Device for producing capsules, spheres, beads or filaments optionally with encapsulated compounds using earth's gravity, centrifugal force or negative or positive pressure

Abstract

This invention relates to an apparatus for encapsulating a compound into capsules, spheres, beads, or filaments, wherein the apparatus comprises: (A) (i) a first tube open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint and is configured to collect the capsules, spheres, beads, or filaments to be produced and contain a crosslinking solution; (ii) a second tube located at the top of the first tube, open at both the top and bottom, and including one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and the compound to be encapsulated; (iii) one or more outlets of the one or two reservoirs of (ii), through which the polymer solution of (ii) can drip or be extruded into the crosslinking solution of (i) if gravity or centrifugal force is applied; (iv) a tube located between (i) and (ii) or between (i) and (v) and passing through the second tube, wherein the tube enables air exchange between (i) and (v); and (v) A cap located at the top of a second tube, and preferably, wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which a polymer solution and / or compound can be placed into one or both reservoirs of the second tube; or (B) (i) a first tube, open on its top side and closed on its bottom side, wherein the bottom side preferably forms a flat footprint and is configured to collect capsules, spheres, beads or filaments to be produced and to contain a crosslinking solution; (ii) a second tube located at the top of the first tube, open on both its top and bottom sides, and including one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and to hold a compound to be encapsulated; (iii) one or more outlets of one or both reservoirs of (ii), through which the polymer solution of (ii) can drip or be squeezed into the crosslinking solution of (i) if a negative pressure is applied; (iv) a connection configured to be connected to a vacuum pump, the connection being an additional opening in the first tube, allowing negative pressure to be generated from the bottom of the device, preferably by a connected vacuum pump; and (v) A cap located at the top of the second tube, and preferably, wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube;Or (C) (i) a first tube, open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint, and configured to collect the capsules, spheres, beads, or filaments to be produced and contain the crosslinking solution; (ii) a second tube located on top of the first tube, open at both the top and bottom, and including one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and to hold the compound to be encapsulated; (iii) one or more outlets of one or both reservoirs of (ii), through which the polymer solution of (ii) can drip or be extruded into the crosslinking solution of (i) if positive pressure is applied; and (iv) A cap located at the top of the second tube, wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which a polymer solution and / or compound can be placed into one or both reservoirs of the second tube, and the opening is also configured to connect to a connection to an air pump to preferably generate positive pressure from the top of the device via the connected air pump; or wherein the second tube or cap further includes a connection configured to connect to an air pump to preferably generate positive pressure from the top of the device via the connected air pump, and preferably wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which a polymer solution and / or compound can be placed into one or both reservoirs of the second tube.

Description

[0001] This invention relates to an apparatus for encapsulating a compound into capsules, spheres, beads, or filaments, wherein the apparatus comprises: (A) (i) A first tube, open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint and is configured to collect the capsules, spheres, beads or filaments to be produced and contain the crosslinking solution. (ii) A second tube located at the top of the first tube, open on both its top and bottom sides, and comprising one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and to hold the compound to be encapsulated. (iii) One or more outlets of one or both reservoirs of (ii) allow the polymer solution of (ii) to drip or be extruded into the crosslinking solution of (i) if gravity or centrifugal force is applied. (iv) A pipe located between (i) and (ii) or between (i) and (v) and passing through the second pipe, wherein the pipe enables air exchange between (i) and (v), and (v) The cap located at the top of the second tube, and Preferably, the second tube or cap further includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube; or (B) (i) A first tube, open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint and is configured to collect the capsules, spheres, beads or filaments to be produced and contain the crosslinking solution. (ii) A second tube located at the top of the first tube, open on both its top and bottom sides, and comprising one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and to hold the compound to be encapsulated. (iii) One or more outlets of one or both reservoirs of (ii) may, if negative pressure is applied, allow the polymer solution of (ii) to drip or be extruded into the crosslinking solution of (i) through said outlet. (iv) A connection portion configured to connect to a vacuum pump, the connection portion being an additional opening in the first tube, allowing negative pressure to be generated from the bottom of the device, preferably via the connected vacuum pump, and (v) The cap located at the top of the second tube, and Preferably, the second tube or cap further includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube; or (C) (i) A first tube, open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint and is configured to collect the capsules, spheres, beads or filaments to be produced and contain the crosslinking solution. (ii) A second tube located at the top of the first tube, open on both its top and bottom sides, and comprising one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and to hold the compound to be encapsulated. (iii) One or more outlets of one or both reservoirs of (ii), if positive pressure is applied, through which the polymer solution of (ii) may drip or be extruded into the crosslinking solution of (i), and (iv) The cap located at the top of the second tube, and The second tube or cap further includes an opening, optionally having a second cap, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube, and the opening is also configured to connect to a connection to an air pump, so as to preferably generate positive pressure from the top of the device via the connected air pump; or The second tube or cap also includes a connection configured to be connected to an air pump to preferably generate positive pressure from the top of the device via the connected air pump, and preferably, the second tube or cap also includes an opening, optionally having a second cap, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube.

[0002] This specification references numerous documents, including patent applications and manufacturer's manuals. While the disclosures in these documents are not considered to be related to the patentability of this invention, their entire contents are hereby incorporated by reference. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0003] Methods known as immobilization involve producing polymer-based scaffolds that take on a specific shape upon contact with a solution containing a specific compound of a cross-linked polymer. Methods known as encapsulation involve producing spherical polymer-based scaffolds that take on this shape upon contact with a solution containing a specific compound of a cross-linked polymer. These beads are characterized by their shape and the presence of micropores, enabling the exchange of substances (i.e., gases, small particles, other compounds) between the capsule body and its surrounding environment. A traditional example of polymer-based capsules is made from alginate. This polymer can produce capsules that can be used to trap various molecules (i.e., drugs, proteins, DNA, RNA, etc.) or living organisms (bacteria, yeast, mammalian cells), and these capsules can be used in, for example, the food, pharmaceutical, and / or biomedical industries.

[0004] Capsules can be manufactured using a variety of artificial or industrial devices. A relatively simple strategy for producing polymer-based capsules is to sequentially drop small volumes of a solution containing a crosslinkable polymer (such as alginate) into a solution containing a crosslinking agent compound (such as CaCl2). This can be accomplished using syringes, pipettes, micropipettes, or more complex devices, such as commercial microencapsulator devices provided by Büchi and Nisco. These devices use two main systems to drop the polymer-based solution into the crosslinking bath: an air pump (Büchi device), a peristaltic pump (Nisco device), or gravity (Gravity VAR W10, Nisco).

[0005] While some apparatuses and methods for producing capsules, spheres, beads, or filaments already exist in the art, there remains a continued need for further apparatuses and methods, particularly those that are simple in form and readily usable for producing capsules, spheres, beads, or filaments, especially those that encapsulate compounds in capsules, spheres, beads, or filaments by applying gravity, centrifugal force, negative pressure, or positive pressure. This invention addresses this need.

