Patent Application
20190367901
The present invention relates to polymer beads, which are useful for enclosing a cell or a microorganism in the inside.
In recent years, transplantation of a cell or the like encapsulated in a polymer bead or polymer capsule to the body has been studied. As such a capsule, for example, JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS I February 2013 VOL 101B, ISSUE 2, 258-268, which is incorporated herein by reference in its entirety, describes an alginate/poly-L-orthinine/alginate capsules having a structure in which a core composed of alginate is coated with poly-L-orthinine and further coated with alginate.
J Mater Sci: Mater Med (2013) 24: 1375-1382, which is incorporated herein by reference in its entirety, describes disulfide-crosslinked polygalacturonic acid hydrogel. J Mater Sci: Mater Med (2013) 24: 1375-1382 describes a film formed from the hydrogel but does not describe a bead or capsule formed from the hydrogel.
Nature Biotechnology, Vol 34, No. 3 (2016), 345-352, which is incorporated herein by reference in its entirety, describes a hydrogel of alginate bonded to an amine having an inhibitory action on a foreign-body reaction.
The alginate (the second layer)/poly-L-orthinine (the first layer)/alginate capsule described in JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS I February 2013 VOL 101B, ISSUE 2, 258-268 has a problem that the alginate layer in the second layer is decomposed in vivo to expose poly-/o L-orthinine in the first layer, as a result of which an inflammation reaction occurs. The present invention has been made by taking note of such circumstances and an object thereof is to provide a polymer bead superior in durability.
This and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that a bead formed from a polymer containing uronic acid units having mercapto groups (—SH) has a disulfide bond (—S—S—) formed partly or entirely by the mercapto groups therein and shows superior durability even under low calcium ion conditions.
Thus present invention provides the following.
(1) A bead comprising a polymer comprising uronic acid units having mercapto groups, the mercapto groups partly or entirely forming a disulfide bond.
(2) The bead of the aforementioned (1) wherein the uronic acid unit having the mercapto group comprises a uronic acid residue and a residue of a compound represented by the formula (A1):
wherein La1 is a single bond or a C1-3 alkylene group and La2 is a C1-4 alkylene group, or a compound represented by the formula (A2):
bonded to each other via an amide bond.
(3) The bead of the aforementioned (1) or (2) wherein the uronic acid is at least one selected from the group consisting of galacturonic acid, mannuronic acid and guluronic acid.
(4) The bead of the aforementioned (1) or (2) wherein the uronic acid is at least one selected from the group consisting of mannuronic acid and guluronic acid, and the polymer comprising the uronic acid units having the mercapto groups is alginic acid having mercapto groups.
(5) The bead of the aforementioned (1) wherein the polymer comprising the uronic acid units having the mercapto groups is a polymer comprising galacturonic acid units having mercapto groups.
(6) The bead of the aforementioned (5) wherein the galacturonic acid unit having the mercapto group comprises a galacturonic acid residue and an amino acid residue having a mercapto group bonded to each other via an amide bond.
(7) The bead of the aforementioned (6) wherein the amino acid having the mercapto group is cysteine.
(8) The bead of any one of the aforementioned (5) to (7) wherein the polymer comprising the galacturonic acid units is polygalacturonic acid.
(9) The bead of any one of the aforementioned (1) to (8) wherein the polymer comprising the uronic acid units having the mercapto groups is further bonded to a compound represented by the formula (B):
wherein Rb1 is a functional group capable of bonding to a mercapto group,
Lb1 and Lb2 are each independently a single bond or a alkylene group,
Qb1 is a single bond, a phenylene group, or a C4-8 cycloalkanediyl group,
Lb3 is a single bond or *—(OCH2CH2)n—** (wherein * shows a bonding position to Qb1, ** shows a bonding position to Qb2, and n is an integer of 1 to 10),
Qb2 is a divalent triazole ring group,
Lb4 is a single bond, a C1-6 alkylene group, or a C1-6 alkylene-oxy group, and
Qb3 is an optionally substituted phenyl group, an optionally substituted monovalent 5- or 6-membered heterocyclic group, or an optionally substituted C4-8 cycloalkyl group.
(10) The bead of the aforementioned (9) wherein the compound represented by the formula (B) is at least one selected from the group consisting of a compound represented by the formula (B1):
a compound represented by the formula (B2):
and
a compound represented by the formula (B3):
(11) The bead of the aforementioned (9) wherein the compound represented by the formula (B) is a compound represented by the formula (B1):
(12) The bead of any one of the aforementioned (9) to (11) wherein the compound represented by the formula (B) is a compound having an inhibitory action on a foreign-body reaction.
(13) The bead of any one of the aforementioned (1) to (12) wherein a proportion of the uronic acid unit having the mercapto group in the total constitutional units of the polymer comprising the uronic acid units having the mercapto groups is 0.1 to 50 mol %.
(14) The bead of any one of the aforementioned (1) to (13) wherein a proportion of the mercapto group forming the disulfide bond in the total mercapto groups is 10 to 100 mol %.
(15) The bead of any one of the aforementioned (1) to (14) wherein a number average molecular weight of the polymer comprising the uronic acid units having the mercapto groups is 25,000 to 500,000.
(16) The bead of any one of the aforementioned (1) to (15) wherein the polymer comprises a divalent metal ion.
(17) The bead of the aforementioned (16) wherein the divalent metal ion is at least one selected from the group consisting of calcium ion, barium ion and strontium ion.
(18) The bead of any one of the aforementioned (1) to (17) further comprising a first layer and a second layer as outer layers, wherein the first layer is formed on the bead and the second layer is formed on the first layer.
(19) The bead of the aforementioned (18) wherein the uronic acid is at least one selected from the group consisting of mannuronic acid and guluronic acid,
the polymer comprising the uronic acid units having the mercapto groups is alginic acid having mercapto groups, and
the uronic acid unit having the mercapto group comprises a uronic acid residue and a residue of a compound represented by the formula (A1):
wherein La1 is a single bond or a C1-3 alkylene group and La2 is a C1-4 alkylene group, or a compound represented by the formula (A2):
bonded to each other via an amide bond.
(20) The bead of the aforementioned (19) wherein the compound represented by the formula (A1) or the compound represented by the formula (A2) is cysteine.
(21) The bead of any one of the aforementioned (18) to (20) wherein the first layer is formed from at least one selected from the group consisting of water-soluble chitosan and polyornithine.
(22) The bead of any one of the aforementioned (18) to (21) wherein the second layer is formed from at least one selected from the group consisting of polygalacturonic acid and polygalacturonic acid having mercapto groups.
(23) The bead of the aforementioned (22) wherein said at least one selected from the group consisting of the polygalacturonic acid and polygalacturonic acid having mercapto groups is further bonded to a compound represented by the formula (b):
H2N-Lb2-Qb1-Lb3-Qb2-Lb4-Qb3 (b)
wherein Lb2 is a single bond or a C1-6 alkylene group,
Qb1 is a single bond, a phenylene group, or a C4-8 cycloalkanediyl group,
Lb3 is a single bond or *—(OCH2CH2)n—** (wherein * shows a bonding position to Qb1, ** shows a bonding position to Qb2, and n is an integer of 1 to 10),
Qb2 is a divalent triazole ring group,
Lb4 is a single bond, a C1-6 alkylene group, or a C1-6 alkylene-oxy group, and
Qb3 is an optionally substituted phenyl group, an optionally substituted monovalent 5- or 6-membered heterocyclic group, or an optionally substituted C4-8 cycloalkyl group.
(24) The bead of the aforementioned (23) wherein the compound represented by the formula (b) is at least one selected from the group consisting of a compound represented by the formula (b1):
a compound represented by the formula (b2):
and
a compound represented by the formula (b3):
(25) The bead of the aforementioned (23) wherein the compound represented by the formula (b) is a compound represented by the formula (b1):
(26) The bead of any one of the aforementioned (23) to (25) wherein the compound represented by the formula (b) is a compound having an inhibitory action on a foreign-body reaction.
(27) The bead of any one of the aforementioned (1) to (26) for use for enclosing a cell or a microorganism in the inside.
(28) The bead of any one of the aforementioned (1) to (26) enclosing the cell or microorganism in the inside.
According to the present invention, a polymer bead superior in durability can be obtained.
The present invention provides a bead having a polymer containing uronic acid units having mercapto groups (hereinafter sometimes to be referred to as “mercapto group-containing uronic acid-based polymer”) and having a disulfide bond formed partly or entirely by the mercapto groups. The bead of the present invention is preferably used for enclosing a cell or microorganism in the inside thereof. Thus, the m present invention also provides a bead enclosing a cell or microorganism in the inside.
The term “uronic acid” means a carboxylic acid obtained by conversion of a hydroxymethyl group (—CH2OH) of monosaccharide to a carboxy group by oxidation. Examples thereof include galacturonic acid, mannuronic acid, guluronic acid, arabinonic acid, fructuronic acid, tagaturonic acid, glucuronic acid, iduronic acid and the like. Only one kind or two or more kinds of the uronic acid may be used. As the uronic acid, galacturonic acid, mannuronic acid or guluronic acid is preferable, and galacturonic acid is more preferable.
In the present invention, the “bead” means an internally-filled sphere. In the pertinent field, a bead used for enclosing a cell or microorganism in the inside is sometimes called a “capsule”.
Examples of the cell to be encapsulated in the bead of the present invention include cells for transplantation and cells for culture. Examples of the cell for transplantation include cells derived from mammals. Examples of the cell derived from mammal include cells derived from human and cells derived from swine. Examples of the cell derived from human and the cell derived from swine include hormone secreting cells thereof. Examples of the hormone secreting cell include pancreatic cell and pituitary cell. Examples of the cell for culture include stem cells such as IFS cell (induced pluripotent stem cell), ES cell (embryonic stem cell), MSC cell (mesenchymal stem cell) and the like. The microorganism to be encapsulated in the bead of the present invention may be any of aerobic bacteria and anaerobic bacteria.
The bead of the present invention may contain a polymer other than a mercapto group-containing uronic acid-based polymer. Examples of such other polymer include alginic acid free of a mercapto group and a salt thereof (i.e., alginate), chitosan, hyaluronic acid, gelatin, carboxymethylcellulose, gellan gum, glucomannan and the like. Only one kind or two or more kinds of such other polymers may be used. As the other polymer, alginic acid free of a mercapto group or a salt thereof is preferable, and alginate free of a mercapto group is more preferable. When the bead of the present invention contains other polymer, the amount thereof is preferably 1 to 80 wt %, more preferably 10 to 70 wt %, further preferably 20 to 60 wt %, per total weight of the polymers contained in the bead. The bead of the present invention particularly preferably contains a mercapto group-containing uronic acid-based polymer alone as a polymer constituting the bead. That is, the polymer constituting the bead is particularly preferably composed of a mercapto group-containing uronic acid-based polymer.
Only one kind or two or more kinds of the polymer containing uronic acid units (i.e., polymer constituting the main chain of the mercapto group-containing uronic acid-based polymer) may be used. The polymer containing uronic acid units is preferably at least one selected from the group consisting of a galacturonic acid unit, a mannuronic acid unit and a guluronic acid unit, more preferably at least one selected from the group consisting of a polymer containing galacturonic acid units and a polymer containing mannuronic acid units and guluronic acid units, further preferably a polymer containing galacturonic acid units.
