Modified polymer and use thereof

Abstract

The present disclosure relates to a modified polymer and a use thereof. Specifically, the present disclosure provides a modified monomer represented by formula I, an alginate polymer containing same, a hydrogel, a capsule, and a corresponding use. The alginate of the present disclosure is used for cell encapsulation, exerting an antifibrotic effect and reducing foreign body reactions.

Description

Modified polymers and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to a modified polymer and uses thereof for encapsulation of cells to reduce foreign body response. BACKGROUND

[0002] Upon implantation of biomaterials into the human body, a series of cell and cytokine related reactions are triggered, known as foreign body response (FBR). During the progression of FBR, macrophages modulate inflammation and healing by polarizing into pro-inflammatory or pro-healing phenotypes and recruit fibroblasts by secreting cytokines. Under the stimulation of biomaterials, fibrotic capsules are eventually formed. The implants and their newly formed capsules introduce various mechanical signals that affect cell functions. Mechanical sensing proteins, such as integrins or ion channels, transduce extracellular mechanical signals into cytoplasmic biochemical signals to respond to mechanical stimuli. As a result, the morphology, migration pattern, function, and polarization state of cells are affected. Under the regulation of different intracellular signaling pathways and their cross-talk, the expression of fibrotic genes increases with the activation of fibroblasts and the transition of fibroblasts to myofibroblasts under rigid or external force stimulation.

[0003] A variety of materials have been widely used for the study of cell encapsulation, such as inorganic nanoparticles, metal-organic frameworks, hydrogels, etc. In comparison, hydrogels are a hydrophilic network composed of cross-linked polymer chains, with a water content of about 90%, and a stiffness similar to the cell matrix. The use of hydrogels to encapsulate cells can provide a physical microenvironment similar to the cell matrix for cells, and can achieve more functions or provide a more balanced biochemical environment through biological, chemical or physical modification methods. In addition, hydrogels can protect encapsulated cells from immune rejection, and their porous framework can enable the diffusion of oxygen, nutrients, secreted substances, and metabolic waste, allowing cells to survive and function.

[0004] However, alginate triggers a fibrotic response that worsens with the encapsulation of cells or xenogeneic donor tissues. The fibrotic tissue on the surface of alginate cuts off the diffusion of nutrients and oxygen to the encapsulated cells, leading to cell necrosis. Therefore, there is a need in the art for polymeric materials that do not trigger FBR and promote the health of encapsulated cells or tissues. SUMMARY

[0005] The modified monomer, the polymer containing the same, the hydrogel and the capsule of the present disclosure can be used for encapsulating cells, playing an anti-fibrotic role and reducing foreign body response.

[0006] The present disclosure provides a modified monomer shown in formula I,

[0007] wherein A is selected from a unit comprising a sugar and a unit comprising a polyvinyl alcohol;

[0008] X is selected from -0-, -S-, or -NR 2 -;

[0009] R 2 is hydrogen or C 1-4 alkyl, said C 1-4 alkyl is optionally substituted with one or more halogen, oxo, hydroxyl, amino, cyano, or C 1-6 alkoxy;

[0010] L1is selected from C 1-30 alkylene, C 1-30 heteroalkylene; wherein said C 1-30 alkylene, C 1-30 heteroalkylene is optionally substituted with one or more R L1 , R L1 is selected from halogen, oxo, hydroxyl, amino, cyano, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy;

[0011] L2is selected from

[0012] R 3 is selected from hydrogen or C 1-6 alkyl;

[0013] L3is selected from C 1-10 alkylene, said C 1-10 alkylene is optionally substituted with one or more R L3 , R L3 is selected from halogen, oxo, hydroxyl, amino, cyano, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy;

[0014] R 1 is selected from

[0015] R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from hydrogen or C 1-6 alkyl.

[0016] In some embodiments, in the modified monomer of Formula I, A is selected from a sugar- comprising unit.

[0017] In some embodiments, in the modified monomer of Formula I, A is selected from a unit comprising a monosaccharide, a unit comprising a disaccharide, or a unit comprising an oligosaccharide.

[0018] In some embodiments, in the modified monomer of Formula I, A is selected from a monosaccharide selected from the group consisting of a substituted or unsubstituted hexose, hexulose, hexuronic acid, pentose, pentulose, and penturonic acid.

[0019] In some embodiments, in the modified monomer of Formula I, A is selected from a unit comprising a mannuronic acid and a unit comprising a guluronic acid.

[0020] In some embodiments, in the modified monomer of Formula I, A is a is connected to X; Y1and Y2are each independently hydrogen or -PO(OR y )2; R y is hydrogen or alkyl.

[0021] In some embodiments, in the modified monomer of Formula I, A is a is connected to X.

[0022] In some embodiments, in the modified monomer of Formula I, A is a is connected to X; Y1and Y2are each independently hydrogen or -PO(OR 6 )2; R 6 is hydrogen or C 1-6 alkyl.

[0023] In some embodiments, in the modified monomer of Formula I, A is a is connected to X.

[0024] In some embodiments, in the modified monomer of Formula I, A is a is connected to X.

[0025] In some embodiments, the modified monomer of Formula I is a modified monomer of Formula II,

[0026] wherein X, L1, L2, L3, and R 1 are as defined in Formula I; Y1and Y2are each independently selected from hydrogen or -PO(OR y )2; R y is selected from hydrogen or C 1-6 alkyl.

[0027] In some embodiments, Y1and Y2are selected from hydrogen.

[0028] In some embodiments, the modifying monomer represented by Formula II is the modifying monomer represented by Formula II-1.

[0029] Y1, Y2, X, L1, L2, L3 and R 1 As defined by any scheme in Equation II.

[0030] In some embodiments, X is -NR in the modified monomers represented by Formula I, II, or II-1. 2 -

[0031] In some embodiments, X is -NH- in the modified monomers represented by Formula I, II, or II-1.

[0032] In some embodiments, in the modified monomers represented by Formula I, II, or II-1, L1 is C 1-30 Alkylene, the C 1-30 Alkylenes are optionally subjected to one or more R L1 Replaced by, R L1 As defined in Equation I.

[0033] In some implementations, L1 is C 1-10 Alkylene, the C 1-10 Alkylenes are optionally subjected to one or more R L1 Replaced by, R L1 As defined in Equation I.

[0034] In some embodiments, in the modified monomers represented by Formula I, II, or II-1, L1 is C 1-6 Alkylene, the C 1-6 Alkylenes are optionally subjected to one or more R L1 Replaced by, R L1 As defined in Equation I.

[0035] In some embodiments, in the modifying monomers represented by Formula I, II, or II-1, L1 is selected from methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, or tert-butylene, optionally modified by one or more R L1 Replaced by, R L1 As defined in Equation I.

[0036] In some embodiments, in the modifying monomers represented by Formula I, II, or II-1, L1 is selected from C having 1-5 heteroatoms selected from N, O, or S. 1-30 Heteroalkyl groups, which are optionally subjected to one or more R L1 Replaced by, R L1 As defined in Equation I.

[0037] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from C 1-10 heteroalkylene optionally substituted with one or more R L1 heteroalkylene optionally substituted with one or more R L1 as defined in Formula I.

[0038] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from -(CH2CH20) m1 -, ml is an integer between 1-15 (e.g., 1, 2, 3, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) optionally substituted with one or more R L1 heteroalkylene optionally substituted with one or more R L1 as defined in Formula I, left side attached to X.

[0039] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from -(CH2CH20) m1 -, ml is an integer between 1-10 optionally substituted with one or more R L1 heteroalkylene optionally substituted with one or more R L1 as defined in Formula I, left side attached to X.