[0006] Therefore, the present invention relates in a first aspect to an apparatus for encapsulating a compound into capsules, spheres, beads, or filaments, wherein the apparatus comprises: (A) (i) a first tube open at its top and closed at its bottom, wherein the bottom preferably forms a flat floor area, and configured to collect the capsules, spheres, beads, or filaments to be produced and contain a crosslinking solution; (ii) a second tube located at the top of the first tube, open at both its top and bottom sides, and including one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and the compound to be encapsulated; (iii) one or more outlets of the one or two reservoirs of (ii), through which the polymer solution of (ii) can drip or be extruded into the crosslinking solution of (i) if gravity or centrifugal force is applied; (iv) a tube located between (i) and (ii) or between (i) and (v) and passing through the second tube, wherein the tube enables air exchange between (i) and (v); and (v) A cap located at the top of a second tube, and preferably, wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which a polymer solution and / or compound may be placed into one or both reservoirs of the second tube; or (B) (i) a first tube, open on its top side and closed on its bottom side, wherein the bottom side preferably forms a flat footprint and is configured to collect capsules, spheres, beads or filaments to be produced and to contain a crosslinking solution; (ii) a second tube located at the top of the first tube, open on both its top and bottom sides, and including one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and to hold a compound to be encapsulated; (iii) one or more outlets of one or both reservoirs of (ii), through which the polymer solution of (ii) may drip or be squeezed into the crosslinking solution of (i) if a negative pressure is applied; (iv) a connection configured to be connected to a vacuum pump, the connection being an additional opening in the first tube, allowing negative pressure to be generated from the bottom of the device, preferably by a connected vacuum pump; and (v) A cap located at the top of the second tube, and preferably, wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube;Or (C) (i) a first tube, open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint, and configured to collect the capsules, spheres, beads, or filaments to be produced and contain the crosslinking solution; (ii) a second tube located on top of the first tube, open at both the top and bottom, and including one or two reservoirs configured to hold a polymer solution crosslinkable by the crosslinking solution of (i) and to hold the compound to be encapsulated; (iii) one or more outlets of one or both reservoirs of (ii), through which the polymer solution of (ii) can drip or be extruded into the crosslinking solution of (i) if positive pressure is applied; and (iv) A cap located at the top of the second tube, wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which a polymer solution and / or compound can be placed into one or both reservoirs of the second tube, and the opening is also configured to connect to a connection to an air pump to preferably generate positive pressure from the top of the device via the connected air pump; or wherein the second tube or cap further includes a connection configured to connect to an air pump to preferably generate positive pressure from the top of the device via the connected air pump, and preferably wherein the second tube or cap further includes an opening, optionally having a second cap on the opening, through which a polymer solution and / or compound can be placed into one or both reservoirs of the second tube.

[0007] An apparatus is an object produced for a specific purpose. According to the invention, the purpose is to encapsulate a compound into capsules, spheres, beads, or filaments, or, in the absence of such a compound, to produce capsules, spheres, beads, or filaments (without encapsulated compound). The apparatus according to (A) relies on gravity or centrifugal force when used for said purpose, the apparatus of (B) relies on negative pressure, and the apparatus of (C) relies on positive pressure.

[0008] The device according to (A) is preferred over the devices according to (B) and (C).

[0009] There are no particular limitations on the compounds that can be encapsulated, as long as they are suitable and can be absorbed by the capsules, spheres, beads, or filaments formed when the claimed device is used to encapsulate the compound into capsules, spheres, beads, or beads. The compounds can be organic or inorganic. Fats, nucleic acids, carbohydrates, proteins (such as enzymes), and hydrocarbon fuels are examples of organic molecules. Nonmetals, salts, metals, acids, and bases are examples of inorganic compounds. Further and preferred examples of compounds will be described below.

[0010] In contexts relating to capsules, spheres, or beads, "enclosed" or "enclosing" can be further defined as "encapsulated" or "encapsulating." Encapsulating a compound into capsules, spheres, beads, or filaments also immobilizes the compound within those capsules, spheres, beads, or filaments.

[0011] Beads are closed, compact, solid spheres made of one or more polymers, which can encapsulate compounds.

[0012] A capsule is a closed shell or envelope that can encapsulate a compound if it is present.

[0013] Filament refers to a closed, compact solid rope or closed-shell rope or encapsulation rope made of one or more polymers, which may contain a compound to be encapsulated.

[0014] A sphere is an object that exhibits a three-dimensional circular shape. If a compound is present, it can be encapsulated inside a spherical sphere.

[0015] It should be understood that the properties of capsules, spheres, and beads may overlap, so the same object may be classified as both capsules and beads at the same time.

[0016] Among capsules, spheres, beads, or filaments, capsules, spheres, and beads are preferred, with capsules being the most preferred. The accompanying examples illustrate the production of capsules containing encapsulation / encapsulation compounds.

[0017] Capsules, spheres, or beads are preferably micron or nanocapsules, micron or nanospheres, or micron or nanobeads. The term "micron or nano" indicates that the size (e.g., maximum diameter) of the capsule, sphere, or bead is in the range of microns (i.e., 1 µm to 1000 µm) or nanometers (i.e., 1 nm to 1000 nm). Preferred sizes of capsules, spheres, or beads will be provided below.

[0018] The filaments are preferably micrometer or nanometer filaments. The term “micrometer or nanometer” indicates that the diameter of the filament (e.g., the shortest distance between the surfaces of these structures) is in the range of micrometers (i.e., 1 µm to 1000 µm) or nanometers (i.e., 1 nm to 1000 nm).

[0019] While it is preferable that the bottom side of the first tube forms a flat footprint so that the device can stand independently, it is also preferable that the bottom side forms a cone or semicircle, especially a cone or semicircle suitable for the centrifuge tube pickup structure of a centrifuge.

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

[0021] The shape of the tubes (first tube and second tube) is not particularly limited; the tubes can be, for example, triangular, square, hexagonal, or circular. Circular tubes are preferred, and they can also be referred to as cylindrical tubes.

[0022] One or both reservoirs of the second tube are configured to hold a polymer solution that can be crosslinked by a crosslinking solution, and a compound to be encapsulated into capsules, spheres, beads, or filaments. In the case of a single reservoir, the polymer solution and the compound can be located in one solution within the same reservoir. In the case of two reservoirs, the polymer solution can be located in one reservoir, while the compound, in solution form, can be located in the second reservoir. In the latter case, one or more outlets of each of the two reservoirs converge, such that the polymer solution and the compound to be encapsulated can be in one solution before contacting the crosslinking solution in the first tube.

[0023] Therefore, according to the invention, in one alternative, the reservoir contains both the polymer solution and the compound to be encapsulated in the same reservoir (one) before the crosslinking solution is released into the first tube. Alternatively, the polymer solution and the compound to be encapsulated are held in separate reservoirs (two) before the crosslinking solution is released into the first tube. As previously mentioned, in this case, they come into contact via an outlet. The option of a single reservoir is preferred.

[0024] Therefore, a reservoir is a container capable of receiving and subsequently including a polymer solution. The reservoir can also receive and subsequently include the compound to be encapsulated. As can be seen from the embodiments, the device can also include more than one reservoir, particularly two, which allows for the parallel encapsulation of two or more compounds into different capsules, spheres, beads, or filaments—in the latter case, each of the two reservoirs includes both a polymer solution and the compound to be encapsulated.