Examples of the polymer containing the galacturonic acid units include polygalacturonic acid, pectin (i.e., polymer in which carboxy groups of polygalacturonic acid are partly methylesterified) and the like. A preferable polymer containing uronic acid units having mercapto groups is, for example, a polymer containing galacturonic acid units having mercapto groups. Only one kind or two or more kinds of the aforementioned polymer may be used. The aforementioned polymer is more preferably polygalacturonic acid having mercapto groups.
A preferable polymer containing mannuronic acid units and guluronic acid units is, for example, alginic acid. That is, a preferable polymer containing uronic acid units having mercapto m groups is, for example, alginic acid having mercapto groups.
The uronic acid unit having a mercapto group is preferably one in which a uronic acid residue and a residue of a compound represented by the formula (A1):
wherein Lal is a single bond or a C1-3 alkylene group, and La2 is a C1-4 alkylene group, or a compound represented by the formula (A2):
are bonded via an amide bond (peptide bond). In the following, “compound represented by the formula (A1)” is sometimes to be abbreviated as “compound (A1)”. Also, compounds represented by other formulas are sometimes to be abbreviated similarly. In addition, compound (A1) and compound (A2) are sometimes collectively indicated as “compound (A)”.
Only one kind or two or more kinds of the uronic acid units having mercapto groups may be used. Therefore, the mercapto group-containing uronic acid-based polymer may have, as the uronic acid units having mercapto groups, only a unit constituted of a uronic acid residue and a residue of compound (A1), or only a unit constituted of a uronic acid residue and a residue of compound (A2), or both a unit constituted of a uronic acid residue and a residue of compound (A1) and a unit constituted of a uronic acid residue and a residue of compound (A2). Only one kind or two or more kinds of compound (A1) may be used.
The carboxy group of the mercapto group-containing uronic acid-based polymer contributes to the water-solubility of the polymer and maintenance of the strength of the obtained bead. Therefore, it is preferable to use the above-mentioned compound (A) having both the mercapto group and the carboxy group to introduce a mercapto group into a polymer containing uronic acid units without reducing the amount of the carboxy group.
The aforementioned amide bond is preferably formed from a carboxy group in a uronic acid unit and an amino group of compound (A). That is, the mercapto group-containing uronic acid-based polymer is preferably a polymer formed by direct bonding of the amino group of compound (A) and the carboxy group of the polymer containing uronic acid units. A mercapto group-containing uronic acid-based polymer having directly bonded compound (A) shows less steric hindrance compared to a mercapto group-containing uronic acid-based polymer in which compound (A) is bonded via a linker such as polyethylene glycol chain or the like and does not inhibit gelling of polymers, thus producing a stronger bead.
In the present specification, the “Cx-y” means that the carbon atom number is not less than x and not more than y (x, y: integer).
In the present specification, the alkylene group may be linear or branched chain. The alkylene group is preferably linear. In the present specification, examples of the “C1-6 alkylene group” include —CH2—, −(CH2)2—, (CH2)3—, —(CH2)4—, —(CH2)5—, —(CH2)6—, —CH(CH3)—, —C(CH3)2—, —CH(C2H5)—, —CH(C3H7)—, —CH(CH(CH3)2)—, —(CH(CH3))2—, —CH2—CH(CH3)—, —CH(CH3)—CH2—, —CH2—CH2—C(CH3)2—, —C(CH3)2—CH2—CH2—, —CH2—CH2—CH2—C(CH3)2—, and —C(CH3)2—CH2—CH2—CH2—. In the present specification, examples of the “C1-2 alkylene group”, “C1-3 alkylene group” and “C1-4 alkylene group” respectively include those mentioned above and having 1 to 2 carbon atoms, 1 to 3 carbon atoms and 1 to 4 carbon atoms.
La1 is preferably a single bond or a C1-2alkylene group, more preferably a single bond or —CH2—, further preferably a single bond.
La2 is preferably a C1-3 alkylene group, more preferably a C1-2 alkylene group, further preferably —CH2—.
Specific examples of compound (A1) (i.e., amino acid having mercapto group) include the following.
Compound (A1) is available from, for example, KANTO CHEMICAL CO., INC., FCH Group and the like. Compound (A2) is available from FCH Group and the like. Of compound (A1) and compound (A2), compound (A1) is preferable, cysteine and homocysteine are more preferable, and cysteine is further preferable.
A carboxy group of the polymer containing uronic acid units can be condensed with an amino group of compound (A) under conditions well known to those of ordinary skill in the art. The aforementioned polymer can be produced, for example, according to the method described J Mater Sci: Mater Med (2013) 24: 1375-1382, which is incorporated herein by reference in its entirety.
It is preferable to use a condensing agent for the aforementioned condensation of the carboxy group and the amino group. Examples of the condensing agent include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) and the like. When EDC-HCl is used, N-hydroxysulfosuccinimide sodium is preferably used in combination.
After the aforementioned condensation, the reaction mixture is purified by a known means such as dialysis and the like, whereby a mercapto group-containing uronic acid-based polymer is preferably obtained.
A polymer containing uronic acid units having mercapto groups is preferably a polymer containing galacturonic acid units having mercapto groups, more preferably polygalacturonic acid having mercapto groups. In these embodiments, the galacturonic acid unit having a mercapto group more preferably has a galacturonic acid residue and an amino acid residue having a mercapto group bonded to each other via an amide bond (peptide bond).
The aforementioned amide bond is more preferably formed from a carboxy group in the galacturonic acid unit and an amino group of an amino acid having a mercapto group. That is, a polymer containing galacturonic acid units having mercapto groups is further preferably a polymer formed from an amino group of an amino acid having a mercapto group and a carboxy group of a polymer containing galacturonic acid units directly bonded to each other.
A carboxy group of a polymer containing galacturonic acid units can be condensed with an amino group of amino acid under conditions well known to those of ordinary skill in the art. The aforementioned polymer can be produced, for example, according to the method described J Mater Sci: Mater Med (2013) 24: 1375-1382, which is incorporated herein by reference in its entirety.
Specific examples of the amino acid having a mercapto group include the aforementioned specific examples of compound (A1). Only one kind or two or more kinds of amino acid having a mercapto group may be used. The amino acid having a mercapto group is preferably at least one selected from the group consisting of cysteine and homocysteine, more preferably cysteine.
The mercapto group-containing uronic acid-based polymer is further preferably bonded to a compound represented by the formula (B):
wherein Rb1 is a functional group capable of bonding to a mercapto group,
Lb1 and Lb2 are each independently a single bond or a C1-6 alkylene group,
Qb1 is a single bond, a phenylene group, or a C4-8 cycloalkanediyl group,
Lb3 is a single bond or *—(OCH2CH2)n—** (wherein * shows a bonding position to Qb1, ** shows a bonding position to Q**2, and n is an integer of 1 to 100),
Qb2 is a divalent triazole ring group,
Lb4 is a single bond, a C1-6 alkylene group, or a C1-6 alkylene-oxy group, and
Qb3 is an optionally substituted phenyl group, an optionally substituted monovalent 5- or 6-membered heterocyclic group, or an optionally substituted C4-8 cycloalkyl group.
In the present specification, examples of the “functional group capable of bonding to a mercapto group” include 1-maleimidyl group, chloromethylcarbonyl group, bromomethylcarbonyl group, vinylsulfonyl group, (meth)acryloyl group, disulfide bond, and organic groups containing any of these groups. Specific examples of the functional group capable of bonding to a mercapto group include the following (in the following formulas, * shows a bonding position).
In the present specification, examples of the “C4-8 cycloalkyl group” include cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, and cyclooctyl group.
In the present specification, examples of the “C4-8 cycloalkanediyl group” include cyclobutane-1,3-diyl group, cyclopentane-1,3-diyl group, cyclohexane-1,4-diyl group, cycloheptane-1,4-diyl group, and cyclooctane-1,5-diyl group.
In the present specification, examples of the “monovalent 5- or 6-membered heterocyclic group” include monovalent groups formed by removing one hydrogen bond from the following heterocycles.
In the present specification, the “divalent triazole ring group” means a divalent monovalent groups formed by removing two hydrogen bonds from a triazole ring.
In the present specification, examples of the substituent that the phenyl group optionally has include halogen atom and amino group.
In the present specification, examples of the substituent that the monovalent 5- or 6-membered heterocyclic group optionally has include halogen atom and oxo group.
In the present specification, examples of the substituent that the C4-8 cycloalkyl group optionally has include halogen atom and oxo group.
Rb1 is preferably a 1-maleimidyl group.
Lb1 is preferably —(CH2)2—.
Lb2 is preferably —CH2— or —(CH2)2—, more preferably —(CH2)2—.
Qb1 is preferably a single bond or a 1,4-phenylene group, more preferably a single bond.
The n in *—(OCH2CH2)n—** for Lb1 is preferably an integer of 1 to 6, more preferably 3. Lb3 is preferably a single bond or *—(OCH2CH2)n—** (wherein n is an integer of 1 to 6), more preferably a single bond or *—(OCH2CH2)3—**, further preferably *—(OCH2CH2)3—**.
Qb2 is preferably a 1H-1,2,3-triazole-1,4-diyl group.
Lb4 is preferably a single bond, —CH2— or —CH2—O, more preferably a single bond or —CH2—, further preferably —CH2—.
Qb3 is preferably a 1,1-dioxothiomorpholin-4-yl group, a 4-aminophenyl group or a tetrahydropyran-2-yl group, more preferably a 1,1-dioxothiomorpholin-4-yl group or a 4-aminophenyl group, further preferably a 1,1-dioxothiomorpholin-4-yl group.
Compound (B) is preferably at least one selected from the group consisting of compound (B1), compound (B2) and compound (B3), which are represented by the following formulas, more preferably compound (B1).
Compound (B1) is a compound of the formula (B) wherein Rb1 is a 1-maleimidyl group, Lb1 is —(CH2)2—, Lb2 is —(CH2)2—, Qb1 is a single bond, Lb3 is *—(OCH2CH2)3—**, Qb2 is a 1H-1,2,3-triazole-1,4-diyl group, Lb4 is —CH2— and Qb3 is a 1,1-dioxothiomorpholin-4-yl group.
Compound (B2) is a compound of the formula (B) wherein Rb1 is a 1-maleimidyl group, Lb1 is —(CH2)2—, Lb2 is —(CH2)2—, Qb1 is a single bond, Lb3 is *—(OCH2CH2)3—**, Qb2 is a 1H-1,2,3-triazole-1,4-diyl group, Lb4 is a single bond and Qb3 is a 4-aminophenyl group.
Compound (B3) is a compound of the formula (B) wherein Rb1 is a 1-maleimidyl group, Lb1 is —(CH2)2—, Lb2 is —CH2—, Qb1 is a 1,4-phenylene group, Lb3 is a single bond, Lb2 is a 1H-1,2,3-triazole-1,4-diyl group, Lb4 is —CH2—O—, and Qb3 is a tetrahydropyran-2-yl group.
Compound (B) is preferably a compound having an inhibitory action on a foreign-body reaction. As used herein, the term “inhibitory action on foreign-body reaction” means an action to inhibit adhesion of an inflammatory cell to a surface of a bead and an action to inhibit formation of a fibrous layer after death of the inflammatory cell adhered to a surface of a bead. Examples of the compound having an inhibitory action on a foreign-body reaction include the aforementioned compound (B1) to compound (B3).