[0040] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from -(CH2CH20) m1 -, ml is an integer between 1-6 optionally substituted with one or more R L1 heteroalkylene optionally substituted with one or more R L1 as defined in Formula I, left side attached to X.

[0041] In some embodiments, in the modified monomer of Formula I, II, or II-1, P in L2is attached to L1.

[0042] In some embodiments, in the modified monomer of Formula I, II, or II-1, L2is

[0043] In some embodiments, in the modified monomer of Formula I, II, or II-1, R 3 is hydrogen.

[0044] In some embodiments, in the modified monomer of Formula I, II, or II-1, L3is selected from C 1-6 alkylene.

[0045] In some embodiments, in the modified monomer of Formula I, II, or II-1, L3is selected from methylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene, sec-butylene, or t-butylene.

[0046] In some embodiments, in the modified monomer of Formula I, II, or II-1, L3is ethylene (-CH2CH2-).

[0047] In some embodiments, in the modified monomer of Formula I, II, or II-1, R 1 is selected from R 7 , R 8 , and R 9 are each independently selected from hydrogen or C 1-6 alkyl.

[0048] In some embodiments, in the modified monomer of Formula I, II, or II-1, R 1 is selected from R 7 , R 8 , and R 9 are each independently selected from hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, or t-butyl.

[0049] In some embodiments, in the modified monomer of Formula I, II, or II-1, R 1 is selected from R 7 , R 8 , and R 9 are each methyl.

[0050] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from C 1-30 alkylene.

[0051] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from C 1-10 alkylene.

[0052] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from C 1-6 alkylene.

[0053] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from methylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene, sec-butylene, or t-butylene.

[0054] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from C 1-30 heteroalkylene.

[0055] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from C 1-30Heteroalkylene.

[0056] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from C 1-10 Heteroalkylene.

[0057] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from -(CH2CH2O) m1 -, m1 is an integer between 1-15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).

[0058] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from -(CH2CH2O) m1 -, m1 is an integer between 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0059] In some embodiments, in the modified monomer of Formula I, II, or II-1, L1is selected from -(CH2CH2O) m1 -, m1 is an integer between 1-6 (e.g., 1, 2, 3, 4, 5, or 6).

[0060] In some embodiments, the modified monomer of Formula I is a modified monomer of Formula III,

[0061] wherein A, X, L3, R 7 , R 8 , and R 9 are as defined in any of Formula I; m1 is an integer selected from 1-15.

[0062] In some embodiments, the modified monomer of Formula I is a modified monomer of Formula IV,

[0063] wherein X, L3, R 7 , R 8 , and R 9 are as defined in any of Formula I; Y1and Y2are each independently selected from hydrogen or -PO(OR y )2, R y is selected from hydrogen or C 1-6 alkyl; m1 is an integer selected from 1-15.

[0064] In some embodiments, in the modified monomer of Formula IV, Y1and Y2are selected from hydrogen.

[0065] In some embodiments, the modified monomer of Formula IV is a modified monomer of Formula IV-1,

[0066] wherein Y1, Y2, X, L3, R 7 , R 8 , R 9 and m1 are as defined for Formula IV.

[0067] In some embodiments, the modified monomer of Formula I is a structure,

[0068] A is a is attached to -NH-.

[0069] The present disclosure provides a polymer comprising the modified monomer described above.

[0070] In some embodiments, at least 0.5% (e.g., 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and any number in between) of the monomers of the polymer are the modified monomer described above, in terms of mole percentage. In some embodiments, 0.5%-50%, 10%-90%, 10%-50%, or 25%-75% of the monomers of the polymer are the modified monomer described above, in terms of mole percentage. In some embodiments, 5%-50% of the monomers of the polymer are the modified monomer described above, in terms of mole percentage. In some embodiments, 10%-30% of the monomers of the polymer are the modified monomer described above, in terms of mole percentage.

[0071] In some embodiments, the polymer is crosslinked using Ca 2+ , Ba 2+ , or Sr 2+ to form a hydrogel. In some embodiments, the polymer is crosslinked using Ca 2+ to form a hydrogel.

[0072] In some embodiments, the polymer is a polymer of alginic acid or a salt thereof, which is a polysaccharide composed of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). In some embodiments, the alginic acid or salt thereof is a high guluronic acid (G) alginic acid or salt thereof and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more guluronic acid (G). In some embodiments, the alginic acid or salt thereof is a high mannuronic acid (M) alginic acid or salt thereof and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more mannuronic acid (M). In some embodiments, the ratio of G:M is about 1.5. In some embodiments, the ratio of G:M is less than 1.5. In some embodiments, the ratio of G:M is greater than or equal to 1.5.

[0073] In some embodiments, the compound (X, L1, L2, L3, and R 1 is 1.0-5.0% N. The conjugation density is the density at which the polymer comprises the modified monomers described above, with a conjugation density of at least 1.0% and less than 5.0% nitrogen (N) (e.g., 2.0% to 5.0%, 2.0% to 4.0%, 2.0% to 3.0%) nitrogen (N) as determined by combustion analysis for percent nitrogen. In some embodiments, the compound has a conjugation density of about 2.3% N.

[0074] The present disclosure provides a polymer of Formula V,

[0075] wherein B is

[0076] A, X, L1, L2, L3, and R 1 as defined in any of Formula I;

[0077] k1 is selected from an integer greater than or equal to 1;

[0078] k2 is independently selected from 0 or 1, and at least one k2 is 1.

[0079] In some embodiments, the polymer of Formula V, B is X, L3, R 7 , R 8 , and R 9 as defined in any of Formula III; and m1 is selected from an integer between 1-15.

[0080] In some embodiments, k1 in the polymer of Formula V is selected from any integer from 5 to 10,000. In some embodiments, k1 in the polymer of Formula V is selected from any integer from 10 to 5,000. In some embodiments, k1 in the polymer of Formula V is selected from any integer from 10 to 500. In some embodiments, k1 in the polymer of Formula V is selected from any integer from 500 to 1,000. In some embodiments, k1 in the polymer of Formula V is selected from any integer from 1,000 to 5,000.

[0081] In some embodiments, the number or quantity of "k2 is 1" is Q, where Q is at least 1. In some embodiments, the percentage of k1 that is Q is at least 0.5% (e.g., 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and any value in between). In some embodiments, the percentage of k1 that is Q is from 5% to 50%. In some embodiments, the percentage of k1 that is Q is from 10% to 30%.

[0082] The present disclosure provides a hydrogel comprising the polymer described above.

[0083] In some embodiments, the polymer is crosslinked using Ca 2+ , Ba 2+ , or Sr 2+ to form a hydrogel. In some embodiments, the polymer is crosslinked using Ca 2+ to form a hydrogel.

[0084] The present disclosure also provides a capsule comprising the hydrogel described above.

[0085] In some embodiments, the hydrogel encapsulates a therapeutic agent.

[0086] In some embodiments, the therapeutic agent is a cell. In some embodiments, the cell comprises a cell from a xenogenic tissue or cell, a heterologous tissue or cell, a tissue or cell from a cadaver, a stem cell, a cell derived from a stem cell, a cell from a cell line, a primary cell, a reprogrammed cell, a reprogrammed stem cell, a cell derived from a reprogrammed stem cell, a genetically engineered cell, or a combination thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is an insulin-producing cell. In some embodiments, the cell is an islet cell. In some embodiments, the cell comprises, but is not limited to, an embryonic stem cell, a mesenchymal stem cell, and an islet beta cell. In some embodiments, the cell is an islet beta cell.