[0025] While the reservoir can be configured to include both the polymer solution and the compound to be encapsulated, this does not require that both items must always be present in the reservoir or be present simultaneously. Instead, it is preferable to add the polymer solution to the reservoir first, followed by the compound.

[0026] One or two reservoirs and one or more outlets are configured such that the solution in one or two reservoirs can only reach the polymer solution in the first tube through one or more outlets.

[0027] The polymer solution can be crosslinked by a crosslinking solution. In this respect, it should be understood that when the crosslinking solution is in the first tube and the polymer solution is in the reservoir of the second tube, and gravity, centrifugal force, negative pressure, or positive pressure are applied to the device, droplets or extrudates of the polymer solution enter the crosslinking solution through one or more outlets of the reservoir, causing the polymer solution to be crosslinked, thereby forming capsules, spheres, beads, or filaments. When the reservoir of the second tube also includes the compound to be encapsulated, the formed capsules, spheres, beads, or filaments encapsulate these compounds.

[0028] The polymer solution (with or without the compound) has a viscosity that ensures it can drip or be extruded into the crosslinked solution under the influence of gravity or centrifugal force. There are no particular limitations on the viscosity of the polymer solution, provided this viscosity requirement is met; the polymer solution can be non-crosslinked or partially crosslinked.

[0029] The polymer solution comes into contact with the crosslinking solution in the form of drops, droplets, or filaments. It is worth noting in this respect that drops are larger than droplets. Furthermore, both terms refer to fluid spheres or filaments that have accumulated sufficient volume and weight to fall. Drops or droplets can be oil droplets or oil droplets, or water droplets or water droplets, to produce an emulsion (depending on the type of crosslinking solution and polymer solution used). Thus, both the crosslinking solution and the polymer solution can be water-based or oil-based, or can be an emulsion. Whether the polymer solution drips or is extruded into the crosslinking solution can be adjusted by the distance between one or more outlets of the reservoir and the crosslinking solution. If the distance is large, polymer solution droplets that can fall into the crosslinking solution are formed; if the distance is narrow, extruded polymer solution is formed. Droplets are preferred over extruded solutions.

[0030] These droplets, small droplets, or filaments are formed by applying Earth's gravity, centrifugal force, negative pressure, or positive pressure to the device.

[0031] To apply gravity, the device according to (A) simply needs to be placed on a surface or support to form droplets, small droplets, or filaments. To apply centrifugal force, the device according to (A) is typically placed in a centrifuge. Ultracentrifugation can also be used to produce a double emulsion (note that a double emulsion, also known as a water-in-oil emulsion (W / O / W emulsion), consists of water droplets or small water droplets dispersed in oil or oil / polymer spheres, which themselves are ultimately dispersed in an aqueous phase).

[0032] Furthermore, the device according to (A) includes a tube passing through the second tube. Air can flow through this tube between the first tube and the cap. The option of the tube being located between (i) and (ii) or between (i) and (v) refers to the possible length of the tube. The length is not limited as long as air can flow between the first tube and the cap. The tube is positioned above the liquid level of the solution in one or both reservoirs, such that no solution can enter the liquid. Therefore, the tube passing through the second tube can also be referred to as an "airflow tube". This airflow is important because otherwise, when gravity or centrifugal force is applied to the device according to (A), the pressure difference within the closed device may prevent the polymer solution from dripping or being extruded into the crosslinking solution.

[0033] To apply negative pressure (also known in the art as underpressure), the device according to (B) is typically connected to a vacuum pump. The negative pressure is applied to a first tube, and preferably from the bottom of the device, such that the polymer solution is absorbed / drawn / drawn into the first tube. For this purpose, the first tube, and preferably its bottom, includes a connection to the vacuum pump, which is or includes an additional opening in the first tube containing the crosslinked solution.

[0034] To apply positive pressure, the device according to (C) is typically connected to an air pump. The positive pressure is applied to the cap or second tube, and preferably from the top of the device, such that the polymer solution is forced from one or more reservoirs into the first tube. For this purpose, the cap or second tube includes a connection to the air pump. This connection may be an opening or an additional connection to the cap or second tube.

[0035] The cap sits atop the second tube and seals the device, preventing impurities from entering the reservoir. The cap forms the top seal of the device. For this reason, the device can also be sterile. For this purpose, the device or its components (before assembly) are sterilized, and within the sealed device, capsules, spheres, beads, or filaments can be formed under sterile conditions. Means and methods for sterilizing the device or its sub-components are well known in the art and include autoclaving (autoclave), dry heat (oven), chemical sterilizing agents (ozone, ethanol, or methanol), or physical agents (radiation).

[0036] Furthermore, in the device, the second tube (component (ii)) or cap (component (v)) preferably includes an opening, optionally with a second cap on the opening. The polymer solution and / or compound can be placed into the reservoir of the second tube through this opening. When the device is in use, the cap can be open or closed. The cap is preferably closed to prevent impurities that may enter through the opening, for example, when operating under aseptic conditions.

[0037] The preferred opening is technically advantageous for the apparatus according to (A) and (B) because it allows the polymer solution and / or compound to be placed in the reservoir of the fully assembled apparatus. This is particularly advantageous for operation under aseptic conditions.

[0038] The opening may optionally be closed by a second cover. The opening may also be closed by a membrane. Furthermore, the opening may include a filter or screen for trapping impurities or biological contaminants, and allow only the polymer solution and / or compound to enter the reservoir.

[0039] Therefore, the second cap preferably comprises a membrane with a pore size equal to or less than 0.45 µm, and preferably about 0.22 µm, to prevent contamination (viruses, bacteria, yeast) from entering, but still maintains the possibility of air exchange when using the device to encapsulate compounds into capsules, spheres, beads, or filaments, or to form capsules, spheres, beads, or filaments. The membrane in question can be punctured to place the polymer solution and / or compound into a reservoir. Furthermore, when using the device to encapsulate compounds or form capsules, spheres, beads, or filaments, the second cap can be opened if present, particularly in connection with the device according to (A).

[0040] In such Figure 1 In the device shown (i.e., the device according to (A)), the cover further includes an opening having a second cover, through which the polymer solution and / or compound can be placed into the reservoir of the second tube.

[0041] In such Figure 4 In the illustrated device (i.e., the device according to (A)), the second tube includes an opening (at its top) through which a polymer solution and / or compound can be placed into a reservoir within the second tube. This opening can be closed by a cap. Figure 4 The cap in the middle has no opening; it is only used to seal the second tube.

[0042] As can be seen from the above, the device of the present invention offers many technical advantages. The device has a simple structure, and can be easily assembled from a few components. The device is flexible because it can use any kind of polymer and corresponding crosslinking solutions to encapsulate a wide variety of possible compounds. The size of the device can be adjusted as needed, particularly to suit commercial centrifuges for use with centrifuge tubes of different sizes (e.g., from 0.1 ml to 250 mL). The device is also easy to use because the formation of capsules, spheres, beads, or filaments that can encapsulate compounds of interest can be initiated simply by applying gravity, centrifugal force, negative pressure, or positive pressure. Furthermore, as mentioned earlier, the device can operate under aseptic conditions if desired. Therefore, the device can be, and preferably is, a sterile device.