Compound (B1) can be produced as described in the below—mentioned Production Examples 7 and 8. Compound (B2) and compound (B3) can be produced according to Nature Biotechnology, Vol 34, No. 3 (2016), 345-352, which is incorporated herein by reference in its entirety, (particularly experiment therein) and in the same manner as in compound (B1) except that amine having the corresponding inhibitory action on a foreign-body reaction is prepared. Compound (B) can be produced by, for example, the following reactions (in the following formula, X is a leaving group (e.g., halogen atom, substituted or unsubstituted phenyloxy group, maleimidyloxy group etc.), and other symbols are as defined above).
Compound (3) can be conveniently synthesized by a Click reaction of compound (1) having an azido group (—N3) and compound (2) having an ethynyl group (—C≡CH) in water or an organic solvent. Compound (3) can be purified by silica gel chromatography and the like. Then, compound (3) is reacted with compound (4) having a leaving group X to synthesize compound (B). Compound (B) can be purified by silica gel chromatography and the like.
Compound (B) wherein Rb1 is a 1-maleimidyl group or organic group containing a 1-maleimidyl group shows high reactivity with a mercapto group. Thus, a bead can be bonded to the aforementioned compound (B) by merely adding a solution of the aforementioned compound (B) to a mixture containing a bead of a mercapto group-containing uronic acid-based polymer, water and the like and standing the obtained mixture. Examples of the solvent for the aforementioned solution include water, dimethylfomamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile and the like. The concentration of the solution of the aforementioned compound (B) is, for example, 1 μmM to 100 mM. The standing time of the mixture after addition of the aforementioned solution of compound (B) is, for example, 10 min to 5 hr, and the temperature thereof is, for example, 10 to 100° C.
The reaction between compound (B) wherein Rb1 is a group other than a 1-maleimidyl group or organic group containing a 1-maleimidyl group and a mercapto group-containing uronic acid-based polymer can be performed by adding a solution of compound (B) to a mixture of a mercapto group-containing uronic acid-based polymer bead, water and the like. Examples of the solvent for the aforementioned solution include water, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile and the like. Where necessary, a dilute solution of sodium hydroxide may be added to the reaction system to adjust the pH thereof to 6 to 7. The reaction time is 10 min to 5 hr and the temperature thereof is 10 to 100° C.
In the bead of the present invention, the proportion of the uronic acid unit having a mercapto group in the total constitutional units of the mercapto group-containing uronic acid-based polymer is preferably 0.1 to 50 mol %, more preferably 0.1 to 30 mol %, further preferably 1 to 10 mol %. This proportion can be calculated from a ratio of a peak area of proton on a carbon atom bonded to a mercapto group (—SH) and a peak area of proton on the carbon skeleton of uronic acid, which is obtained when a sample is measured by a nuclear magnetic resonance apparatus.
In the bead of the present invention, a disulfide bond (—S—S—) is formed from mercapto groups (—SH) by natural oxidation. Due to this disulfide bond, the bead of the present invention shows superior durability. In the bead of the present invention, a proportion of the mercapto group forming the disulfide bond in the total mercapto groups is preferably 10 to 100 mol %, more preferably 50 to 100 mol %, further preferably 70 to 100 mol %. This proportion can be calculated by measuring the amount of mercapto group immediately after bead production when the disulfide bond is considered to be nil and the amount of mercapto group after progress of a given time from the production of a bead considered to be formed by the disulfide bond by the Elleman method and comparing them.
The mercapto group-containing uronic acid-based polymer preferably contains a carboxy group in addition to the mercapto group. This carboxy group may be in a free acid (—COOH) form or an anion (—COO−) form. The amount of the carboxy group in the mercapto group-containing uronic acid-based polymer is preferably 80 to 100 mol, more preferably 90 to 100 mol, further preferably 95 to 100 mol, relative to 100 moles of the total constitutional units of the mercapto group-containing uronic acid-based polymer. This amount can be measured by a ratio of a peak area of proton on a carbon atom bonded to a mercapto group (—SH) and a peak area of proton on the carbon skeleton of uronic acid, which is obtained when a sample is measured by a nuclear magnetic resonance apparatus.
The number average molecular weight of the mercapto group-containing uronic acid-based polymer is preferably 25,000 to 500,000, more preferably 25,000 to 300,000, further preferably 25,000 to 100,000. The number average molecular weight of a polymer crosslinked by forming a disulfide bond cannot be measured. Therefore, this number average molecular weight is a value of a polymer not forming a disulfide bond. This number average molecular weight can be measured by gel permeation chromatography (GPC).
The bead of the present invention can be produced by adding a mercapto group-containing uronic acid-based polymer to an aqueous solution containing a divalent metal ion. The thus-produced bead of the present invention contains a divalent metal ion.
Examples of the divalent metal ion include alkaline earth metal ion and the like. Among these, calcium ion, barium ion and strontium ion are preferable, and calcium ion is more preferable. Only one kind or two or more kinds of the divalent metal ion may be used. The amount of the divalent metal ion in the bead of the present invention is preferably 1 to 300 mmol, more preferably 10 to 200 mmol, further preferably 20 to 100 mmol, per 1 L of the bead. This amount can be measured by atomic absorption analysis.
Examples of the aqueous solution containing a divalent metal ion include aqueous calcium chloride solution, aqueous barium chloride solution, aqueous strontium chloride solution and the like. Among these, aqueous calcium chloride solution and aqueous barium chloride solution are preferable, and aqueous calcium chloride solution is more preferable. The concentration of the divalent metal ion in the aqueous solution containing a divalent metal ion is preferably 10 to 200 mM, more preferably 20 to 100 mM.
When the mercapto group-containing uronic acid-based polymer has a carboxy group, it is preferable to prepare an aqueous solution of the mercapto group-containing uronic acid-based polymer by converting the carboxy group to an anion form with an alkali metal hydroxide. The aforementioned alkali metal hydroxide is preferably sodium hydroxide.
It is preferable to form a bead by adding an aqueous solution of the mercapto group-containing uronic acid-based polymer to an aqueous solution containing the aforementioned divalent metal ion. The concentration of the mercapto group-containing uronic acid-based polymer in the aqueous solution to be added is preferably not less than 0.5 w/v %, more preferably not less than 1 w/v %, further preferably not less than 2 w/v %, preferably not more than 30 w/v %, more preferably not more than w/v %, further preferably not more than 10 w/v %.
It is preferable to use a syringe when adding an aqueous solution containing the mercapto group-containing uronic acid-based polymer. The inner diameter of the needle of the syringe is preferably 0.14 to 2.27 mm, more preferably 0.14 to 0.52 mm.
The temperature when adding an aqueous solution of the mercapto group-containing uronic acid-based polymer (i.e., temperature of the aforementioned aqueous solution and temperature of aqueous solution containing divalent metal ion) is preferably 4 to 37° C., more preferably 10 to 30° C.
The bead of the present invention may further contain a monovalent metal ion. Examples of the monovalent metal ion include sodium ion, potassium ion and the like.
The average particle diameter of the bead of the present invention is preferably 0.01 to 20 mm, more preferably 0.1 to 5 mm, further preferably 0.2 to 2 mm. In the present invention, “average particle diameter of bead” means, unless particularly described, an average maximum diameter of randomly selected 5 beads. This average particle diameter can be measured using a microscope and digital camera. To be specific, the average particle diameter of the bead can be calculated by taking a microphotograph of the beads at 4× magnification using a microscope and a digital camera, randomly selecting 5 beads, and calculating the maximum diameters of the 5 selected beads in the photograph with the software attached to the digital camera.
The bead of the present invention preferably contains water. The water amount of the bead of the present invention is preferably not less than 80 wt %, more preferably not less than 90 wt %, further preferably not less than 95 wt %, preferably not more than 99.5 wt %, more preferably not more than 99 wt %, further preferably not more than 98 wt %, particularly preferably not more than 97 wt %. This water content can be calculated by comparing the bead weight before and after drying. To be specific, 50 beads are randomly selected, surface moisture is removed, and the total weight thereof before drying is measured. Then, these are dried in a thermostatic dryer at 100° C. for 3 hr and the total weight thereof after drying is measured. The water amount of one bead can be calculated by comparing the obtained total bead weights before and after drying.
The amount of the mercapto group-containing uronic acid-based polymer in the bead of the present invention is preferably not less than 0.5 wt %, more preferably not less than 1 wt %, further preferably not less than 2 wt %, particularly preferably not less than 3 wt %, preferably not more than 20 wt %, more preferably not more than 10 wt %, further preferably not more than 5 wt %.
The bead of the present invention enclosing a cell or microorganism in the inside can be produced, for example, by adding a suspension containing a cell or microorganism and the mercapto group-containing uronic acid-based polymer to an aqueous solution containing a divalent metal ion. The explanation of the divalent metal ion (kind and concentration of ion) is as mentioned above.
When the mercapto group-containing uronic acid-based polymer has a carboxy group, the aforementioned polymer is preferably a salt with an alkali metal (particularly salt with alkali metal hydroxide). The aforementioned alkali metal is preferably sodium and the aforementioned alkali metal hydroxide is preferably sodium hydroxide.
The concentration of the aforementioned polymer in a suspension containing a cell or microorganism and the mercapto group-containing uronic acid-based polymer is preferably 1 to w/v %, more preferably 2 to 10 w/v %. When the aforementioned suspension contains a cell, the cell mass is preferably 1.0×10 to 1.0×109 cells/mL, more preferably 1.0×102 to 1.0×107 cells/mL. The cell mass can be measured by MTT assay using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). When the aforementioned suspension contains a microorganism, the microbial biomass is preferably 1.0×10 to 1.0×109 microorganisms/mL, more preferably 1.0×102 to 1.0×107 microorganisms/mL. The microbial biomass can be measured by the surface plate method.
It is preferable to use a syringe when adding a suspension containing a cell or microorganism and the mercapto group-containing uronic acid-based polymer. The inner diameter of the needle of the syringe is preferably 0.14 to 2.27 mm, more preferably 0.14 to 0.52 mm.
The temperature when adding a suspension containing a cell or microorganism and the mercapto group-containing uronic acid-based polymer (i.e., temperature of the aforementioned suspension and temperature of the aqueous solution containing a divalent metal ion) is preferably 4 to 37° C., more preferably 10 to 37° C.
The average particle diameter of the cell or microorganism-enclosing bead of the present invention is preferably 0.01 to 20 mm, more preferably 0.1 to 5 mm, further preferably 0.2 to 2 mm. This average particle diameter can be measured using a microscope and digital camera, as mentioned above.
The cell mass of the cell-enclosing bead of the present invention is preferably 1.0×10 to 1.0×109 cells/mL, more preferably 1.0×102 to 1.0×107 cells/mL. The cell mass can be measured by MTT assay. The microbial biomass of the microorganism-enclosing bead of the present invention is preferably 1.0×10 to 1.0×109 microorganisms/mL, more preferably 1.0×102 to 1.0×107 microorganisms/mL. The microbial biomass can be measured by the surface plate method.
The bead of the present invention optionally further has at least one outer layer (e.g., two outer layers). Examples of such bead include one further having the first layer and the second layer as outer layers, in which the first layer is formed on the bead of the present invention and the second layer is formed on the first layer. In the following, the bead of the present invention having the first layer and the second layer is sometimes referred to as the core-shell type bead of the present invention.