[0087] In some embodiments, the hydrogel encapsulates a single cell or a plurality of cells in the capsule.

[0088] In some embodiments, the capsule is spherical. In some embodiments, the average diameter of the capsule is 0.01 mm-10 mm (e.g., 0.01 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 5.5 mm, 6.0 mm, 6.5 mm, 7.0 mm, 7.5 mm, 8.0 mm, 8.5 mm, 9.0 mm, 9.5 mm, 10.0 mm, and any value in between). In some embodiments, the average diameter of the capsule is 0.1 mm-10 mm. In some embodiments, the average diameter of the capsule is 1 mm-8 mm. In some embodiments, the average diameter of the capsule is 1 mm-5 mm. In some embodiments, the average diameter of the capsule is 1 mm-3 mm. In some embodiments, the average diameter of the capsule is 1 mm-2 mm. In some embodiments, the average diameter of the capsule is about 2 mm. In some embodiments, the average diameter of the capsule is about 1.5 mm.

[0089] In some embodiments, in the alginate-containing capsule, the amount of alginate (e.g., in actual weight of alginate as a percentage of the weight of the capsule) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w / w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.

[0090] The present disclosure also provides a pharmaceutical composition comprising the modified monomer of Formula I described above, the polymer described above, or the hydrogel described above, and one or more pharmaceutically acceptable excipients.

[0091] The present disclosure also provides a pharmaceutical composition comprising the capsule described above, and one or more pharmaceutically acceptable excipients.

[0092] In some embodiments, the capsule (e.g., the therapeutic agent therein) can be in a therapeutically effective amount.

[0093] In some embodiments, the unit dose of the pharmaceutical composition can be 0.001 mg-1000 mg.

[0094] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the capsule described above, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of the capsule described above. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the capsule. In some embodiments, the pharmaceutical composition contains 1%-99% of the capsule described above. In some embodiments, the pharmaceutical composition contains 2%-98% of the capsule described above.

[0095] The present disclosure further provides use of the modified monomer described above as shown in Formula I, the polymer described above, the hydrogel described above, the capsule described above, or the pharmaceutical composition described above in the manufacture of a medicament for treating a disease, including but not limited to diabetes, hemophilia, skin injury, spinal cord injury, heart injury, bone and joint inflammation.

[0096] In some embodiments, the disease is diabetes.

[0097] The present disclosure further provides a method of treating a disease, comprising administering the capsule or the pharmaceutical composition described above to a subject. In some embodiments, the disease includes but is not limited to diabetes, hemophilia, skin injury, spinal cord injury, heart injury, bone and joint inflammation. In some embodiments, the disease is diabetes.

[0098] The present disclosure further provides a method of treating a disease, comprising implanting the capsule or the pharmaceutical composition described above into a subject. In some embodiments, the disease includes but is not limited to diabetes, hemophilia, skin injury, spinal cord injury, heart injury, bone and joint inflammation. In some embodiments, the disease is diabetes.

[0099] The present disclosure further provides a method of delivering a therapeutic agent to a subject, comprising administering the capsule or the pharmaceutical composition described above to the subject.

[0100] The present disclosure further provides a device comprising the capsule described above or the pharmaceutical composition described above.

[0101] The present disclosure further provides a package comprising the capsule described above or the pharmaceutical composition described above.

[0102] The present disclosure further provides an apparatus comprising the capsule described above or the pharmaceutical composition described above.

[0103] Definitions of terms

[0104] Where the disclosure does not specify particular configurations, the compounds of the disclosure can exist in particular geometric or stereoisomeric forms. The disclosure contemplates all such compounds, including cis- and trans-forms, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, individual isomers, and mixtures of isomers, e.g., racemic mixtures, enantiomeric mixtures, and diastereomeric mixtures, all in any combination. Additional asymmetric carbon atoms can be present in a substituent group. All such isomers, as well as mixtures thereof, are included within the scope of the disclosure.

[0105] In addition, the compounds and intermediates of the disclosure can exist in different tautomeric forms, and all such forms are embraced within the scope of the disclosure. The term "tautomers" or "tautomeric forms" refers to different energy structures that can interconvert via a low energy barrier. For example, prototropic tautomers (also known as proton-shift tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerization.

[0106] The compounds of the disclosure can be asymmetric, e.g., having one or more stereocenters. Unless otherwise specified, all stereoisomers are included, such as enantiomers and diastereomers. Compounds of the disclosure having asymmetric carbon atoms can be isolated in optically active form or as racemic mixtures. Optically active forms can be obtained, for example, by resolution of a racemic mixture or by synthesis from optically active starting materials.

[0107] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomer. Alternatively, when a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group) is present in the molecule, a diastereomeric salt form of the compound of the disclosure is formed with an appropriate optically active acid or base, and then resolved into its component parts by conventional means known to those of skill in the art. Additionally, separation of the enantiomers and diastereomers is typically accomplished by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g., formation of a carbamate from an amine).

[0108] The present disclosure also includes certain isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl, and the like.

[0109] Unless otherwise stated, when a position is designated specifically as deuterium (D), the position is understood to have deuterium in an abundance of at least 1000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 10% deuterium incorporation). The example compound having deuterium in an abundance of at least 1000 times greater than the natural abundance of deuterium can be deuterium in an abundance of at least 2000 times greater than the natural abundance of deuterium, deuterium in an abundance of at least 3000 times greater than the natural abundance of deuterium, deuterium in an abundance of at least 4000 times greater than the natural abundance of deuterium, deuterium in an abundance of at least 5000 times greater than the natural abundance of deuterium, deuterium in an abundance of at least 6000 times greater than the natural abundance of deuterium, or greater. The present disclosure also includes various deuterated forms of the compounds of Formula I. Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom. One skilled in the art would be able to synthesize deuterated forms of the compounds of Formula I by reference to the relevant literature. Commercially available deuterated starting materials can be used in the preparation of deuterated forms of the compounds of Formula I, or can be synthesized using conventional techniques employing deuterated reagents including, but not limited to, deuterated borane, tritiated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide, and deuterated methyl iodide, among others.

[0110] In the chemical structures of the compounds of the present disclosure, the bond indicates that the configuration is not specified, i.e., if chiral isomers are present in the chemical structure, the bond may be or or both and configurations are present. If atropisomers are present in the chemical structure, the bond may be denotes towards the outside of the paper. Although all the above structural formulae are drawn in some isomer form for the sake of simplicity, the present disclosure can include all isomers, such as tautomers, rotamers, atropisomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structure of the compounds described in the present disclosure, the configuration of the bond is not specified, i.e. the configuration of the bond may be E or Z, or both E and Z.

[0111] Unless otherwise indicated, the symbols used herein denote that they can be attached to one or more of any of the groups described herein within the scope of the disclosure described herein.

[0112] The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight chain or branched chain group comprising 1 to 30 carbon atoms (C 1-30 alkyl), preferably 1 to 12 carbon atoms (C 1-12 alkyl), more preferably 1 to 10 carbon atoms (C 1-10 alkyl), more preferably 1 to 6 carbon atoms (C 1-6 alkyl), further preferably 1 to 4 carbon atoms (C 1-4 alkyl). Among them, C 1-4 alkyl includes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl. The alkyl group can be substituted or unsubstituted, when substituted, the substituents can be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl, which alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted with halogen, hydroxyl, nitro, cyano or amino.