[0043] According to the preferred embodiment of the first aspect, the compound to be encapsulated is a cell, a small molecule, a micron or nano-metal compound, a micron or nano-ceramic compound, a micron or nano-electronic device, a virus, an intracellular organelle, a (poly)peptide (e.g., an enzyme, a growth factor, a hormone), or a nucleic acid molecule.

[0044] The cells can be prokaryotic or eukaryotic, with eukaryotic cells preferred. Eukaryotic cells can be, for example, plant cells, yeast cells, or animal cells, with animal cells preferred. Animal cells are preferably human cells.

[0045] The term "small molecule" as used herein is preferably an organic molecule. Organic molecules involve or belong to a class of compounds with a carbon basis, where carbon atoms are linked together by carbon-carbon bonds. The original definition of the term "organic" relates to the source of the compound; organic compounds are carbon-containing compounds obtained from plant, animal, or microbial sources, while inorganic compounds are obtained from mineral sources. Organic compounds can be natural or synthetic. In a preferred embodiment, the organic molecule is an aromatic molecule, more preferably a heteroaromatic molecule. In organic chemistry, the term aromaticity is used to describe cyclic (cyclic), planar (flat) molecules with resonant rings that exhibit greater stability than other geometric or interconnected arrangements with the same set of atoms. Aromatic molecules are very stable and do not readily decompose or react with other substances. In heteroaromatic molecules, at least one atom in the aromatic ring is an atom other than carbon, such as N, S, or O. For all the above-described organic molecules, the molecular weight is preferably in the range of 200 Da to 1500 Da, more preferably in the range of 300 Da to 1000 Da.

[0046] Alternatively, the "small molecule" according to the invention is an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds that do not contain carbon atoms (except carbon dioxide, carbon monoxide, and carbonates). Preferably, the molecular weight of the small molecule is less than about 2000 Da, or less than about 1000 Da, for example less than about 500 Da, and even more preferably less than about Da. The size of the small molecule can be determined by methods well known in the art, such as mass spectrometry.

[0047] Small molecules can be designed, for example, based on the crystal structure of the target molecule, where the sites presumed to be responsible for biological activity can be identified and validated in vivo assays (such as high-throughput screening (HTS) assays).

[0048] The term “micrometer or nanometer” also refers to the size (e.g., maximum diameter) of a compound or device in the range of micrometers (i.e., 1 µm to 1000 µm) or nanometers (i.e., 1 nm to 1000 nm).

[0049] A metallic compound is any compound made of or comprising a metal or alloy. An alloy is a substance that has metallic properties and is composed of two or more elements, at least one of which is a metal. Gold, silver, platinum, aluminum, titanium, copper, tin, iron, and their alloys are preferred.

[0050] Ceramic compounds are any compounds made of or including ceramics. They are any of a variety of hard, brittle, heat-resistant, and corrosion-resistant materials produced by molding inorganic non-metallic materials (such as clay) and then firing them at high temperatures. Common examples are pottery, porcelain, and brick.

[0051] Electronic devices, such as transistors, capacitors, and resistors, are typically housed within active chips and require some degree of environmental protection, as well as electrical and mechanical connections to surrounding components. Electronic devices can include transistors, capacitors, inductors, resistors, diodes, insulators, and / or conductors. Viruses typically exist in the form of viral particles. Viral particles, also known as virions, are composed of a viral genome, which can be single-stranded or double-stranded DNA or RNA, surrounded by a protein coat, or capsid.

[0052] Intracellular organelles are organized structures with unique morphology and function that occur within the cell. Intracellular organelles include the nucleus, mitochondria, plastids, vacuoles, vesicles, lysosomes, ribosomes, and chloroplasts.

[0053] The term "protein," used interchangeably with "polypeptide" herein, describes a linear molecular chain of amino acids, including single-chain proteins or fragments thereof containing at least 50 amino acids. The term "peptide," as used herein, describes a group of molecules consisting of up to 49 amino acids, while the term "polypeptide" (also referred to as "protein") describes a group of molecules consisting of at least 50 amino acids. The term "peptide," as used herein, describes a group of molecules consisting of preferably at least 15, at least 20, at least 25, and at least 40 amino acids. Peptides and groups of polypeptides are collectively referred to using the term "(poly)peptide." (Poly)peptides can further form oligomers consisting of at least two identical or different molecules. The corresponding higher-order structures of such polymers are accordingly referred to as homodimers or heterodimers, homodimers or heterotrimers, etc. Furthermore, peptide analogs of such proteins / (poly)peptides in which amino acids and / or peptide bonds have been replaced by functional analogs are also covered in this invention. Such functional analogs include all known amino acids except for 20 genetically encoded amino acids, such as selenocysteine. The terms “(poly)peptide” and “protein” also refer to naturally modified (poly)peptides and proteins, wherein the modifications are affected by, for example, glycosylation, acetylation, phosphorylation and similar modifications well known in the art.

[0054] (Poly)peptides can be, for example, enzymes, antibodies, growth factors, or hormones.

[0055] The term "nucleic acid molecule" according to the present invention includes DNA (such as cDNA or double-stranded or single-stranded genomic DNA) and RNA. In this respect, "DNA" (deoxyribonucleic acid) refers to any chain or sequence of chemical building blocks adenine (A), guanine (G), cytosine (C), and thymine (T) (called nucleotide bases) linked together by a deoxyribose sugar backbone. DNA may have one nucleotide base chain or two complementary chains that can form a double helix structure. "RNA" (ribonucleic acid) refers to any chain or sequence of chemical building blocks adenine (A), guanine (G), cytosine (C), and uracil (U) (called nucleotide bases) linked together by a ribose sugar backbone. RNA typically has one nucleotide base chain, such as mRNA. Single-stranded and double-stranded hybrid molecules are also included, namely DNA-DNA, DNA-RNA, and RNA-RNA. Nucleic acid molecules can also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “capping,” substitution of one or more naturally occurring nucleotides with analogs, and internucleotide modifications, such as modifications with uncharged linkages (e.g., methylphosphonates, triphosphates, phosphoramides, carbamates, etc.) and charged linkages (e.g., thiophosphates, dithiophosphates, etc.). Nucleic acid molecules (hereinafter also referred to as polynucleotides) may include one or more additional covalently linked groups, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents (e.g., metals, radioactive metals, iron, metal oxides, etc.), and alkylating agents. Polynucleotides can be derivatized by forming methyl or ethyl phosphate triesters or alkyl phosphoramide linkages. Also included are nucleic acid mimics known in the art, such as synthetic or semi-synthetic derivatives of DNA or RNA, and mixed polymers. Nucleic acid mimics or derivatives according to the present invention include phosphate-thiocyanate nucleic acids, phosphoramide nucleic acids, 2'-O-methoxyethyl ribonucleic acid, morpholinonucleotides, hexitol nucleic acids (HNA), peptide nucleic acids (PNA), and locked nucleic acids (LNA) (see Braasch and Corey, Chem Biol 2001, 8: 1). An LNA is a modified RNA derivative in which the ribose ring is bound by a methylene linker between the 2'-oxygen and 4'-carbon. Nucleic acids containing modified bases, such as thiouracil, thioguanine, and fluorouracil, are also included. Nucleic acid molecules typically carry genetic information, including information about cellular machinery used to produce proteins and / or peptides. Nucleic acid molecules may also include promoters, enhancers, response elements, signal sequences, polyadenylated sequences, introns, 5'- and 3'-uncoding regions, etc.