The explanation of the bead (core) of the core-shell type bead of the present invention is as mentioned above unless particularly described. In a polymer containing uronic acid units having mercapto groups contained in the bead, the aforementioned uronic acid is at least one selected from the group consisting of mannuronic acid and guluronic acid, and the polymer containing uronic acid units having mercapto groups is preferably an alginic acid having mercapto groups.
The aforementioned uronic acid unit having a mercapto group is preferably one in which a uronic acid residue and a residue of compound (A1) or compound (A2) is bonded via an amide bond. The explanation of the compound (A1) and compound (A2) in the core-shell type bead of the present invention is as mentioned above unless particularly described. Of these compound (A1) and compound (A2), compound (A1) is preferable, cysteine and homocysteine are more preferable, and cysteine is further preferable.
The thickness of the first layer (one of shells) of the core-shell type bead of the present invention is preferably 0.1 to 200 μm, more preferably 1 to 100 μm. The first layer is preferably formed from at least one selected from the group consisting of water-soluble chitosan and polyornithine, more preferably water-soluble chitosan or polyornithine, further preferably water-soluble chitosan. Water-soluble chitosan is advantageous in that it does not easily induce an inflammation reaction in the body compared with polyornithine.
The number average molecular weight of the water-soluble chitosan is preferably 10,000 to 1,000,000, more preferably 50,000 to 500,000. The number average molecular weight can be measured by gel permeation chromatography (GPC). The number average molecular weight of polyornithine is preferably 10,000 to 300,000, more preferably 10,000 to 100,000. This number average molecular weight can be measured by gel permeation chromatography (GPC).
Examples of the water-soluble chitosan include glycol chitosan obtained by bonding glycol to chitosan and aldonic acid-modified chitosan obtained by bonding aldonic acid to chitosan. Examples of the glycol include ethylene glycol, propylene glycol, diethylene glycol and the like. Examples of the aldonic acid include threonic acid, xylonic acid, gluconic acid and the like. The second layer is particularly preferably formed from glycol chitosan.
Ornithine constituting polyornithine may be any of L-form and D-form, and preferably L-form. Polyomithine may be in the form of a salt with an inorganic acid. The acid for forming the polyornithine salt may be any of an organic acid and an inorganic acid, preferably an inorganic acid. Examples of the inorganic acid include hydrobromic acid, hydrochloric acid and the like. Among these, hydrobromic acid is preferable. Polyornithine for forming the first layer is preferably poly-L-ornithine hydrobromide.
Water-soluble chitosan is available from, for example, Sigma-Aldrich. Polyomithine is available from, for example, Sigma-Aldrich.
The thickness of the second layer (one of shells) of the core-shell type bead of the present invention is preferably 0.1 to 200 μm, more preferably 1 to 100 μm. The second layer is preferably formed from at least one selected from the group consisting of polygalacturonic acid and polygalacturonic acid having mercapto groups (hereinafter sometimes to be referred to as “polygalacturonic acid and the like”). Polygalacturonic acid does not permit easy proliferation of immunocytes such as macrophage and the like thereon. Using this as the second layer (the outermost layer), a foreign-body reaction in the body can be inhibited.
The number average molecular weight of the polygalacturonic acid for forming the second layer is preferably 25,000 to 500,000, more preferably 25,000 to 300,000, further preferably 25,000 to 100,000. This number average molecular weight can be measured by gel permeation chromatography (GPC).
The polygalacturonic acid and the like for forming the second layer preferably contains a divalent metal ion. The polygalacturonic acid and the like can form the second layer even if they do not contain a divalent metal ion. Examples of the divalent metal ion include alkaline earth metal ion and the like. Among these, calcium ion, barium ion and strontium ion are preferable, and calcium ion is more preferable. Only one kind or two or more kinds of divalent metal ion may be used. The amount of the divalent metal ion in polygalacturonic acid and the like is preferably 1 to 300 mmol, more preferably 1 to 200 mmol, further preferably 1 to 100 mmol, per 1 L of the bead. This amount can be measured by atomic absorption analysis.
The polygalacturonic acid having mercapto groups for forming the second layer is preferably a galacturonic acid having a galacturonic acid residue and an amino acid residue having the mercapto group bonded to each other via an amide bond. Specific examples of the amino acid having a mercapto group include the aforementioned specific examples of compound (A1). Only one kind or two or more kinds of amino acid having a mercapto group may be used. The amino acid having a mercapto group is preferably at least one selected from the group consisting of cysteine and homocysteine, more preferably cysteine.
The proportion of the galacturonic acid unit having a mercapto group in the total constitutional units of the polygalacturonic acid having mercapto groups for forming the second layer is preferably 0.1 to 50 mol %, more preferably 0.1 to 30 mol %, further preferably 1 to 10 mol %. This proportion can be calculated from a ratio of a peak area of proton on a carbon atom bonded to a mercapto group (—SH) and a peak area of proton on the carbon skeleton of galacturonic acid, which is obtained when a sample is measured by a nuclear magnetic resonance apparatus.
The polygalacturonic acid and polygalacturonic acid having mercapto groups for forming the second layer is preferably a salt with an alkali metal (particularly salt with alkali metal hydroxide). The aforementioned alkali metal is preferably sodium and the aforementioned alkali metal hydroxide is preferably sodium hydroxide.
To form the second layer, at least one selected from the is group consisting of polygalacturonic acid bonded to a compound represented by the formula (b):
H2N-Lb2-Qb1-Lb3-Qb2-Lb4-Qb3 (b)
wherein Lb2 is a single bond or a C1-6 alkylene group,
Qb1 is a single bond, a phenylene group, or a C4-8 cycloalkanediyl group,
Lb3 is a single bond or *—(OCH2CH2)n—** (wherein * shows a bonding position to Qb1, ** shows a bonding position to Qb2, and n is an integer of 1 to 10),
Qb2 is a divalent triazole ring group,
Lb4 is a single bond, a C1-6 alkylene group, or a C1-6 alkylene-oxy group, and
Qb3 is an optionally substituted phenyl group, an optionally substituted monovalent 5- or 6-membered heterocyclic group, or an optionally substituted C4-8 cycloalkyl group, and polygalacturonic acid having mercapto groups bonded to compound (b) may also be used.
Compound (b) is the same as compound (B) except that Rb1-Lb1-CO— is not bonded to amino group. The explanation of the groups in compound (b) is the same as that for the groups in the aforementioned compound (B).
Compound (b) is preferably at least one selected from the group consisting of compound (b1), compound (b2) and compound (b3), which are represented by the following formulas, more preferably compound (b1).
Confound (b) is preferably a compound having an inhibitory action on a foreign-body reaction. Examples of the compound having an inhibitory action on a foreign-body reaction include the aforementioned compound (b1) to compound (b3). Compound (b) can be produced by a known method (e.g., method described in Nature Biotechnology, Vol 34, No. 3 (2016), 345-352, which is incorporated herein by reference in its entirety). In addition, compound (b) can be bonded to polygalacturonic acid and the like by condensing the amino group of compound (b) and the carboxy group of polygalacturonic acid and the like under conditions well known to those of ordinary skill in the art.
The bead free of an outer layer of the present invention and the bead having an outer layer of the present invention (particularly core-shell type bead of the present invention) are both useful for enclosing a cell or microorganism in the inside. Explanation of the cell mass and the average particle diameter of the bead (core) and the like in the bead having an outer layer of the present invention (particularly core-shell type bead of the present invention) is the same as that for the bead free of an outer layer of the present invention.
The core-shell type bead of the present invention can be produced by, for example, as shown below. First, the bead free of an outer layer of the present invention is produced as mentioned above, the obtained bead are mixed with an aqueous solution of the polymer for forming the first layer, the bead is allowed to stand in the aforementioned aqueous solution, the bead is taken out, the first layer is formed on the bead, the obtained bead is mixed with an aqueous solution of a polymer for forming the second layer, the bead is allowed to stand in the aforementioned aqueous solution, the bead is taken out, and the second layer is formed on the first layer, whereby the core-shell type bead of the present invention can be obtained.
When polygalacturonic acid and the like are used as a polymer for forming the second layer, a bead having the first layer is mixed with an aqueous solution of polygalacturonic acid and the like, the bead is allowed to stand in the aforementioned aqueous solution, the bead is taken out, and the obtained bead is mixed with an aqueous solution containing a divalent metal ion, whereby polygalacturonic acid and the like preferably contain a divalent metal ion.
The present invention is explained more specifically in the following by referring to Production Examples and the like. However, the present invention is not limited by the following Production Examples and the like. The present invention can be practiced with appropriate modifications as long as they can be adapted to the above-mentioned and below-mentioned gists, all of which are encompassed in the technical scope of the present invention. The room temperature indicated below means 25° C.
The following reagents were used in the below-mentioned Production Examples and so on.
polygalacturonic acid (manufactured by Sigma-Aldrich, number average molecular weight: 25,000 to 50,000)
sodium alginate (“sodium alginate 300-400” manufactured by Wako Pure Chemical Industries, Ltd., viscosity of 1 w/v % aqueous solution at 25° C.: 300 to 400 cp)
sodium alginate (“192-09995” manufactured by Wako Pure Chemical Industries, Ltd.)
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (“WSCI.HCl” manufactured by WATANABE CHEMICAL INDUSTRIES, LTD.)
N-hydroxysulfosuccinimide sodium (manufactured by Tokyo Chemical Industry Co., Ltd.)
cysteine hydrochloride monohydrate (manufactured by KANTO CHEMICAL CO., INC.)
calcium chloride dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.)
ethanol (manufactured by KANTO CHEMICAL CO., INC.)
trisodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.)
poly-L-ornithine hydrobromide (manufactured by Sigma-Aldrich, number average molecular weight: 30,000 to 70,000)
glycol chitosan (manufactured by Sigma-Aldrich, polymer obtained by bonding ethylene glycol to chitosan with number average molecular weight of 10,000 to 300,000)
For the production of beads in the below-mentioned Examples 1, 2 and 6 to 9, and Comparative Examples 1, 2 and 5, a metal needle “SNA-30G-B” (inner diameter: 0.14 mm, hereinafter to be referred to as “30G needle”) manufactured by Musashi Engineering, Inc. was used.
For the production of beads in the below-mentioned Examples 3 to 5 and Comparative Examples 3 and 4, a metal needle “SNA-28G-B” (inner diameter: 0.18 mm, hereinafter to be referred to as “28G needle”) manufactured by Musashi Engineering, Inc. was used.
For the production of beads in the below-mentioned Example 10, a syringe needle 27G “NN2725R.B” (inner diameter: 0.40 mm, hereinafter to be referred to as “27G needle”) manufactured by Terumo Corporation was used.
Polygalacturonic acid (5 g) was suspended in pure water prepared by a pure water production equipment manufactured by Millipore (hereinafter to be referred to as “pure water”) (24 mL). 1N Aqueous sodium hydroxide solution (24 mL) was slowly added to the obtained suspension at room temperature with stirring. After confirmation of complete dissolution of polygalacturonic acid, the obtained aqueous solution was freeze-dried to give sodium polygalacturonate as a white powder (5.6 g).