[0113] The term "alkylene" is the moiety remaining after further removing one hydrogen atom from "alkyl", the definition of which is referred to above. The alkylene group can be a straight chain or branched chain group comprising 1 to 30 carbon atoms (C 1-30 alkylene), preferably 1 to 10 carbon atoms (C 1-10 alkylene), more preferably 1 to 6 carbon atoms (C 1-6 alkylene), further preferably 1 to 4 carbon atoms (C 1-4 alkylene). Among them, C 1-4 alkylene includes methylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene, t-butylene, sec-butylene. The alkylene group can be substituted or unsubstituted, the definition of the substituents is the same as "alkyl".

[0114] The term "heteroalkyl" refers to saturated aliphatic hydrocarbon groups that are straight-chain or branched, and contain 1 to 30 carbon atoms, and contain one or more heteroatoms selected from N, O, S. Preferably, heteroalkyl groups contain 1 to 30 carbon atoms, 1-5 heteroatoms selected from N, O, S; more preferably, heteroalkyl groups contain 1 to 20 carbon atoms, 1-5 heteroatoms selected from N, O, S; further preferably, heteroalkyl groups contain 1 to 10 carbon atoms, 1-5 heteroatoms selected from N, O, S. Heteroalkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available attachment point, preferably one or more of the following groups, independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0115] The term "heteroalkyl" refers to saturated aliphatic hydrocarbon groups that are straight-chain or branched, and contain 1 to 30 carbon atoms, and contain one or more heteroatoms selected from N, O, S. Preferably, heteroalkyl groups contain 1 to 30 carbon atoms, 1-5 heteroatoms selected from N, O, S; more preferably, heteroalkyl groups contain 1 to 20 carbon atoms, 1-5 heteroatoms selected from N, O, S; further preferably, heteroalkyl groups contain 1 to 10 carbon atoms, 1-5 heteroatoms selected from N, O, S. Heteroalkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available attachment point, preferably one or more of the following groups, independently selected from halogen, hydroxyl, oxo, cyano, amino, C m -, m is any integer (e.g., an integer from 1 to 15). Heteroalkylene groups can be substituted or unsubstituted, and the definition of substituents is the same as "heteroalkyl".

[0116] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propyloxy, isopropoxy, n-butyloxy, isobutyloxy, sec-butyloxy, or t-butyloxy. Alkenyl groups used in any context herein are optionally substituted in the same manner as alkyl groups.

[0117] The term "alkenyl" refers to unsaturated aliphatic straight-chain or branched hydrocarbon groups, and contain one or more carbon-carbon double bonds. Exemplary alkenyl groups include C2-C10, C2-C8, C2-C7, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl groups. Included, but not limited to, ethenyl (i.e., vinyl), 1-propenyl, 2-propenyl (i.e., allyl), 2-methyl-1-propenyl, 1-butenyl, 2-butenyl (i.e., crotyl), and the like. Alkenyl groups used in any context herein are optionally substituted in the same manner as alkyl groups.

[0118] The term "alkynyl" refers to unsaturated aliphatic straight chain or branched chain hydrocarbon groups and contain one or more carbon-carbon triple bonds. Exemplary alkynyl groups include C2-C10, C2-C8, C2-C7, C2-C6, C2-C4, C3-C12, and C3-C6 alkynyl groups. Included are, but are not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl. Alkynyl groups used in any context herein are optionally substituted in the same manner as alkyl groups.

[0119] The term "cycloalkyl" refers to saturated monocyclic or polycyclic cyclic hydrocarbon substituents, the cycloalkyl ring comprising 3 to 20 carbon atoms (also referred to as 3 to 20 membered), preferably monocyclic cycloalkyl groups comprising 3 to 6 carbon atoms (also referred to as 3 to 6 membered). Non-limiting examples of 3 to 6 membered cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted, when substituted, the substituents can be substituted at any available point of attachment, preferably one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3 to 6 membered cycloalkyl, or 3 to 6 membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halogen, hydroxyl, nitro, cyano, or amino. The term "heterocycloalkyl" refers to saturated monocyclic or polycyclic cyclic hydrocarbon substituents, comprising 3 to 20 ring atoms (also referred to as 3 to 20 membered), wherein one or more ring atoms are heteroatoms selected from N, O, S, P, C(=O), or S(=O) m ring atoms are carbon. Preferably monocyclic heterocycloalkyl groups comprising 3 to 12 ring atoms (also referred to as 3 to 12 membered), wherein 1 to 4 are heteroatoms; preferably comprising 3 to 8 ring atoms (also referred to as 3 to 8 membered), wherein 1 to 3 are heteroatoms; more preferably comprising 3 to 6 ring atoms (also referred to as 4 to 6 membered).

[0120] Heterocycloalkyl groups can be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3 to 6 membered cycloalkyl, or 3 to 6 membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0121] The term "aryl" refers to 6 to 14 membered all-carbon monocyclic or fused polycyclic (that is, rings which share adjacent pairs of carbon atoms) groups with a conjugated pi-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl.

[0122] Aryl can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halo, hydroxy, nitro, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halo, hydroxy, nitro, cyano, or amino.

[0123] The term "heteroaromatic ring" or "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 12 membered, preferably 5-10 membered, more preferably 5 membered or 6 membered. Non-limiting examples include: imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, and the like.

[0124] Heteroaryl can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halo, hydroxy, nitro, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halo, hydroxy, nitro, cyano, or amino.

[0125] The terms "cycloalkylene", "heterocycloalkylene", "arylene", and "heteroarylene" refer to the moiety remaining after the further removal of one hydrogen atom from a "cycloalkyl", "heterocycloalkyl", "aryl", and "heteroaryl" group, respectively, the definitions of which are given above. Cycloalkylene, heterocycloalkylene, arylene, and heteroarylene groups can be substituted or unsubstituted, the substituents being as defined for "cycloalkyl", "heterocycloalkyl", "aryl", and "heteroaryl".

[0126] The term "hydroxy" refers to -OH.

[0127] The term "halo" refers to fluorine, chlorine, bromine, or iodine.

[0128] The term "cyano" refers to -CN.

[0129] The term "amino" refers to -NH2.

[0130] The term "nitro" refers to -NO2.

[0131] The term "oxo" refers to the =O substituent.

[0132] "Optionally" or "optional" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted with halo or cyano" means that the alkyl group can or can not be substituted with halo or cyano, and that the description includes instances where the alkyl group is substituted with halo or cyano and instances where the alkyl group is not substituted with halo or cyano. 1-6 "Alkyl" means that halo or cyano can or can not be present, and that the description includes instances where the alkyl group is substituted with halo or cyano and instances where the alkyl group is not substituted with halo or cyano.

[0133] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3, of a group are independently of each other replaced with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which can or cannot be possible, can be determined by a person skilled in the art without undue effort (experimentally or theoretically).

[0134] "Substituted with one or more" means that a single or multiple substituents can be present. When multiple substituents are present, these can be multiple identical substituents or a combination of one or multiple different substituents.

[0135] The term "linked", when referring to the association between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are associated via non-covalent bonds (e.g., hydrogen bonds or ionic bonds), including direct and indirect connections.

[0136] The term "directly linked" means that a first compound or group is connected to a second compound or group without any intervening atoms or atom groups. The term "indirectly linked" means that a first compound or group is connected to a second compound or group via an intermediate group, compound or molecule (e.g., a linker group).

[0137] Unless otherwise specified, the "modified monomers", "polymers" of the present disclosure can exist in the form of a salt, including acid addition salts and base addition salts (e.g., sodium salts). These salts can be prepared by methods known in the art.

[0138] The term "alginate or salt thereof" refers to the general term for polysaccharides composed of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G) in any M / G ratio and salts and derivatives thereof. The beta-D-mannuronic acid (M) monomer and the alpha-L-guluronic acid (G) monomer in the present disclosure have the structures shown below:

[0139] The term "alginate or salt thereof" used in the present disclosure includes any polymer or salt thereof having the structure shown below.