[0056] Nucleic acid molecules can also be in the form of vectors. According to the invention, the term "vector" preferably refers to plasmids, granules, viruses, bacteriophages, or another vector, such as those conventionally used in genetic engineering to carry nucleic acid molecules. Nucleic acid molecules can, for example, be inserted into several commercially available vectors. Non-restrictive examples include prokaryotic plasmid vectors such as the pUC series, pBluescript (Stratagene), pET series expression vectors (Novagen), or pCRTOPO (Invitrogen), as well as vectors compatible with mammalian cell expression, such as pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all from Invitrogen).

[0057] The nucleic acid molecule to be inserted into the vector can be synthesized, for example, by standard methods, or isolated from natural sources. The linking of the coding sequence to transcriptional regulatory elements and / or other amino acid coding sequences can also be performed using established methods. Transcriptional regulatory elements (part of the expression cassette) that ensure expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements include regulatory sequences that ensure transcription initiation (e.g., translation start codons, promoters such as naturally associated or heterologous promoters and / or insulators; see above), internal ribosome entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476), and optionally, poly-A signals that ensure transcription termination and transcript stability. Additional regulatory elements may include transcription and translation enhancers. Preferably, the polynucleotide encoding the polypeptide / protein or fusion protein is operatively linked to such expression control sequences that allow expression in prokaryotes or eukaryotic cells. The vector may also include nucleic acid sequences encoding secretion signals as further regulatory elements. Such sequences are well known to those skilled in the art. Furthermore, depending on the expression system used, a lead sequence capable of guiding the expressed peptide into a cellular compartment can be added to the coding sequence of the polynucleotide. Such lead sequences are well known in the art.

[0058] In addition, preferred vectors include selection markers. Examples of selection markers include genes encoding resistance to neomycin, ampicillin, hygromycin, and kanamycin. Specially designed vectors allow DNA to shuttle between different hosts, such as bacterial-fungal cells or bacterial-animal cells (e.g., the Gateway system provided by Invitrogen). Expression vectors can direct the replication and expression of polynucleotides as well as encoded peptides or fusion proteins.

[0059] According to another preferred embodiment of the first aspect, one or more outlets of one or two reservoirs of (ii) (through which the polymer solution of (ii) may drip or be extruded into the crosslinked solution of (i)) include or are connected to a syringe needle, hollow tube or mesh, wherein the connection is optionally made by an adapter connecting one or more outlets of one or two reservoirs and their syringe needle, hollow tube or mesh.

[0060] The syringe needle, hollow tube, or mesh should be configured such that the polymer solution (optionally containing the compound to be encapsulated) contacts the crosslinking solution in the form of single drops, small drops, or extrusions.

[0061] The diameter, length, and shape of the syringe needle can be adjusted to regulate the size, formation speed, and / or frequency of the droplet or droplet or extrudate. The aperture of the mesh can also be adjusted to regulate the size, formation speed, and / or frequency of the droplet or droplet or extrudate.

[0062] The needle is preferably made of metal, and the hollow tube or mesh is preferably made of polymer (preferably a plastic polymer).

[0063] According to a more preferred embodiment of the first aspect, the syringe needle or hollow tube is curved, or includes a ring, or includes a helix, or has a different radius, or has a conical shape.

[0064] These syringe needle shapes can be used to adjust the size of the droplet, droplet, or filament, the forming or extrusion speed, and / or frequency. The rings are illustrated in the accompanying examples.

[0065] According to a further preferred embodiment of the first aspect, the device has a size suitable for a centrifuge.

[0066] As mentioned above, in order to initiate or accelerate the formation of droplets, small droplets, or filaments (compared to Earth's gravity), one option is to apply centrifugal force. In this regard, many different centrifuges are commercially available. For such centrifuges, a range of centrifuge tubes with standardized sizes are also provided; see, for example, Table 1 from the supplier TEquipment and Table 2 from Chen et al. (Aerosol Science and Technology 38(9):926-937).

[0067] Table 1. Centrifuge tube series from 10 mL to 250 mL

[0068] Table 2 Series of 1.0 mL to 2.0 mL microcentrifuge tubes

[0069] The tubes in Table 2 are suitable for microcentrifuges. Microcentrifuges are devices for 0.1 ml to 2.0 ml tubes (microtubes) and up to 96-well plates. They typically feature a compact design, small footprint, and can achieve speeds up to 30,000 g. According to the preferred embodiment of the first aspect, the length of the device is between about 2.5 cm and about 16 cm, and the diameter is between about 1 cm and about 6 cm.

[0070] As can be seen from Tables 1 and 2, these preferred dimensions of the device of the present invention correspond to the dimensions of commercially available centrifuge tubes (including microcentrifuge tubes).

[0071] The term “about” used in this article refers to the increased preference of ±20%, ±10%, and ±5%.

[0072] According to another preferred embodiment of the first aspect, the first cylindrical tube, the second cylindrical tube, and the cap are connected to each other by insertion, bolting, magnetic connection, and / or molded together.

[0073] Although plug-in and bolted connections are illustrated in the accompanying embodiments for assembling the components of the device into a complete device, magnetic and / or molded connections may also be used.

[0074] Molded connections are typically irreversible, while plug-in, bolted, and magnetic connections are reversible.

[0075] According to a further preferred embodiment of the first aspect, one or both reservoirs of the second cylindrical tube are funnel-shaped.

[0076] While there are no particular restrictions on the shape of the reservoir, the shape is preferably a funnel, wherein one or more tips of the funnel are themselves one or more outlets of one or more reservoirs, or are connected to one or more outlets of one or more reservoirs.

[0077] According to another further preferred embodiment of the first aspect, the first cylindrical tube, the second cylindrical tube, and / or the cap are made of a material selected from plastics, metals, Teflon, printed polymers, ceramic materials, glass, or any combination thereof, wherein the plastic is preferably polypropylene, polyethylene terephthalate, or polystyrene.

[0078] The surfaces of the device should generally be smooth, non-stick, and durable. All materials meeting these criteria can be used to manufacture components of the device. Non-limiting but preferred examples include plastics, metals, Teflon (i.e., tetrafluoroethylene), printed polymers, ceramic materials, and glass. The preferred material for the first tube, cylindrical tube, and / or cap is plastic, preferably polypropylene, polyethylene terephthalate, or polystyrene.

[0079] According to a further preferred embodiment of the first aspect, at least one of the one or more outlets has two or more openings connected to one or two reservoirs into which the polymer solution of (ii) can drip or be extruded into the crosslinking solution of (i).

[0080] The option of having at least one outlet with two or more openings (which connect to the reservoir and drain into one opening) is a further means of adjusting the size, formation speed, and / or frequency of the droplets, droplets, or extrudates, in addition to the grids and syringe needles discussed above.

