A microcapsule and methods of making and using same

Patent metadata

Field	Value
Publication number	US-2026055363-A1
Application number	US-202218715650-A
Country	US
Kind code	A1
Filing date	Dec 1, 2022
Priority date	Dec 1, 2021
Publication date	Feb 26, 2026
Grant date	—

Abstract

The present disclosure provides in one aspect a microcapsule comprising: (a) a core comprising a polyhydroxy compound and/or an antichaotropic agent; and (b) a semi-permeable shell surrounding the core; wherein the semi-permeable shell comprises a gel formed from a polyampholyte, wherein the polyampholyte in the gel is covalently cross-linked. The present disclosure provides further aspects relating to a method of making a microcapsule, methods of using the microcapsule and kits for making a microcapsule.

First claim

1 .- 86 . (canceled)

87 . A microcapsule comprising:
(a) a core comprising a polyhydroxy compound and/or an antichaotropic agent; and (b) a semi-permeable shell concentrically surrounding the core; wherein the semi-permeable shell comprises a gel formed from a polyampholyte, wherein the polyampholyte in the gel is covalently cross-linked, wherein the microcapsule further comprises at least one biological entity.

88 . The microcapsule of claim 87 , wherein the at least one biological entity is selected from a cell, a microorganism, a bacterium, a virus, or a nucleic acid.

89 . The microcapsule of claim 88 , wherein the at least one cell is at least one prokaryotic cell, such as a bacterial cell or an archaea cell, or at least one eukaryotic cell.

90 . The microcapsule of claim 87 , wherein the microcapsule comprises at least one biological entity and the at least one biological entity is at least one nucleic acid, optionally wherein the at least one nucleic acid is at least 100 nucleotides long.

91 . The microcapsule of claim 87 , wherein the microcapsule comprises at least one solid particle, optionally wherein the at least one solid particle is a metal nanoparticle, a mineral particle, a polymer particle, a fluorescent nanoparticle, a magnetic nanoparticle or a composite particle.

92 . The microcapsule of claim 87 , wherein the polyampholyte is a single type of biopolymer, such as polypeptide, or is a mixture of two or more polyampholytes or a mixture of at least one polyampholyte and at least one of polyelectrolyte, polysaccharide, oligosaccharide, sugar, or synthetic polymer, such as poly(ethylene glycol), or is a thermo-responsive polymer capable of forming a thermo-reversible gel in response to a temperature change.

93 . The microcapsule of claim 87 , wherein the polyampholyte is:
(a) is a biopolymer, a modified biopolymer or a synthetic polymer, wherein the modified biopolymer is the polyampholyte modified with one or more chemically cross-linkable groups for the covalent cross-linking, wherein the polyampholyte has a degree of substitution of 10 to 90%, preferably 40 to 90%, more preferably 60 to 80%. (b) comprises peptide bonds, and/or (c) is a peptide, a polypeptide, an oligopeptide or a protein.

94 . The microcapsule of claim 87 , wherein the semi-permeable shell comprises peptide bonds, and which are biodegradable with a protease to release the inner content of the microcapsule.

95 . The microcapsule of claim 94 , wherein the polyampholyte comprises amino acids, and where in at least 10% of amino acids in the polyampholyte are disorder-promoting amino acids, preferably wherein at least 30% of amino acids in the polyampholyte are disorder-promoting amino acids, where the disorder promoting amino acids are selected from proline, glycine, glutamic acid/glutamate, serine, lysine, alanine, arginine, and glutamine.

96 . The microcapsule of claim 87 , wherein the polyampholyte is selected from the group consisting of collagen, mucin, laminin, elastin, elastin-like polypeptides, fibrin, silk fibrion, fibronectin, vimentin, glycinin, gluten, casein, or hydrolyzed forms thereof, such as gelatin.

97 . The microcapsule of claim 87 , wherein the gel is formed from a polyampholyte modified with a chemical group, selected from the group consisting of acrydite, acrylate, methacryloyl, acrylamide, methacrylamide, bisacrylamide, methacrylate, methacrylic acid, acrylic acid, polyacrylic acid, methacrylic anhydride, acryloyl, vinyl, vinylsulfone, vinylpyrrolidone, thiol, disulphide, cystamine, carboxyl, amine, imine, azide, triazole, tetrazine, azidophenylalanine, alkynyl, alkenyl, alkynes, diisocyanate, hydroxypropionic acid, hydroxy phenol, azobenzene, methylcyclopropene, trans-cyclooctene (TCO), norbornene, diacrylcyclooctyne (DBCO) or cyclooctanyl moieties and/or reagents.