[0140] The term "alginate or salt thereof" in the present disclosure means represents

[0141] The term "pharmaceutical composition" means a composition comprising one or more entities described herein (e.g., a capsule encapsulating a therapeutic agent), as well as other components such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to a subject, and to enhance the effectiveness of the active ingredient in achieving a biological activity.

[0142] The term "pharmaceutical composition" means a composition comprising one or more entities described herein (e.g., a capsule encapsulating a therapeutic agent), as well as other components such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to a subject, and to enhance the effectiveness of the active ingredient in achieving a biological activity.

[0143] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" includes, but is not limited to, any auxiliary, carrier, glidant, sweetening, diluting, preservative, dye / colorant, flavoring, surfactant, wetting, dispersing, suspending, stabilizing, isotonic, solvent, or emulsifying agent that has been approved by the U.S. Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0144] The term "capsule" or "hydrogel capsule" refers to a particle formed from a crosslinked hydrogel, having a core of crosslinked hydrogel surrounded by one or more polymeric shells, having one or more layers of crosslinked hydrogel, having a coating of crosslinked hydrogel, or a combination thereof. The capsule can have any shape suitable for encapsulation of a therapeutic agent (e.g., a cell), such as a spherical shape. The capsule can contain one or more cells dispersed in the crosslinked hydrogel, thereby "encapsulating" the cells. References herein to "capsules" relate to and include microcapsules, unless the context clearly dictates otherwise. In some embodiments, the average diameter of the capsule is selected from the group consisting of 0.01 mm - 10 mm (e.g., 0.01 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 5.5 mm, 6.0 mm, 6.5 mm, 7.0 mm, 7.5 mm, 8.0 mm, 8.5 mm, 9.0 mm, 9.5 mm, 10.0 mm, and any value in between).

[0145] The term "islet cell" refers to any naturally occurring or any synthetically produced or modified cell that is intended to partially or wholly reproduce, mimic, or otherwise express part or all of the function of a cell of a Langerhans islet. The term "islet cell" includes glucose-responsive insulin-producing cells that are derived from stem cells, such as from an induced pluripotent stem cell line.

[0146] ​The term "spherical" refers to particles having curved surfaces that form a spherical shape (e.g., a perfectly round sphere) or a spheroid shape. Spheres and spheroids can be mathematically defined by a rotation about each of three perpendicular axes a, b, and c, either circular, elliptical, or a combination. For a sphere, the three axes a, b, and c are of equal length. Generally, a spheroid shape is an ellipsoid having semi-axes a, b, and c that are within 10%, or 5% or 2.5% of each other (for its average surface). The diameter of a spherical or spheroid shape is the average diameter, e.g., the average of the semi-axes.

[0147] The term "administering" refers to implanting, absorbing, ingesting, injecting, or otherwise introducing an entity described herein (e.g., a capsule comprising an encapsulated therapeutic agent, or a pharmaceutical composition comprising the capsule), or providing it to a subject.

[0148] The term "subject" refers to a human or non-human animal. In some embodiments, the subject is a human (i.e., a male or female, e.g., of any age group, a pediatric subject (e.g., an infant, child, adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or elderly adult)). In some embodiments, the subject is a non-human animal, e.g., a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey)). In some embodiments, the subject is a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog) or bird (e.g., a commercially relevant bird, e.g., a chicken, duck, goose, or turkey). In some embodiments, the animal is a mammal. The animal can be male or female and at any stage of development. The non-human animal can be a transgenic animal.

[0149] The term "effective amount," "effective dose," "effective therapeutic amount," or "therapeutically effective amount" refers to the amount of a drug, compound, or pharmaceutical composition that is necessary to achieve one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying onset of a disease, disorder or condition, including biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, and intermediate pathological phenotypes presenting during development of the disease.

[0150] The term "treatment" refers to one or more of alleviating, reversing, remitting, delaying the onset of, or inhibiting the progression of one or more symptoms, manifestations, or underlying causes of a disease, disorder, or condition.

[0151] The term "inhibit," can be used interchangeably with "reduce," "silence," "down- regulate," "repress," and other similar terms, and includes inhibition at any level. Inhibition can be assessed by a decrease in absolute or relative levels of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., buffer control or inert agent control) treated subject, cell, or sample.

[0152] In the present disclosure, the numerical values such as the data measured and calculated for the content of the substance inevitably have a certain degree of error. In general, ±10% is within a reasonable error range. Depending on the context in which it is used, there is a certain degree of error variation, which does not exceed ±10%, which can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%. BRIEF DESCRIPTION OF DRAWINGS

[0153] Figure 1 is the effect of alginate on the viability of HeLa cell line.

[0154] Figure 2 is a comparison of the number of macrophages recruited in the peritoneal cavity of mice by the capsules of Example 2 v / s and the capsules of Comparative Example 1.

[0155] Figure 3 is a comparison of the number of neutrophils recruited in the peritoneal cavity of mice by the capsules of Example 2 v / s and the capsules of Comparative Example 1.

[0156] Figure 4 is the change in blood glucose of mice over a period of 66 days after transplanting the human pancreatic beta cells encapsulated in the capsules of Example 2 into the peritoneal cavity of mice model for type 1 diabetes. The control is a type 1 diabetes model mouse. DETAILED DESCRIPTION

[0157] The present disclosure is further described in connection with the following examples, which are not intended to limit the scope of the disclosure. Unless otherwise indicated, the methods of the present disclosure were performed in accordance with conventional techniques or as recommended by the manufacturer of the reagent or product. Unless otherwise indicated, the reagents used in the following examples were commercially available.

[0158] Unless otherwise indicated, the reagents used in the following examples were commercially available.

[0159] Reagent and supplies information:

[0160] In the following examples, statistical significance is marked as: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0161] Preparation of compound 1 of Example 1

[0162] Step 1) Preparation of compound 1b

[0163] 1 -amino-3, 6, 9-trioxa-11 -undecanol (50.2 g, 259.78 mmol, purchased from Leyen) was dissolved in ethyl acetate (250 mL) and water (250 mL), sodium bicarbonate (32.73 g, 389.67 mmol) was added, then benzyl chloroformate (46.37 g, 272.77 mmol, 38.26 mL) was added. After addition, it was stirred at room temperature overnight. After the reaction was completed, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate (200 mL). The combined organic phases were dried, filtered, and concentrated. Purification by column chromatography (dichloromethane / methanol = 15 / 1) gave 74.8 g of product, yield: 87.9%.

[0164] MS m / z (ESI): 328.3 [M+1] + .

[0165] 1 H NMR (400 MHz, CDC13) δ 7.36-7.28 (m, 5H), 5.07 (s, 2H), 3.86-3.61 (m, 10H), 3.55-3.51 (m, 4H), 3.31-3.28 (m, 2H).

[0166] Step 2) Preparation of compound 1c

[0167] Compound 1b (25 g, 76.37 mmol) was dissolved in toluene (1.5 L), and triethylamine (15.45 g, 152.73 mmol, 21.29 mL) was added. It was cooled to 0°C. 2-chloro-2-oxo-1,3,2-dioxaphospholane (14.14 g, 99.28 mmol, 9.13 mL) was added dropwise. After addition, it was stirred for 5 minutes. It was stirred at room temperature overnight. It was suction filtered and washed with toluene (100 mL). The filtrate was concentrated to give a crude product of 30.5 g, yield, 92.1%. It was used directly in the next step.