[0081] It should be understood that when dripped or extruded into the crosslinking solution, the polymer solution includes the compound to be encapsulated.

[0082] According to a further preferred embodiment of the first aspect, the first tube includes a crosslinking solution, wherein the crosslinking solution preferably includes divalent or trivalent cations, such as Mg2+, Ca2+, Ba2+, Cu2+, Fe2+, Zn2+, Cr3+ and / or Fe3+.

[0083] The most common method for cross-linking polymers is to use divalent or trivalent cations to bind lone pairs of electrons from oxygen atoms in the alginate chain, thus establishing a large coordination network. For example, the actual cross-linking process varies depending on the cations involved, and different cations have different binding preferences based on different combinations of residue blocks present within the alginate molecule.

[0084] Calcium ions are preferably used for cross-linking alginate. All cations are preferably used at a concentration of about 0.05 M to about 1.5 M.

[0085] According to 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.

[0086] In the case of two (or three) polymers, all these different polymers are preferably used in a ratio of about 1:1 (w / v), and / or the concentration of each polymer is preferably between about 0.3% w / v and about 2.0% w / v. This is particularly suitable for alginate and gelatin.

[0087] According to a further preferred embodiment of the first aspect, the diameter of the capsule, sphere, bead, or filament to be produced is 1 nm to 1 cm, preferably 5 nm to 5 mm, and most preferably 50 nm to 1000 µm.

[0088] As described above, the dimensions of the entire device, particularly the dimensions and / or shape of the various means discussed above for adjusting the size of droplets, droplets, or extrudates, can be adjusted to achieve the size of droplets, droplets, or extrudates, thereby producing capsules, spheres, beads, or filaments within the scope of the preferred embodiments described above.

[0089] In the case of the most preferred size of 50 nm to about 1000 µm in diameter, capsules, spheres, beads or filaments may also be referred to as nano or micro capsules, spheres or beads or filaments.

[0090] The invention relates in a second aspect to a method for encapsulating a compound into a capsule, sphere, bead, or filament using the apparatus of the first aspect, wherein a first tube comprises a crosslinking solution and a compound to be encapsulated, and one or both reservoirs of a second tube comprise a polymer solution crosslinkable by the crosslinking solution, wherein the method comprises: (a) dripping or extruding a polymer solution containing the compound to be encapsulated into the crosslinking solution by applying gravity or centrifugal force or preferably a negative pressure from the bottom or preferably a positive pressure from the top.

[0091] The present invention relates in a related third aspect to a method for producing capsules, spheres, beads or filaments by means of the apparatus of the first aspect, wherein a first tube includes a crosslinking solution and a reservoir of a second tube includes a polymer solution that can be crosslinked by the crosslinking solution, wherein the method includes: (a) dripping or extruding the polymer solution into the crosslinking solution by applying gravity or centrifugal force or preferably applying negative pressure from the bottom or preferably applying positive pressure from the top.

[0092] The definitions and preferred embodiments of the first aspect of the present invention are applicable to the second and third aspects to the extent applicable.

[0093] According to the second aspect, one or two reservoirs include a crosslinking solution and a compound to be encapsulated, such that the method results in the formation of capsules, spheres, beads, or filaments in which the compound is encapsulated.

[0094] According to the third aspect, one or two reservoirs include a crosslinking solution but not the compound to be encapsulated, such that the method results in the formation of capsules, spheres, beads, or filaments that do not encapsulate the compound (i.e., capsules, spheres, beads, or filaments that do not encapsulate the compound). Such capsules, spheres, beads, or filaments are significant, for example, for studying the polymerization process and the properties of several materials (such as gel strength or diffusion) in spherical geometries.

[0095] According to a further preferred embodiment of the second and third aspects, the method further includes (b) separating the capsules, spheres, beads or filaments formed in step (a) from the first tube.

[0096] The means and methods for separating capsules, spheres or beads are well known, and include, for example, centrifugation, filtration and precipitation.

[0097] According to another preferred embodiment of the second and third aspects, the method further includes (a') adding a crosslinking solution and / or (if present) the compound to be encapsulated to the first tube, and / or adding a polymer solution that can be crosslinked by the crosslinking solution to one or both reservoirs of the second tube before step (a).

[0098] As described above, the crosslinking solution and / or (if present, for example, in the case of the second aspect) the compound to be encapsulated and the polymer solution can be placed in the device during device assembly, or placed in the assembled device through the opening in question. On the other hand, the crosslinking solution cannot be placed in the first tube using this opening, but can only be placed in the first tube during device assembly.

[0099] According to a further preferred embodiment of the second and third aspects, the method further includes (a') adding a crosslinking solution and / or (if present) before step (a), and the method further includes sterilizing the device, preferably autoclaving, before step (a) or (a').

[0100] As described above, means and methods for sterilizing devices or their sub-components are well known in the art and include autoclaving (autoclave), dry heat (oven), chemical sterilizing agents (ozone, ethanol, methanol) or physical agents (radiation).

[0101] The invention relates in a fourth aspect to a parts kit comprising components (i) to (iv) of one or more devices as a single part or a partial preforming device in the first aspect.

[0102] The definitions and preferred embodiments of the first aspect of this invention are applicable to the fourth aspect within the scope of their applicability.

[0103] In particular, for the case discussed where the crosslinking solution and / or (if present) the compound to be encapsulated and the polymer solution are placed in the device during assembly, a kit can be provided to the user instead of a pre-assembled device.

[0104] The kit also allows for the individual sterilization of single parts or pre-molded components.

[0105] According to a preferred embodiment of the fourth aspect, the kit further includes a crosslinking solution and / or a polymer solution that can be crosslinked by the crosslinking solution.

[0106] The crosslinking solution and / or polymer solution are preferably provided in separate containers or vials. They may also be provided in unit doses, wherein the volume of the crosslinking solution and / or polymer solution is such that it fits into a device.

[0107] With regard to the embodiments characterized in this specification, and particularly in the claims, it is intended to combine each embodiment mentioned in a dependent claim with each embodiment of each claim (independent or dependent) upon which the dependent claim depends. For example, in cases where independent claim 1 lists three alternatives A, B, and C, dependent claim 2 lists three alternatives D, E, and F, and claim 3 depends on claims 1 and 2 and lists three alternatives G, H, and I, it should be understood that the specification expressly 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 otherwise specified.

[0108] Similarly, where no alternatives are listed in the independent and / or dependent claims, it should be understood that if a dependent claim references multiple prior claims, any combination of the subject matter covered by them is considered to be explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 referencing claim 1, and dependent claim 3 referencing both claims 2 and 1, the combination of subject matter of claims 3 and 1, and the combination of subject matter of claims 3, 2, and 1 are thus clearly and explicitly disclosed. If there is a further dependent claim 4 referencing any one of claims 1 to 3, then the combination of subject matter of claims 4 and 1, claims 4, 2 and 1, claims 4, 3 and 1, and claims 4, 3, 2, and 1 are thus clearly and explicitly disclosed. Attached Figure Description

[0109] Figure 1 : Overall design of a miniature encapsulator. (I) Photograph of a miniature encapsulator manufactured by 3D printing. (II) Drawings of the four main parts of the miniature encapsulator. (III) Internal dimensions of the miniature encapsulator device.