By reference to the method described in J Mater Sci: Mater Med (2013) 24: 1375-1382, which is incorporated herein by reference in its entirety, polygalacturonic acid having a cysteine residue (hereinafter sometimes to be abbreviated as “cysteine-polygalacturonic acid”) was produced. To be specific, sodium polygalacturonate (250 mg) obtained in Production Example 1 was dissolved in pure water (12.5 mL) at room temperature. With stirring the aqueous solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (120 mg) and N-hydroxysulfosuccinimide sodium (54 mg) were added at room temperature. Then, 1N hydrochloric acid (200 μL) was added to the obtained mixture at room temperature to adjust the pH thereof to 5, and the mixture was further stirred at room temperature. The mixture was stirred for 1 hr and cysteine hydrochloride monohydrate (125 mg) was added to the mixture at room temperature. 1N Aqueous sodium hydroxide solution (520 μL) was added to adjust the pH thereof to 4, and the mixture was further stirred at room temperature. The mixture was stirred for 2 hr, 1N aqueous sodium hydroxide solution (200 μL) was added to adjust the pH thereof to 6, and the mixture was further stirred at room temperature. The mixture was stirred for 1 hr and ethanol (50 mL) was added slowly with a pipette at room temperature to allow for precipitation. The suspension containing the precipitate was adjusted to pH 4 with 1N hydrochloric acid (400 μL).
The suspension containing the precipitate was dispensed to two centrifugal tubes and centrifuged (2500 rpm, 3 min) to allow for complete precipitation. Using a pipette, the supernatant liquid in the centrifugal tube was discarded, water (10 mL) was newly added to the centrifugal tube and the precipitate was dissolved at room temperature. Ethanol (20 mL) was added to the obtained aqueous solution at room temperature to allow for precipitation. The suspension containing the precipitate was centrifuged (2500 rpm, 3 min) to allow for complete precipitation. This centrifugation operation was performed 3 times in total.
The precipitates in the two centrifugal tubes were dissolved in water, the obtained aqueous solution was filled in a dialysis tube (Biotech CE Tubing MWCO: 8-10 kD), and dialysis was performed at room temperature using the following dialysis solution. The first dialysis was performed using a 0.15 M aqueous sodium chloride solution for 9 hr. The second dialysis was performed using a 0.15 M aqueous sodium chloride solution for 16 hr. The third dialysis was performed using 1 mM hydrochloric acid for 6 hr. After these dialyses, the aqueous solution in the dialysis tube was freeze-dried to give cysteine-polygalacturonic acid as a white powder (95 mg). The results of 1H-NMR (proton nuclear magnetic resonance) measured by the below-mentioned method are shown below.
1H-NMR (400 MHz, D2O) δ (ppm): 2.75-3.0 (m), 3.50-3.75 (m), 3.75-4.0 (m), 4.20-4.40 (m), 4.80-5.25 (m)
To confirm the effect of purification in Production Example 2, the same condensation and purification operation was performed using the same materials as in Production Example 2 except that a condensing agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was not used to avoid a condensation reaction to obtain polygalacturonic acid as a white powder (106 mg). The results of 1H-NMR (proton nuclear magnetic resonance) measured by the below-mentioned method are shown below.
1H-NMR (400 MHz, D2O) δ (ppm): 3.50-3.75 (m), 3.75-4.0 (m), 4.20-4.40 (m), 4.80-5.25 (m)
In the same manner as in Production Example 2 except that sodium alginate (“sodium alginate 300-400” manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of sodium polygalacturonate, alginic acid having a cysteine residue (hereinafter sometimes to be abbreviated as “cysteine-alginic acid”) was obtained as a white powder (100 mg). The results of 1H-NMR (proton nuclear magnetic resonance) measured by the below-mentioned method are shown below.
1H-NMR (400 MHz, D2O) δ (ppm): 2.75-2.90 (m), 3.25-4.30 (m), 4.30-5.80 (m)
To confirm the effect of purification in Production Example 4, the same condensation and purification operation was performed using the same materials as in Production Example 4 except that a condensing agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was not used to avoid a condensation reaction to obtain alginic acid as a white powder (76 mg). The results of 1H-NMR (proton nuclear magnetic resonance) measured by the below-mentioned method are shown below.
1H-NMR (400 MHz, D2O) δ (ppm): 3.25-4.30 (m), 4.30-5.80 (m)
Sodium alginate (manufactured by Wako Pure Chemical Industries, Ltd. “192-09995”) (500 mg) was dissolved in pure water (12.5 mL) at room temperature. With stirring the aqueous solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (240 mg) and N-hydroxysulfosuccinimide sodium (108 mg) were added at room temperature. Then, 1N hydrochloric acid (400 μL) was added to the obtained mixture at room temperature to adjust the pH thereof to 5, and the mixture was further stirred at room temperature. The mixture was stirred for 1 hr and cysteine hydrochloride monohydrate (1000 mg) was added to the mixture at room temperature. 1N Aqueous sodium hydroxide solution (4.5 mL) was added to adjust the pH thereof to 4, and the mixture was further stirred at room temperature. The mixture was stirred for 2 hr, 1N aqueous sodium hydroxide solution (1200 μL) was added to adjust the pH thereof to 6, and the mixture was further stirred at room temperature. The mixture was stirred for 1 hr and 1N hydrochloric acid (600 μL) was added to adjust the pH thereof to 4. Ethanol (100 mL) was added to the mixture slowly with a pipette at room temperature to allow for precipitation.
The suspension containing the precipitate was dispensed to 4 centrifugal tubes and centrifuged (2500 rpm, 3 min) to allow for complete precipitation. The supernatant liquid in the centrifugal tube was discarded, water (10 mL) was newly added to the centrifugal tube and the precipitate was dissolved by shaking well. Ethanol (20 mL) was added to the obtained aqueous solution at room temperature to allow for precipitation. The suspension containing the precipitate was centrifuged (2500 rpm, 3 min) to allow for complete precipitation. This centrifugation operation was performed 3 times in total.
The precipitates in the 4 centrifugal tubes were dissolved in water, the obtained aqueous solution was filled in two dialysis tubes (Biotech CE Tubing MWCO: 8-10 kD), and dialysis was performed at room temperature using the following dialysis solution. The first dialysis was performed using a 0.15 M aqueous sodium chloride solution and 1 mM hydrochloric acid for 8 hr. The second dialysis was performed using a 0.15 M aqueous sodium chloride solution and 1 mM hydrochloric acid for 17 hr. The third dialysis was performed using 1 mM hydrochloric acid for 6 hr. After these dialyses, the aqueous solutions in the dialysis tubes were freeze-dried to give cysteine-alginic acid as a white powder (304 mg). The results of 1H-NMR measured by the below-mentioned method are shown below.
1H-NMR (400 MHz, D2O) δ (ppm): 2.75-2.90 (m), 3.25-4.30 (m), 4.30-5.80 (m)
Compound (b1) (i.e., 2-[2-[2-[2-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]triazol-1-yl]ethoxy]ethoxy]ethoxy]ethaneamine) was produced as described in the experiment of Nature Biotechnology, Vol 34, No. 3 (2016), 345-352, which is incorporated herein by reference in its entirety.
To a solution of compound (b1) (0.965 g, 2.46 mmol) in dimethylformamide (DMF) (20 mL) were added N,N-diisopropylethylamine (0.637 mL, 3.70 mmol) and compound (b2) (i.e., 3-maleimidopropionic acid N-hydroxysuccinimide ester) (0.655 g, 2.46 mmol), and the mixture was stirred at room temperature overnight. To the reaction mixture was added 0.1N hydrochloric acid (20 mL) and the mixture was extracted with ethyl acetate. The solvent was evaporated and the obtained residue was purified by high performance liquid chromatography (water-acetonitrile, each containing 0.1% by volume trifluoroacetic acid) to give compound (B1) (i.e., 2-[3-[2-[2-[2-[2-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]triazol-1-yl]ethoxy]ethoxy]ethoxy]ethylamino]but-3-enyl]cyclopenta-4-en-1,3-dione) (0.398 g, 0.733 mmol, yield 30%). The results of MS (mass spectrum) and 1H-NMR measured by the below-mentioned methods are described below.
MS(ESI) m/z 543 (M+H)+
1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.00 (s, 1H), 4.51 (t, J=5.3 HZ, 1H), 3.82 (t, J=5.3 Hz, 1H), 3.77 (s, 1H), 3.60 (dd, J=7.9, 6.6 Hz, 1H), 3.54-3.50 (m, 1H), 3.50-3.46 (m, 3H), 3.41-3.30 (m, 4H), 3.20-3.07 (m, 3H), 2.96-2.87 (m, 2H), 2.72 (s, 1H), 2.67-2.43 (m, 12H), 2.37 (s, 1H), 2.33 (m, 1H).
Alginic acid bonded to compound (b1) was produced under the conditions described in the experiment of Nature Biotechnology, Vol 34, No. 3 (2016), 345-352, which is incorporated herein by reference in its entirety. The aforementioned alginic acid was purified by the operation described in Production Example 2. The results of 1H-NMR measured by the below-mentioned method are shown below.
1H-NMR (400 MHz, D2O) δ (ppm): 8.06-7.92 (m), 5.29-4.31 (m), 4.22-3.27 (m), 3.26-3.13 (m), 3.07-2.91 (m)
Sodium polygalacturonate (250 mg) was dissolved in 0.1 M MES buffer (50 mL, pH:6.0) at room temperature. While stirring the obtained aqueous solution at room temperature, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (241 mg) and N-hydroxysulfosuccinimide sodium (137 mg) were added and the mixture was further stirred for 1 hr to give solution 1.
Then, compound (b1) (125 mg) was dissolved in a mixed solvent of pure water (1.5 mL) and acetonitrile (1.5 mL) to prepare solution 2. Solution 2 wad added to the aforementioned solution 1 and the obtained mixture was stirred at room temperature for 20 hr. After stirring, ethanol (80 mL) was added slowly to the mixture with a pipette at room temperature to allow for precipitation to give a suspension.
The obtained suspension was dispensed to two centrifugal tubes and centrifuged (2500 rpm, 3 min) to allow for complete precipitation. Using a pipette, the supernatant liquid in the centrifugal tube was discarded, water (10 mL) was newly added to the centrifugal tube and the precipitate was dissolved at room temperature. The obtained aqueous solution was filled in a dialysis tube (Biotech CE Tubing MWCO: 8-10 kD) and dialysis was performed 3 times at room temperature using a dialysis solution of a mixture of 0.15 M aqueous sodium chloride solution and 1 mM hydrochloric acid. Respective dialysis times were 4 hr, 18 hr and 6 hr. Then using 1 mM hydrochloric acid as a dialysis solution, dialysis was performed for 7 hr. After these dialyses, the aqueous solution in the dialysis tube was freeze-dried to give polygalacturonic acid bonded to compound (b1) as a white powder (260 mg).
The results of 1H-NMR measured by the below-mentioned method are shown below.
1H-NMR (400 MHz, D2O) δ (ppm): 8.10-7.90 (m), 5.10-4.90 (m), 4.90-4.40 (m), 4.40-4.10 (m), 4.10-3.80 (m), 3.80-3.65 (m), 3.65-3.45 (m), 3.26-3.20 (m), 3.05-2.90 (m)
Each white powder (20 mg) obtained in Production Examples 2 to 6, compound (B1) (20 mg) obtained in Production Example 8, compound (b1)-bonded alginic acid (20 mg) obtained in Production Example 9 and compound (b1)-bonded polygalacturonic acid bonded obtained in Production Example 10 was suspended in D2O (1 mL). 1N NaOD solution (solvent: D2O) (40 μL) was added to the obtained suspension and the white powder was dissolved. 1H-NMR of the obtained solution was measured using 400 MHz nuclear magnetic resonance analysis device manufactured by Bruker.