[0168] MS m / z (ESI): 434.3 [M+1] + .

[0169] Step 3) Preparation of compound 1d

[0170] Compound 1c (15.25 g, 35.19 mmol), was added to a solution of trimethylamine in acetonitrile (2M, 329.08 mmol, 164.54 mL) in a sealed tube and heated to 70 °C with stirring overnight. Cooled to room temperature. Added methanol, dissolved, concentrated to get crude, purified by column chromatography (dichloromethane / methanol (4 / 1-1 / 1) to get 12 g of product, further purified by reverse phase chromatography (trifluoroacetic acid / water / acetonitrile, 0.5%) to get 9.6 g of product, yield: 55.3%.

[0171] MS m / z (ESI): 493.4 [M+1] + .

[0172] 1 H NMR (400 MHz, CD3OD) δ 7.37 - 7.31 (m, 5H), 5.09 (s, 2H), 4.35 - 4.31 (m, 2H), 4.05 - 4.01 (m, 2H), 3.69 - 3.62 (m, 12H), 3.55 (t, J = 5.6 Hz, 2H), 3.33 - 3.30 (m, 2H), 3.21 (s, 9H).

[0173] Step 4) Preparation of compound 1

[0174] Compound 1d (7.7 g, 15.63 mmol) was dissolved in methanol (100 mL) under nitrogen protection, added Pd / C (1.45 g). Then, after hydrogen balloon replacement, stirred at 25 °C overnight. After the reaction was completed, the filter was prepared with diatomite, and the filtrate was concentrated to obtain the product, light yellow oil 5.44 g, yield: 97.09%.

[0175] MS m / z (ESI): 359.3 [M+1] + .

[0176] 1 H NMR (400 MHz, CD3OD) δ 4.33 - 4.29 (m, 2H), 4.05 - 4.01 (m, 2H), 3.73 - 3.65 (m, 14H), 3.25 (s, 9H), 3.08 (t, J = 5.2 Hz, 2H).

[0177] Example 2 Chemical modification of sodium alginate

[0178] Low molecular weight sodium alginate (1.0 g, 5.05 mmol, purchased from Qingdao Mingyue, viscosity 13 mPa.s, G:M ratio > 1.5) was dissolved in water (30 mL), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (1.05 g, 3.79 mmol, purchased from adamas), an aqueous solution of compound 1 (1.36 g, 3.79 mmol) obtained from example 1 (15 mL) were added, heated to 55 °C, stirred overnight, cooled to room temperature, precipitated by adding ethanol (360 mL), suction filtered, the filter cake was dissolved in physiological saline (50 mL), precipitated by adding ethanol (400 mL), suction filtered, the filter cake was dissolved in water (30 mL), freeze-dried to obtain 1.1 g of product.

[0179] The conjugation density of the chemically modified sodium alginate was measured by combustion analysis for nitrogen, the conjugation density of compound 1 was 2.3% N.

[0180] The control example 1 was synthesized by referring to CN112203646A example 2, the conjugation density was 5.5% N.

[0181] Test example 1: in vitro biocompatibility evaluation

[0182] To test the in vitro biocompatibility of the chemically modified alginate, its cytotoxicity to HeLa cells was evaluated. Example 2 (dissolved in 0.8% physiological saline at a weight / volume ratio of 2%) was loaded into a tissue culture 96-well plate. As controls, low molecular weight sodium alginate (dissolved in 0.8% physiological saline at a weight / volume ratio of 2%), control example 1 (dissolved in 0.8% physiological saline at a weight / volume ratio of 2%) and physiological saline were also loaded into the wells of the 96-well plate, and treated with a 100 mM CaCl2crosslinking solution. After aspirating the excess crosslinking agent, 200 microliters of cell culture medium were added and incubated in a 37 °C, humidity-controlled incubator for 24 hours. Subsequently, the medium was added to wells pre-seeded with HeLa cells and incubated in a 37 °C, humidity-controlled incubator for 3 days.

[0183] Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After aspirating the medium from all wells, 100 microliters of phenol red-free DMEM medium and 10 microliters of 5 mg / ml MTT solution were added to each well. The plate was incubated in a 37 °C, humidity-controlled incubator for 4 hours. After the incubation was completed, 85 microliters of solution were aspirated and 10 microliters of DMSO were added. Before measurement, the purple formazan crystals in the wells were dissolved by pipetting up and down. After incubating the plate at 37 °C for 10 minutes, the air bubbles generated by stirring were removed, and the cells seeded in the wells without alginate were normalized.

[0184] The cell viability results are shown in Figure 1, which shows the effect of alginate on the viability of the HeLa cell line. The cell viability for the low molecular weight alginate was 97% compared to the negative control (saline), the cell viability for Control Example 1 alginate was 80%, and the cell viability for Example 2 was 90%, which is significantly better than Control Example 1, showing lower cytotoxicity. This test identified a modified alginate polymer that showed lower cytotoxicity relative to Control Example 1, and Example 2 alginate was selected for further analysis.

[0185] Test Example 2: Preparation of alginate hydrogel capsules

[0186] All buffers were sterilized by autoclave, and the alginate solutions were sterilized by filtration through a 0.2 micron filter. The sterilized solutions were manipulated aseptically in a Class A2 biological safety cabinet to ensure the sterility of the capsules / balls prepared. The steps for preparing the hydrogel capsules were as follows:

[0187] (1) Low molecular weight sodium alginate (VLVG): 5.05 mmol, purchased from Qingdao Mingyue, viscosity 13 mPa.s, G:M ratio > 1.5, dissolved in 0.8% saline at a weight / volume ratio of 5%;

[0188] (2) High molecular weight sodium alginate: purchased from Qingdao Mingyue, viscosity 143 mPa.s, G:M ratio > 1.5, dissolved in 0.8% saline at a weight / volume ratio of 3%;

[0189] (3) v / s hydrogel capsule preparation: mixed at a volume ratio of 70% low molecular weight sodium alginate and 30% high molecular weight sodium alginate to prepare the v / s hydrogel capsules.

[0190] (4) Control Example 1 hydrogel capsule preparation: Control Example 1 was first dissolved in 0.8% saline at a weight / volume ratio of 5%, and then mixed at a volume ratio of 50% Control Example 1 and 50% high molecular weight sodium alginate to prepare the Control Example 1 hydrogel capsules.

[0191] (5) Example 2 hydrogel capsule preparation: Example 2 was first dissolved in 0.8% saline at a weight / volume ratio of 5%, and then mixed at a volume ratio of 50% Example 2 and 50% high molecular weight sodium alginate to prepare the Example 2 hydrogel capsules.

[0192] Test Example 3: Verification of anti-fibrosis activity

[0193] (1) Capsule preparation: refer to Test Example 2 above;

[0194] (2) Implantation of hydrogel capsules

[0195] This section uses immunocompetent male C57BL / 6 mice provided by Vivotecnia as experimental subjects. The entire procedure complies with the ethical norms for animal experimentation. Before the procedure, the mice were subcutaneously injected with 0.05 mg / kg of butorphanol as a preoperative analgesic, and with 0.3 mL of 0.9% saline solution to prevent dehydration. The mice were anesthetized with a mixture of 3% isoflurane and oxygen, and their abdomen was shaved and disinfected with iodophor and isopropyl alcohol. A 0.5 cm incision was made along the median line of the abdomen, and the peritoneum was exposed using blunt dissection techniques. The peritoneal wall was then grasped with forceps, and a 0.5-1 cm incision was made at the white line. The desired volume of capsules was loaded into a sterile pipette and implanted into the abdominal cavity through the incision. Subsequently, the incision was sutured with 5-0 conical tip polydioxanone (PDS II) absorbable suture, and the skin was closed with wound clips and tissue glue.