[0110] Figure 2 Details of components “b” and “e” of the miniature encapsulator device.

[0111] Figure 3 Details of components “a” and “d” of the miniature encapsulator device.

[0112] Figure 4 : Overall design of the S-Pocket Miniature Encapsulator. The left image shows the new device. The middle and left photos depict the dimensions of each part of the microencapsulator.

[0113] Figure 5 The overall design of parts "a" and "b" of the S-Pocket Miniature Encapsulator. Image B1 shows an overview of part "b" and depicts the internal parts of the device. Cartoons B2, B3, and B4 describe three options for part "b", differing in the angle that allows the hydrogel to flow from part "b" to part "c".

[0114] Figure 6 : Overall design of the 2X-pocket miniature encapsulator. The left image shows the 2X-pocket miniature encapsulator as seen from the outside. The right image depicts the internal parts of components "a" and "b" of this new device.

[0115] Figure 7 The design of parts “b” and “g” of the 2X-pocket miniature encapsulator. Image “I” shows a top view of part “b” used in the 2X-pocket miniature encapsulator. Image “II” depicts the internal parts of part “b”. Photograph “III” shows the internal parts of the 2X-pocket miniature encapsulator, as described in Figure “IV”.

[0116] Figure 8 : A spiral injector designed to improve the performance of pocket-sized miniature encapsulators.

[0117] Some of the features in the attached figures are summarized in Table 3.

[0118] Table 3 Detailed Implementation

[0119] Example 1: Miniature Encapsulator: Conceptual Design and Technical Details Operational concept As previously mentioned, an alternative to dropping an alginate solution into a CaCl2 solution is gravity-mediated. The pocket-sized microencapsulator is designed to produce microcapsules using normal Earth gravity (or G-force; 1.0 G) or enhanced gravity mediated by a centrifuge (i.e., a laboratory centrifuge).

[0120] Chief Designer The miniature encapsulator is 10.5 mm long and 28 mm in diameter. These dimensions allow it to fit into commercial laboratory centrifuges.

[0121] The edges of the pocket-sized miniature encapsulator are flat so as not to touch the lid when used in a laboratory centrifuge and to keep the device upright, thus allowing the device to be operated on any flat surface (i.e., a sterile hood).

[0122] The miniature encapsulator consists of five main parts (named "a", "b", "c", "d", and "e"). Figure 1 ).

[0123] Component "c" is a cylindrical tube with an opening on one side. This part serves as a reservoir for a solution containing a cross-linked compound (i.e., CaCl2). Component "b" is a cylindrical tube open at both ends with a funnel-shaped internal feature. This enables it to function as a reservoir for a polymer-based solution (i.e., alginate), allowing it to drip or be extruded onto component "e" via runoff.

[0124] In addition, component "b" includes a hollow tube (blue, see [link]) passing through component "b". Figure 1 -III). This thin tube allows for partial air exchange between "b" and "c". This is necessary to allow pressure balance between the two compartments. Figure 2 ).

[0125] Regarding component "e", it is a hollow conical cylinder installed in a hole located at the bottom of component "b". It allows the polymer-based solution to flow out between parts "b" and "c". In addition, component "e" allows the generation of individual polymer-based droplets or droplets, which fall into part "c" containing the crosslinked solution (by simple gravity or centrifugal force).

[0126] Finally, the bottom of component "e" is designed to fit commercial syringes (in... Figure 1 (Named "f" in Chinese). This allows users / customers to change the needle diameter as needed, thereby changing the size of the capsule to be executed.

[0127] Regarding component "a", this component corresponds to the main cap of the miniature encapsulator and has a cylindrical shape. One side of this component is closed. When the cap is opened, it allows the polymer-based solution to be loaded from the top of component "b".

[0128] At the top of component "a", there is a small circular conduit serving as the upper orifice of the component. This small orifice enables the maintenance of a constant pressure throughout the entire miniature encapsulator, preventing the creation of vacuum or negative pressure areas within the device. Furthermore, it is a second alternative to using a commercial syringe to load the polymer-based solution into component "b".

[0129] The orifice is sized to fit the dimensions of component "d". Component "d" corresponds to the small circular cap and is installed in the conduit at the top of component "a". Furthermore, component "d" is designed to maintain the microencapsulator's seal, preventing contamination (i.e., bacteria, yeast, viruses). Detailed descriptions of components "a" and "d" can be found [link to detailed description]. Figure 3 .

[0130] Parts “a”, “b”, and “c” can be joined and locked using a screw system. The coils are visible at the bottom of part “a”, the top of part “c”, and both edges of part “b”.

[0131] dripping conditions When a single drop of a polymer-based solution comes into contact with a crosslinking solution, microcapsules form. Depending on the chemical compounds used to generate the polymer-based microcapsules, the crosslinking process can take anywhere from milliseconds to minutes, and the generation of spherical microcapsules depends on five main parameters: 1) Concentration of the polymer-based solution used to manufacture microcapsules.

[0132] 2) The distance between the droplet (i.e., the needle tip) and the surface of the crosslinking solution, or the volume of the crosslinking solution placed in the pocket miniature encapsulator part "c".

[0133] 3) Centrifugal speed or g force used to drop polymer-based solutions onto crosslinked solutions.

[0134] 4) The inner diameter of the drip portion (part "e") or commercial needle (part "f") used to drop the polymer-based solution onto the crosslinked solution.

[0135] 5) Concentration of the crosslinking solution Regarding the size of the capsules produced by the device, they can range from as small as 20 µm (diameter) to as large as 1.0 cm (diameter).

[0136] Table 4 shows examples of parameters used with different alginate gelatin mixtures.

[0137] Table 4: Experimental working parameters for performing experiments on polymer-based microcapsules made from an alginate-gelatin mixture (1:1) as the polymer-based solution and CaCl2 as the crosslinking solution.

[0138] Miniature Encapsulator: Production The miniature encapsulators can be made from different materials. We have already manufactured a Teflon version and a 3D-printed version using BioMedClear V1 ink (FormLabs industries).

[0139] The same prototype can also be made of metal and / or glass.

[0140] Example 2 - A smaller version of the pocket miniature encapsulator: conceptual design and technical details.

[0141] Operational concept To reduce the size of the miniature encapsulator and allow for the use of less biological / chemical materials, a smaller version of the device was designed and implemented. It will be named the S-miniature encapsulator (i.e., the S-device).

[0142] The S-device consists of three parts (named "a", "b", and "c"), as follows: Figure 4 As shown. The S-pocket micro-encapsulator is approximately 5 cm high and 1.5 cm wide. It is designed for use in benchtop centrifuges (i.e., centrifuges for tubes with a maximum volume of 2.0 mL).

[0143] The device has a small cover (in) Figure 4 (Displayed in red), closed by a double-click sealing system ( Figure 5 The hydrogel's reservoir is circular and, similar to a miniature encapsulator, includes two conduits inside. One allows for air exchange, and the second allows the polymer to flow from part "b" to "c". The device does not use needles because part "b" incorporates its own needle in its design.