A peak of cysteine was not observed in 1H-NMR of the white powders of polygalacturonic acid and alginic acid obtained in Production Examples 3 and 5. Therefrom it was confirmed that the material (free cysteine) used for producing cysteine-polygalacturonic acid or cysteine-alginic acid can be completely removed by the purification operation performed in Production Examples 2 and 4.
A peak of cysteine was observed in δ 2.75-3.00 in 1H-NMR of the white powder obtained in Production Example 2. Complete removal of free cysteine by purification was confirmed from the 1H-NMR results of Production Example 3 (control synthesis). Thus, the aforementioned peak is derived from the cysteine residue covalently bonded to the galacturonic acid.
These results confirm that the white powder obtained in Production Example 2 was polygalacturonic acid having a cysteine residue.
From the results of 1H-NMR, the proportion of the galacturonic acid unit having a mercapto group in the total constitutional units of cysteine-polygalacturonic acid was calculated to be 4 mol %. From this proportion and the number JO average molecular weight of polygalacturonic acid as the material, the number average molecular weight of cysteine-polygalacturonic acid was calculated to be 25,600 to 51,300.
A peak of cysteine was observed in δ 2.70-2.90 in 1H-NMR of the white powder obtained in Production Example 4. Complete removal of free cysteine by purification was confirmed from the 1H-NMR results of Production Example 5 (control synthesis). Thus, the aforementioned peak is derived from the cysteine residue covalently bonded to the alginic acid. These results confirm that the white powder obtained in Production Example 4 was alginic acid having a cysteine residue.
A solution of compound (B1) obtained in Production Example 8 in dimethyl sulfoxide (DMSO) (concentration: 100 μM) was prepared. To the obtained solution (10 μL) was added acetonitrile (330 μL) to prepare a diluted solution. Using the obtained diluted solution and ACQUITY UPLC/SQD system (manufactured by WATERS), MS of compound (B1) was measured.
Sodium alginate was dissolved in pure water to prepare a 2 w/v % aqueous sodium alginate solution. The obtained 2 w/v % aqueous sodium alginate solution was placed in a 5 mL syringe. A 30G needle was set to the syringe and droplets of the aqueous sodium alginate solution were added dropwise to 100 mM aqueous calcium chloride solution at room temperature to produce alginate beads.
Pure water (20 mL) was added to polygalacturonic acid (1.0 g) at room temperature to prepare a suspension. 1N Aqueous sodium hydroxide solution (4.7 mL) was slowly added at room temperature to the obtained suspension with stirring to prepare a transparent aqueous sodium polygalacturonate solution. The obtained aqueous solution was placed in a 5 mL syringe. A 30G needle was set to the syringe and droplets of the aqueous sodium polygalacturonate solution were added dropwise to 100 mM aqueous calcium chloride solution (25 mL) at room temperature to produce polygalacturonate beads.
Cysteine-polygalacturonic acid (40 mg) was suspended in pure water (1 mL) at room temperature. 1N Aqueous sodium hydroxide solution (150 μL) was added to the obtained suspension, and the mixture was stirred at room temperature until a transparent aqueous solution was obtained to prepare an aqueous sodium cysteine-polygalacturonate solution. A 30G needle was set to 1 mL syringe and droplets of the aqueous sodium cysteine-polygalacturonate solution were slowly added dropwise to 100 mM aqueous calcium chloride solution at room temperature to produce cysteine-polygalacturonate beads.
An average particle diameter of the obtained cysteine-polygalacturonate bead was 1.6 mm. The average particle diameter was measured by the below-mentioned microscope and a digital camera. To be specific, using the microscope and a digital camera (magnification: ×4), microphotographs of the beads were taken, 5 beads were randomly selected, the maximum diameters in the photograph of the selected 5 beads were calculated with the software attached to the digital camera, and the average particle diameter of the beads (average maximum diameter of 5 beads) was calculated.
Cysteine-alginic acid (20 mg) was suspended in pure water (1 mL) at room temperature. 1N Aqueous sodium hydroxide solution (150 μL) was added to the obtained suspension, and the mixture was stirred at room temperature until a transparent aqueous solution was obtained to prepare an aqueous sodium cysteine-alginate solution. A 30G needle was set to 1 mL syringe and droplets of the aqueous sodium cysteine-alginate solution were added dropwise to 100 mM aqueous calcium chloride solution at room temperature to produce sodium cysteine-alginate beads. The average particle diameter of the cysteine-alginate bead measured in the same manner as in Example 1 was 1.7 mm.
100 mM Aqueous calcium chloride solution was added by 160 μL to a 96-well plate. 4 w/v % Aqueous sodium cysteine-polygalacturonate solution or 4 w/v % aqueous sodium polygalacturonate solution was added by 20 to each well to produce one bead in each well. Immediately after (standing for 0 hr) bead production at room temperature and after standing for 24 hr from bead production, PBS solution for SH group measurement [mixed solution of Dulbecco's phosphate buffered saline (D-PBS(−)) (15 mL), pure water (25 mL) and 1N NaOH aqueous solution (50 μL)] (160 μL) and a solution (20 μL) obtained by dissolving 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (39.6 mg) in Dulbecco's phosphate buffered saline (D-PBS(−)) (10 mL) were added to each well, and the mixture was further allowed to stand in a dark place for 1 hr. Thereafter, the supernatant (50 μL) was recovered from each well, diluted with pure water (150 μL), and the absorbance was determined (λmax=412 nm) using microplate reader Benchmark Plus (BIO-RAD). The mercapto group concentration was calculated using an analytical curve obtained by measuring various concentrations of cysteine by a similar method. In this Experimental Example, mercapto group concentration of 8 wells was measured and an average thereof was determined.
In the well in which a bead was produced from 4 w/v % aqueous sodium polygalacturonate solution and aqueous calcium chloride solution, the mercapto group was not detected at all both at stand 0 and after 24 hr.
In the well in which a bead was produced from 4 w/v % aqueous sodium cysteine-polygalacturonate solution and aqueous calcium chloride solution, the mercapto group concentration was 0.58 μM at stand 0 hr. On the other hand, after standing for 24 hr, the mercapto group concentration decreased to 0.17 μM. Therefrom it was clarified that the mercapto groups (—SH) changed to a disulfide bond (—S—S—) and the polymer chain was crosslinked for cysteine-polygalacturonic acid. It is considered that a disulfide bond is not formed at stand 0 hr. Thus, the proportion of the mercapto group forming the disulfide bond in the total mercapto groups is calculated to be 100×(0.58−0.17)/0.58=70.6 mol % in the cysteine-polygalacturonic acid bead after standing for 24 hr.
For the production of an alginate bead and the like, a divalent metal ion, particularly Ca2, is often used. On the other hand, the Ca2+ concentration in the body is about 2 mM at maximum. Thus, when alginate bead containing Ca2+ and the like are transplanted into a living organism, the Ca2+ concentration in the bead is considered to decrease to 2 mM due to equilibrium reaction. As a result, the strength of the bead may decrease in the body. Accordingly, durability of the beads of Comparative Examples 1 and 2 and Examples 1 and 2 under low Ca2+ conditions was evaluated.
Ca2+ in the beads can be removed using sodium citrate since sodium citrate has the ability to bind to divalent metal ion. Thus, the obtained beads were immersed in 55 mM aqueous sodium citrate solution and the shape of the beads was observed. To be specific, 5 beads each of Comparative Examples 1 and 2 and Examples 1 and 2 were put in the wells of a 24-well plate, and 55 mM aqueous sodium citrate solution (1 mL) was added to each well such that the beads were entirely immersed in the aqueous sodium citrate solution. The 24-well plate was shaken for up to 30 hr on a rotary shaker and the shape of the beads was observed.
The following microscope (magnification: ×4) and a digital camera were used for observation of the beads. The bead area in the microphotographs taken with the digital camera (hereinafter to be referred to as “bead area”) was measured with the software attached to the digital camera. The area of beads each of the beads obtained in Comparative Examples 1 and 2 and Examples 1 and 2 was measured and an average was calculated. The results thereof are shown in Table 1.
culture microscope “CKX41” manufactured by OLYMPUS
microscope digital camera “DP22” manufactured by OLYMPUS
The alginate bead of Comparative Example 1 dissolved and completely disappeared after shaking for 30 min in 55 mM aqueous sodium citrate solution.
The polygalacturonate beads of Comparative Example 2 had a bead area of 3.52 mm2 before shaking in the 55 mM aqueous sodium citrate solution. The bead area increased to 4.37 mm2 after shaking for 30 min, and increased to 6.82 mm2 after JO shaking for 18 hr. After shaking for 24 hr, the bead was further swelled and deformed and the bead area could not be measured.
The cysteine-polygalacturonate beads of Example 1 had a bead area of 2.49 mm2 before shaking in the aqueous sodium citrate solution. The bead area was not more than 2.5 mm2 and did not increase even after shaking for 30 min, 3 hr, 18 hr, 24 hr and 30 hr, and the bead shape remained unchanged. As is clear from these results, the cysteine-polygalacturonate bead did not swell at all under low Ca2+ conditions.
The cysteine-alginate beads of Example 2 had a bead area of 2.49 mm2 before shaking in the 55 mM aqueous sodium citrate solution. The bead area increased to 5.76 mm2 after shaking for 30 hr but the bead shape was maintained. On the other hand, the alginate bead of Comparative Example 1 dissolved and disappeared after shaking for 30 min, and the polygalacturonate bead of Comparative Example 2 was swelled and deformed after shaking for 24 hr. As is clear from these results, swelling thereof can be inhibited by introducing a cysteine residue into the alginate bead.
As is clear from the comparison of Example 1 and Example 2, a bead showing still more superior durability can be obtained using a polymer containing galacturonic acid units (particularly, polygalacturonic acid) as a polymer containing uronic acid units.
Cells stably expressing Gα15-tarans48 LD derived from PEAKRAPID (PRG48) and maintained using DMEM/Ham's F-12 (manufactured by NACALAI TESQUE, INC.) containing 10 v/v % fetal bovine serum (manufactured by Nichirei Corporation) and 1 v/v % penicillin-streptomycin (manufactured by NACALAI TESQUE, INC.) were washed with D-PBS(−) (manufactured by NACALAI TESQUE, INC.) and the cells were recovered from 150 mm TC-treated Culture Dish (manufactured by CORNING) by using trypsin-EDTA solution [solution obtained by adding 0.25 g/L aqueous trypsin-1 mmol/L EDTA (manufactured by NACALAI TESQUE, INC.) solution to D-PBS(−) (manufactured by NACALAI TESQUE, INC.) at 20 v/v %]. The cell suspension was centrifuged (1,200 rpm, 3 min), the supernatant was removed, and the cells obtained by centrifugation were suspended in D-PBS(−) (manufactured by NACALAI TESQUE, INC.). The obtained suspension was centrifuged (1,200 rpm, 3 min), the supernatant was removed, and the cells were suspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (115 mM NaCl, 5 mM KCl, 5 mM D-glucose, 15 mM HEPES, pH 7.4) at 1.0×107 cells/mL. The obtained cell suspension (200 μL) was added to 4 w/v % aqueous sodium cysteine-polygalacturonate solution (1.8 mL) at room temperature to give a suspension (polymer concentration: 3.6 w/v %, cell mass: 1.0×107 cells/mL). The obtained suspension was placed in a 1 mL syringe (Terumo) with 28G needle and added dropwise to 100 mM aqueous calcium chloride solution (25 mL) at room temperature to give beads enclosing the cells in the inside (cell mass in bead: 1.0×107 cells/mL).