[0196] (3) Recovery of materials

[0197] On day 14 post-implantation, the mice were euthanized by CO2 inhalation, followed by cervical dislocation. First, the skin and peritoneal wall were incised along the abdomen using forceps and scissors, and the tip of a wash bottle was inserted into the abdominal cavity to rinse all the material capsules inside the abdominal cavity with Krebs buffer (SOLABIO, Cat. #G0430) and collect them into a petri dish. After ensuring that all capsules were rinsed out or manually removed, if there were capsules directly adhered to the peritoneal tissue fibrosis, they were transferred to a 50 mL conical tube for subsequent processing and imaging.

[0198] (4) Cell recovery and flow cytometry (FACS) analysis

[0199] The recovered material capsules were first gently washed with Krebs buffer (SOLABIO, Cat. #G0430) and then transferred to a 15 mL conical tube. Then, the excess buffer was aspirated, 5 mL of 0.25% trypsin (Thermo, Cat. #25200056) was added, and incubated at 37°C for 5 minutes, and the reaction was terminated by adding 5 mL of sterile 1x PBS. The remaining cells were centrifuged at 300-400 g at 4°C and resuspended in a small amount (about 65 μL) of eBioscience staining buffer (Thermo, Cat. #00-4222), then 15 μL was aspirated and the cell count was performed using a cell counter (Countstar, Cat. #IN030101), and the remaining 50 μL was incubated with antibodies.

[0200] All samples were co-stained with two fluorescently labeled monoclonal antibodies for 25 minutes at 4°C in the dark. The antibodies included CD68 (1 μΐ per sample, 0.5 μg; CD68-Alexa 647, Clone FA-11, Cat #11-5931, BioLegend), Ly-6G (Gr-1) (1 μΐ per sample, 0.5 μg; Ly-6G-Alexa-647, Clone RB6-8C5, Cat #108418, BioLegend), and CD11b (0.4 μΐ per sample, 0.2 μg; CD11b-Alexa-488, Clone M1 / 70, Cat #101217, BioLegend). Then 2 mL of eBioscience Flow Cytometry Staining Buffer (Thermo, Cat #00-4222) was added and the samples were centrifuged at 400-500 g for 5 minutes at 4°C. The supernatant was aspirated and the washing step was repeated twice with staining buffer. After the third wash, each sample was resuspended in 500 μΐ of Flow Cytometry Staining Buffer and filtered through a 40 μιη filter (Thermo, Cat #22363547) and finally analyzed by flow cytometry using an Attune Flow Cytometer (Thermo, Cat #A24863).

[0201] At 14 days post-transplantation, the retrieved capsules were analyzed by flow cytometry (FACS) to characterize the different immune cell populations recruited by the Example 2 capsules in comparison to v / s and the Control Example 1 capsules in the peritoneal cavity. The results are shown in Figures 2 and 3, which demonstrate that the Example 2 capsules had significantly lower numbers of macrophages and neutrophils than the v / s and the Control Example 1 capsules, indicating their advantage in reducing the inflammatory response.

[0202] (5) Statistical Analysis

[0203] Data are presented as mean ± SEM, with N = 12 biological replicates per treatment group in the HeLa cell line viability experiments. In the in vivo biocompatibility tests, N = 4 mice per treatment group. All animals were included in the analysis unless unforeseen illness or death occurred. The animal population was randomly selected. The investigators were not blinded during experiments. Statistical significance analysis of FACS data was performed by unpaired two-tailed t-test or one-way ANOVA with Bonferroni’s multiple comparison correction, unless otherwise stated, which was implemented in GraphPad Prism 5.

[0204] Statistical significance is marked as: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0205] Test Example 4: Encapsulation for xenogenic cell transplantation

[0206] Transplantation of pancreatic beta cells is a promising therapeutic strategy that is expected to achieve functional cure of type 1 diabetes. We used the molecule of Example 2 for cell encapsulation in this system to extend the survival time of exogenous cells using its anti-fibrosis activity.

[0207] (1) Configuration of sodium alginate solution: Refer to Test Example 2 to configure the V / S, Control Example 1, and Example 2 sodium alginate solutions.

[0208] (2) Cell culture: Human pancreatic beta cells were obtained by differentiating embryonic stem cells (hPSCs) according to the method in the literature (Hogrebe NJ. Et al, Generation of insulin-producing pancreatic beta cells from multiple human stem cell lines. Nat Protoc. 2021, 16(9):4109-4143. doi:10.1038 / s41596-021-00560-y.). The specific steps are as follows:

[0209] Embryonic stem cell culture (Stage 0): Human H9 embryonic stem cell lines were expanded in Matrigel (Corning, 354277)-coated culture dishes, and mTeSR1 (stemcell, 1000276) medium was used for culture until the cells reached 90% confluence. Cells were digested into single-cell suspension by TrypLE and inoculated into new culture dishes at an appropriate density (usually 0.8 x 10^5 cells / cm 2 ) to prepare for starting differentiation.

[0210] Definitive endoderm induction (Stage 1): After starting differentiation, cells were cultured in BE1 basal medium containing Activin A (RD, 338-AC-CF-050, 100 ng / mL) and CHIR99021 (MCE, HY-10182G, 3 mM) for 24 hours, and then continued to culture for 3 days using only Activin A. This stage aims to induce definitive endoderm, and cells should express endoderm markers FOXA2 and SOX17.

[0211] Primitive gut tube generation (Stage 2): Definitive endoderm cells were cultured in BE2 medium containing KGF (RD, 251-KG-CF-050, 50 ng / mL) for 2 days to further differentiate into primitive gut tube cells.

[0212] Pancreatic Progenitor Generation (Stage 3 and Stage 4): Cells were cultured in BE3 medium containing KGF, SANT1 (sigma, S4572), TPPB (santa cruz, SC-204424), LDN193189 (MCE, HY-12071G), and Retinoic Acid (sigma, R2625) to first induce PDX1+ pancreatic progenitor cells (Stage 3), followed by promoting NKX6-1 expression by reducing the RA concentration (Stage 4). The key of this stage is to generate PDX1+ / NKX6-1+ double positive cells, which are the precursors of functional pancreatic islet beta cells.

[0213] Endocrine induction (Stage 5): Pancreatic progenitor cells were cultured in S5 medium containing SANT1, RA, XXI (Millipore, 565790), ALK5i II (Enzo, ALX-270-445), T3 (RD, 133.32), and latrunculin A (Cayman Chemical, 10010630) for 7 days to induce NEUROG3 expression and promote endocrine cell differentiation. Latrunculin A was used in the first 24 hours to relieve the inhibition of cytoskeleton on endocrine differentiation.

[0214] Islet beta cell maturation (Stage 6): Endocrine cells were cultured in serum-rich medium (ESFM) for more than 14 days to further mature into functional islet beta cells.

[0215] (3) Cell encapsulation

[0216] The cells were counted, and 2x10 6 Islet beta cells were placed in a 1.5 mL centrifuge tube, and after standing for 30 s, the cell mass was settled, the culture medium was aspirated, and 500 μL of the sodium alginate solution of Example 2 was added. After mixing by blowing, the mixture of cells and sodium alginate solution was transferred to a 1 mL screw syringe, the bubbles were discharged, a 30G needle was installed, and the cells were manually extruded drop by drop at a distance of about 20 cm from the liquid surface into 20 mM BaCl2. Crosslinking and hardening for 20 min, the BaCl2 crosslinking liquid was discarded, and after washing, the Example 2 capsule was obtained. The same method was used to prepare the control example 1 capsule by mixing an equal amount of cells with the control example 1 solution.