[0144] Regarding part "b", Figure 5 The image shows three different designs. The difference between these three options lies in the angle of the reservoir used to hold the hydrogel, allowing it to flow from part "b" of the device to part "c".

[0145] Regarding the "c," this corresponds to the reservoir for the crosslinking solution, used to collect the polymer-based capsules after crosslinking for loading into a benchtop centrifuge.

[0146] Example 3: A pocket-sized miniature encapsulator with dual needle tips: conceptual design and technical details To enable the device to be used with two different solutions and / or two different compositions, a combined system was designed and implemented. This device differs from the original device in part "b", as follows: Figure 6 As shown. The new device will be named the 2X-Pocket Miniature Encapsulator.

[0147] Unlike traditional versions of the pocket miniature encapsulator, the 2X-pocket miniature encapsulator consists of a modular system, with two small reservoirs in part "b", such as... Figure 6 -I and Figure 6 -II is shown. These two reservoirs are used to hold two different hydrogels / solutions, which are mixed in a portion of "g", as shown. Figure 6 As shown in -III and 6-IV. Similar to the previous design, the system has a tube passing through section "b" to keep the pressure equal in different parts of the device.

[0148] In this scenario, two different syringes converge at a single point to produce a droplet combining both solutions. This droplet cross-links upon contact with the solution placed in portion "c," as... Figure 7 As shown. Device "g" is detachable and can be combined with different syringe sizes, thereby enabling the mixing of solutions with different physical and / or rheological properties.

[0149] Example 4: Twisted Injector Besides efforts to improve the performance of the miniature encapsulator, another option for improving the device is a new drip system, which includes the use of a spiral injector ( Figure 8 This type of syringe, also known as a twisting syringe, is used for dripping solutions with extremely low viscosity or low surface tension because of its ability to slow down fluid movement during dripping. The helical syringe was tested with polymer-based solutions, as shown in Table 4.

Claims

1. An apparatus for encapsulating a compound into a capsule, sphere, bead, or filament, wherein the apparatus comprises: （A） (i) The first tube, its It is open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint. Configured to collect capsules, spheres, beads, or filaments to be produced and contain cross-linking solutions, (ii) The second pipe located at the top of the first pipe, Both the top and bottom sides are open, and It includes one or two reservoirs configured to hold a polymer solution crosslinkable by (i) and to hold the compound to be encapsulated. (iii) One or more outlets of one or both reservoirs of (ii) allow the polymer solution of (ii) to drip or be extruded into the crosslinking solution of (i) if gravity or centrifugal force is applied. (iv) A pipe located between (i) and (ii) or between (i) and (v) and passing through a second pipe, wherein the pipe enables air exchange between (i) and (v), and (v) The cap located at the top of the second tube, and Preferably, the second tube or cap further includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube; or （B） (i) The first tube, its It is open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint. Configured to collect capsules, spheres, beads, or filaments to be produced and contain cross-linking solutions, (ii) The second pipe located at the top of the first pipe, Both the top and bottom sides are open, and It includes one or two reservoirs configured to hold a polymer solution crosslinkable by (i) and to hold the compound to be encapsulated. (iii) One or more outlets of one or both reservoirs of (ii), if negative pressure is applied, allow the polymer solution of (ii) to drip or be extruded into the crosslinking solution of (i) through said outlet. (iv) A connection portion configured to connect to a vacuum pump, the connection portion being an additional opening in the first tube, allowing negative pressure to be generated from the bottom of the device, preferably via the connected vacuum pump, and (v) The cap located at the top of the second tube, and Preferably, the second tube or cap further includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube; （C） (i) The first tube, its It is open at the top and closed at the bottom, wherein the bottom preferably forms a flat footprint. Configured to collect capsules, spheres, beads, or filaments to be produced and contain cross-linking solutions, (ii) The second pipe located at the top of the first pipe, Both the top and bottom sides are open, and It includes one or two reservoirs configured to hold a polymer solution crosslinkable by (i) and to hold the compound to be encapsulated. (iii) One or more outlets of one or both reservoirs of (ii), if positive pressure is applied, through which the polymer solution of (ii) may drip or be extruded into the crosslinking solution of (i), and (iv) The cap located at the top of the second tube, and The second tube or cap further includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube, and the opening is also configured to connect to a connection to an air pump to preferably generate positive pressure from the top of the device via the connected air pump; or The second tube or cap also includes a connection configured to be connected to an air pump to preferably generate positive pressure from the top of the device via the connected air pump, and preferably, the second tube or cap also includes an opening, optionally having a second cap on the opening, through which the polymer solution and / or compound can be placed into one or both reservoirs of the second tube.

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

3. The apparatus according to claim 1 or 2, wherein one or more outlets of one or both reservoirs of (ii) (through which the polymer solution of (ii) may drip or be extruded into the crosslinked solution of (i)) include or are connected to a syringe needle, hollow tube or mesh, wherein the connection is optionally made by an adapter connecting one or more outlets of one or both reservoirs and their syringe needle, hollow tube or mesh.

4. The apparatus according to any one of claims 1 to 3, wherein the apparatus has a size suitable for a centrifuge.

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

6. The device according to any one of claims 1 to 5, wherein the first tube, the second tube, and the cap are connected to each other by plug-in connection, bolt connection, magnetic connection, and / or molded together.

7. The apparatus according to any one of claims 1 to 6, wherein one or both reservoirs of the second tube are funnel-shaped.

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

9. The apparatus according to any one of claims 1 to 8, wherein at least one of the one or more outlets has two or more openings connected to one or two reservoirs into which the reservoirs drain, and the polymer solution of (ii) can drip or be extruded through said opening into the crosslinking solution of (i).

10. The apparatus according to any one of claims 1 to 9, wherein the first tube comprises a crosslinking solution, wherein the crosslinking solution preferably comprises divalent or trivalent cations, such as Mg2+, Ca2+, Ba2+, Cu2+, Fe2+, Zn2+, Cr3+ and / or Fe3+.

11. The apparatus according to any one of claims 1 to 10, wherein one or both reservoirs of the second tube comprise 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 encapsulating a compound into a capsule, sphere, bead, or filament using the apparatus according to any one of claims 1 to 11, wherein a first tube comprises a crosslinking solution and the compound to be encapsulated, and one or both reservoirs of a second tube comprise a polymer solution crosslinkable by the crosslinking solution, wherein the method comprises: (a) The polymer solution containing the compound to be encapsulated is dripped or extruded into the crosslinking solution by applying gravity or centrifugal force, or preferably negative pressure from the bottom or positive pressure from the top.

13. A method for producing capsules, spheres, beads, or filaments using the apparatus according to any one of claims 1 to 11, wherein a first tube comprises a crosslinking solution, and one or both reservoirs of a second tube comprise a polymer solution crosslinkable by the crosslinking solution, wherein the method comprises: (a) The polymer solution is dripped or extruded into the crosslinking solution by applying gravity or centrifugal force, or preferably negative pressure from the bottom or positive pressure from the top.

14. The method according to claim 12 or 13 further comprises sterilizing the device prior to step (a) or (a'), preferably autoclaving.

15. A parts kit comprising components (i) to (iv) of one or more devices according to any one of claims 1 to 11, which are individual parts or partial preforms.

Images