The average particle diameter of the obtained bead enclosing the cells in the inside was 1.8 mm. This was measured in the same manner as in Example 1.
The obtained bead was washed 3 times with DMEM/Ham's F-12 (manufactured by NACALAI TESQUE, INC.), and the bead enclosing cell in the inside was placed in DMEM/Ham's F12 (manufactured by NACALAI TESQUE, INC.) and the cell was cultured (37° C. 5% CO2) for 14 days.
In the same manner as in Example 3 except that 4 w/v % aqueous sodium alginate solution was used instead of 4 w/v % aqueous sodium cysteine-polygalacturonate solution, a bead enclosing cell in the inside was obtained. In the same manner as in Example 3, the cell was cultured for 14 days.
Five cell-enclosing beads of Example 3 and Comparative Example 3 after cell culture on day 1 and day 14 were placed in each well of a 48-well plate and washed twice with D-PBS(−) (manufactured by NACALAI TESQUE, INC.) (300 μL). Thereafter, to each well were added D-PBS(−) (manufactured by NACALAI TESQUE, INC.) (150 μL), fluorescein diacetate (FDA) solution (solution obtained by adding 0.5 mg/mL FDA solution (solvent: dimethyl sulfoxide (DMSO)) (10 μL) to D-PBS(−) (manufactured by NACALAI TESQUE, INC.) (5 mL)) (150 μL), and propidium iodide (PI) solution (solution obtained by adding 1.0 mg/mL aqueous PI solution (10 μL) to D-PBS(−) (manufactured by NACALAI TESQUE, INC.) (5 mL)) (150 μL), and the mixture was allowed to stand under 37° C. 5% CO2 conditions for 30 min. Living cells were stained green and dead cells were stained red. After standing, each well was washed with D-PBS(−) and observed under a fluorescence microscope BZ—X700 (manufactured by KEYENCE).
The proportion of the living cells after 14 days of culture to the living cells after 1 day of culture was 95% for the cells in the cell-enclosing cysteine-polygalacturonate bead obtained in Example 3. On the other hand, the proportion for the cells in the cell-enclosing alginate bead obtained in Comparative Example 4 was 60%. It was confirmed from these results that the cells in the cell-enclosing cysteine-polygalacturonate bead could survive at least equal to the cells in the cell-enclosing alginate bead up to day 14 of culture.
Cysteine-polygalacturonic acid (45 mg) obtained in the same manner as in Production Example 2 was suspended in pure water (2.84 mL) at room temperature. 1N Aqueous sodium hydroxide solution (160 μL) was added to the obtained suspension, and the mixture was stirred at room temperature until a transparent aqueous solution was obtained to prepare a 1.5 w/v % aqueous sodium cysteine-polygalacturonate solution. Using a 1 mL syringe with a 28G needle, the obtained 1.5 w/v % aqueous sodium cysteine-polygalacturonate solution was added by μL to 100 mM aqueous calcium chloride solution (20 mL) to produce cysteine-polygalacturonate beads.
After production as mentioned above, a mixture of bead, water and the like was allowed to stand at room temperature for 24 hr, a solution (20 μL) of 100 mM compound (B1) in dimethyl sulfoxide (DMSO) was added to the mixture, and the obtained mixture was allowed to stand at room temperature for 24 hr to produce a bead of cysteine-polygalacturonate bonded to compound (B1).
In the same manner as in Example 4 except that 1.5 w/v % aqueous sodium cysteine-alginate solution was prepared using cysteine-alginic acid obtained in the same manner as in Production Example 6 instead of cysteine-polygalacturonic acid, a cysteine-alginate bead and a bead of cysteine-alginate bonded to compound (B1) were produced.
Immediately after (standing for 0 hr) bead production at room temperature, after standing for 24 hr (standing for 24 hr) from bead production, and after standing for 24 hr at room temperature from addition of compound (B1) (24 hr from addition of compound (B1)), one bead was placed in each well of a 96-well plate, PBS solution for SH group measurement [mixed solution of Dulbecco's phosphate buffered saline (D-PBS(−)) (15 mL), pure water (25 mL) and 1N NaOH aqueous solution (50 μL)](160 μL) and a solution (20 μL) obtained by dissolving 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (39.6 mg) in Dulbecco's phosphate buffered saline (D-PBS(−)) (10 mL) were added to each well, and the mixture was further allowed to stand in a dark place for 1 hr. Thereafter, the supernatant (50 μL) was recovered from each well, diluted with pure water (150 μL), and the absorbance was determined (λmax=412 nm) using microplate reader Benchmark Plus (BIO-RAD). The mercapto group concentration was calculated using an analytical curve obtained by measuring various concentrations of cysteine by a similar method. In this Experimental Example, the mercapto group concentration of 4 wells was measured and an average thereof was determined. The results thereof are shown in Tables 2 and 3.
Both cysteine-polygalacturonate bead and cysteine-alginate bead showed a decrease in the mercapto group concentration 24 hr after production of the bead. The decrease indicates that the mercapto groups (—SH) in the bead changed to a disulfide bond (—S—S—).
The bead after 24 hr from the addition of compound (B1) to the mixture containing cysteine-polygalacturonate bead or cysteine-alginate bead showed a further decrease in the mercapto group concentration. The decrease shows that a functional group capable of bonding to a mercapto group in compound (B1) (i.e., 1-maleimidyl group) and mercapto group in the bead reacted and a bead bonded to compound (B1) was obtained.
Compound (b1)-bonded alginic acid (15 mg) was suspended in pure water (1 mL) at room temperature. 1N Aqueous sodium hydroxide solution (80 μL) was added to the obtained suspension, and the mixture was stirred at room temperature until a transparent aqueous solution was obtained to prepare an aqueous solution of sodium alginate bonded to compound (b1). A 28G needle was set to 1 mL syringe and droplets of the aqueous solution of sodium alginate bonded to compound (b1) were added dropwise to 100 mM aqueous calcium chloride solution at room temperature to produce beads of alginate bonded to compound (b1).
An alginate bead produced in the same manner as in Comparative Example 1 except that 1.5 w/v % aqueous sodium alginate solution was prepared, a bead of alginate bonded to compound (b1) and produced in Comparative Example 4, and a bead of cysteine-alginate bonded to compound (B1) and produced in Example 5 were allowed to stand in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) at 4° C. overnight. In the same manner as in Experimental Example 2 except that the area of 3 beads was measured, average bead areas after standing for 0 hr and 20 hr were determined. The results thereof are shown in Table 4.
As shown in Table 4, the bead of cysteine-alginate bonded to compound (B1) has a smaller bead area after standing for 20 hr and shows superior durability compared to the alginate bead and the bead of alginate bonded to compound (b1).
Sodium alginate was dissolved in pure water to prepare a 1.5 w/v % aqueous sodium alginate solution. The obtained 1.5 w/v % aqueous sodium alginate solution was placed in a 1 mL syringe. A 30G needle was set to the syringe and droplets of the aqueous sodium alginate solution were added dropwise to 100 mM aqueous calcium chloride solution at room temperature to produce alginate beads.
After 10 min from dropwise addition of the aqueous sodium alginate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). Then, an aqueous solution (5 mL) of 0.1 w/v % poly-L-ornithine hydrobromide was added to the washed beads. After standing for 10 min from the addition, the supernatant liquid of the suspension containing the beads was discarded, and the obtained polyornithine salt (first layer)/alginate beads were washed 3 times with pure water (5 mL).
Successively, 0.25 w/v % aqueous sodium alginate solution (5 mL) was added to the washed beads. After standing for 10 min from the addition, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). Then, 100 mM aqueous calcium chloride solution (5 mL) was added to the obtained beads to give alginate (second layer)/polyornithine salt (first layer)/alginate bead.
Cysteine-alginic acid (90 mg) was suspended in pure water (6 mL) at room temperature. 1N Aqueous sodium hydroxide solution (140 μL) was added to the obtained suspension to prepare 1.5 w/v % aqueous sodium cysteine-alginate solution with pH 6. The obtained aqueous solution was placed in a 1 mL syringe. A 30G needle was set to the syringe and droplets of the aqueous sodium cysteine-alginate solution were added dropwise to 100 mM aqueous calcium chloride solution (25 mL) at room temperature to produce cysteine-alginate beads.
After 10 min from dropwise addition of the aqueous sodium cysteine-alginate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). Then, an aqueous solution (5 mL) of 0.1 w/v % poly-L-omithine hydrobromide was added to the washed beads. After standing for min from the addition, the supernatant liquid of the suspension containing the beads was discarded, and the obtained polyornithine salt (first layer)/cysteine-alginate beads were washed 3 times with pure water (5 mL).
Successively, 0.1 w/v % aqueous sodium polygalacturonate solution (5 mL) was added to the washed beads. This aqueous sodium polygalacturonate solution was prepared by suspending polygalacturonic acid (10 mg) in pure water (10 mL) and adding 1N aqueous sodium hydroxide solution (25 μL) to the obtained suspension.
After standing for 10 min from the addition of the aqueous sodium polygalacturonate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). A 100 mM aqueous calcium chloride solution (5 mL) was added to the obtained beads to give polygalacturonate (second layer)/polyornithine salt (first layer)/cysteine-alginate beads.
Cysteine-alginic acid (90 mg) was suspended in pure water (6 mL) at room temperature. 1N Aqueous sodium hydroxide solution (140 μL) was added to the obtained suspension to prepare 1.5 w/v % aqueous sodium cysteine-alginate solution with pH 6. The obtained aqueous solution was placed in a 1 mL syringe. A 30G needle was set to the syringe and droplets of the aqueous sodium cysteine-alginate solution were added dropwise to 100 mM aqueous calcium chloride solution at room temperature to produce cysteine-alginate beads.
After 10 min from dropwise addition of the aqueous sodium cysteine-alginate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). Then, 0.1 w/v % aqueous glycol chitosan solution (5 mL) was added to the obtained beads. After standing for 10 min from the addition, the supernatant liquid of the suspension containing the beads was discarded, and the obtained glycol chitosan (first layer)/cysteine-alginate beads were washed 3 times with pure water (5 mL).
Successively, 0.1 w/v % aqueous sodium polygalacturonate solution (5 mL) was added to the washed beads. This aqueous sodium polygalacturonate solution was prepared by suspending polygalacturonic acid (10 mg) in pure water (10 mL) and adding 1N aqueous sodium hydroxide solution (25 μL) to the obtained suspension.
After standing for 10 min from the addition of the aqueous sodium polygalacturonate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). A 100 mM aqueous calcium chloride solution (5 mL) was added to the obtained beads to give polygalacturonate (second layer)/glycol chitosan (first layer)/cysteine-alginate beads.
