or two reservoirs configured to harbor a polymer solution that can be cross-linked by the cross-linking solution of (i) and to harbor the compound to be enclosed,(iii) one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) if positive pressure forces are applied, and(iv) a lid positioned on top of the second tube, and wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube and which opening is also a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump; or wherein the second tube or the lid further comprises a connection being configured to be connected to an air pump, to generate positive pressure forces preferably from the top of the device via a connected air pump, and preferably wherein the second tube or the lid further comprises an opening, optionally with a second lid on said opening, through which opening the polymer solution and / or the compound can be placed into the one or two reservoirs of the second tube.

2. The device of claim 1 , wherein the compound to be enclosed is a cell, a small molecule, a micro- or nano-metallic compound, a micro- or nano-ceramic compound, micro- or nano-electronic device, virus, intracellular organelle, a (poly)peptide (e.g. enzyme, growth factor, hormone) or a nucleic acid molecule.

3. The device of claim 1 or 2, wherein the one or more outlets of the one or two reservoirs of (ii) through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i) comprises or is connected to a syringe needle, hollow tube or mesh, wherein the connection is optionally via an adapter connecting the one or more outlets of the one or two reservoirs and their syringe needle, hollow tube or mesh.

4. The device of any one of claims 1 to 3, wherein the device has dimensions that fit into a centrifuge.

5. The device of any one of claims 1 to 4, wherein the device has a length of between 2.5 cm and 16 cm and a diameter of between 1 cm and 6 cm.

6. The device of any one of claims 1 to 5, wherein the first tube, the second tube and, the lid are connected with each other by plug connection(s), bolted connection^), magnetic connection^) and / or are molded into each other.

7. The device of any one of claims 1 to 6, wherein the one or two reservoirs of the second tube is / are funnel shaped.

8. The device of any one of claims 1 to 7, wherein the first tube, the second tube and / or the lid are made of material that is selected from plastics, metal, Teflon, printed polymers, ceramic materials, glass or any combination thereof, wherein the plastics is preferably polypropylene, polyethylene terephthalate or polystyrene.

9. The device of any one of claims 1 to 8, wherein at least one of the one or more outlets has two or more openings that are connected to the one or two reservoirs which empty into one opening through which the polymer solution of (ii) can drop or extrude into the cross-linking solution of (i).

10. The device of any one of claims 1 to 9, wherein the first tube contains a cross-linking solution, wherein the cross-linking solution preferably comprises divalent or trivalent cations, such as Mg2+, Ca2+, Ba2+, Cu 2+, Fe2+, Zn2+, Cr3+, and / or Fe3+.

11. The device of any one of claims 1 to 10, wherein the one or two reservoirs of the second tube comprises a polymer solution, wherein the polymer solution preferably comprises alginate, or gelatin and alginate, or chitosan and gelatin, or silk and polyethylene glycol and gelatin, or alginate and fibronectin.

12. A method for the enclosure of a compound into capsules, spheres, beads, or filaments by the device of any one of claims 1 to 11 , wherein the first tube contains a cross-linking solution and the compound to be enclosed, and the one or two reservoirs of the second tube comprises a polymer solution that can be cross-linked by the cross-linking solution, wherein the method comprises(a) dropping or extruding the polymer solution with the compound to be enclosed into the cross-linking solution by applying earth gravitational forces or centrifugation forces or negative pressure preferably from the bottom or positive pressure preferably from the top.

13. A method for the generation of capsules, spheres, beads or filaments by the device of any one of claims 1 to 11 , wherein the first tube contains a cross-linking solution, and the one or two reservoirs of the second tube comprises a polymer solution that can be cross-linked by the crosslinking solution, wherein the method comprises(a) dropping or extruding the polymer solution into the cross-linking solution by applying earth gravitational forces or centrifugation forces or negative pressure preferably from the bottom or positive pressure preferably from the top.

14. The method of claims12 or 13, further comprising prior to step (a) or (a’) sterilizing, preferably autoclaving the device.

15. A kit of parts comprising parts (i) to (iv) of one or more devices of any one of claims 1 to 11 as single parts or partially preformed devices.

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