98 . The microcapsule of claim 87 , wherein the microcapsule comprise at least one biological entity in the core and the shell is permeable to a reagent, an enzyme, a nutrient, and/or a substrate but not permeable to the biological entity of interest.

99 . The microcapsule of claim 87 , wherein the semi-permeable shell is permeable to smaller molecular weight compounds having a molecular weight of 200,000±100,000 Da or less through the shell, while mostly impermeable to larger molecular weight compounds, preferably wherein the semi-permeable shell allows for diffusion of smaller molecular weight compounds of having a molecular weight of 120,000±60,000 Da or less through the shell, while retaining larger molecular weight compounds.

100 . The microcapsule of claim 87 , wherein the core is a liquid, a semi-liquid, a polymer, or a hydrogel and comprises the polyhydroxy biodegradable compound selected from a polyelectrolyte, polysaccharide, a carbohydrate, an oligosaccharide, or a sugar, which can be natural or synthetic.

101 . The microcapsule of claim 87 , wherein the polyhydroxy compound has a molecular weight of 300 Da to 5000 kDa, preferably 10 kDa to 800 kDa, and even more preferably 400 kDa to 600 kDa.

102 . The microcapsule of claim 87 , wherein the polyhydroxy compound can be hydrolyzed upon treatment with a hydrolase enzyme (e.g., a glycosidase, a dextranase, an amylase, a sucrase, or a cellulase) to reduce the viscosity of the core.

103 . The microcapsule of claim 87 , wherein the antichaotropic agent is a kosmotropic salt such as a sulphate, a phosphate or a citrate, preferably ammonium sulphate.

104 . The microcapsule of claim 87 , wherein the microcapsule is from 1 μm to 100,000 μm in diameter, preferably between 1 μm and 500 μm in diameter, and more preferably between 20 μm and 200 μm in diameter and the shell of the microcapsule is from 0.2 to 100 μm thick, preferably from 1 to 10 μm thick.

105 . The microcapsule of claim 87 , wherein the microcapsule does not disintegrate during PCR, thermocycling or an incubation at an elevated temperature, such as at least 10 minutes at 50±10° C. or even at 90±10° C. and retains encapsulate biological species of interest during cryopreservation.

106 . The microcapsule of claim 87 , wherein the microcapsule is formed by (a) changing the temperature of a droplet, wherein the droplet is a water-in-oil droplet, a water-in-water droplet or a water-in-air droplet, comprising a core enriched in the polyhydroxy compound and/or antichaotropic agent and a shell enriched in the polyampholyte, to induce gelation of the polyampholyte; and (b) and covalently cross-linking the gelled polyampholyte

107 . A composition comprising one or more microcapsule of claim 87 in a carrier oil, or in an aqueous solution.

108 . The composition of claim 107 , wherein the aqueous solution is a cell culture medium, or a cell storage medium or an aqueous buffer, optionally wherein the aqueous buffer is a buffer for washing cells or lysing the encapsulated cells.

109 . An in vitro method for cultivating at least one cell encapsulated in or attached to an inner surface or an outer surface of a microcapsule according to claim 87 , comprising incubating the microcapsule in an aqueous environment suitable to allow for cell survival, cell growth and/or cell proliferation.

110 . The in vitro method of claim 109 , wherein the aqueous environment is a suitable growth media comprising one or more nutrients needed for encapsulated cell survival and/or growth, wherein where the cell is encapsulated the method comprising allowi

Assignees

• Univ Vilnius

Inventors

• Mazutis Linas
• Leonaviciene Greta

Classifications

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N2533/40

  Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers · CPC title

• C12N2513/00

  3D culture · CPC title

• C12N15/88

  using microencapsulation, e.g. using {amphiphile} liposome vesicle · CPC title

• C12N1/04

  Preserving or maintaining viable microorganisms (immobilised microorganisms C12N11/00) · CPC title