Cysteine-alginic acid (90 mg) was suspended in pure water (6 mL) at room temperature. 1N Aqueous sodium hydroxide solution (140 μL) was added to the obtained suspension to prepare 1.5 w/v % aqueous sodium cysteine-alginate solution with pH 6. The obtained 1.5 w/v % aqueous sodium cysteine-alginate solution was placed in a 1 mL syringe. A 30G needle was set to the syringe and droplets of the aqueous sodium cysteine-alginate solution were added dropwise to 100 mM aqueous calcium chloride solution (25 mL) at room temperature to produce cysteine-alginate beads.
After 10 min from dropwise addition of the aqueous sodium alginate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). Then, 0.1 w/v % aqueous glycol chitosan solution (5 mL) was added to the obtained beads. After standing for 10 min from the addition, the supernatant liquid of the suspension containing the beads was discarded, and the obtained glycol chitosan (first layer)/cysteine-alginate beads were washed 3 times with pure water (5 mL).
Successively, 0.1 w/v % aqueous sodium cysteine-polygalacturonate solution (5 mL) was added to the washed beads. This aqueous sodium cysteine-polygalacturonate solution was prepared by suspending cysteine-polygalacturonic acid (10 mg) in pure water (10 mL) and adding 1N aqueous sodium hydroxide solution (25 μL) to the obtained suspension.
After standing for 10 min from the addition of the aqueous sodium cysteine-polygalacturonate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). A 100 mM aqueous calcium chloride solution (5 mL) was added to the obtained beads to give cysteine-polygalacturonate (second layer)/glycol chitosan (first layer)/cysteine-alginate beads.
Cysteine-alginic acid (90 mg) was suspended in pure water (6 mL) at room temperature. 1N Aqueous sodium hydroxide solution (140 μL) was added to the obtained suspension to prepare 1.5 w/v % aqueous sodium cysteine-alginate solution with pH 6. The obtained 1.5 w/v % aqueous sodium cysteine-alginate solution was placed in a 1 mL syringe. A 30G needle was set to the syringe and droplets of the aqueous sodium cysteine-alginate solution were added dropwise to 100 mM aqueous calcium chloride solution at room temperature to produce cysteine-alginate beads.
After 10 min from dropwise addition of the aqueous sodium alginate solution, the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). Then, 0.1 w/v % aqueous glycol chitosan solution (5 mL) was added to the obtained beads. After standing for 10 min from the addition, the supernatant liquid of the suspension containing the beads was discarded, and the obtained glycol chitosan (first layer)/cysteine-alginate beads were washed 3 times with pure water (5 mL).
Successively, aqueous solution (5 mL) of 0.1 w/v % sodium polygalacturonate bonded to compound (b1) was added to the washed beads. This aqueous solution of sodium polygalacturonate bonded to compound (b1) was prepared by suspending compound (b1)-bonded polygalacturonic acid (10 mg) in pure water (10 mL) and adding 1N aqueous sodium hydroxide solution (50 μL) to the obtained suspension.
After standing for 10 min from the addition of the aqueous solution of sodium cysteine-polygalacturonate bonded to compound (b1), the supernatant liquid of the suspension containing the beads was discarded and the obtained beads were washed 3 times with pure water (5 mL). A 100 mM aqueous calcium chloride solution (5 mL) was added to the obtained beads to give cysteine-polygalacturonate bonded to compound (b1) (second layer)/glycol chitosan (first layer)/cysteine-alginate beads.
Experimental Example 6: Durability Evaluation Respective beads obtained in Comparative Example 5 and Examples 6 to 9 were immersed in 55 mM aqueous sodium citrate solution or 10 mM aqueous EDTA solution and the shape of the beads was observed. To be specific, 3 beads each were put in a 48-well plate, and 55 mM aqueous sodium citrate solution or 10 mM aqueous EDTA solution (each 400 μL) was added to each well such that the beads were entirely immersed in the solution. The 48-well plate was shaken for up to 180 min on a rotary shaker and the shape of the beads was observed.
The following microscope (magnification: ×4) and a digital camera were used for observation of the beads. The bead area in the microphotographs taken with the digital camera (hereinafter to be referred to as “bead area”) was measured with the software attached to the digital camera. The area of 3 beads each was measured and an average was calculated.
culture microscope “CKX41” manufactured by OLYMPUS
microscope digital camera “DP22” manufactured by OLYMPUS
The swelling rate after shaking was calculated from an average bead area before shaking and an average bead area after shaking and by the following formula:
swelling rate (%) after shaking=100×average bead area after shaking/average bead area before shaking
The results of the swelling rate after shaking for each time are shown in Tables 5 and 6.
Human mesenchymal stem cells (hMSC) (manufactured by Lonza Japan Ltd.) maintained using DMEM medium (manufactured by Sigma Chemical Company) containing 10 v/v % fetal bovine serum (manufactured by Nichirei Corporation) and 1 v/v % penicillin-streptomycin (manufactured by NACALAI TESQUE, INC.) were washed with D-PBS(−) (manufactured by NACALAI TESQUE, INC.), the cells were recovered from 175 mm2 flask (manufactured by Falcon) by using TrypLE™ Select (manufactured by Life Technologies), and an equal amount or more of a culture medium was added. The cell suspension was centrifuged (1,000 rpm, 3 min), the supernatant was removed, and the cells obtained by centrifugation were suspended in D-PBS(−) (manufactured by NACALAI TESQUE, INC.). The obtained suspension was centrifuged (1,000 rpm, 3 min), the supernatant was removed, and the cells were suspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (115 mM NaCl, 5 mM KCl, 5 mM D-glucose, 15 mM HEPES, pH 7.4) at 1.0×107 cells/mL. The obtained cell suspension (200 μL) was added to 1.5 w/v % aqueous sodium cysteine-alginate solution (1.8 mL) at room temperature to give a suspension (cell mass: 1.0×105 cells/mL). The obtained suspension was placed in a 1 mL syringe (Terumo) with 27G needle and added dropwise to 100 mM aqueous calcium chloride solution (25 mL) at room temperature to give beads enclosing the cells in the inside (cell mass in bead: 1.0×105 cells/mL).
The obtained bead was washed twice with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (5 mL), 0.1 w/v % glycol chitosan solution was added to the washed beads and the mixture was allowed to stand for 10 min. The 0.1 w/v % aqueous glycol chitosan solution was removed, and the obtained bead was washed twice with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (5 mL). The 0.1 w/v % glycol chitosan solution was prepared by dissolving glycol chitosan (3 mg) in 3 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (115 mM NaCl, 5 mM KCl, 5 mM D-glucose, 15 mM HEPES, pH 7.4).
A 0.15 w/v % aqueous sodium polygalacturonate solution was added to the washed beads and the mixture was allowed to stand for 5 min. The 0.15 w/v % aqueous sodium polygalacturonate solution was removed and the obtained bead was washed 3 times with DMEM/Ham's F-12 (manufactured by NACALAI TESQUE, INC.) to give polygalacturonate (second layer)/glycol chitosan (first layer)/cysteine-alginate bead enclosing human mesenchymal stem cell (hMSC) in the inside.
The beads enclosing the cells in the inside obtained as mentioned above were placed in DMEM medium (manufactured by Sigma Chemical Company) containing 10 v/v % fetal bovine serum (manufactured by Nichirei Corporation) and 1 v/v % penicillin-streptomycin (manufactured by NACALAI TESQUE, INC.) and the cells were cultured (37° C. 5% CO2) for 14 days.
The proportion of the living cells after 14 days of culture to the living cells after 1 day of culture was not less than 95% for the cells in the obtained bead.
A 1 w/v % aqueous sodium alginate solution (50 μL) or 1.5 w/v % aqueous sodium polygalacturonate solution (50 μL) was added to a 96-well poly-L-lysine coated plate (manufactured by IWAKI & CO., LTD.) and the mixture was allowed to stand (37° C., 5% CO2) for 1 hr. A 100 mM aqueous calcium chloride solution (150 μL) was added to each well of the plate, and the mixture was allowed to stand at room temperature for 10 min. The aqueous calcium chloride solution in each well was removed and the cells were washed twice with DMEM/Ham's F-12 (manufactured by NACALAI TESQUE, INC.) (200 μL) containing 10 v/v % fetal bovine serum (manufactured by Nichirei Corporation) and 1 v/v % penicillin-streptomycin (manufactured by NACALAI TESQUE, INC.).
RAW264.7 cells (manufactured by DS Pharma Biomedical Co., Ltd.) maintained using DMEM/Ham's F-12 (manufactured by Sigma Chemical Company) containing 10 v/v % fetal bovine serum (manufactured by Nichirei Corporation) and 1 v/v % penicillin-streptomycin (manufactured by NACALAI TESQUE, INC.) were washed with D-PBS(−) (manufactured by NACALAI TESQUE, INC.), the cells were recovered from 100 mm TC-treated culture dish (manufactured by IWAKI & CO., LTD.) by using 0.25 g/L trypsin-1 mmol/L EDTA (manufactured by NACALAI TESQUE, INC.), and an equal amount or more of a medium was added. The cell suspension was centrifuged (1,200 rpm, 3 min), the supernatant was removed, and the cells obtained by centrifugation were suspended in a culture medium (manufactured by NACALAI TESQUE, INC.) at 1.0×104 cells/mL. The suspension was added by 100 μL to each well of a prepared 96-well plate and the cells were cultured (37° C. 5% CO2) for 4 days.
Thereafter, a cell count reagent mixture (mixture of “Cell Count Reagent SF” manufactured by NACALAI TESQUE, INC. (20 μL) and culture medium (80 μL)) (100 μL) was added to each well, and the cells were cultured (37° C. 5% CO2) for 3.5 hr. Thereafter, 0.1N hydrochloric acid was added to each well (10 μL/well) and the supernatant (100 μL) was recovered from each well and the absorbance was determined (λmax=450 nm) using microplate reader Benchmark Plus (BIO-RAD).
The viable cell count was calculated using an analytical curve obtained by measuring various concentrations of cell suspension by a similar method. In this Experimental Example, the viable cell count of 4 wells was measured and an average thereof was determined. The proliferation rate was calculated from a viable cell count (average) when sodium alginate or sodium polygalacturonate was used and the control viable cell count (average) when none of sodium alginate and sodium polygalacturonate was used and by the following formula proliferation rate (%)=100×viable cell count (average) using sodium alginate or sodium polygalacturonate/viable cell count (average) of control
The results are shown in Table 7.
The proliferation rate of RAW264.7 cells when sodium polygalacturonate was used is lower than that when sodium alginate was used. From this result, it is assumed that a core-shell type bead on which cells do not proliferate easily can be produced using sodium polygalacturonate as the second layer (outermost layer).
The beads of the present invention can be used for enclosing cell or microorganism in the inside thereof. Therefore, the beads of the present invention are useful for protection and culture of cell or microorganism.
Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.
As used herein the words “a” and “an” and the like carry the meaning of “one or more.”
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
All patents and other references mentioned above are incorporated in full herein by this reference, the same as if set forth at length.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-027246 | Feb 2017 | JP | national |
| 2017-225903 | Nov 2017 | JP | national |
This application is a continuation of International Patent Application No. PCT/JP2018/005156, filed on Feb. 15, 2018, and claims priority to Japanese Patent Application No. 2017-027246, filed on Feb. 16, 2017, and Japanese Patent Application No. 2017-225903, filed on Nov. 24, 2017, all of which are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2018/005156 | Feb 2018 | US |
| Child | 16541570 | US |