[0217] (4) Type 1 diabetes model of C57 / 6J mice

[0218] Male C57BL / 6J mice (Vantianlihua) weighing 23-25 g and 8-9 weeks of age were purchased and acclimated for one week before modeling. One day before modeling, the mice were fasted overnight (12-16 h). On the day of modeling, a citric acid buffer solution was prepared. Specifically, 2.1 g of citric acid (Mw: 210.14) was weighed into 100 mL of double-distilled water to fully dissolve and prepare solution A; 2.94 g of sodium citrate (Mw: 294.10) was weighed into 100 mL of double-distilled water to fully dissolve and prepare solution B; solutions A and B were mixed in a ratio of 1:1, and the pH was measured and adjusted to 4.2-4.5 using a pH meter. The buffer solution was used to dissolve STZ at a concentration of 15 mg / mL, and the first injection dose was 150 mg / kg. The second injection was performed three days later, and the preparation method was the same as the first time, and the dose was 100 mg / kg.

[0219] (5) Intraperitoneal transplantation of capsules: The method was the same as in Test Example 3.

[0220] (6) Blood glucose monitoring: The tail tip blood of the mice was taken every week, and the Roche blood glucose meter (Zhaozhaojin sampling) was used to detect the random blood glucose.

[0221] (7) Sample analysis: The mice were sacrificed for sample collection 66 days and 87 days after transplantation, and the intraperitoneal capsules were recovered, and the method was the same as in Test Example 3. The recovered capsules were subjected to bright field imaging to observe the FBR effect on their surface. The recovered capsules were stained using a live and dead staining kit (Solabio, CA1630), and the survival of beta cells was analyzed. Calcein AM in the kit is a live cell dye, which emits green fluorescence, and PI is a dead cell dye, which emits red fluorescence. The specific operation was carried out according to the kit instructions. After staining, a fluorescence microscope (Thermo, EVOS M5000) was used for imaging.

[0222] (8) Statistical analysis

[0223] The data are expressed as mean ± standard error (mean ± SEM), N = 2 mice per treatment group, and all animals are included in the analysis. The animal population was randomly selected.

[0224] The results show that the beta cells encapsulated using the molecule of Example 2 can effectively control the blood glucose to about 15 mM for a detection period of up to 66 days, while the average blood glucose of the modeling group is about 25 mM (see Figure 4). The sample analysis of the recovered capsules shows that most of the transplanted islet beta cells are still in a survival state, which indicates that the molecule of Example 2 can still effectively provide anti-fibrosis activity after encapsulating the cells, thereby prolonging the survival of the exogenous transplanted cells.

Claims

1. A modified monomer of the formula I ###0001### I wherein A is selected from the group consisting of a unit comprising a sugar; X is selected from -0-, -S- or -NR 2 -; R 2 is hydrogen or C 1-4 alkyl, said C 1-4 alkyl is optionally substituted by one or more halogen, oxo, hydroxy, amino, cyano or C 1-6 alkoxy; L1 is selected from C 1-30 Alkylene, C 1-30 Heteroalkylene; wherein, the C 1-30 Alkylene, C 1-30 Heteroalkyl groups are optionally substituted with one or more R L1 Replaced by, R L1 Selected from halogen, oxo, hydroxyl, amino, cyano, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy; L2is selected from R 3 selected from hydrogen or C 1-6 alkyl; L3is selected from C 1-10 alkylene, said C 1-10 alkylene is optionally substituted by one or more R L3 substituents, R L3 is selected from halogen, oxo, hydroxy, amino, cyano, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy; R 1 selected from R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from hydrogen or C 1-6 alkyl.

2. The modification monomer of claim 1, wherein, A is selected from the group consisting of a unit comprising a monosaccharide, a unit comprising a disaccharide or a unit comprising an oligosaccharide; A is preferably a monosaccharide selected from the group consisting of a substituted or unsubstituted hexose, hexulose, hexuronic acid, pentose, pentulose and penturonic acid; A is further preferably selected from the group consisting of a unit comprising a mannuronic acid and a unit comprising a guluronic acid.

3. The modification monomer according to claim 1 or 2, wherein, the modified monomer of Formula I is a modified monomer of Formula II, wherein X, L1, L2, L3and R 1 as defined in claim 1 ; Y1and Y2are each independently selected from hydrogen or -PO(OR y )2; R y is selected from hydrogen or C 1-6 alkyl.

4. The modification monomer according to any one of claims 1-3, wherein, X is -NR 2 - 5. The modification monomer according to any one of claims 1-4, wherein, L1is selected from C with 1-5 heteroatoms selected from N, O or S 1-30 heteroalkylene, optionally substituted with one or more R L1 substituents, R L1 as defined in claim 1 ; preferably, L1is selected from -(CH2CH20) m1 -, m1is selected from an integer between 1-15, optionally substituted with one or more R L1 substituents, R L1 as defined in claim 1 ; more preferably, L1is selected from -(CH2CH20) m1 -, m1is selected from an integer between 1-10, optionally substituted with one or more R L1 substituents, R L1 as defined in claim 1.

6. The modification monomer according to any one of claims 1-5, wherein, L2 is 7. The modification monomer according to any one of claims 1-6, wherein, L3 is C 1-6 alkylene, preferably methylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene, sec-butylene or t-butylene.

8. The modification monomer of any one of claims 1-7, wherein, R 1 selected from R 7 , R 8 and R 9 are each independently selected from hydrogen or C 1-6 alkyl, preferably, R 7 , R 8 and R 9 are each independently selected from hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl or t-butyl.

9. The modification monomer of any one of claims 1-8, wherein, the modified monomer of Formula I is a modified monomer of Formula III, wherein A, X, L3, R 7 , R 8 and R 9 are as defined in claim 1 ; m1 is selected from an integer between 1 and 15.

10. The modified monomer of claims 1-9, wherein, the modified monomer of Formula I is a modified monomer of Formula IV, wherein X, L3, R 7 , R 8 and R 9 are as defined in any one of claims 1-8; Y1and Y2are each independently selected from hydrogen or -PO(OR y )2; R y is selected from hydrogen or C 1-6 alkyl; m1 is selected from an integer between 1 and 15.

11. The modified monomer of claims 1-9, wherein, The modified monomer of Formula I is the following structure, A is a end is attached to -NH-.

12. A polymer comprising the modified monomer of formula I according to any one of claims 1 to 11.

13. A hydrogel comprising the polymer according to claim 12.

14. A capsule comprising the hydrogel according to claim 13; preferably, the hydrogel encapsulates a therapeutic agent, preferably a cell, preferably an islet cell.

15. The capsule according to claim 14, which is spherical; The average diameter of the capsule is preferably between 0.01 mm and 10 mm, more preferably between 0.1 mm and 10 mm, further preferably between 1 mm and 8 mm, more further preferably between 1 mm and 2 mm.

16. A pharmaceutical composition comprising the capsule according to claim 14 or 15 and one or more pharmaceutically acceptable excipients.

17. Use of the modified monomer according to any one of claims 1 to 11, the polymer according to claim 12, the hydrogel according to claim 13, the capsule according to claim 14 or 15 or the pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment of a disease selected from the group consisting of diabetes, haemophilia, skin damage, spinal cord injury, heart damage, osteoarthritic inflammation, preferably diabetes.

18. A device comprising the capsule according to claim 14 or 15 or the pharmaceutical composition according to claim 16.

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